Age

(years) Sex Mechanism

4 38 M Fall from

of injury

1 80 M Fall C2/Type II Frankel C

2 65 M Fall C2/Type I Frankel E

3 60 M Fall C6 – C7/A.3.1.1 Frankel E

5 67 M Fall C6-C7/B.3.2.2 Frankel C

6 69 F Fall C6 – C7/C.2.2.1 Frankel A

7 55 M Fall C6 – C7/A.3.1.1 Frankel E

8 39 M Fall T5 – T6/ A.3.3.1 Frankel D

9 23 F Fall T8/A.3.2.3 Frankel E

10 53 M Fall T8 – T9/B.2.2.2 Frankel C

11 65 F Fall T8 – T9/B.1.1.1 Frankel E

12 57 M Fall T9/A.3.2.3 Frankel E

13 64 M Fall T10 – T11/C.2.2.1 Frankel A

14 79 M Fall T10 – T11/A.3.2.1 Frankel B

15 40 M Fall T10 – T11/C.2.1.3 Frankel A

Accident T11 – T12/B.1.1.1 Frankel E

16 52 M Car

experience of spinal fractures occurring in patients suffering from AS and to highlight the difficulties that exist as far as both diagnosis and surgical management. In this study, twenty patients suffering from ankylosing spondylitis were operated due to a spinal fracture. The fracture was located at the cervical spine in 7 cases, at the thoracic spine in 9, at the thoracolumbar junction in 3 and at the lumbar spine in one case (Table 1). Three of the cervical fractures were managed by both anterior and posterior approaches while all the rest were managed only by posterior approach, having no intra-operative complications, but one case with superficial wound infection and two cases (patients with cervical injuries) with loosening of posterior screws without loss of stability. Early mobilization was encouraged in all the patients. Cervical collars were used for 3-6 months, and thoracolumbar spinal orthoses were used for 6-12 months. Neurological defects were revealed in 10 patients. In four of them, neurological signs were progressively developed after a time period of 4 to 15 days. The initial radiological study was negative for a spinal fracture in twelve patients (60%). Authors noted that there was a statistically significant improvement of Frankel neurological classification between the preoperative and postoperative evaluation, only 35% of patients presented an improvement (10% from Frankel B to Frankel D, 10% from Frankel C to Frankel D, 10% from Frankel C to Frankel E and 5% from Frankel D to Frankel E) while 65% of patients were in stable condition (15% from Frankel A to Frankel A and 50% from Frankel E to Frankel E). The authors concluded that operative treatment for AS is useful and effective. It usually succeeds the improvement of the patients' neurological status. They also stated that taking into consideration the cardiovascular problems that these patients have, anterior and posterior stabilization aren't always possible, and in such cases, posterior approach can be performed and give excellent results, while total operation time, blood loss and other complications are decreased.

Olerud et al., (1996) believe that in the cervical spine, where implant loosening is a considerable problem, the failure of support is presented mainly in cases where only anterior or only posterior stabilization was applied because the stabilizing system may not be able to confront the forces which act on it. Thus, both anterior and posterior stabilization of the spine should be applied, especially for the cervical and the thoraco-lumbar spine. Nevertheless, in everyday practice posterior stabilization is usually performed. This is in order to reduce the possible causal factors of intra-operative and postoperative complications, taking into consideration that the most of these patients have cardiovascular and pulmonary disorders caused by restrictive ankylosis of the thoracic cage and prolonging the operating time by performing double stabilization and thoracotomy aggravates cardiovascular function. Moreover, the anterior approach to the cervical-thoracic junction is extremely difficult in these patients due to the great inclination and the kyphosis that exists at this region.

Long stabilizing systems that offer support to a greater area of the spine and the parallel use of braces postoperatively have been used in order to strengthen the stabilization. Serin et al., (2004) showed that four levels posterior fixation is superior to two levels posterior fixation and a four levels fixation plus offset hook is the most stable. Tezeren and Kuru (2005) demonstrated that final outcome regarding sagittal index and anterior body compression is better in the long segment instrumentation group than in the short segment instrumentation group.

experience of spinal fractures occurring in patients suffering from AS and to highlight the difficulties that exist as far as both diagnosis and surgical management. In this study, twenty patients suffering from ankylosing spondylitis were operated due to a spinal fracture. The fracture was located at the cervical spine in 7 cases, at the thoracic spine in 9, at the thoracolumbar junction in 3 and at the lumbar spine in one case (Table 1). Three of the cervical fractures were managed by both anterior and posterior approaches while all the rest were managed only by posterior approach, having no intra-operative complications, but one case with superficial wound infection and two cases (patients with cervical injuries) with loosening of posterior screws without loss of stability. Early mobilization was encouraged in all the patients. Cervical collars were used for 3-6 months, and thoracolumbar spinal orthoses were used for 6-12 months. Neurological defects were revealed in 10 patients. In four of them, neurological signs were progressively developed after a time period of 4 to 15 days. The initial radiological study was negative for a spinal fracture in twelve patients (60%). Authors noted that there was a statistically significant improvement of Frankel neurological classification between the preoperative and postoperative evaluation, only 35% of patients presented an improvement (10% from Frankel B to Frankel D, 10% from Frankel C to Frankel D, 10% from Frankel C to Frankel E and 5% from Frankel D to Frankel E) while 65% of patients were in stable condition (15% from Frankel A to Frankel A and 50% from Frankel E to Frankel E). The authors concluded that operative treatment for AS is useful and effective. It usually succeeds the improvement of the patients' neurological status. They also stated that taking into consideration the cardiovascular problems that these patients have, anterior and posterior stabilization aren't always possible, and in such cases, posterior approach can be performed and give excellent results, while total operation time,

Olerud et al., (1996) believe that in the cervical spine, where implant loosening is a considerable problem, the failure of support is presented mainly in cases where only anterior or only posterior stabilization was applied because the stabilizing system may not be able to confront the forces which act on it. Thus, both anterior and posterior stabilization of the spine should be applied, especially for the cervical and the thoraco-lumbar spine. Nevertheless, in everyday practice posterior stabilization is usually performed. This is in order to reduce the possible causal factors of intra-operative and postoperative complications, taking into consideration that the most of these patients have cardiovascular and pulmonary disorders caused by restrictive ankylosis of the thoracic cage and prolonging the operating time by performing double stabilization and thoracotomy aggravates cardiovascular function. Moreover, the anterior approach to the cervical-thoracic junction is extremely difficult in these patients due to the great inclination and the kyphosis

Long stabilizing systems that offer support to a greater area of the spine and the parallel use of braces postoperatively have been used in order to strengthen the stabilization. Serin et al., (2004) showed that four levels posterior fixation is superior to two levels posterior fixation and a four levels fixation plus offset hook is the most stable. Tezeren and Kuru (2005) demonstrated that final outcome regarding sagittal index and anterior body compression is better in the long segment instrumentation group than in the short segment instrumentation

blood loss and other complications are decreased.

that exists at this region.

group.


Surgical Treatment After Spinal Trauma in Patients with Ankylosing Spondylitis 67

that these patients have, anterior and posterior stabilization aren't always possible. There is a need for wider multicenter studies to get a correct picture of the incidence and the

Bechterew, VM. (1979). The classic stiffening of the spine in flexion, a special form of

Belanger, TA.; Milam, RA IV.; Roh. JS.; & Bohlman, HH. (2005). Cervicothoracic extension

Bohlman. HH. (1979). Acute fractures and dislocation of the cervical spine. An analysis of

Bridwell, KH.; Lewis, SJ.; Edwards, C.; Lenke, LG.; Iffrig, TM.; Berra, A.; Baldus, C.; &

Bridwell, KH.; Lewis SJ.; Rinella A.; Lenke LG.; Baldus C.; & Blanke K. (2004). Pedicle

Broom MJ.; & Raycroft, JF. (1988). Complications of fractures of the cervical spine in

Calin A. (1985). Ankylosing spondylitis. *Clinics in Rheumatic Diseases*, Vol.11, No.1 (April

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Deutsch, H.; & Haid Jr, RW. (2008). Cervical ankylosing spondylitis, in Mummaneni PV.;

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*Anaesthesiologica Scandinavica,* Vol.49, No.2, (February 2005), pp. 264–266 Chin, KR.; & Ahn, J. (2007). Controlled cervical extension osteotomy for ankylosing

*1976),* Vol.32, No.17, (August 2007), pp. 1926–1929

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disease. *Clinical Orthopaedics and Related Research*, Vol.143, (September 1979), pp. 4-7

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ankylosing spondylitis. *Spine (Phila, Pa 1976),* Vol.13, No.7, (July 1988), pp. 763–766

patient with a large cervical anterior osteophyte: a case report. *Acta* 

spondylitis utilizing the Jackson operating table: technical note. *Spine (Phila Pa* 

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problems encountered in management of vertebral column trauma in AS.

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**5. References** 

pp. 2093–2101

1985), Review, pp. 41-60

978-1-57626-189-7

44–50


Table 1. Patients' data. (Adapted from Sapkas et al, BMC Musculoskeletal Disorders 2009 2, 10(1), 96)

#### **3.2.1 Perioperative complications**


#### **4. Conclusions**

Even minor injuries may cause fractures in an ankylosing spine. Patients with AS who sustain injuries of the spine are at greater risk of developing neurological impairment. These neurological disorders may be established at the time of injury but it is not unusual for them to become progressively, with several days delay. It is not an exaggeration to say that new back pain in patients with AS should be assumed to be caused by a fracture until proven otherwise. Thus, thorough clinical and radiological assessment should be performed in these patients and should be repeated for the first few weeks, especially if the patient complains of indefinable pain or if neurological disorders are noted. Accident and Emergency physicians should always bear in mind that simple radiological evaluation of these injuries may not be able to reveal fractures at first. CT and MRI are valuable tools in order to reveal these fractures.

The operative treatment of these injuries is useful and effective for these patients. It usually succeeds the improvement of the patients' neurological status, apart from cases where paraplegia is already established. However, the operative treatment is very demanding, especially when the cervical spine is concerned. Both anterior and posterior stabilization offer better support. Taking into consideration the cardiovascular and pulmonary problems that these patients have, anterior and posterior stabilization aren't always possible. There is a need for wider multicenter studies to get a correct picture of the incidence and the problems encountered in management of vertebral column trauma in AS.

#### **5. References**

66 Clinical and Molecular Advances in Ankylosing Spondylitis

Table 1. Patients' data. (Adapted from Sapkas et al, BMC Musculoskeletal Disorders 2009 2,

1. Intraoperative blood loss: Operations involving patients with AS have been associated with increased perioperative blood loss (Nash and Brown, 1979; Palm et al., 2002). It may partly be caused by high intra-abdominal pressures due to difficulties in patient

2. Trauma to dura mater: Due to chronic inflammation of the disease adhesions between dura meter, ligamentum flavum and bone may exist, making easier possible lacerations

3. Poor bone quality and internal fixation: The spine in AS is osteoporotic, due to the chronic inflammation and the bone atrophy. The consumption of corticosteroid drugs in the long run takes a serious part in this process making implant loosening a

Even minor injuries may cause fractures in an ankylosing spine. Patients with AS who sustain injuries of the spine are at greater risk of developing neurological impairment. These neurological disorders may be established at the time of injury but it is not unusual for them to become progressively, with several days delay. It is not an exaggeration to say that new back pain in patients with AS should be assumed to be caused by a fracture until proven otherwise. Thus, thorough clinical and radiological assessment should be performed in these patients and should be repeated for the first few weeks, especially if the patient complains of indefinable pain or if neurological disorders are noted. Accident and Emergency physicians should always bear in mind that simple radiological evaluation of these injuries may not be able to reveal fractures at first. CT and MRI are valuable tools in

The operative treatment of these injuries is useful and effective for these patients. It usually succeeds the improvement of the patients' neurological status, apart from cases where paraplegia is already established. However, the operative treatment is very demanding, especially when the cervical spine is concerned. Both anterior and posterior stabilization offer better support. Taking into consideration the cardiovascular and pulmonary problems

Posterior instrumentation/ T10-L2

Posterior instrumentation/ T10-L2

Posterior instrumentation/ T10-L2

Posterior instrumentation/ T12-L4

Frankel E

Frankel E

Frankel E

Frankel D

17 69 F Fall T12 – L1/A.3.2.3 Frankel E

18 38 M Fall T12 – L1/A.3.2.3 Frankel E

19 40 M Fall T12 – L1/B.1.1.1 Frankel E

20 55 M Fall L1 – L2/ B.1.1.1 Frankel B

**3.2.1 Perioperative complications** 

positioning (de Kleuver, 2006).

and tears of the dura.

considerable problem.

order to reveal these fractures.

**4. Conclusions** 

10(1), 96)


Surgical Treatment After Spinal Trauma in Patients with Ankylosing Spondylitis 69

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**Part 2** 

**HLA and Non-MHC Genes, Immune Response,** 

**and Gene Expression Studies** 


### **Part 2**

### **HLA and Non-MHC Genes, Immune Response, and Gene Expression Studies**

70 Clinical and Molecular Advances in Ankylosing Spondylitis

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**5** 

Wen-Chan Tsai

*Taiwan* 

*Kaohsiung Municipal Ta-Tung Hospital,* 

*Kaohsiung Medical University* 

**HLA-B27 and Ankylosing Spondylitis** 

Ankylosing spondylitis (AS) is a chronic inflammatory disease with potential disabling outcomes. Clinically, patients presented with inflammatory lower back pain, enthesis and alternated buttock pain (van der Linden & van der Heijde, 1998). Bernard Corner (1666- 1698) was the first physician who published the clinical features of AS in his medical thesis (Baker & Weisman, 2006). In the late 19th century, 3 independent physicians: Marie, Strupell, and Bechterew were able to describe the specific radiographic change in those patients by the help of the invention of radiology (Bywaters, 1983). But, still clinically, the boundary between AS and rheumatoid arthritis was unclear. Thanks to the discovery of rheumatoid factor which was strongly associated with rheumatoid arthritis, the distinction between these two arthritides became crystal clear. In those patients with inflammatory lower back pain and seronegative for rheumatoid factor, the diagnosis of ankylosing spondylitis became more popular in the early 1960 (Zeider et al., 2011). In 1963, American Rheumatism Association proposed a new nomenclature and classification for the rheumatic diseases. In this new edition, AS was specified as a complete different disease entity from rheumatoid arthritis (Blumberg et al., 1964). In addition to those different clinical characteristics, such as bone proliferation in enthesis site and sacroiliitis, in AS patients from those of patients with rheumatoid arthritis, AS is also known for its high association with HLA-B27. It has been known for more than 30 years since this association was discovered at 1973 (Schlosstein, 1973; Brewerton et al., 1973), although afterward, researchers found several HLA antigens were associated with other diseases (Invernizzi, 2011; McElroy, 2011; Piga, 2011), the strongest of any HLA antigens associated with human disease is HLA-B27 molecule. Hence, the roles of HLA-B27 in the pathogenesis and clinical manifestation of ankylosing spondylitis were among most frequent discussed topics in the past three

HLA-B27 is one of the HLA class I molecules which are highly polymorphic and plays major role in protective immunity against intracellular parasites including virus and bacteria (Bjorkman et al., 1987). Traditionally, HLA class I molecule is considered to present peptide antigens to cytotoxic (CD8+) T cells. X-ray crystallographic studies revealed that extracellular structure of heavy chain of class I molecule contained three components: α-1, α-2 and α-3 domains. α-1 and α-2 together with a β pleated intervening sequence to form a peptide

**1. Introduction** 

decades.

**2. Structure, subtypes and epidemiology of HLA-B27** 

### **HLA-B27 and Ankylosing Spondylitis**

#### Wen-Chan Tsai

*Kaohsiung Municipal Ta-Tung Hospital, Kaohsiung Medical University Taiwan* 

#### **1. Introduction**

Ankylosing spondylitis (AS) is a chronic inflammatory disease with potential disabling outcomes. Clinically, patients presented with inflammatory lower back pain, enthesis and alternated buttock pain (van der Linden & van der Heijde, 1998). Bernard Corner (1666- 1698) was the first physician who published the clinical features of AS in his medical thesis (Baker & Weisman, 2006). In the late 19th century, 3 independent physicians: Marie, Strupell, and Bechterew were able to describe the specific radiographic change in those patients by the help of the invention of radiology (Bywaters, 1983). But, still clinically, the boundary between AS and rheumatoid arthritis was unclear. Thanks to the discovery of rheumatoid factor which was strongly associated with rheumatoid arthritis, the distinction between these two arthritides became crystal clear. In those patients with inflammatory lower back pain and seronegative for rheumatoid factor, the diagnosis of ankylosing spondylitis became more popular in the early 1960 (Zeider et al., 2011). In 1963, American Rheumatism Association proposed a new nomenclature and classification for the rheumatic diseases. In this new edition, AS was specified as a complete different disease entity from rheumatoid arthritis (Blumberg et al., 1964). In addition to those different clinical characteristics, such as bone proliferation in enthesis site and sacroiliitis, in AS patients from those of patients with rheumatoid arthritis, AS is also known for its high association with HLA-B27. It has been known for more than 30 years since this association was discovered at 1973 (Schlosstein, 1973; Brewerton et al., 1973), although afterward, researchers found several HLA antigens were associated with other diseases (Invernizzi, 2011; McElroy, 2011; Piga, 2011), the strongest of any HLA antigens associated with human disease is HLA-B27 molecule. Hence, the roles of HLA-B27 in the pathogenesis and clinical manifestation of ankylosing spondylitis were among most frequent discussed topics in the past three decades.

#### **2. Structure, subtypes and epidemiology of HLA-B27**

HLA-B27 is one of the HLA class I molecules which are highly polymorphic and plays major role in protective immunity against intracellular parasites including virus and bacteria (Bjorkman et al., 1987). Traditionally, HLA class I molecule is considered to present peptide antigens to cytotoxic (CD8+) T cells. X-ray crystallographic studies revealed that extracellular structure of heavy chain of class I molecule contained three components: α-1, α-2 and α-3 domains. α-1 and α-2 together with a β pleated intervening sequence to form a peptide

HLA-B27 and Ankylosing Spondylitis 75

Till July 2011, 82 HLA-B27 subtypes were described based on nucleotide differences (International IMunoGene Tics information system [IMGT], 2011). Most nucleotide changes locate at exons 2 and 3 which encode the α-1 and α-2 domains. HLA-B\*27:05 is the most prevalent subtype and present in almost every population in the world. It was thought that HLA-B\*27:05 was the ancestor subtype, all other subtypes could have evolved from HLA-B\*27:05 by point mutation (B\*27:03), reciprocal recombination (B\*27:07, B\*27:09) and gene conversion (B\*27:01, B\*27:02, B\*27:04, B\*27:06). Following the ethnic migration and genetic evolution, HLA-B27 evolved into three ancestral pathways. Each pathway developed into a specific pattern. The first pattern was characterized by amino acid substitutions in the α-1 domain. HLA-B\*27:02 was the most frequent allele, followed by HLA-B\*27:03. This pattern is found largely in Africa, Middle Eastern and European groups. The second pattern contains a constant substitution at α-1 domain and variable substitutions at α-2 domain. HLA-B27:04 was the most prevalent subtype. This pattern is largely found in Eastern Asian such as Chinese, Thai and Korean. The third pattern contains a similar α-1 domain as HLA-B\*27:05 and variable substitution at α-2 domain. In which, HLA-B\*27:07 is the most prevalent subtype. This pattern is largely found in Middle East, but also in Turkey and

Fig. 2. Phylogenetic trees for the most common HLA-B27 subtypes (Adapted and modified

It is interesting to note that not all subtypes are associated with AS. In addition to B\*27:05, most alleles such as B\*27:01, B\*27:02, B\*27:03, B\*27:04, B\*27:10, B\*27:13, B\*27:14, B\*27:15 are documented to be associated with ankylosing spondylitis (Taurog, 2007). In Chinese

Greece (Reveille & Maganti, 2009) (Figure 2).

from Blanco-Gelaz et al., 2001).

binding groove. α-3 domain is the membrane-proximal portion of the heavy chain which interact with CD8 of cytotoxic T cells. Besides binding peptides, class I molecule must associate non-covalently with beta2-microglobulin to form the tri-molecule complex on the cell surface. In lack of any one of these molecules, the molecular stability of this tri-molecule complex will be weak and easy to be degraded (Natarajan et al., 1999; Madden et al., 1991). Through their different amino acid compositions at binding groove, different HLA class I antigens has their own specific selectivity of binding peptides (Madden et al., 1992). In addition, to the selectivity of binding peptides, differences in the amino acid composition also influence the strength of association between heavy chain and beta2-microglobulin. (David, 1997).

HLA-B27 is a unique HLA class I molecule, not only because of its high association with AS but also has characteristically different amino acid composition from other class I molecules. In brief, there are two important characteristic structures which are different from others: the presence of B pocket and the free thiol group of Cys67 (Madden, 1995; Powis et al, 2009). In the presence of B pocket in the binding groove, B27 anchoring peptides had a very specific P2 residue: arginine. Free thiol Cys67 residue made B27 molecule easy to form homodimer in the extracellular domain which has great impact on its physiological role (Allen et al, 1999). There is an astonishing distribution of HLA-B27 gene among world population, with highest prevalence in northern territory of the earth, Eskimos and Native American in the circumpolar area and north Canada were known for their high carrier rate and some of the world's highest prevalence rates of spondyloarthropathies are described in these groups (Peschken & Esdaile, 1999; Boyer et al., 1997). It was shown that the distribution of HLA-B27 had a tendency of a decreasing north-south gradient of prevalence and was speculated that the peculiar geographic distribution of HLA-B27 might reflect a genetic selection for better survival from microbial infection (Piazza et al., 1980) (Figure 1).

Fig. 1. The structure of HLA-B27 molecule. E45 and C67 are shared between all predisposing alleles. The presence of unpaired C67 made B27 molecule easy to form homodimers. In addition, the presence of H9 in the floor of β pleated sheet is critical for the stability of the heavy chain/β2-microglobulin complex.

binding groove. α-3 domain is the membrane-proximal portion of the heavy chain which interact with CD8 of cytotoxic T cells. Besides binding peptides, class I molecule must associate non-covalently with beta2-microglobulin to form the tri-molecule complex on the cell surface. In lack of any one of these molecules, the molecular stability of this tri-molecule complex will be weak and easy to be degraded (Natarajan et al., 1999; Madden et al., 1991). Through their different amino acid compositions at binding groove, different HLA class I antigens has their own specific selectivity of binding peptides (Madden et al., 1992). In addition, to the selectivity of binding peptides, differences in the amino acid composition also influence the strength of

HLA-B27 is a unique HLA class I molecule, not only because of its high association with AS but also has characteristically different amino acid composition from other class I molecules. In brief, there are two important characteristic structures which are different from others: the presence of B pocket and the free thiol group of Cys67 (Madden, 1995; Powis et al, 2009). In the presence of B pocket in the binding groove, B27 anchoring peptides had a very specific P2 residue: arginine. Free thiol Cys67 residue made B27 molecule easy to form homodimer in the extracellular domain which has great impact on its physiological role (Allen et al, 1999). There is an astonishing distribution of HLA-B27 gene among world population, with highest prevalence in northern territory of the earth, Eskimos and Native American in the circumpolar area and north Canada were known for their high carrier rate and some of the world's highest prevalence rates of spondyloarthropathies are described in these groups (Peschken & Esdaile, 1999; Boyer et al., 1997). It was shown that the distribution of HLA-B27 had a tendency of a decreasing north-south gradient of prevalence and was speculated that the peculiar geographic distribution of HLA-B27 might reflect a genetic selection for better survival from microbial infection (Piazza et al., 1980) (Figure 1).

Fig. 1. The structure of HLA-B27 molecule. E45 and C67 are shared between all predisposing alleles. The presence of unpaired C67 made B27 molecule easy to form homodimers. In addition, the presence of H9 in the floor of β pleated sheet is critical for the stability of the

heavy chain/β2-microglobulin complex.

association between heavy chain and beta2-microglobulin. (David, 1997).

Till July 2011, 82 HLA-B27 subtypes were described based on nucleotide differences (International IMunoGene Tics information system [IMGT], 2011). Most nucleotide changes locate at exons 2 and 3 which encode the α-1 and α-2 domains. HLA-B\*27:05 is the most prevalent subtype and present in almost every population in the world. It was thought that HLA-B\*27:05 was the ancestor subtype, all other subtypes could have evolved from HLA-B\*27:05 by point mutation (B\*27:03), reciprocal recombination (B\*27:07, B\*27:09) and gene conversion (B\*27:01, B\*27:02, B\*27:04, B\*27:06). Following the ethnic migration and genetic evolution, HLA-B27 evolved into three ancestral pathways. Each pathway developed into a specific pattern. The first pattern was characterized by amino acid substitutions in the α-1 domain. HLA-B\*27:02 was the most frequent allele, followed by HLA-B\*27:03. This pattern is found largely in Africa, Middle Eastern and European groups. The second pattern contains a constant substitution at α-1 domain and variable substitutions at α-2 domain. HLA-B27:04 was the most prevalent subtype. This pattern is largely found in Eastern Asian such as Chinese, Thai and Korean. The third pattern contains a similar α-1 domain as HLA-B\*27:05 and variable substitution at α-2 domain. In which, HLA-B\*27:07 is the most prevalent subtype. This pattern is largely found in Middle East, but also in Turkey and Greece (Reveille & Maganti, 2009) (Figure 2).

Fig. 2. Phylogenetic trees for the most common HLA-B27 subtypes (Adapted and modified from Blanco-Gelaz et al., 2001).

It is interesting to note that not all subtypes are associated with AS. In addition to B\*27:05, most alleles such as B\*27:01, B\*27:02, B\*27:03, B\*27:04, B\*27:10, B\*27:13, B\*27:14, B\*27:15 are documented to be associated with ankylosing spondylitis (Taurog, 2007). In Chinese

HLA-B27 and Ankylosing Spondylitis 77

developed earlier in patients who were HLA-B27+ than those HLA-B27- (Wu et al., 2009). B-27+ patients had higher incidence of anterior uveitis and hip joint involvement (Khan et al., 1977; Feldtkeller et al., 2003). The level of HLA-B27 mRNA had been claimed to be correlated with clinical disease activity in Chinese patients (Liu, 2006). It is interesting to find that in animal study, the copy number of HLA-B27 genes seemed to be a critical factor in determining the expression of a arthritis phenotype (Mammer et al, 1990). However, the effect of B27 homozygosity on the risk of disease development was controversial (van der Linden et al., 1984; Kim et al., 2009; Jaakkola et al., 2006). On the other hand, only a small percentage of B27-carriers developed AS. Twin studies suggest that susceptibility to AS is more than 90% inherited. HLA-B27 accounts for more than 50 % of this inheritance (Brown et al., 2000; Brown et al., 1997). Other genes must be involved in the disease development. A genome-wide study identified several other candidate genes such as ERAP1, IL-23R, IL1R2, ANTXR2, TNFSF15, TNFR1 and TRADD (Australo-Anglo-American Spondyloarthritis consortium [TSAC]; Reveille et al., 2010; Wellcome Trust Case Control Consortium [TASC]; Burton et al., 2010). Many of these candidate genes are hot topics for research recently

(Campbell et al., 2011; Chen et al., 2011; Layh-Schmitt & Colbert, 2008; Brown, 2010).

unfolded protein response were considered main streams of hypotheses.

**3.1 Theory of molecular mimicry and arthritogenic peptides** 

According to the above finding, theories of the disease pathogenesis were proposed. Hypotheses including molecular mimicry, arthritogenic peptide, free heavy chain and

A striking finding of the similarity of 6 consecutive amino acids of HLA-B27 to 6 consecutive amino acids of nitrogenase from *Klebsiella pneumonia* made researchers to propose that after the microbial infection, our immune system mis-recognized self-antigens as a target and launched an autoimmune response (Schwimmbeck & Oldstone, 1998). A Finnish group also identified that two bacterial proteins shared homology with HLA-B27, namely YadA (Yersinia adhesin) and OmpH, outer surface proteins of Yersinia and Salmonella, respectively (Lahesmaa et al., 1991). Further support of this hypothesis is the finding that HLA class II antigen associated with rheumatoid arthritis shared the same amino acid sequences with some viral antigens ( Albani & Carson, 1996 ). Data from serologic studies also indicated that patients with AS had high incidence of antibodies against microbial pathogens (Ewing et al., 1990). This study indicate that AS patient sera contain antibodies which were reactive to K. pneumoniae nitrogenase peptides and HLA B27.1 peptides, and that there are at least two epitopes in the alpha 1 domain at the groove region, that are autoantigenic. However, not all reports are consistent, later reports did not support the

finding of antibodies against self-antigens in patients with AS (de Vries et al., 1992).

As mentioned before, the unique amino acid composition, especially in the peptide –binding groove, made B27 molecule distinct. The presence of B pocket fits only arginine inside. This finding together with the knowledge of highly polymorphism of B27 alleles which were differentially associated with AS pave a new way to search for the presence of arthritogenic peptides with the ability to provoke arthritis. All these alleles differed from each other by only one or few amino acid changes, but interestingly, the association with AS were quite different. Most HLA-B27 alleles are found at a very low frequency, their association with AS are largely unknown. B\*27:05 is an ancestral type and associated with AS in almost every population in

population, B\*27:04 seemed to play major role in the pathogenesis of AS. Meta-analysis results showed a positive association between B\*27:04 and susceptibility to AS in Han population (Zang et al., 2011). Also in the Taiwanese population, susceptibility to AS was determined by the presence of HLA-B\*27:04 (Hou et al., 2007). In contrast, two subtypes, B\*27:06 and B\*27:09 have been reported not to be associated with AS and even considered to play a protective role. B\*27:06 is a common subtype in countries such as Indonesia, Singapore and Thailand. In Singapore Chinese, B\*27:06 had a significant negative association with AS (Ren et al., 1997). The same result also was reported from Taiwanese patients (Chen et al., 2002). Similarly, B\*27:09, a rare subtype primarily found in Sardinia island and southern Italy, was found to have negative association with AS (Fiorillo et al., 2003). In addition to B\*27:06 and B\*27:09, other HLA-B27 alleles such as B\*27:08 in Venezuela and B\*27:07 in Cyprus were claimed not to be associated with AS (Armas et al., 1999). But these results are not universal, in other populations the same alleles have been found in AS patients (Cipriani et al., 2003; Varnavidou-Nicolaidou et al., 2004; Paladini et al., 2005). (Table 1)


Table 1. The ethnic distribution and ankylosing spondylitis association of most frequent subtypes of HLA-B27.

#### **3. Hypotheses of the role of HLA-B27 in the pathogenesis of ankylosing spondylitis**

Since the discovery of the high association of HLA-B27 with AS, models have been proposed to explain the role of B27 in the pathogenesis of AS. Several aspects of research were made including epidemiology studies, analysis of molecular structure, transgenic animal models and analysis of environmental factors. Using these tools, during the past three decades, several hypotheses had been raised successfully to explain some aspects of this association, but still, like the story of blind men and elephant, each hypothesis touch tangentially different appendages of the animal. The correct description fit all aspects of the elephant remained unsolved.

In most population, B27 carry-rate was more than 80%in patients with ankylosing spondylitis. However, B27 carry-rate in patients with ankylosing spondylitis was less than 50% in some areas. For example, among African Americans, 50% of patients with ankylosing spondylitis possess HLA-B27 (Akkoc & Khan, 2006). Focus had been put on the effect and level of gene expression of B27 on disease presentation. It was reported that disease

population, B\*27:04 seemed to play major role in the pathogenesis of AS. Meta-analysis results showed a positive association between B\*27:04 and susceptibility to AS in Han population (Zang et al., 2011). Also in the Taiwanese population, susceptibility to AS was determined by the presence of HLA-B\*27:04 (Hou et al., 2007). In contrast, two subtypes, B\*27:06 and B\*27:09 have been reported not to be associated with AS and even considered to play a protective role. B\*27:06 is a common subtype in countries such as Indonesia, Singapore and Thailand. In Singapore Chinese, B\*27:06 had a significant negative association with AS (Ren et al., 1997). The same result also was reported from Taiwanese patients (Chen et al., 2002). Similarly, B\*27:09, a rare subtype primarily found in Sardinia island and southern Italy, was found to have negative association with AS (Fiorillo et al., 2003). In addition to B\*27:06 and B\*27:09, other HLA-B27 alleles such as B\*27:08 in Venezuela and B\*27:07 in Cyprus were claimed not to be associated with AS (Armas et al., 1999). But these results are not universal, in other populations the same alleles have been found in AS patients (Cipriani et al., 2003; Varnavidou-Nicolaidou et al., 2004; Paladini et al.,

B27 subtype **Ethnic distribution Association with AS** 

Table 1. The ethnic distribution and ankylosing spondylitis association of most frequent

**3. Hypotheses of the role of HLA-B27 in the pathogenesis of ankylosing** 

Since the discovery of the high association of HLA-B27 with AS, models have been proposed to explain the role of B27 in the pathogenesis of AS. Several aspects of research were made including epidemiology studies, analysis of molecular structure, transgenic animal models and analysis of environmental factors. Using these tools, during the past three decades, several hypotheses had been raised successfully to explain some aspects of this association, but still, like the story of blind men and elephant, each hypothesis touch tangentially different appendages of the animal. The correct description fit all aspects of the

In most population, B27 carry-rate was more than 80%in patients with ankylosing spondylitis. However, B27 carry-rate in patients with ankylosing spondylitis was less than 50% in some areas. For example, among African Americans, 50% of patients with ankylosing spondylitis possess HLA-B27 (Akkoc & Khan, 2006). Focus had been put on the effect and level of gene expression of B27 on disease presentation. It was reported that disease

+ + + + - + + -

Caucasians, Central American, American Indians

2005). (Table 1)

subtypes of HLA-B27.

elephant remained unsolved.

**spondylitis** 

Most ethnic groups

Sardinian, Italy

Caucasians, Cyprus, middle east Caucasians, Central Americans

Africans Asians Asians

**B\*27:05 B\*27:02 B\*27:03 B\*27:04 B\*27:06 B\*27:07 B\*27:08 B\*27:09**  developed earlier in patients who were HLA-B27+ than those HLA-B27- (Wu et al., 2009). B-27+ patients had higher incidence of anterior uveitis and hip joint involvement (Khan et al., 1977; Feldtkeller et al., 2003). The level of HLA-B27 mRNA had been claimed to be correlated with clinical disease activity in Chinese patients (Liu, 2006). It is interesting to find that in animal study, the copy number of HLA-B27 genes seemed to be a critical factor in determining the expression of a arthritis phenotype (Mammer et al, 1990). However, the effect of B27 homozygosity on the risk of disease development was controversial (van der Linden et al., 1984; Kim et al., 2009; Jaakkola et al., 2006). On the other hand, only a small percentage of B27-carriers developed AS. Twin studies suggest that susceptibility to AS is more than 90% inherited. HLA-B27 accounts for more than 50 % of this inheritance (Brown et al., 2000; Brown et al., 1997). Other genes must be involved in the disease development. A genome-wide study identified several other candidate genes such as ERAP1, IL-23R, IL1R2, ANTXR2, TNFSF15, TNFR1 and TRADD (Australo-Anglo-American Spondyloarthritis consortium [TSAC]; Reveille et al., 2010; Wellcome Trust Case Control Consortium [TASC]; Burton et al., 2010). Many of these candidate genes are hot topics for research recently (Campbell et al., 2011; Chen et al., 2011; Layh-Schmitt & Colbert, 2008; Brown, 2010).

According to the above finding, theories of the disease pathogenesis were proposed. Hypotheses including molecular mimicry, arthritogenic peptide, free heavy chain and unfolded protein response were considered main streams of hypotheses.

#### **3.1 Theory of molecular mimicry and arthritogenic peptides**

A striking finding of the similarity of 6 consecutive amino acids of HLA-B27 to 6 consecutive amino acids of nitrogenase from *Klebsiella pneumonia* made researchers to propose that after the microbial infection, our immune system mis-recognized self-antigens as a target and launched an autoimmune response (Schwimmbeck & Oldstone, 1998). A Finnish group also identified that two bacterial proteins shared homology with HLA-B27, namely YadA (Yersinia adhesin) and OmpH, outer surface proteins of Yersinia and Salmonella, respectively (Lahesmaa et al., 1991). Further support of this hypothesis is the finding that HLA class II antigen associated with rheumatoid arthritis shared the same amino acid sequences with some viral antigens ( Albani & Carson, 1996 ). Data from serologic studies also indicated that patients with AS had high incidence of antibodies against microbial pathogens (Ewing et al., 1990). This study indicate that AS patient sera contain antibodies which were reactive to K. pneumoniae nitrogenase peptides and HLA B27.1 peptides, and that there are at least two epitopes in the alpha 1 domain at the groove region, that are autoantigenic. However, not all reports are consistent, later reports did not support the finding of antibodies against self-antigens in patients with AS (de Vries et al., 1992).

As mentioned before, the unique amino acid composition, especially in the peptide –binding groove, made B27 molecule distinct. The presence of B pocket fits only arginine inside. This finding together with the knowledge of highly polymorphism of B27 alleles which were differentially associated with AS pave a new way to search for the presence of arthritogenic peptides with the ability to provoke arthritis. All these alleles differed from each other by only one or few amino acid changes, but interestingly, the association with AS were quite different. Most HLA-B27 alleles are found at a very low frequency, their association with AS are largely unknown. B\*27:05 is an ancestral type and associated with AS in almost every population in

HLA-B27 and Ankylosing Spondylitis 79

microglobulin, the tri-molecular complex is not stable enough. Only HLA-B27 was found to be able to express as free form. Amino acid residue 9 in the floor of β pleated sheet is critical for the stability of the heavy chain/β2-microglobulin complex. All the HLA-B27 subtypes contain histidine at this site, interestingly, two other classs I molecules, HLA-B73 and HLA-B40, which had been reported in a few cases of spondyloarthropathies, were found to have histidine at amino acid residue 9 (David, 1997). Histidine at this position was claimed to weaken the non-covalent interaction between heavy chain and β2-microglobulin. The unstable structure made HLA-B27 dissociate from beta2-microglobulin and presented as free form on the cell surface. It was proposed that free HLA-B27 bound different peptides from those stable forms. Higher percentage of free heavy chain-carrying monocytes was found in the peripheral blood and synovial fluid in patients with AS compared to normal population. The level of free heavy is correlated with sedimentation rate (Tsai et al., 2002). In addition, as mentioned before, in the presence of unpaired Cys67 free heavy chains have been shown to form homodimer. Expression of heavy chain homodimer on the surface of cell lines and AS patients' peripheral blood mononuclear cells was observed. (Kollnberger et

This HC homodimer was found to bind to NK inhibitory receptors KIR3DL1 and KIR3DL2 and LILRA1, LILRB2 alleles on the surface of NK, T and B cells. Patients with ankylosing spondylitis have higher level of Th17 cells expressing KIR3DL2 and responsive to B27 HC homodimer (Bowness et al., 2011). This hypothesis postulates that through this interaction,

In the last decade, another new theory was proposed calling "unfolded protein response". In this theory, researcher proposed that due to its unique structure, i.e.; the presence of B pocket and free form, HLA-B27 molecule is not properly folded in the endoplasmic reticulum. The accumulation of unfolded proteins in the endoplasmic reticulum induced stress reaction in the organelle and hence triggered inflammatory response. Most evidences came from animal study. Khare et al found high incidence of joint inflammation and ankylosis when HLA-B27 was transgenic into β2-microglobulin deficient mice (Khare et al., 1995). Later, investigator found a deficiency in class I molecule expression either due lack of

joint disease (Kinsbury et al, 2000). In animal study, transgenic rats were found to have increased IL-23 secretion when unfolded protein response was triggered by B27 molecule in the presence of pattern recognition receptor agonist (Colbert et al., 2010). This finding reminds us that IL-23R was found to be a susceptibility gene from genome-wide scan. Another observation from genome-wide scan shows that one of the peptide-trimming peptidase: ERAP1 is associated with AS. Defect in the function of ERAP1 might delay the folding process of HLA class I molecules (Evans et al., 2011). Patients with AS were found to have high level of chaperon proteins which were related to the folding process of class I

In conclusion, the high association of HLA-B27 with ankylosing spondylitis paved the path to the resolution of pathogenesis of this disease. Identification of this important finding

molecules in their macrophage derived from peripheral joint (Dong et al., 2002).

/TAP-) or β2-microglobulin was able to induce spontaneous inflammatory

NK cells, B cells and T cells were activated to induce the inflammatory reaction.

al., 2002)

peptides (TAP-

**4. Conclusion** 

**3.3 Unfolded protein response theory** 

the world. B\*27:04 is very prevalent in Asian country and also is thought to make individual susceptible to AS. On the contrary, B\*27:03, B\*27:06 and B\*27:09 were considered to play a protective role. Rare incidence of these allele-carriers developed AS. B\*27:03 was initially thought not to be associated with AS and only was prevalent in Black African population where B\*27:05 is also not associated with AS (Hill et al., 1991). Certainly, other genes might be involved in the pathogenesis. Recently, AS patients possessing B\*27:03 were found (Reveille et al., 2000). B\*27:09 were found in healthy inhabitants of Sardinian island but not in patients, who only carry B\*27:05. Although a case of AS possess B\*27:09 together with B\*14:03, another AS-associated allele in black Africans reported (Cauli et al., 2007). The amino acid sequence of B\*27:09 differed from that of B\*27:05 only in residue 116 (His vs Asp) .(de Castro, 2009) . Selfpeptide pVIPR binds to B\*27:05 in dual conformation but only one conventional form can be bound to B\*27:09 ( Hulsmeyer M et al., 2004). This different binding link might provoke different T cell response. B\*27:06 is found mainly in Southeastern Asia among healthy control, while other B27 alleles were associated with AS. B\*27:03 differs from B\*27:05 by the Y59H change located in the A pocket. B\*27:04 and B\*27:06 are closely related differ only by two amino acid changes, namely H114D and D116Y. Different amino acid sequences in the binding groove will change the polar-nonpolar interaction of heavy chain with peptides, hence causes different peptides anchoring to the groove. It was postulated that some disease-causing alleles of HLA-B27 selectively bound arthritogenic peptides derived from several intracellular parasites which were claimed to be triggering agents in reactive arthritis. Even more, some investigators found that peptides derived from self antigen, including peptide from HLA-B27 itself and cartilage were found to trigger CD8+ T cell response (Kuhne et al., 2009; Atagunduz et al., 2005). Three self peptides derived from cartilage/bone proteins showed homology to sequence of protein from arthritogenic bacteria. One of them, peptide PRGLLAWISR derived from chondoitin sulfate N-acetygalatosaminyltransferase 1 shared 8 amino acids with FhuB protein from *Yersinia Enterocolitica* and 7 amino acids with intracellular attenuator protein A from *Salmonella Typhimurium* (Dror LB et al., 2010). The presence of self peptide in the HLA binding groove with homologous sequence from arthritogenic bacteria forms the cornerstone of molecular mimicry. It is interesting to note that in addition to HLA-B27, HLA-B39 had similar B pocket was found to be associated with ankylosing spondylitis in those who were HLA-B27 negative. It was considered to harbor same peptide repertoire with HLA-B27 (Yamaguchi et al, 1995). Another observation is that HLA-B\*14:03 is a major MHC molecule associated with AS in Africa, it differs from HLA-B\*27:05 at 18 positions and shares only 3-5% peptide repertoires (Lopez-Larrea et al., 2002). Two important papers showed that CD8+ cytotoxic T cells are not essential for the arthritis to develop. In these observations, May et al use monoclonal antibody to deplete CD8+ T cells from peripheral circulation, however, arthritis and colitis still develop in the HLA-B27 transgenic rat (May et al.,2003). In addition, the same conclusion was obtained by the chemical deletion of CD8a gene expression which eliminated CD8+ T cells from peripheral blood (Taurog et al, 2009). The other observation revealed that CD4+ T cells, when transferred to athymic nude rat which had high level of HLA-B27/h2m expression in the bone marrow, developed arthritis (Taurog et al., 1999).

#### **3.2 Free heavy chain theory**

Another models focus on the molecular stability of tri-molecular complex indicating that due to unique amino acid composition at interface between HLA-B27 and beta2-

the world. B\*27:04 is very prevalent in Asian country and also is thought to make individual susceptible to AS. On the contrary, B\*27:03, B\*27:06 and B\*27:09 were considered to play a protective role. Rare incidence of these allele-carriers developed AS. B\*27:03 was initially thought not to be associated with AS and only was prevalent in Black African population where B\*27:05 is also not associated with AS (Hill et al., 1991). Certainly, other genes might be involved in the pathogenesis. Recently, AS patients possessing B\*27:03 were found (Reveille et al., 2000). B\*27:09 were found in healthy inhabitants of Sardinian island but not in patients, who only carry B\*27:05. Although a case of AS possess B\*27:09 together with B\*14:03, another AS-associated allele in black Africans reported (Cauli et al., 2007). The amino acid sequence of B\*27:09 differed from that of B\*27:05 only in residue 116 (His vs Asp) .(de Castro, 2009) . Selfpeptide pVIPR binds to B\*27:05 in dual conformation but only one conventional form can be bound to B\*27:09 ( Hulsmeyer M et al., 2004). This different binding link might provoke different T cell response. B\*27:06 is found mainly in Southeastern Asia among healthy control, while other B27 alleles were associated with AS. B\*27:03 differs from B\*27:05 by the Y59H change located in the A pocket. B\*27:04 and B\*27:06 are closely related differ only by two amino acid changes, namely H114D and D116Y. Different amino acid sequences in the binding groove will change the polar-nonpolar interaction of heavy chain with peptides, hence causes different peptides anchoring to the groove. It was postulated that some disease-causing alleles of HLA-B27 selectively bound arthritogenic peptides derived from several intracellular parasites which were claimed to be triggering agents in reactive arthritis. Even more, some investigators found that peptides derived from self antigen, including peptide from HLA-B27 itself and cartilage were found to trigger CD8+ T cell response (Kuhne et al., 2009; Atagunduz et al., 2005). Three self peptides derived from cartilage/bone proteins showed homology to sequence of protein from arthritogenic bacteria. One of them, peptide PRGLLAWISR derived from chondoitin sulfate N-acetygalatosaminyltransferase 1 shared 8 amino acids with FhuB protein from *Yersinia Enterocolitica* and 7 amino acids with intracellular attenuator protein A from *Salmonella Typhimurium* (Dror LB et al., 2010). The presence of self peptide in the HLA binding groove with homologous sequence from arthritogenic bacteria forms the cornerstone of molecular mimicry. It is interesting to note that in addition to HLA-B27, HLA-B39 had similar B pocket was found to be associated with ankylosing spondylitis in those who were HLA-B27 negative. It was considered to harbor same peptide repertoire with HLA-B27 (Yamaguchi et al, 1995). Another observation is that HLA-B\*14:03 is a major MHC molecule associated with AS in Africa, it differs from HLA-B\*27:05 at 18 positions and shares only 3-5% peptide repertoires (Lopez-Larrea et al., 2002). Two important papers showed that CD8+ cytotoxic T cells are not essential for the arthritis to develop. In these observations, May et al use monoclonal antibody to deplete CD8+ T cells from peripheral circulation, however, arthritis and colitis still develop in the HLA-B27 transgenic rat (May et al.,2003). In addition, the same conclusion was obtained by the chemical deletion of CD8a gene expression which eliminated CD8+ T cells from peripheral blood (Taurog et al, 2009). The other observation revealed that CD4+ T cells, when transferred to athymic nude rat which had high level of HLA-B27/h2m expression in the bone marrow, developed arthritis (Taurog et al., 1999).

Another models focus on the molecular stability of tri-molecular complex indicating that due to unique amino acid composition at interface between HLA-B27 and beta2-

**3.2 Free heavy chain theory** 

microglobulin, the tri-molecular complex is not stable enough. Only HLA-B27 was found to be able to express as free form. Amino acid residue 9 in the floor of β pleated sheet is critical for the stability of the heavy chain/β2-microglobulin complex. All the HLA-B27 subtypes contain histidine at this site, interestingly, two other classs I molecules, HLA-B73 and HLA-B40, which had been reported in a few cases of spondyloarthropathies, were found to have histidine at amino acid residue 9 (David, 1997). Histidine at this position was claimed to weaken the non-covalent interaction between heavy chain and β2-microglobulin. The unstable structure made HLA-B27 dissociate from beta2-microglobulin and presented as free form on the cell surface. It was proposed that free HLA-B27 bound different peptides from those stable forms. Higher percentage of free heavy chain-carrying monocytes was found in the peripheral blood and synovial fluid in patients with AS compared to normal population. The level of free heavy is correlated with sedimentation rate (Tsai et al., 2002). In addition, as mentioned before, in the presence of unpaired Cys67 free heavy chains have been shown to form homodimer. Expression of heavy chain homodimer on the surface of cell lines and AS patients' peripheral blood mononuclear cells was observed. (Kollnberger et al., 2002)

This HC homodimer was found to bind to NK inhibitory receptors KIR3DL1 and KIR3DL2 and LILRA1, LILRB2 alleles on the surface of NK, T and B cells. Patients with ankylosing spondylitis have higher level of Th17 cells expressing KIR3DL2 and responsive to B27 HC homodimer (Bowness et al., 2011). This hypothesis postulates that through this interaction, NK cells, B cells and T cells were activated to induce the inflammatory reaction.

#### **3.3 Unfolded protein response theory**

In the last decade, another new theory was proposed calling "unfolded protein response". In this theory, researcher proposed that due to its unique structure, i.e.; the presence of B pocket and free form, HLA-B27 molecule is not properly folded in the endoplasmic reticulum. The accumulation of unfolded proteins in the endoplasmic reticulum induced stress reaction in the organelle and hence triggered inflammatory response. Most evidences came from animal study. Khare et al found high incidence of joint inflammation and ankylosis when HLA-B27 was transgenic into β2-microglobulin deficient mice (Khare et al., 1995). Later, investigator found a deficiency in class I molecule expression either due lack of peptides (TAP- /TAP-) or β2-microglobulin was able to induce spontaneous inflammatory joint disease (Kinsbury et al, 2000). In animal study, transgenic rats were found to have increased IL-23 secretion when unfolded protein response was triggered by B27 molecule in the presence of pattern recognition receptor agonist (Colbert et al., 2010). This finding reminds us that IL-23R was found to be a susceptibility gene from genome-wide scan. Another observation from genome-wide scan shows that one of the peptide-trimming peptidase: ERAP1 is associated with AS. Defect in the function of ERAP1 might delay the folding process of HLA class I molecules (Evans et al., 2011). Patients with AS were found to have high level of chaperon proteins which were related to the folding process of class I molecules in their macrophage derived from peripheral joint (Dong et al., 2002).

#### **4. Conclusion**

In conclusion, the high association of HLA-B27 with ankylosing spondylitis paved the path to the resolution of pathogenesis of this disease. Identification of this important finding

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**6** 

*Mexico* 

**Humoral Immune Response** 

**with Ankylosing Spondylitis** 

*1Unidad Académica de Ciencias Químico Biológicas y Farmacéuticas-Universidad Autónoma de Nayarit, 2Unidad de Reumatología-Instituto Mexicano del Seguro Social HGZ No. 1 Tepic, Nayarit,* 

**to** *Salmonella* **Antigens and Polymorphisms** 

**in Receptors for the Fc of IgG in Patients** 

Angélica N. Rodríguez-Ocampo1, Juan Manuel Agraz-Cibrian1, Salvador Peña-Virgen2 and José Francisco Zambrano-Zaragoza1

Ankylosing Spondylitis (AS) is the prototype of an interrelated group of rheumatic diseases now named spondyloarthritides (SpA), otherwise known as spondyloarthropathies. Clinical features of this disease include inflammatory back pain, asymmetrical peripheral oligoarthritis, enthesitis, and specific organ involvement, such as anterior uveitis, psoriasis

AS is a chronic inflammatory disease primarily affecting the spine. Its major clinical features include sacroiliitis, loss of spinal mobility and spinal inflammation. The chronic inflammation leads to fibrosis and ossification, where bridging spurs of bone known as syndesmophytes form, especially at the edges of the inter-vertebral discs, thus producing

AS is a disease that affects more men than women, with a ratio of 2:1 (Feldtkeller *et al.*, 2003). The prevalence of the disease is between 0.1 and 1.4 %in general population. Studies conducted in different countries have shown that the incidence of AS is between 0.5 and 14

The diagnosis of AS is based more on clinical features than laboratory tests. Table 1 shows the criteria for diagnosing AS, according to the modified New York criteria (van der Linden et al., 1984). Further, in 1990, Amor and colleagues proposed the first set of classification criteria for the entire group of spondyloarthritis, allowing a patient to be classified as having spondyloarthritis whatever the presenting symptoms (Amor *et al.*, 1990). A different set of criteria for the entire group of spondyloarthritis was developed by the European Spondyloarhropathy Study Group (Dougados *et al.*, 1991), with inflammatory back pain and

and chronic inflammatory bowel disease (Braun & Sieper, 2007).

the ankylosing (Ebringer & Wilson, 2000).

per 100,000 people per year (Braun & Sieper, 2007).

**1. Introduction** 

Ma. de Jesús Durán-Avelar1, Norberto Vibanco-Pérez1,


### **Humoral Immune Response to** *Salmonella* **Antigens and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis**

Ma. de Jesús Durán-Avelar1, Norberto Vibanco-Pérez1, Angélica N. Rodríguez-Ocampo1, Juan Manuel Agraz-Cibrian1, Salvador Peña-Virgen2 and José Francisco Zambrano-Zaragoza1 *1Unidad Académica de Ciencias Químico Biológicas y Farmacéuticas-Universidad Autónoma de Nayarit, 2Unidad de Reumatología-Instituto Mexicano del Seguro Social HGZ No. 1 Tepic, Nayarit, Mexico* 

#### **1. Introduction**

84 Clinical and Molecular Advances in Ankylosing Spondylitis

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ankylosing spondylitis in Han populations: a meta-analysis. Clin Exp Rheumatol.

Ankylosing Spondylitis (AS) is the prototype of an interrelated group of rheumatic diseases now named spondyloarthritides (SpA), otherwise known as spondyloarthropathies. Clinical features of this disease include inflammatory back pain, asymmetrical peripheral oligoarthritis, enthesitis, and specific organ involvement, such as anterior uveitis, psoriasis and chronic inflammatory bowel disease (Braun & Sieper, 2007).

AS is a chronic inflammatory disease primarily affecting the spine. Its major clinical features include sacroiliitis, loss of spinal mobility and spinal inflammation. The chronic inflammation leads to fibrosis and ossification, where bridging spurs of bone known as syndesmophytes form, especially at the edges of the inter-vertebral discs, thus producing the ankylosing (Ebringer & Wilson, 2000).

AS is a disease that affects more men than women, with a ratio of 2:1 (Feldtkeller *et al.*, 2003). The prevalence of the disease is between 0.1 and 1.4 %in general population. Studies conducted in different countries have shown that the incidence of AS is between 0.5 and 14 per 100,000 people per year (Braun & Sieper, 2007).

The diagnosis of AS is based more on clinical features than laboratory tests. Table 1 shows the criteria for diagnosing AS, according to the modified New York criteria (van der Linden et al., 1984). Further, in 1990, Amor and colleagues proposed the first set of classification criteria for the entire group of spondyloarthritis, allowing a patient to be classified as having spondyloarthritis whatever the presenting symptoms (Amor *et al.*, 1990). A different set of criteria for the entire group of spondyloarthritis was developed by the European Spondyloarhropathy Study Group (Dougados *et al.*, 1991), with inflammatory back pain and

Humoral Immune Response to *Salmonella* Antigens

of IgG).

**spondylitis** 

role in the disease.

(Dominguez-Lopez *et al.*, 2000).

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 87

In addition, evidence of the importance of HLA-B27-bacteria interaction comes from work in animals, where HLA-B27-transgenic rats developed SpA-like features, but many transgene copies are needed to transfer the disease. Environmental factors also play a role, since HLA-B27-transgenic rats bred in a germ-free environment do not develop the disease, though gut

On the other hand, about 10-20% of HLA-B27-positive patients with reactive arthritis develop AS after 10-20 years. A possible central role of bacteria in the pathogenesis of SpA is further supported by the relation between Crohn's disease, HLA-B27 positivity, and ankylosing spondylitis, as 54% of HLA-B27-positive patients with Crohn's disease develop AS, but only 2.6% of HLA-B27-negative patients develop this disease. Leakage of the gut mucosa, as a result of the inflammation caused by colitis such as that found in Crohn's disease, leads to an interaction of the immune system with gut bacteria. In about 50% of patients with AS, chronic macroscopic or microscopic mucosal lesions resembling Crohn's

In this study, we provide support for the hypothesis of the interaction between an environmental factor (a bacterial antigen) and a genetic factor (a receptor for the Fc fragment

It has been postulated that infectious agents play a crucial role as triggering factors for some autoimmune diseases, such as rheumatoid arthritis and ankylosing spondylitis. However, the mechanisms by which these microbial antigens become involved in the aetiopathogenesis of

Humoral immune responses against bacteria such as *Klebsiella pneumoniae*, *Salmonella typhimurium*, *Shigella flexneri*, *Yersinia enterocolitica* and *Campylobacter jejuni* have been analyzed in patients with SpA, and it has been suggested that some microbial agents have a

*Klebsiella pneumoniae* has been considered the main microbial agent implicated as a triggering factor for the aetiopathogenesis of AS (Rashid & Ebringer, 2007). It has been reported that IgA antibodies to *Klebsiella pneumoniae* are significantly elevated in AS patients compared to healthy subjects (Blankenberg-Sprenkels *et al.*, 1998; Tani *et al.*, 1997). Moreover, an association between the heat shock protein (HSP) of 60 kDa from *Klebsiella pneumoniae* and AS has been evidenced because of the significantly higher levels of IgG antibodies in proportion to this protein observed in AS patients compared to control groups (Cancino-Diaz *et al.*, 1998; Parra-Campos *et al.*, 1996), while the cellular immune response, measured as lymphoproliferation (LP) against this protein, has also been reported

Other HSPs have been associated with HLA-B27-positive subjects, because of the higher levels of IgG antibodies observed, compared to HLA-B27-negative subjects, in particular HSP60 from *Klebsiella pneumoniae* and *Salmonella typhi* (Dominguez-Lopez *et al.*, 2002).

The antibody response against the lipopolisaccharide (LPS) of *Klebsiella pneumoniae*, *Escherichia coli*, *Salmonella typhimurium* and *Salmonella enteritidis* has been evaluated by

**2. Environmental factors involved in the pathogenesis of ankylosing** 

the disease remain unknown, though abundant data suggest this possibility.

flora contribute to the development of colitis (Braun & Sieper, 2007).

disease have been detected in the gut mucosa (Braun & Sieper, 2007).

peripheral arthritis as major entry criteria. Recognition of the drawbacks of criteria focused on a specific subtype, the Assessment of Spondyloarthritis International Society did a large cross-sectional study to propose new criteria on the basis of the two main clinical features identified in daily practice—eg, axial symptoms and peripheral involvement (Dougados & Baeten, 2011).

Clinical criteria




Radiological criterion


Definite ankylosing spondylitis is present if the radiological criterion is associated with at least one clinical criterion

Table 1. Modified New York criteria, 1984, for ankylosing spondylitis (Braun & Sieper, 2007).

Sacroiliitis on imaging\* plus one or more features of spondyloarthritis†

Or

HLA-B27 plus two or more other features of spondyloarthritis†

\*Active (acute) inflammation on MRI highly suggestive of sacroiliitis associated with spondyloarthritis or definite radiographic sacroiliitis according to modified New York criteria.

†Inflammatory back pain, arthritis, enthesitis (heel), uveitis, dactylitis, psoriasis, Crohn's disease or ulcerative colitis, good response to non-steroidal anti-inflammatory drugs, family history for spondyloarthritis, HLA-B27, or elevated C-reactive protein (a spondyloarthritis feature in the context of chronic back pain).

Table 2. Assessment of Spondyloarthritis International Society (ASAS) classification criteria for axial spondyloarthritis in patients with back pain for 3 months or more and age at onset younger than 45 years (Dougados & Baeten, 2011).

Although AS is of unknown aetiology, it is considered an autoimmune disease in which environmental and genetic factors are involved. There is a strong association with HLA-B27, as approximately 95% of AS patients are positive for this antigen. However, this association does not explain the cause of the disease. It has been reported that the risk of developing AS is about 5% for HLA-B27-positive subjects, but substantially higher for HLA-B27-positive relatives. However, most HLA-B27-positive individuals remain healthy. The HLA-B27 subtypes most clearly associated with AS are HLA-B\*2705 B\*2702, B\*2704 and B\*2707. The HLA-B\*2706 and B\*2709 subtypes do not appear to be associated with AS (Reveille & Arnett, 2005).

peripheral arthritis as major entry criteria. Recognition of the drawbacks of criteria focused on a specific subtype, the Assessment of Spondyloarthritis International Society did a large cross-sectional study to propose new criteria on the basis of the two main clinical features identified in daily practice—eg, axial symptoms and peripheral involvement (Dougados &



Definite ankylosing spondylitis is present if the radiological criterion is associated with at

Sacroiliitis on imaging\* plus one or more features of spondyloarthritis†

Or

HLA-B27 plus two or more other features of spondyloarthritis†

\*Active (acute) inflammation on MRI highly suggestive of sacroiliitis associated with spondyloarthritis or definite radiographic sacroiliitis according to modified New York

†Inflammatory back pain, arthritis, enthesitis (heel), uveitis, dactylitis, psoriasis, Crohn's disease or ulcerative colitis, good response to non-steroidal anti-inflammatory drugs, family history for spondyloarthritis, HLA-B27, or elevated C-reactive protein (a

Table 2. Assessment of Spondyloarthritis International Society (ASAS) classification criteria for axial spondyloarthritis in patients with back pain for 3 months or more and age at onset

Although AS is of unknown aetiology, it is considered an autoimmune disease in which environmental and genetic factors are involved. There is a strong association with HLA-B27, as approximately 95% of AS patients are positive for this antigen. However, this association does not explain the cause of the disease. It has been reported that the risk of developing AS is about 5% for HLA-B27-positive subjects, but substantially higher for HLA-B27-positive relatives. However, most HLA-B27-positive individuals remain healthy. The HLA-B27 subtypes most clearly associated with AS are HLA-B\*2705 B\*2702, B\*2704 and B\*2707. The HLA-B\*2706 and B\*2709 subtypes do not appear to be associated with AS (Reveille &

Table 1. Modified New York criteria, 1984, for ankylosing spondylitis (Braun & Sieper,

Baeten, 2011).

2007).

criteria.

Arnett, 2005).

Clinical criteria

Radiological criterion

least one clinical criterion

but are not relieved by rest


spondyloarthritis feature in the context of chronic back pain).

younger than 45 years (Dougados & Baeten, 2011).

In addition, evidence of the importance of HLA-B27-bacteria interaction comes from work in animals, where HLA-B27-transgenic rats developed SpA-like features, but many transgene copies are needed to transfer the disease. Environmental factors also play a role, since HLA-B27-transgenic rats bred in a germ-free environment do not develop the disease, though gut flora contribute to the development of colitis (Braun & Sieper, 2007).

On the other hand, about 10-20% of HLA-B27-positive patients with reactive arthritis develop AS after 10-20 years. A possible central role of bacteria in the pathogenesis of SpA is further supported by the relation between Crohn's disease, HLA-B27 positivity, and ankylosing spondylitis, as 54% of HLA-B27-positive patients with Crohn's disease develop AS, but only 2.6% of HLA-B27-negative patients develop this disease. Leakage of the gut mucosa, as a result of the inflammation caused by colitis such as that found in Crohn's disease, leads to an interaction of the immune system with gut bacteria. In about 50% of patients with AS, chronic macroscopic or microscopic mucosal lesions resembling Crohn's disease have been detected in the gut mucosa (Braun & Sieper, 2007).

In this study, we provide support for the hypothesis of the interaction between an environmental factor (a bacterial antigen) and a genetic factor (a receptor for the Fc fragment of IgG).

#### **2. Environmental factors involved in the pathogenesis of ankylosing spondylitis**

It has been postulated that infectious agents play a crucial role as triggering factors for some autoimmune diseases, such as rheumatoid arthritis and ankylosing spondylitis. However, the mechanisms by which these microbial antigens become involved in the aetiopathogenesis of the disease remain unknown, though abundant data suggest this possibility.

Humoral immune responses against bacteria such as *Klebsiella pneumoniae*, *Salmonella typhimurium*, *Shigella flexneri*, *Yersinia enterocolitica* and *Campylobacter jejuni* have been analyzed in patients with SpA, and it has been suggested that some microbial agents have a role in the disease.

*Klebsiella pneumoniae* has been considered the main microbial agent implicated as a triggering factor for the aetiopathogenesis of AS (Rashid & Ebringer, 2007). It has been reported that IgA antibodies to *Klebsiella pneumoniae* are significantly elevated in AS patients compared to healthy subjects (Blankenberg-Sprenkels *et al.*, 1998; Tani *et al.*, 1997). Moreover, an association between the heat shock protein (HSP) of 60 kDa from *Klebsiella pneumoniae* and AS has been evidenced because of the significantly higher levels of IgG antibodies in proportion to this protein observed in AS patients compared to control groups (Cancino-Diaz *et al.*, 1998; Parra-Campos *et al.*, 1996), while the cellular immune response, measured as lymphoproliferation (LP) against this protein, has also been reported (Dominguez-Lopez *et al.*, 2000).

Other HSPs have been associated with HLA-B27-positive subjects, because of the higher levels of IgG antibodies observed, compared to HLA-B27-negative subjects, in particular HSP60 from *Klebsiella pneumoniae* and *Salmonella typhi* (Dominguez-Lopez *et al.*, 2002).

The antibody response against the lipopolisaccharide (LPS) of *Klebsiella pneumoniae*, *Escherichia coli*, *Salmonella typhimurium* and *Salmonella enteritidis* has been evaluated by

Humoral Immune Response to *Salmonella* Antigens

Zaragoza *et al.*, 2009).

band.

band from *S. typhimurium* were recognized.

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 89

Considering that *Salmonella typhimurium* is in fact *Salmonella enterica* serovar *typhimurium*, we asked if the antigen recognized by patients with AS (p30) is an antigen specific to

To answer this question, a group of 28 patients with AS treated with non-steroidal antiinflammatory drugs and sulfazalasine, but without receiving tumour necrosis alpha blockers, and 28 non-AS-related healthy subjects were included to analyze the IgG and IgA humoral immune response against *Salmonella enterica* serovar *enteritidis* (*S. enteritidis)* by western-blot, using similar conditions, and protocols previously reported by us (Zambrano-

Our results show that 14/28 AS patients recognized a band with a relative molecular mass of 10 kDa (p10) from *S. enteritidis* with IgG antibodies, but that none of the subjects in the healthy group did (p<0.001, Figure 1). However, no differences in the recognition of *S. enteritidis* antigens were found when IgA antibodies were detected (Figure 2); suggesting that the antigenic behaviour of *S. enteritidis* and *S. typhimurium* are different, and only some antigens from each serovar could be important for the etiopathogenesis of AS. Additionally, it appear that only IgG antibodies anti- *S. enteritidis* could be important in the association with AS. These results are in agreement with Ahmadi et al, who did not find differences in

the antibody levels against *S. enteritidis* in patients with AS (Ahmadi *et al.*, 1998).

Fig. 1. Representative Western-blot strips showing antigens of *S. enteritidis* recognized by IgG antibodies in AS patients and healthy subjects. The bars on the left indicate the molecular masses of standard markers (kDa); the arrow indicates the recognized 10 kDa

In spite of the controversial role of bacterial antigens in the etiopathogenesis of AS, specifically the Gram negative bacterial antigens, we found a second candidate to be associated with AS, the p10 that could be a relevant antigen for patients with AS. Moreover, we also found that the humoral immune response to *S. typhimurium* is different to those for *S. enteritidis*, the antigens recognized and the antibody isotypes produced against those bacteria are different, because neither the 30 kDa band from *S. enteritidis* nor the 10 kDa

*Salmonella typhimurium*, or if it can be found in another serovar of *Salmonella enterica*.

ELISA, indicating that only the LPS from *Klebsiella pneumonaiae* and *Escherichia coli* are associated with AS, on account of the higher levels of IgG and IgA antibodies observed (Ahmadi *et al.*, 1998).

The association of *Salmonela spp*. with AS is supported by the presence of DNA from *Salmonella sp.* in the synovial fluid of patients with SpA (Pacheco-Tena *et al.*, 2001). On the other hand, the behaviour of *Salmonella typhimurium* is modified by the presence of HLA-B27 in transfected cells, because of the increased production of IL-6, IL-8 and IL-10 and the lower production of TNFα (Ekman *et al.*, 2002; Saarinen *et al.*, 2002).

In our laboratory, we have previously found that 71.4% of patients with AS and 14.3% of healthy subjects recognized a 30 kDa band (p30) from of *S. typhimurium* (p<0.001) by using anti-human IgG in a western-blot analysis. Moreover, the levels of IgA and IgG against a crude extract of *S. typhimurium* were significantly higher in AS patients than in healthy subjects, though no differences in IgM levels were found. When the antibody levels against electroeluted p30 were analyzed, we found that IgG and IgA against p30 were statistically higher in AS patients than in healthy subjects; however, as in the case of the response to the crude extract of *S. typhimurium*, the absorbance obtained in IgM to this antigen showed no significant differences between groups (Zambrano-Zaragoza *et al.*, 2009).

In the sera from AS patients and controls, all four IgG subclasses were found to be involved in the recognition of the p30 from *S. typhimurium*, but the frequency of IgG3 antibodies to p30 was statistically different in AS patients compared to healthy subjects (Zambrano-Zaragoza *et al.*, 2009).

These results showed that a 30 kDa band from *S. typhimurium* is recognized by the IgG antibodies of most AS patients, compared to healthy subjects, and suggest an association between a particular antigen of *S. typhimurium* (p30) and the disease.

An association of *S. typhimurium* with AS has been reported previously (Brown & Wordsworth, 1997; Leirisalo-Repo *et al.*, 2003), but no specific antigen of this bacterium has been reported until now. Nevertheless, in association with other bacterial antigens, certain proteins have been reported to be implicated as triggers of AS (Lahesmaa *et al.*, 1991). Thus, we have reported that the 30 kDa band from *S. typhimurium* could be differentially recognized by the immune response in AS patients, and hence be involved in the immunopathogenesis of AS (Zambrano-Zaragoza *et al.*, 2009). These findings led us to ask whether antigens from *S. enteritidis* are recognized in the same way by patients with AS, or if this response is specific for *S. typhimurium.*

The interaction between HLA-B27 and *S. enteritidis* has been reported by using mouse fibroblasts transfected with HLA-B27, HLA-B7, or beta2-microglobulin only. Although *S. enteritidis* invaded all three of these transfected cells with the same efficiency, more living intracellular *Salmonella* organisms were found in the HLA-B27 transfectants than in the other transfected cell lines, suggesting that the bactericidal effect is impaired in these cells. Moreover, impaired NO production in HLA-B27-transfected cells was indicated as a possible mechanism (Virtala *et al.*, 1997).

Another study, one using transfected human monocytic U937 cell lines, demonstrated that the expression of the HLA-B27 antigen does not influence the uptake of *S. enteritidis* into U937 cells in vitro. It is interesting to note that HLA-B27 markedly impaired the elimination of *S. enteritidis* in the HLA-B27-transfected U937 cells (Laitio *et al.*, 1997).

ELISA, indicating that only the LPS from *Klebsiella pneumonaiae* and *Escherichia coli* are associated with AS, on account of the higher levels of IgG and IgA antibodies observed

The association of *Salmonela spp*. with AS is supported by the presence of DNA from *Salmonella sp.* in the synovial fluid of patients with SpA (Pacheco-Tena *et al.*, 2001). On the other hand, the behaviour of *Salmonella typhimurium* is modified by the presence of HLA-B27 in transfected cells, because of the increased production of IL-6, IL-8 and IL-10 and the

In our laboratory, we have previously found that 71.4% of patients with AS and 14.3% of healthy subjects recognized a 30 kDa band (p30) from of *S. typhimurium* (p<0.001) by using anti-human IgG in a western-blot analysis. Moreover, the levels of IgA and IgG against a crude extract of *S. typhimurium* were significantly higher in AS patients than in healthy subjects, though no differences in IgM levels were found. When the antibody levels against electroeluted p30 were analyzed, we found that IgG and IgA against p30 were statistically higher in AS patients than in healthy subjects; however, as in the case of the response to the crude extract of *S. typhimurium*, the absorbance obtained in IgM to this antigen showed no

In the sera from AS patients and controls, all four IgG subclasses were found to be involved in the recognition of the p30 from *S. typhimurium*, but the frequency of IgG3 antibodies to p30 was statistically different in AS patients compared to healthy subjects (Zambrano-

These results showed that a 30 kDa band from *S. typhimurium* is recognized by the IgG antibodies of most AS patients, compared to healthy subjects, and suggest an association

An association of *S. typhimurium* with AS has been reported previously (Brown & Wordsworth, 1997; Leirisalo-Repo *et al.*, 2003), but no specific antigen of this bacterium has been reported until now. Nevertheless, in association with other bacterial antigens, certain proteins have been reported to be implicated as triggers of AS (Lahesmaa *et al.*, 1991). Thus, we have reported that the 30 kDa band from *S. typhimurium* could be differentially recognized by the immune response in AS patients, and hence be involved in the immunopathogenesis of AS (Zambrano-Zaragoza *et al.*, 2009). These findings led us to ask whether antigens from *S. enteritidis* are recognized in the same way by patients with AS, or

The interaction between HLA-B27 and *S. enteritidis* has been reported by using mouse fibroblasts transfected with HLA-B27, HLA-B7, or beta2-microglobulin only. Although *S. enteritidis* invaded all three of these transfected cells with the same efficiency, more living intracellular *Salmonella* organisms were found in the HLA-B27 transfectants than in the other transfected cell lines, suggesting that the bactericidal effect is impaired in these cells. Moreover, impaired NO production in HLA-B27-transfected cells was indicated as a

Another study, one using transfected human monocytic U937 cell lines, demonstrated that the expression of the HLA-B27 antigen does not influence the uptake of *S. enteritidis* into U937 cells in vitro. It is interesting to note that HLA-B27 markedly impaired the elimination

of *S. enteritidis* in the HLA-B27-transfected U937 cells (Laitio *et al.*, 1997).

lower production of TNFα (Ekman *et al.*, 2002; Saarinen *et al.*, 2002).

significant differences between groups (Zambrano-Zaragoza *et al.*, 2009).

between a particular antigen of *S. typhimurium* (p30) and the disease.

if this response is specific for *S. typhimurium.*

possible mechanism (Virtala *et al.*, 1997).

(Ahmadi *et al.*, 1998).

Zaragoza *et al.*, 2009).

Considering that *Salmonella typhimurium* is in fact *Salmonella enterica* serovar *typhimurium*, we asked if the antigen recognized by patients with AS (p30) is an antigen specific to *Salmonella typhimurium*, or if it can be found in another serovar of *Salmonella enterica*.

To answer this question, a group of 28 patients with AS treated with non-steroidal antiinflammatory drugs and sulfazalasine, but without receiving tumour necrosis alpha blockers, and 28 non-AS-related healthy subjects were included to analyze the IgG and IgA humoral immune response against *Salmonella enterica* serovar *enteritidis* (*S. enteritidis)* by western-blot, using similar conditions, and protocols previously reported by us (Zambrano-Zaragoza *et al.*, 2009).

Our results show that 14/28 AS patients recognized a band with a relative molecular mass of 10 kDa (p10) from *S. enteritidis* with IgG antibodies, but that none of the subjects in the healthy group did (p<0.001, Figure 1). However, no differences in the recognition of *S. enteritidis* antigens were found when IgA antibodies were detected (Figure 2); suggesting that the antigenic behaviour of *S. enteritidis* and *S. typhimurium* are different, and only some antigens from each serovar could be important for the etiopathogenesis of AS. Additionally, it appear that only IgG antibodies anti- *S. enteritidis* could be important in the association with AS. These results are in agreement with Ahmadi et al, who did not find differences in the antibody levels against *S. enteritidis* in patients with AS (Ahmadi *et al.*, 1998).

Fig. 1. Representative Western-blot strips showing antigens of *S. enteritidis* recognized by IgG antibodies in AS patients and healthy subjects. The bars on the left indicate the molecular masses of standard markers (kDa); the arrow indicates the recognized 10 kDa band.

In spite of the controversial role of bacterial antigens in the etiopathogenesis of AS, specifically the Gram negative bacterial antigens, we found a second candidate to be associated with AS, the p10 that could be a relevant antigen for patients with AS. Moreover, we also found that the humoral immune response to *S. typhimurium* is different to those for *S. enteritidis*, the antigens recognized and the antibody isotypes produced against those bacteria are different, because neither the 30 kDa band from *S. enteritidis* nor the 10 kDa band from *S. typhimurium* were recognized.

Humoral Immune Response to *Salmonella* Antigens

were higher in the AS group.

the Fc of IgG.

(Carter, 2010).

differences were found in both studies.

**3. Receptors for the Fc of IgG** 

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 91

the body, and its role in the inflammatory response observed. We found that in both patients and controls recognition of the p30 of *S. typhimurium* by serum antibodies was due to all of the IgG subclasses, but that the frequency and levels of IgG3 antibodies against p30

These results suggest that in the humoral immune response against a microbial antigen (p30) in a susceptible individual, the IgG3 antibodies produced against this protein could be involved in the pathogenesis of AS, and that the relationship between the humoral immune response observed and the inflammatory process could be explained by differences in the expression of specific polymorphisms of receptors for the Fc of IgG. However, this suggestion must be explored much more thoroughly. In this case, the p30 antigen could cross-react with a putative auto-antigen, and the resulting antibody response could be responsible for maintaining the inflammatory process, probably through the receptors for

Host genetics are better understood in AS compared to other types of SpAs. The strong link between AS and HLA-B27 has been known for years; the HLA-B27 is the major risk factor associated with AS, this molecule is present in many genetically diverse population, however other genes than HLA-B27 have been analyzed and associate with AS, such as endoplasmic reticulum endopeptidase I (ERAP1), interleukin 23 receptor (IL23R), and tumour necrosis factor receptor 1 (TNFR1) (Dougados & Baeten, 2011). Perhaps the definitive missing link lies in the recently discovered genetic contributions of AS and how these genes might co-localise with HLA-B27 in the presence of certain stool microflora

Fig. 3. Antibody levels against a crude extract of *S. enteritidis* in AS patients and healthy subjects. Levels of IgG (panel A) and IgA (panel B). The graph shows the median A490nm value for each group and the percentile at 25 and 75%. Non-statistically significant

Specific receptors for most immunoglobulin isotypes have been described. IgG represents the dominant antibody in plasma, while the receptors for the Fc of IgG (FcγR) play important roles in the initiation and regulation of many immunological and inflammatory

Considering that western blot is not a quantitative test, as it shows only the frequencies of recognition of some antigens, so a quantitative test must be used to determine the possible association of this antigen (p10) with AS.

Fig. 2. Representative Western-blot strips showing antigens from *S. enteritidis* recognized by IgA antibodies in AS patients and healthy subjects. The bars on the left indicate the molecular masses of standard markers (kDa).

To do this, we analyzed the IgG and IgA antibody levels against either a crude extract or the electroeluted 10 kDa band (p10) from *S. enteritidis* using ELISA, as we have done for *S. typhymurium* (Zambrano-Zaragoza *et al.*, 2009).

We did not find any statistically significant differences in the antibody levels against either the crude extract or p10 (Figures 3 and 4), indicating that neither the total antigens nor the p10 of *S. enteritidis* are associated with AS.

Although different bacteria have been associated as possible triggers of AS, in this study we argue that the antigenic differences present in related bacteria, such as *S. typhimurium* and *S. enteritidis*, could be differentiated by the immune response of patients with AS, and thus be involved in the aetiopathogenesis of the disease.

These results indicate that not all species of *Salmonella* are associated with the disease. As has been reported, the HSP60 of *S. typhi* (Dominguez-Lopez *et al*., 2009), and *S. typhimurium*, in particular, the p30 (Zambrano-Zaragoza *et al.*, 2009), are indeed associated with the illness. However, the relationship between the antibody levels observed and the mechanisms involved in the pathogenesis of AS has not yet been elucidated.

Considering that the differences in the IgG immune response observed could be due to a IgG subclass, we explored the IgG subclass that recognizes the p30 of *S. typhimurium* and found that patients with AS produce more IgG3 than healthy subjects (Zambrano-Zaragoza *et al.*, 2009), which suggests that the humoral immune response and, in particular, the IgG3 antibody levels, could play a role in the pathogenesis of AS.

IgG subclasses have been shown to be involved in autoimmunity, because of differences in the expression of Fc gamma receptors that explain the clearance of immune complexes from

Considering that western blot is not a quantitative test, as it shows only the frequencies of recognition of some antigens, so a quantitative test must be used to determine the possible

Fig. 2. Representative Western-blot strips showing antigens from *S. enteritidis* recognized by

To do this, we analyzed the IgG and IgA antibody levels against either a crude extract or the electroeluted 10 kDa band (p10) from *S. enteritidis* using ELISA, as we have done for *S.* 

We did not find any statistically significant differences in the antibody levels against either the crude extract or p10 (Figures 3 and 4), indicating that neither the total antigens nor the

Although different bacteria have been associated as possible triggers of AS, in this study we argue that the antigenic differences present in related bacteria, such as *S. typhimurium* and *S. enteritidis*, could be differentiated by the immune response of patients with AS, and thus be

These results indicate that not all species of *Salmonella* are associated with the disease. As has been reported, the HSP60 of *S. typhi* (Dominguez-Lopez *et al*., 2009), and *S. typhimurium*, in particular, the p30 (Zambrano-Zaragoza *et al.*, 2009), are indeed associated with the illness. However, the relationship between the antibody levels observed and the

Considering that the differences in the IgG immune response observed could be due to a IgG subclass, we explored the IgG subclass that recognizes the p30 of *S. typhimurium* and found that patients with AS produce more IgG3 than healthy subjects (Zambrano-Zaragoza *et al.*, 2009), which suggests that the humoral immune response and, in particular, the IgG3

IgG subclasses have been shown to be involved in autoimmunity, because of differences in the expression of Fc gamma receptors that explain the clearance of immune complexes from

mechanisms involved in the pathogenesis of AS has not yet been elucidated.

antibody levels, could play a role in the pathogenesis of AS.

IgA antibodies in AS patients and healthy subjects. The bars on the left indicate the

association of this antigen (p10) with AS.

molecular masses of standard markers (kDa).

*typhymurium* (Zambrano-Zaragoza *et al.*, 2009).

involved in the aetiopathogenesis of the disease.

p10 of *S. enteritidis* are associated with AS.

the body, and its role in the inflammatory response observed. We found that in both patients and controls recognition of the p30 of *S. typhimurium* by serum antibodies was due to all of the IgG subclasses, but that the frequency and levels of IgG3 antibodies against p30 were higher in the AS group.

These results suggest that in the humoral immune response against a microbial antigen (p30) in a susceptible individual, the IgG3 antibodies produced against this protein could be involved in the pathogenesis of AS, and that the relationship between the humoral immune response observed and the inflammatory process could be explained by differences in the expression of specific polymorphisms of receptors for the Fc of IgG. However, this suggestion must be explored much more thoroughly. In this case, the p30 antigen could cross-react with a putative auto-antigen, and the resulting antibody response could be responsible for maintaining the inflammatory process, probably through the receptors for the Fc of IgG.

Host genetics are better understood in AS compared to other types of SpAs. The strong link between AS and HLA-B27 has been known for years; the HLA-B27 is the major risk factor associated with AS, this molecule is present in many genetically diverse population, however other genes than HLA-B27 have been analyzed and associate with AS, such as endoplasmic reticulum endopeptidase I (ERAP1), interleukin 23 receptor (IL23R), and tumour necrosis factor receptor 1 (TNFR1) (Dougados & Baeten, 2011). Perhaps the definitive missing link lies in the recently discovered genetic contributions of AS and how these genes might co-localise with HLA-B27 in the presence of certain stool microflora (Carter, 2010).

Fig. 3. Antibody levels against a crude extract of *S. enteritidis* in AS patients and healthy subjects. Levels of IgG (panel A) and IgA (panel B). The graph shows the median A490nm value for each group and the percentile at 25 and 75%. Non-statistically significant differences were found in both studies.

#### **3. Receptors for the Fc of IgG**

Specific receptors for most immunoglobulin isotypes have been described. IgG represents the dominant antibody in plasma, while the receptors for the Fc of IgG (FcγR) play important roles in the initiation and regulation of many immunological and inflammatory

Humoral Immune Response to *Salmonella* Antigens

antibodies and the pathogenesis of the diseases is still unclear.

**3.1.1 Polymorphisms in FcγR and autoimmunity** 

can contribute to the development of autoimmune diseases.

*et al.*, 2001).

inflammation.

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 93

These haplotypes have distinct affinities for the different IgG subclasses. In this context, FcγRIIa-131H has a higher affinity for human IgG3 than FcγRIIa-131R. FcγRIIIa-158V has a higher affinity for IgG1 and IgG3 than FcγRIIIa-158-F, and FcγRIIIb-NA1 internalizes human IgG1- or IgG3-opsonized particles more efficiently than FcγRIIIb-NA2 (Dijstelbloem

Interest in the FcγR in the context of autoimmunity is rooted in mechanisms of immune complex handling (Salmon & Pricop, 2001), although these molecules participate in a much broader range of cell functions. In the case of rheumatic diseases such as AS, the antibody immune response has been described in several studies. However, the link between these

The FcγR offer a link between the humoral and inflammatory responses because of their differences in affinity for the different IgG subclasses and cellular distribution. Hence, the hypothesis is that if AS patients develop an immune response against bacterial antigens that depends predominantly on IgG3 antibodies, as we have reported previously (Zambrano-Zaragoza *et al.*, 2009), then these antibodies will bind to a haplotype of FcγR to promote

FcγR have been implicated in the pathogenesis of autoimmunity in different ways: 1) by maintaining the level of self-tolerance and increasing the activation threshold of autoreactive B cells; 2) by facilitating the elimination of some autoreactive B cells during development; 3) FcγR ligation can affect dendritic cell maturation; and, 4) FcγR are critical

On the other hand, an allelic variant of FcγR has been reported to be present in normal populations, but without exercising any impact on the normal functions of the immune system. However, some isophorms present in certain environmental and genetic contexts

Genetic variants of FcγR have been associated with different autoimmune and infectious diseases. Evidence supporting the role of the heterogeneity of FcγR in Systemic Lupus Erythematosus (SLE) has been controversial. SLE is the prototype of immune-complexmediated autoimmune diseases and several studies on associations with FcγRIIa and

In cases of myasthenia gravis, patients have been found to have a higher frequency of the FcγRIIIb-NA1 allele than FcγRIIb-NA2, compared to a control group (Raknes *et al.*, 1998).

In other autoimmune diseases, such as Wegener's granulomatosis, it has been reported that either FcγRIIa-131-RH or FcγRIIIa-158-VF represent an inheritable risk factor for the development of the disease (Dijstelbloem *et al.*, 1999), while other authors have reported an

In the case of rheumatic autoimmune diseases, it has been reported that FcγRIIIa-158VV is overrepresented in RA patients, FcγRIIIa-158VF was higher in healthy controls, and FcγRIIIa-158-FF is equally distributed in both populations. However, no association

for the elimination of circulating IgG immune complexes (Willcocks *et al.*, 2009).

FcγRIIIb have been published (Hong *et al.*, 2005; Michel *et al.*, 2000; Ni *et al.*, 2000).

between FcγRRIIIa-158-VV and clinical parameters was found (Nieto *et al.*, 2000).

association with FcγRIIIb-NA1 (van der Pol & van de Winkel, 1998).

processes, thus providing a crucial link between humoral and cellular immune responses. Ligation of these receptors triggers a variety of signals to develop effectors of the immune response, such as macrophage phagocytosis, antibody dependent cellular cytotoxicity (ADCC), neutrophil activation, cytokine release, degranulation and the inhibition of B cell activation (Dijstelbloem *et al.*, 2001; Ravetch & Bolland, 2001; van der Pol & van de Winkel, 1998).

Fig. 4. Antibody levels against the p10 of *S. enteritidis* in AS patients and healthy subjects. Levels of IgG (panel A) and IgA (panel B). Antibodies determined by ELISA, assayed with 0.3 g of antigen/well, and sera diluted to 1/400 in triplicate. The graph shows the median A490nm value for each group and the percentile at 25 and 75%. Non-statistically significant differences were found in both studies.

Three classes of FcγR have been reported on the surface of immune system cells FcγRI (CD64) that can bind to monomeric IgG, FcγRII (CD32) and FcγRIII (CD16), which then bind to immune-complexes.

These receptors can be divided into two types: activating (FcγRI, FcγRIIa and FcγRIIIa), characterized by the presence of an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain; and inhibiting (FcγRIIb), which contain an immunoreceptor tyrosine-based inhibition motif (ITIM) in their cytoplasmic domain (Dijstelbloem *et al.*, 2001).

Most cell types express both activating and inhibitory receptors. Therefore the cellular response depends on the relative expression of activating and inhibitory receptors. This ratio is also influenced by the cytokine environment (Cohen-Solal *et al.*, 2004). Moreover, the presence of allelic variants, with different affinities for selective the antibody isotypes support their role in the regulation of the inflammatory process due to the humoral immune response.

Three polymorphisms in the FcγR genes that affect the IgG binding affinities have been described: first, a G>A point mutation in FcγRIIa (rs1801274) that causes an H-131-R substitution; second, a T>G SNP at nucleotide 559 (rs396991) in FcγRIIIa that results in a V-158-F substitution; and, third, two combinations of five nucleotides (141, 147, 227, 277 and 349) in the exon 3 of IIIb that encode two isophorms, called FcγRIIIb-NA1 and FcγRIIIb-NA2 (Willcocks *et al.*, 2009).

These haplotypes have distinct affinities for the different IgG subclasses. In this context, FcγRIIa-131H has a higher affinity for human IgG3 than FcγRIIa-131R. FcγRIIIa-158V has a higher affinity for IgG1 and IgG3 than FcγRIIIa-158-F, and FcγRIIIb-NA1 internalizes human IgG1- or IgG3-opsonized particles more efficiently than FcγRIIIb-NA2 (Dijstelbloem *et al.*, 2001).

Interest in the FcγR in the context of autoimmunity is rooted in mechanisms of immune complex handling (Salmon & Pricop, 2001), although these molecules participate in a much broader range of cell functions. In the case of rheumatic diseases such as AS, the antibody immune response has been described in several studies. However, the link between these antibodies and the pathogenesis of the diseases is still unclear.

The FcγR offer a link between the humoral and inflammatory responses because of their differences in affinity for the different IgG subclasses and cellular distribution. Hence, the hypothesis is that if AS patients develop an immune response against bacterial antigens that depends predominantly on IgG3 antibodies, as we have reported previously (Zambrano-Zaragoza *et al.*, 2009), then these antibodies will bind to a haplotype of FcγR to promote inflammation.

#### **3.1.1 Polymorphisms in FcγR and autoimmunity**

92 Clinical and Molecular Advances in Ankylosing Spondylitis

processes, thus providing a crucial link between humoral and cellular immune responses. Ligation of these receptors triggers a variety of signals to develop effectors of the immune response, such as macrophage phagocytosis, antibody dependent cellular cytotoxicity (ADCC), neutrophil activation, cytokine release, degranulation and the inhibition of B cell activation (Dijstelbloem *et al.*, 2001; Ravetch & Bolland, 2001; van der Pol & van de Winkel,

Fig. 4. Antibody levels against the p10 of *S. enteritidis* in AS patients and healthy subjects. Levels of IgG (panel A) and IgA (panel B). Antibodies determined by ELISA, assayed with 0.3 g of antigen/well, and sera diluted to 1/400 in triplicate. The graph shows the median A490nm value for each group and the percentile at 25 and 75%. Non-statistically significant

Three classes of FcγR have been reported on the surface of immune system cells FcγRI (CD64) that can bind to monomeric IgG, FcγRII (CD32) and FcγRIII (CD16), which then bind

These receptors can be divided into two types: activating (FcγRI, FcγRIIa and FcγRIIIa), characterized by the presence of an immunoreceptor tyrosine-based activation motif (ITAM) in the cytoplasmic domain; and inhibiting (FcγRIIb), which contain an immunoreceptor tyrosine-based inhibition motif (ITIM) in their cytoplasmic domain (Dijstelbloem *et al.*,

Most cell types express both activating and inhibitory receptors. Therefore the cellular response depends on the relative expression of activating and inhibitory receptors. This ratio is also influenced by the cytokine environment (Cohen-Solal *et al.*, 2004). Moreover, the presence of allelic variants, with different affinities for selective the antibody isotypes support their role in the regulation of the inflammatory process due to the humoral immune

Three polymorphisms in the FcγR genes that affect the IgG binding affinities have been described: first, a G>A point mutation in FcγRIIa (rs1801274) that causes an H-131-R substitution; second, a T>G SNP at nucleotide 559 (rs396991) in FcγRIIIa that results in a V-158-F substitution; and, third, two combinations of five nucleotides (141, 147, 227, 277 and 349) in the exon 3 of IIIb that encode two isophorms, called FcγRIIIb-NA1 and FcγRIIIb-NA2

1998).

differences were found in both studies.

to immune-complexes.

2001).

response.

(Willcocks *et al.*, 2009).

FcγR have been implicated in the pathogenesis of autoimmunity in different ways: 1) by maintaining the level of self-tolerance and increasing the activation threshold of autoreactive B cells; 2) by facilitating the elimination of some autoreactive B cells during development; 3) FcγR ligation can affect dendritic cell maturation; and, 4) FcγR are critical for the elimination of circulating IgG immune complexes (Willcocks *et al.*, 2009).

On the other hand, an allelic variant of FcγR has been reported to be present in normal populations, but without exercising any impact on the normal functions of the immune system. However, some isophorms present in certain environmental and genetic contexts can contribute to the development of autoimmune diseases.

Genetic variants of FcγR have been associated with different autoimmune and infectious diseases. Evidence supporting the role of the heterogeneity of FcγR in Systemic Lupus Erythematosus (SLE) has been controversial. SLE is the prototype of immune-complexmediated autoimmune diseases and several studies on associations with FcγRIIa and FcγRIIIb have been published (Hong *et al.*, 2005; Michel *et al.*, 2000; Ni *et al.*, 2000).

In cases of myasthenia gravis, patients have been found to have a higher frequency of the FcγRIIIb-NA1 allele than FcγRIIb-NA2, compared to a control group (Raknes *et al.*, 1998).

In other autoimmune diseases, such as Wegener's granulomatosis, it has been reported that either FcγRIIa-131-RH or FcγRIIIa-158-VF represent an inheritable risk factor for the development of the disease (Dijstelbloem *et al.*, 1999), while other authors have reported an association with FcγRIIIb-NA1 (van der Pol & van de Winkel, 1998).

In the case of rheumatic autoimmune diseases, it has been reported that FcγRIIIa-158VV is overrepresented in RA patients, FcγRIIIa-158VF was higher in healthy controls, and FcγRIIIa-158-FF is equally distributed in both populations. However, no association between FcγRRIIIa-158-VV and clinical parameters was found (Nieto *et al.*, 2000).

Humoral Immune Response to *Salmonella* Antigens

and the 270 bp to the internal control.

different patterns obtained are shown.

min; and a final extension step at 72°C for 10 min.

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 95

seconds, 63°C for 30 seconds and 72°C for 90 seconds; and a final extension step at 72°C for 10 min. A 980 bp fragment was observed in 2% agarose gels together with the 270 bp

Fig. 5. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIa, showing the different genotypes: 1) molecular weight markers; 2) a homozygotic FcγRIIa-131RR subject; 3) a heterozygotic FcγRIIa-131HR subject; and, 4) a homozygotic FcγRIIa-131HH subject. In all cases, the 980 bp band corresponds to the fragment of FcγRIIa,

In order to determine the FcγRIIIa genotypes, two reactions were carried out for each sample, according to the method described by Van Der Berg et al., (Van Den Berg *et al.*, 2001). PCRs were performed by adding, 100 ng of genomic DNA, 200 µM of each dNTPs, 6 mM MgCl2, 20 ng of each control primer (Ctrl-1 and Ctrl-2), 200 ng of KIM-G(V) or KIM1(F) primer in their respective reaction, and 2.0 U of *Taq* polymerase (Invitrogen) to a 50 µL solution containing PCR buffer 1X (Invitrogen). PCR conditions were as follows: Denaturation for 10 min at 95°C, 37 cycles of 95°C for 30 seconds, 57°C for 20 seconds and 72°C for 25 seconds; and a final extension step at 72°C for 7 min. A 160 bp fragment was detected for the FcγRIIIa and a 270 bp fragment for the internal control used. In Figure 6 the

The FcγRIIIb genotyping was done in one single reaction to amplify the 141 bp (NA1) and/or 219 bp (NA2) fragments of the receptor, and the 480 bp of the internal control (HGH, Figure 7). PCRs were performed by adding, 100 ng of genomic DNA, and 200 ng of each primer (NA1, NA2, and HGHC) to a 25 µL solution containing 1 bead of pure-taq ready to go PCR (GE healthcare). PCR conditions were: PCR conditions were as follows: Denaturation for 3 min at 94°C, 30 cycles of 94°C for 1 min, 57°C for 2 min and 72°C for 1

Genotypes and allele frequencies were obtained by direct count. Differences in genotypes and allele frequencies between patients and controls were compared using the Chi square test. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated to estimate the effect of different alleles. All analyses were carried out using the OpenEpi v 2.0 software, with p≤0.05 set as the level of statistical significance, and results are shown in Table 5.

fragment of the internal control (TCR vα22 gene). Figure 5 shows a typical reaction.

#### **3.1.2 Polymorphisms in FcγR in ankylosing spondylitis**

Considering our previous findings, in which the humoral immune response against *S. typhimurium* was directed principally against p30, and the IgG subclass involved was mainly IgG3, we hypothesized that the relationship between the humoral response observed and the pathogenesis of the disease could be due (at least in part) to FcγR polymorphisms, because of the differences in IgG subclass affinity reported.

To test this hypothesis, the genotypification of FcγRIIa, FcγRIIIa and FcγRIIIb was carried out in a small cohort of 35 patients with AS and 120 non-AS-related individuals using the primers reported (Table 3) in a PCR protocol.


Table 3. PCR primers used for genotypification of FcγR.

As internal controls, primers to either an amplified 270 bp from the TCR Vα22 (Ctrl-1 and Ctrl-2) gene, or a 439 bp fragment of the human growth hormone gene (HGH-1 and HGH-2) were used (Table 4).


Table 4. PCR primers used as internal controls.

All participants were informed as to the nature of the study and written consent was obtained in accordance with the Helsinki Declaration. Blood samples were taken by venipuncture. The study design was previously approved by the local Ethics Committee.

Genomic DNA from each individual tested was extracted from whole peripheral blood using the easy DNA kit (Invitrogen). The FcγRIIa genotypes were determined using the amplification refractory mutation system-PCR (Raknes *et al.*, 1998). For each sample, two independent PCR reactions were carried out. PCRs were performed by adding 100 ng of genomic DNA, 200 µM of each dNTPs, 3 mM MgCl2, 20 ng of each control primer (Ctrl-1 and Ctrl-2), 200 ng of EC2-131-R or EC2-131-H primer and their respective reaction, and 2.0 U of *Taq* polymerase (Invitrogen) to a 50 µL solution containing PCR buffer 1X (Invitrogen). PCR conditions were as follows: Denaturation for 5 min at 94°C, 45 cycles of 94°C for 45

Considering our previous findings, in which the humoral immune response against *S. typhimurium* was directed principally against p30, and the IgG subclass involved was mainly IgG3, we hypothesized that the relationship between the humoral response observed and the pathogenesis of the disease could be due (at least in part) to FcγR polymorphisms,

To test this hypothesis, the genotypification of FcγRIIa, FcγRIIIa and FcγRIIIb was carried out in a small cohort of 35 patients with AS and 120 non-AS-related individuals using the

Receptor Name Sequence Ref

EC2-131R 5′-CCA GAA TGG AAA ATC CCA GAA ATT CTC TCG-3′

EC2-131H 5′-CCA GAA TGG AAA ATC CCA GAA ATT CTC TCA-3′ TM1 5′-CCA TTG GTG AAG AGC TGC CCA TGC TGG GCA-3′

As internal controls, primers to either an amplified 270 bp from the TCR Vα22 (Ctrl-1 and Ctrl-2) gene, or a 439 bp fragment of the human growth hormone gene (HGH-1 and HGH-2)

(Raknes *et al.*, 1998) HGH-2 5'-ATC CAC TCA CGG ATT TCT GTT GTG TTT C-3'

All participants were informed as to the nature of the study and written consent was obtained in accordance with the Helsinki Declaration. Blood samples were taken by venipuncture. The study design was previously approved by the local Ethics Committee.

Genomic DNA from each individual tested was extracted from whole peripheral blood using the easy DNA kit (Invitrogen). The FcγRIIa genotypes were determined using the amplification refractory mutation system-PCR (Raknes *et al.*, 1998). For each sample, two independent PCR reactions were carried out. PCRs were performed by adding 100 ng of genomic DNA, 200 µM of each dNTPs, 3 mM MgCl2, 20 ng of each control primer (Ctrl-1 and Ctrl-2), 200 ng of EC2-131-R or EC2-131-H primer and their respective reaction, and 2.0 U of *Taq* polymerase (Invitrogen) to a 50 µL solution containing PCR buffer 1X (Invitrogen). PCR conditions were as follows: Denaturation for 5 min at 94°C, 45 cycles of 94°C for 45

KIM-1(F) 5'-TCT CTG AAG ACA CAT TTC TAC TCC CTA A-3'

A013 5'-ATA TTT ACA GAA TGG CAC AGG-3'

Name Sequence Ref

NA-REV 5'-ATG GAC TTC TAG CTG CAC-3'

Table 3. PCR primers used for genotypification of FcγR.

HGH-1 5'-CAG TGC CTT CCC AAC CAT TCC CTT A-3'

Ctrl-1 5'-GAT TCA GTG ACC CAG ATG GAA GGG-3' Ctrl-2 5'-AGC ACA GAA GTA CAC CGC TGA GTC-3'

Table 4. PCR primers used as internal controls.

KIM-G(V) 5'-TCT CTG AAG ACA CAT TTC TAC TCC CTA C-3' (Van Den

NA1 5'-CAG TGG TTT CAC AAT GTG AA-3' (Raknes *et al.*, 1998) NA2 5'-CAA TGG TAC AGC GTG CTT-3'

(Raknes *et al.*, 1998)

Berg *et al.*, 2001)

**3.1.2 Polymorphisms in FcγR in ankylosing spondylitis** 

because of the differences in IgG subclass affinity reported.

primers reported (Table 3) in a PCR protocol.

FcγRIIa

FcγRIIIa

FcγRIIIb

were used (Table 4).

seconds, 63°C for 30 seconds and 72°C for 90 seconds; and a final extension step at 72°C for 10 min. A 980 bp fragment was observed in 2% agarose gels together with the 270 bp fragment of the internal control (TCR vα22 gene). Figure 5 shows a typical reaction.

Fig. 5. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIa, showing the different genotypes: 1) molecular weight markers; 2) a homozygotic FcγRIIa-131RR subject; 3) a heterozygotic FcγRIIa-131HR subject; and, 4) a homozygotic FcγRIIa-131HH subject. In all cases, the 980 bp band corresponds to the fragment of FcγRIIa, and the 270 bp to the internal control.

In order to determine the FcγRIIIa genotypes, two reactions were carried out for each sample, according to the method described by Van Der Berg et al., (Van Den Berg *et al.*, 2001). PCRs were performed by adding, 100 ng of genomic DNA, 200 µM of each dNTPs, 6 mM MgCl2, 20 ng of each control primer (Ctrl-1 and Ctrl-2), 200 ng of KIM-G(V) or KIM1(F) primer in their respective reaction, and 2.0 U of *Taq* polymerase (Invitrogen) to a 50 µL solution containing PCR buffer 1X (Invitrogen). PCR conditions were as follows: Denaturation for 10 min at 95°C, 37 cycles of 95°C for 30 seconds, 57°C for 20 seconds and 72°C for 25 seconds; and a final extension step at 72°C for 7 min. A 160 bp fragment was detected for the FcγRIIIa and a 270 bp fragment for the internal control used. In Figure 6 the different patterns obtained are shown.

The FcγRIIIb genotyping was done in one single reaction to amplify the 141 bp (NA1) and/or 219 bp (NA2) fragments of the receptor, and the 480 bp of the internal control (HGH, Figure 7). PCRs were performed by adding, 100 ng of genomic DNA, and 200 ng of each primer (NA1, NA2, and HGHC) to a 25 µL solution containing 1 bead of pure-taq ready to go PCR (GE healthcare). PCR conditions were: PCR conditions were as follows: Denaturation for 3 min at 94°C, 30 cycles of 94°C for 1 min, 57°C for 2 min and 72°C for 1 min; and a final extension step at 72°C for 10 min.

Genotypes and allele frequencies were obtained by direct count. Differences in genotypes and allele frequencies between patients and controls were compared using the Chi square test. Odds ratios (OR) with 95% confidence intervals (95% CI) were calculated to estimate the effect of different alleles. All analyses were carried out using the OpenEpi v 2.0 software, with p≤0.05 set as the level of statistical significance, and results are shown in Table 5.

Humoral Immune Response to *Salmonella* Antigens

to the control group (Dijstelbloem *et al.*, 2001).

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 97

Fig. 7. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIIa, showing the different genotypes: 1) a homozygotic FcγRIIIb-NA1 subject; 2) a heterozygotic FcγRIIIb-NA1/NA2 subject; 3) a homozygotic FcγRIIIb-NA2 subject; and, 4) molecular weight markers. In all cases, the 141 and 219 bp bands correspond to the NA1 and NA2 amplification products, respectively, while the 439 bp indicates the internal control.

substitution at position 131. The 131-H variant has higher affinity for IgG3 than 131R. FcγRIIIa, which is expressed on mononuclear phagocytes and natural killer cells, also has two co-dominantly expressed alleles, which differ at amino acid position 158 in the extracellular domain (valine or phenylalanine, respectively). FcγRIIIa allelic variants differ in IgG1 and IgG3 binding; VV homozygotes bind to IgG1 and IgG3 more avidly than do FF homozygotes, and here we report that AS patients show mostly the VV genotype, compared

As we have reported, all IgG subclasses anti-p30 are produced by AS patients, with higher levels compared to healthy controls (Zambrano-Zaragoza *et al.*, 2009). These data altogether with those of FcγR polymorphisms suggest that IgG3 could be important in maintenance of the inflammatory process observed in AS, because of the presence of the allelic variants FcγRIIa-131-HH and FcγRIIIa-158-VV in the group of AS patients, and then could contribute to the pathogenesis of the disease. Additionally IgG2 anti-p30, that is also produced by AS patients (Zambrano-Zaragoza *et al.*, 2009), could have a synergic action with IgG3 in the

It has been reported that mononuclear cells infiltrate the cartilaginous structures of sacroiliac joints and inter-vertebral discs, leading to destruction and ankylosis (Braun & Sieper, 2007). Thus, inflammation and the cellular immune response could be modulated through the FcγR. Therefore, we propose that one link between the humoral immune response against an environmental antigen such as p30 and AS could be through the FcγR and, more specifically, the 131-HH genotype of FcγRIIa, and the 158-VV genotype of

There is convincing evidence that imbalanced immune responses are responsible for autoimmune diseases such as arthritis, multiple sclerosis, and systemic lupus erythematosus (SLE). It is also widely accepted that many factors, including genetic and environmental components, are involved in the initiation and severity of autoimmune symptoms. Thus,

FcγRIIa, because the higher affinity of the 131-HH allelic variant for IgG2.

FcγRIIIa, which is involved in inflammation mediated by immune complexes.

Fig. 6. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIIa, showing the different genotypes for: 1) a homozygotic FcγRIIIa-158VV subject; 2) a heterozygotic FcγRIIIa-158VF subject; 3) a homozygotic FcγRIIIa-158FF subject; and, 4) molecular weight markers. In all cases, the 160 bp band corresponds to the fragment of FcγRIIIa, and the 270 bp to the internal control.

For FcγRIIa polymorphisms, our results show that 16/35 patients and 29/120 healthy subjects were homozygotic 131-HH (OR = 2.642; IC= 1.205-5.796; p= 0.013); eight out of 35 patients and 42/120 healthy subjects were homozygotic 131-RR (OR=0.5503; IC=0.230-1.318; p=0.1765); and 11/35 patients and 49/120 healthy subjects were heterozygotic 131-HR (OR=0.664; IC=0.298-1.480; p= 0.3170). These results suggest that the homozygotic 131-HH of FcγRIIa allele could be associated with AS. Moreover, the frequency of allele H was significantly higher in AS patients compared to healthy controls (OR= 1.980; 95%CI= 1.149- 3.412, p= 0.0138).

For FcγRIIIa, 7/35 AS patients and 5/120 healthy subjects had the 158-VV genotype (OR= 5.75; IC= 1.698-19.47; p= 0.002); 14/35 AS patients and 51/120 healthy subjects showed the 158-FF genotype (OR= 0.902; IC= 0.419-1.942; p=0.792); and 14/35 and 64/120 showed the 158-VF genotype (OR= 0.737; IC = 0.364-1.569; p= 0.0427). Our results suggest that the homocygotic 158-V of FcγRIIIa could be associated with AS.

In the case of FcγRIIIb, we found that 9 out of 35 AS patients and 16/120 healthy subjects had NA1/NA1 genotype (OR=2.25; IC=0.894-5.662; p= 0.080); 15/35 AS patients and 64/120 healthy subjects had the NA2/NA2 genotype (OR=0.656; IC=0.307-1.402; p= 0.275); and 11/35 AS patients and 40/120 healthy subjects were NA1/NA2 (OR= 0.917; IC=0.408-2.057; p=0.833). No significant association was found between the NA1 and NA2 haplotypes of FcγRIIIb and AS.

In spite of the statistical differences among groups, it is important to emphasize that the study population is small and, therefore, the results must be taken as only indicative of the possible role of these polymorphisms in the pathogenesis of AS; they are by no means conclusive.

FcγRIIa which is expressed in most immune cells has two allelic variants with different affinities for IgG subclasses. The mutation is G519A that results in an amino acid

Fig. 6. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIIa, showing the different genotypes for: 1) a homozygotic FcγRIIIa-158VV subject; 2) a heterozygotic FcγRIIIa-158VF subject; 3) a homozygotic FcγRIIIa-158FF subject; and, 4) molecular weight markers. In all cases, the 160 bp band corresponds to the fragment of

For FcγRIIa polymorphisms, our results show that 16/35 patients and 29/120 healthy subjects were homozygotic 131-HH (OR = 2.642; IC= 1.205-5.796; p= 0.013); eight out of 35 patients and 42/120 healthy subjects were homozygotic 131-RR (OR=0.5503; IC=0.230-1.318; p=0.1765); and 11/35 patients and 49/120 healthy subjects were heterozygotic 131-HR (OR=0.664; IC=0.298-1.480; p= 0.3170). These results suggest that the homozygotic 131-HH of FcγRIIa allele could be associated with AS. Moreover, the frequency of allele H was significantly higher in AS patients compared to healthy controls (OR= 1.980; 95%CI= 1.149-

For FcγRIIIa, 7/35 AS patients and 5/120 healthy subjects had the 158-VV genotype (OR= 5.75; IC= 1.698-19.47; p= 0.002); 14/35 AS patients and 51/120 healthy subjects showed the 158-FF genotype (OR= 0.902; IC= 0.419-1.942; p=0.792); and 14/35 and 64/120 showed the 158-VF genotype (OR= 0.737; IC = 0.364-1.569; p= 0.0427). Our results suggest that the

In the case of FcγRIIIb, we found that 9 out of 35 AS patients and 16/120 healthy subjects had NA1/NA1 genotype (OR=2.25; IC=0.894-5.662; p= 0.080); 15/35 AS patients and 64/120 healthy subjects had the NA2/NA2 genotype (OR=0.656; IC=0.307-1.402; p= 0.275); and 11/35 AS patients and 40/120 healthy subjects were NA1/NA2 (OR= 0.917; IC=0.408-2.057; p=0.833). No significant association was found between the NA1 and NA2 haplotypes of

In spite of the statistical differences among groups, it is important to emphasize that the study population is small and, therefore, the results must be taken as only indicative of the possible role of these polymorphisms in the pathogenesis of AS; they are by no means

FcγRIIa which is expressed in most immune cells has two allelic variants with different affinities for IgG subclasses. The mutation is G519A that results in an amino acid

FcγRIIIa, and the 270 bp to the internal control.

homocygotic 158-V of FcγRIIIa could be associated with AS.

3.412, p= 0.0138).

FcγRIIIb and AS.

conclusive.

Fig. 7. A representative agarose gel (2%) of PCR products obtained from the amplification of FcγRIIIa, showing the different genotypes: 1) a homozygotic FcγRIIIb-NA1 subject; 2) a heterozygotic FcγRIIIb-NA1/NA2 subject; 3) a homozygotic FcγRIIIb-NA2 subject; and, 4) molecular weight markers. In all cases, the 141 and 219 bp bands correspond to the NA1 and NA2 amplification products, respectively, while the 439 bp indicates the internal control.

substitution at position 131. The 131-H variant has higher affinity for IgG3 than 131R. FcγRIIIa, which is expressed on mononuclear phagocytes and natural killer cells, also has two co-dominantly expressed alleles, which differ at amino acid position 158 in the extracellular domain (valine or phenylalanine, respectively). FcγRIIIa allelic variants differ in IgG1 and IgG3 binding; VV homozygotes bind to IgG1 and IgG3 more avidly than do FF homozygotes, and here we report that AS patients show mostly the VV genotype, compared to the control group (Dijstelbloem *et al.*, 2001).

As we have reported, all IgG subclasses anti-p30 are produced by AS patients, with higher levels compared to healthy controls (Zambrano-Zaragoza *et al.*, 2009). These data altogether with those of FcγR polymorphisms suggest that IgG3 could be important in maintenance of the inflammatory process observed in AS, because of the presence of the allelic variants FcγRIIa-131-HH and FcγRIIIa-158-VV in the group of AS patients, and then could contribute to the pathogenesis of the disease. Additionally IgG2 anti-p30, that is also produced by AS patients (Zambrano-Zaragoza *et al.*, 2009), could have a synergic action with IgG3 in the FcγRIIa, because the higher affinity of the 131-HH allelic variant for IgG2.

It has been reported that mononuclear cells infiltrate the cartilaginous structures of sacroiliac joints and inter-vertebral discs, leading to destruction and ankylosis (Braun & Sieper, 2007). Thus, inflammation and the cellular immune response could be modulated through the FcγR. Therefore, we propose that one link between the humoral immune response against an environmental antigen such as p30 and AS could be through the FcγR and, more specifically, the 131-HH genotype of FcγRIIa, and the 158-VV genotype of FcγRIIIa, which is involved in inflammation mediated by immune complexes.

There is convincing evidence that imbalanced immune responses are responsible for autoimmune diseases such as arthritis, multiple sclerosis, and systemic lupus erythematosus (SLE). It is also widely accepted that many factors, including genetic and environmental components, are involved in the initiation and severity of autoimmune symptoms. Thus,

Humoral Immune Response to *Salmonella* Antigens

in the inflammatory process observed in AS.

**4. Conclusion** 

**5. Acknowledgments** 

(PIFI)-2001, 2003, 2008 and 2010.

Ornelas Muñoz for their technical assistance.

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 99

Fig. 8. A hypothetical model of the interaction between the environmental (the p30 from *S. typhimurium*)*,* and the genetic (allelic variants FcγRIIA-131-HH, and FcγRIIIa-158-VV) factor

In this chapter, we proposed a link between the humoral immune response in a susceptible

The immune response against *S. typihumium* appears to be strain-specific, because of the difference observed with the humoral immune response against *S. enteritidis*. Patients with AS produce IgG3 antibodies against the p30 of *S. typhimurium,* in contrast to healthy controls. Moreover, patients with AS have higher frequencies of FcγRIIa-131-HH and FcγRIIIa-158-VV, which suggests a link between these environmental and genetic factors.

This work was supported in part by the *Programa Integral de Fortalecimiento Institucional*

The authors would like to thank Georgina Nataly Ibarra Muro and Alma Senorina de Jesús

individual and a genetic factor, FcγRIIa-131-HH, and FcγRIIIa-158-VV.


Table 5. Genotype and allele frequencies of FcγRIIa, FcγRIIIa and FcγRIIIb polymorphisms in patients with AS and controls.

identifying these components could prove helpful in gaining further insight into these diseases and developing novel immunotherapeutic strategies to interfere with chronic inflammation.

On the basis of these findings, we proposed a hypothetical model of interaction between two factors: one environmental, the other genetic (Figure 8). In this model, the immune response against *S. typhimurium* in a susceptible subject leads to the recognition of p30 and the production of IgG3 antibodies. p30 should cross-react with a putative auto-antigen, such that the humoral immune response is maintained by this putative auto-antigen, and it is responsible of the maintenance of the antibody levels.

Then, in the susceptible subject, the FcγRIIa-131-HH and FcγRIIIa-158-VV polymorphism, which have a higher affinity for IgG3, promote and maintain (at least in part) the inflammatory response observed, as well as the ankylosis that is due mainly to the inflammatory response that trigger new bone formation (Braun & Sieper, 2007). However, it is necessary to enlarge the study population in order to confirm both the genetic data and the association of this polymorphism with AS.

Fig. 8. A hypothetical model of the interaction between the environmental (the p30 from *S. typhimurium*)*,* and the genetic (allelic variants FcγRIIA-131-HH, and FcγRIIIa-158-VV) factor in the inflammatory process observed in AS.

#### **4. Conclusion**

98 Clinical and Molecular Advances in Ankylosing Spondylitis

Controls

HH 16 (45.7%) 29 (24.2%) 0.01347 2.642 1.205-5.796 RR 8 (22.9%) 42 (35%) 0.176 0.550 0.223-1.318 HR 11 (31.4%) 49 (40.8%) 0.317 0.664 0.298-1.480

H 43 (61.4%) 107(44.6%) 0.0138 1.980 1.149-3.412 R 27 (38.6%) 133 (55.4%)

VV 7(20%) 5 (4.2%) 0.002 5.75 1.698-19.47 FF 14(40%) 51 (42.5%) 0.792 0.902 0.419-1.942 VF 14 (40%) 64 (53.3%) 0.427 0.737 0.364-1.569

V 28(40%) 74 (30.8%) 0.151 1.495 0.862-2.595 F 42 (60%) 166 69.2%)

NA1/NA1 9 (25.7%) 16 (13.33%) 0.080 2.25 0.894-5.662 NA2/NA2 15 (42.9%) 64 (53.33%) 0.275 0.656 0.307-1.402 NA1/NA2 11 (31.4%) 40 (33.33%) 0.833 0.917 0.408-2.057

NA1 29 (41.4%) 72(30%) 0.073 1.650 0.952-2.860 NA2 41 (58.6%) 168 (70%)

Table 5. Genotype and allele frequencies of FcγRIIa, FcγRIIIa and FcγRIIIb polymorphisms

identifying these components could prove helpful in gaining further insight into these diseases and developing novel immunotherapeutic strategies to interfere with chronic

On the basis of these findings, we proposed a hypothetical model of interaction between two factors: one environmental, the other genetic (Figure 8). In this model, the immune response against *S. typhimurium* in a susceptible subject leads to the recognition of p30 and the production of IgG3 antibodies. p30 should cross-react with a putative auto-antigen, such that the humoral immune response is maintained by this putative auto-antigen, and it is

Then, in the susceptible subject, the FcγRIIa-131-HH and FcγRIIIa-158-VV polymorphism, which have a higher affinity for IgG3, promote and maintain (at least in part) the inflammatory response observed, as well as the ankylosis that is due mainly to the inflammatory response that trigger new bone formation (Braun & Sieper, 2007). However, it is necessary to enlarge the study population in order to confirm both the genetic data and

(n=120) p OR 95% CI

Polymorphisms AS

FcγRIIa - Genotype


FcγRIIIa - Genotype


FcγRIIIb - Genotype


inflammation.

in patients with AS and controls.

responsible of the maintenance of the antibody levels.

the association of this polymorphism with AS.

(n=35)

In this chapter, we proposed a link between the humoral immune response in a susceptible individual and a genetic factor, FcγRIIa-131-HH, and FcγRIIIa-158-VV.

The immune response against *S. typihumium* appears to be strain-specific, because of the difference observed with the humoral immune response against *S. enteritidis*. Patients with AS produce IgG3 antibodies against the p30 of *S. typhimurium,* in contrast to healthy controls. Moreover, patients with AS have higher frequencies of FcγRIIa-131-HH and FcγRIIIa-158-VV, which suggests a link between these environmental and genetic factors.

#### **5. Acknowledgments**

This work was supported in part by the *Programa Integral de Fortalecimiento Institucional* (PIFI)-2001, 2003, 2008 and 2010.

The authors would like to thank Georgina Nataly Ibarra Muro and Alma Senorina de Jesús Ornelas Muñoz for their technical assistance.

Humoral Immune Response to *Salmonella* Antigens

*Clin Exp Immunol* 86(3): 399-404.

cells. *Eur J Immunol* 27(6): 1331-1338.

*Xue Yi Chuan Xue Za Zhi* 17(6): 409-412.

*Latinoam Microbiol* 38(2): 121-127.

evidence. *Clin Rheumatol* 26(6): 858-864.

14(5): 346-350.

15(4): 408-412.

*Med* 108(7): 580-583.

*Rheum* 43(4): 735-739.

920-927.

176.

and Polymorphisms in Receptors for the Fc of IgG in Patients with Ankylosing Spondylitis 101

Feldtkeller, E., Khan, M. A., van der Heijde, D., van der Linden, S. & Braun, J. (2003). Age at

Hong, C. H., Lee, J. S., Lee, H. S., Bae, S. C. & Yoo, D. H. (2005). The association between

Lahesmaa, R., Skurnik, M., Vaara, M., Leirisalo-Repo, M., Nissila, M., Granfors, K. &

Laitio, P., Virtala, M., Salmi, M., Pelliniemi, L. J., Yu, D. T. & Granfors, K. (1997). HLA-B27

Leirisalo-Repo, M., Hannu, T. & Mattila, L. (2003). Microbial factors in

Michel, M., Piette, J. C., Roullet, E., Duron, F., Frances, C., Nahum, L., Pelletier, N., Crassard,

Ni, P., Shen, F., Meng, W., Jiang, F. & Feng, S. (2000). [The association and linkage analysis

Nieto, A., Caliz, R., Pascual, M., Mataran, L., Garcia, S. & Martin, J. (2000). Involvement of

Pacheco-Tena, C., Alvarado De La Barrera, C., Lopez-Vidal, Y., Vazquez-Mellado, J.,

Parra-Campos, V., Escobar-Gutierrez, A., Dominguez-Lopez, M. L., Cancino-Diaz, M.,

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**7** 

**Genetics in Ankylosing Spondylitis –** 

Bruno Filipe Bettencourt1,3, Iris Foroni1,3, Ana Rita Couto1,3,

*2Grupo de Epidemiologia e Genética Humana do Departamento de Biologia da* 

*3Genetic and Arthritis Research Group (GARG), Institute for Molecular and Cell Biology* 

The association between HLA-B27 and ankylosing spondylitis (AS) has lead to intense research over the last 4 decades. In the course of this research, it was possible to obtain the best example of an association between a disease and a genetic marker. Genetic factors contribute with more than 90% for the susceptibility risk to AS being the MHC region, in particularly HLA-B27, the main contributor (Brown, et al., 1996). This evidence is supported by studies using familial cohorts; segregation and twin studies (Brown, et al., 1997; Jarvinen,

The advent of genotyping technologies, in particular the use of genome-wide linkage studies (GWLS) and whole-genome association studies (WGAS), allowed a wider view of the genetic factors related to AS and supported the presence of non-MHC genetic AS susceptibility factors. The first whole genome wide linkage study identified numerous loci in linkage with the disease, on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q (Laval, et al., 2001). These results prompted to an even more intense research on AS and, consequently, the investigation of other areas outside MHC. Later, three other major studies tried to narrow down the chromosome areas first identified (Consortium TASC, 2010; Consortium TASC/WTCCC2, 2011; Consortium WTCCC/TASC, 2007). This way it was possible to identify specific gene regions that would lead to new insights in the mechanisms underlying the disease susceptibility (Table 1). In this chapter we intend to provide an overview of the main genes, other than HLA-B\*27, which were identified as been associated to AS during the last decade.

At this moment, over 80 subtypes of HLA-B27 (coding and non-coding) are known (http://hla.alleles.org/alleles/class1.html). All variants were originated from the parental

**2. Genes with consistent association to ankylosing spondylitis** 

**1. Introduction** 

1995; Rubin, et al., 1994).

**2.1 Major histocompatibility complex** 

Manuela Lima2,3 and Jácome Bruges-Armas1,3 *1Serviço Especializado de Epidemiologia e Biologia Molecular,* 

*Hospital de Santo Espírito de Angra do Heroísmo* 

**Beyond HLA-B\*27** 

*Universidade dos Açores* 

*Portugal* 

*(IBMC), University of Porto* 


### **Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27**

Bruno Filipe Bettencourt1,3, Iris Foroni1,3, Ana Rita Couto1,3, Manuela Lima2,3 and Jácome Bruges-Armas1,3 *1Serviço Especializado de Epidemiologia e Biologia Molecular, Hospital de Santo Espírito de Angra do Heroísmo 2Grupo de Epidemiologia e Genética Humana do Departamento de Biologia da Universidade dos Açores 3Genetic and Arthritis Research Group (GARG), Institute for Molecular and Cell Biology (IBMC), University of Porto Portugal* 

#### **1. Introduction**

102 Clinical and Molecular Advances in Ankylosing Spondylitis

Reveille, J. D. & Arnett, F. C. (2005). Spondyloarthritis: update on pathogenesis and

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Salmon, J. E. & Pricop, L. (2001). Human receptors for immunoglobulin G: key elements in the pathogenesis of rheumatic disease. *Arthritis Rheum* 44(4): 739-750. Tani, Y., Tiwana, H., Hukuda, S., Nishioka, J., Fielder, M., Wilson, C., Bansal, S. & Ebringer,

Van Den Berg, L., Myhr, K. M., Kluge, B. & Vedeler, C. A. (2001). Fcgamma receptor polymorphisms in populations in Ethiopia and Norway. *Immunology* 104(1): 87-91. van der Linden, S., Valkenburg, H. A. & Cats, A. (1984). Evaluation of diagnostic criteria for

van der Pol, W. & van de Winkel, J. G. (1998). IgG receptor polymorphisms: risk factors for

Virtala, M., Kirveskari, J. & Granfors, K. (1997). HLA-B27 modulates the survival of

Willcocks, L. C., Smith, K. G. & Clatworthy, M. R. (2009). Low-affinity Fcgamma receptors,

Zambrano-Zaragoza, J. F., de Jesus Duran-Avelar, M., Rodriguez-Ocampo, A. N., Garcia-

modulated by HLA-B27. *Rheumatology (Oxford)* 41(6): 651-657.

Granfors, K. (2002). Invasion of Salmonella into human intestinal epithelial cells is

A. (1997). Antibodies to Klebsiella, Proteus, and HLA-B27 peptides in Japanese patients with ankylosing spondylitis and rheumatoid arthritis. *J Rheumatol* 24(1):

ankylosing spondylitis. A proposal for modification of the New York criteria.

Salmonella enteritidis in transfected L cells, possibly by impaired nitric oxide

Latorre, E., Burgos-Vargas, R., Dominguez-Lopez, M. L., Pena-Virgen, S. & Vibanco-Perez, N. (2009). The 30-kDa band from Salmonella typhimurium: IgM, IgA and IgG antibody response in patients with ankylosing spondylitis.

management. *Am J Med* 118(6): 592-603.

*Arthritis Rheum* 27(4): 361-368.

disease. *Immunogenetics* 48(3): 222-232.

*Rheumatology (Oxford)* 48(7): 748-754.

production. *Infect Immun* 65(10): 4236-4242.

autoimmunity and infection. *Expert Rev Mol Med* 11: e24.

109-114.

The association between HLA-B27 and ankylosing spondylitis (AS) has lead to intense research over the last 4 decades. In the course of this research, it was possible to obtain the best example of an association between a disease and a genetic marker. Genetic factors contribute with more than 90% for the susceptibility risk to AS being the MHC region, in particularly HLA-B27, the main contributor (Brown, et al., 1996). This evidence is supported by studies using familial cohorts; segregation and twin studies (Brown, et al., 1997; Jarvinen, 1995; Rubin, et al., 1994).

The advent of genotyping technologies, in particular the use of genome-wide linkage studies (GWLS) and whole-genome association studies (WGAS), allowed a wider view of the genetic factors related to AS and supported the presence of non-MHC genetic AS susceptibility factors. The first whole genome wide linkage study identified numerous loci in linkage with the disease, on chromosomes 1p, 2q, 6p, 9q, 10q, 16q, and 19q (Laval, et al., 2001). These results prompted to an even more intense research on AS and, consequently, the investigation of other areas outside MHC. Later, three other major studies tried to narrow down the chromosome areas first identified (Consortium TASC, 2010; Consortium TASC/WTCCC2, 2011; Consortium WTCCC/TASC, 2007). This way it was possible to identify specific gene regions that would lead to new insights in the mechanisms underlying the disease susceptibility (Table 1). In this chapter we intend to provide an overview of the main genes, other than HLA-B\*27, which were identified as been associated to AS during the last decade.

#### **2. Genes with consistent association to ankylosing spondylitis**

#### **2.1 Major histocompatibility complex**

At this moment, over 80 subtypes of HLA-B27 (coding and non-coding) are known (http://hla.alleles.org/alleles/class1.html). All variants were originated from the parental

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 105

composed by White Caucasians and Mexican Mestizos (Maksymowych, W P, et al., 2000). The prevalence of AS in Sub-Saharan black Africans is very low, a fact that has been related to the low frequency of HLA-B27 in that region. The allele HLA-B\*1403, found exclusively in African or Afro-American populations, was reported in four out of eight AS Togolese patients, a population where HLA-B27 is considered to be virtually absent (Lopez-Larrea, C, et al., 2002). It was the first time that a variant of B\*14 was found to be present in AS patients. However, a confirmation is needed and a mandatory replication of this study in other sub-Saharan populations is still missing. These findings in HLA-B alleles, other than HLA-B27, are not clear enough to understand if there is a direct association with AS or if

Data derived from GWAS for susceptibility loci in AS extended the HLA association in the MHC region between markers D6S276 and DRB1 (Brown, et al., 1998b). This finding has been supported by the results obtained in different ethnic groups (Brown, et al., 1998a; Jaakkola, et al., 2006; Kchir, et al., 2010; Mahfoudh, et al., 2011; Ploski, et al., 1995; Said-Nahal, et al., 2002; Sims, A M, et al., 2007). HLA-DRB1 was found to have a strong association with AS. In British patients the level of this association was increased in DR1 homozygotes, a fact that may suggest a B27-independent association, excluding a possible LD effect (Brown, et al., 1998a; Sims, A M, et al., 2007). On the other hand, in French families the association between DR1 and AS seems to be a consequence of LD with HLA-B27 (Said-Nahal, et al., 2002). Here, it was reported an excess of transmission of HLA-DR4 to patients, independently of its LD with B27. Together with the lack of transmission of DR4 to HLA-B27 positive siblings, this report suggests that the presence of DR4 may contribute to AS simultaneously with B27. The HLA-DR1 is associated to SpA in Mexican patients, however, this finding was not confirmed when only AS patients were analysed (Vargas-Alarcon, et al., 2002). The sample size and the high prevalence of peripheral enthesopathy and arthropathy in the studied group may explain this result, different from the results obtained

Consistent results, on the association of DRB1 alleles with AS, have been difficult to ascertain in different ethnic groups. A Finnish report proposed that, in this specific population, the HLA-DRB1 alleles do not seem to play a strong role in AS susceptibility , but may influence the age of symptom onset (Jaakkola, et al., 2006). The results, in this casecontrol study, are limited due to the sample size. Still, a strong association of DRB1\*08, both independent and as part of a HLA-B27 haplotype, was suggested. HLA-DRB1\*08 had been already reported in juvenile-AS B27-positive individuals, in Norwegian population (Ploski, et al., 1995). Two haplotypes were identified in a British case-control study: B\*27positive /DRB1\*07positive and B\*27negative /DRB1\*03positive (Sims, A M, et al., 2007). The frequency of HLA-DRB1\*15 was found to be increased, in Tunisian AS patients, but this allele was in LD with B27 (Mahfoudh, et al., 2011). Within the same population, a casecontrol study reported a significant increase in HLA-DRB1\*11 frequency among patients. It

was also found a possible protective effect of HLA-DRB1\*13 (Kchir, et al., 2010).

Recently, in a Spanish/Portuguese cohort, it was reported the association between HLA-DPA1 and HLA-DPB1 alleles and AS (Díaz-Peña, et al., 2011). The study was focused on these two MHC genes that lay on a region unrelated to B27. Both AS patients and controls were HLA-B27 positive. This way it was possible to achieve results from an HLA-B27 neutral point of view. The data showed significant results between AS and DPA1\*01:03,

this is the result of LD with another MHC gene.

in the British population.

B\*27:05 as a result of different mechanisms, such as point mutation, gene conversion, reciprocal recombination and interlocus gene conversion (Reveille, J D & Maganti, 2009). The data obtained from studies within same ethnic groups allowed the perception that there are differences in the rank of association of each B27 subtype and AS. The allele HLA-B\*27:05 is the most frequent variant of B27 worldwide, although the frequencies of all the known subtypes diverge when different ethnic groups are compared (Reveille, J D, 2011). For that reason, it has been very difficult to establish a clear ranking of B27 variants and association with AS, that could be applied to all groups (Brown, 2010).

Among European Caucasians, B\*27:05 and B\*27:02 are suggested to be the strongest disease associated variants (Brown, et al., 1996; MacLean, et al., 1993; Reveille, J D, 2011). In Asian cohorts, HLA-B\*27:04 was found to be more strongly associated with AS than B\*27:05 (Hou, et al., 2007; Liu, et al., 2010; Lopez-Larrea, C, et al., 1995). Other B\*27 alleles were already reported in AS cases, namely B\*27:01 (Ball & Khan, 2001), \*27:03 (Gonzalez-Roces, et al., 1997), \*27:07, \*27:08 (Armas, et al., 1999), \*27:10 (Garcia, et al., 1998), \*27:14, \*27:15 (Garcia-Fernandez, et al., 2001), and \*27:19 (Tamouza, et al., 2001).

The information obtained in studies with B27 subtypes confirm this allele as a critical factor on AS susceptibility. However, since it is known that less than 8% of B27 individuals develop AS (van der Linden, et al., 1984), it is difficult to exclude another gene nearby B27 as the one responsible for the AS susceptibility. The high level of linkage disequilibrium (LD) in the MHC region is a major barrier to the identification of any other possible gene directly involved.

New insights were reached inside MHC, despite all the complexity involving this region. HLA-B60 (B\*40:01) was identified as the first non-HLA-B\*27 gene strongly associated to AS susceptibility. The first report of this association stated that HLA-B60 was increased among AS patients HLA-B27 positive, in five independent data sets. On the other hand, the studied variant was not increased in HLA-B27 negative patients with AS. A whole view of the obtained data show that the susceptibility to AS is threefold higher in individuals both B27/B60 positive (Robinson, et al., 1989). These findings were later supported by other studies using different cohorts. In a group of UK patients with AS, not only the association strength of B27/B60 positive was confirmed. The presence of HLA-B60 was observed in HLA-B27 negative patients, suggesting that, despite having a much weaker effect, it may function as an AS susceptibility gene independent of HLA-B27 (Brown, et al., 1996). Moreover, in a group of Taiwan Chinese it was confirmed the association of B60 with AS and also, for the first time, an independent association of B61 was found. Both alleles were strongly increased in HLA-B27 negative patients (Wei, et al., 2004). Although all this replications confirming the relationship between HLA-B60 and AS, the same was not seen in Mexican Mestizos where, instead, HLA-B49 was identified as significantly increased in patients with AS (Vargas-Alarcón, et al., 1994).

Another allele, HLA-B\*39, has been reported to be increased in HLA-B27 negative White Caucasian and Japanese AS patients (Khan, et al., 1980; Yamaguchi, et al., 1995). The studies that provided this data used small cohorts and consequently it is not possible to state if this is an effective association neither the level of its power. Even so, it was formulated a parallelism between B27 and B\*39 components of the peptide-anchoring B pocket as well as peptide-ligand motifs, supporting some potential explanation for the HLA-B27 influence in the development of AS (Yamaguchi, et al., 1995). This results were not confirmed in a cohort

B\*27:05 as a result of different mechanisms, such as point mutation, gene conversion, reciprocal recombination and interlocus gene conversion (Reveille, J D & Maganti, 2009). The data obtained from studies within same ethnic groups allowed the perception that there are differences in the rank of association of each B27 subtype and AS. The allele HLA-B\*27:05 is the most frequent variant of B27 worldwide, although the frequencies of all the known subtypes diverge when different ethnic groups are compared (Reveille, J D, 2011). For that reason, it has been very difficult to establish a clear ranking of B27 variants and

Among European Caucasians, B\*27:05 and B\*27:02 are suggested to be the strongest disease associated variants (Brown, et al., 1996; MacLean, et al., 1993; Reveille, J D, 2011). In Asian cohorts, HLA-B\*27:04 was found to be more strongly associated with AS than B\*27:05 (Hou, et al., 2007; Liu, et al., 2010; Lopez-Larrea, C, et al., 1995). Other B\*27 alleles were already reported in AS cases, namely B\*27:01 (Ball & Khan, 2001), \*27:03 (Gonzalez-Roces, et al., 1997), \*27:07, \*27:08 (Armas, et al., 1999), \*27:10 (Garcia, et al., 1998), \*27:14, \*27:15 (Garcia-

The information obtained in studies with B27 subtypes confirm this allele as a critical factor on AS susceptibility. However, since it is known that less than 8% of B27 individuals develop AS (van der Linden, et al., 1984), it is difficult to exclude another gene nearby B27 as the one responsible for the AS susceptibility. The high level of linkage disequilibrium (LD) in the MHC region is a major barrier to the identification of any other possible gene directly

New insights were reached inside MHC, despite all the complexity involving this region. HLA-B60 (B\*40:01) was identified as the first non-HLA-B\*27 gene strongly associated to AS susceptibility. The first report of this association stated that HLA-B60 was increased among AS patients HLA-B27 positive, in five independent data sets. On the other hand, the studied variant was not increased in HLA-B27 negative patients with AS. A whole view of the obtained data show that the susceptibility to AS is threefold higher in individuals both B27/B60 positive (Robinson, et al., 1989). These findings were later supported by other studies using different cohorts. In a group of UK patients with AS, not only the association strength of B27/B60 positive was confirmed. The presence of HLA-B60 was observed in HLA-B27 negative patients, suggesting that, despite having a much weaker effect, it may function as an AS susceptibility gene independent of HLA-B27 (Brown, et al., 1996). Moreover, in a group of Taiwan Chinese it was confirmed the association of B60 with AS and also, for the first time, an independent association of B61 was found. Both alleles were strongly increased in HLA-B27 negative patients (Wei, et al., 2004). Although all this replications confirming the relationship between HLA-B60 and AS, the same was not seen in Mexican Mestizos where, instead, HLA-B49 was identified as significantly increased in

Another allele, HLA-B\*39, has been reported to be increased in HLA-B27 negative White Caucasian and Japanese AS patients (Khan, et al., 1980; Yamaguchi, et al., 1995). The studies that provided this data used small cohorts and consequently it is not possible to state if this is an effective association neither the level of its power. Even so, it was formulated a parallelism between B27 and B\*39 components of the peptide-anchoring B pocket as well as peptide-ligand motifs, supporting some potential explanation for the HLA-B27 influence in the development of AS (Yamaguchi, et al., 1995). This results were not confirmed in a cohort

association with AS, that could be applied to all groups (Brown, 2010).

Fernandez, et al., 2001), and \*27:19 (Tamouza, et al., 2001).

patients with AS (Vargas-Alarcón, et al., 1994).

involved.

composed by White Caucasians and Mexican Mestizos (Maksymowych, W P, et al., 2000). The prevalence of AS in Sub-Saharan black Africans is very low, a fact that has been related to the low frequency of HLA-B27 in that region. The allele HLA-B\*1403, found exclusively in African or Afro-American populations, was reported in four out of eight AS Togolese patients, a population where HLA-B27 is considered to be virtually absent (Lopez-Larrea, C, et al., 2002). It was the first time that a variant of B\*14 was found to be present in AS patients. However, a confirmation is needed and a mandatory replication of this study in other sub-Saharan populations is still missing. These findings in HLA-B alleles, other than HLA-B27, are not clear enough to understand if there is a direct association with AS or if this is the result of LD with another MHC gene.

Data derived from GWAS for susceptibility loci in AS extended the HLA association in the MHC region between markers D6S276 and DRB1 (Brown, et al., 1998b). This finding has been supported by the results obtained in different ethnic groups (Brown, et al., 1998a; Jaakkola, et al., 2006; Kchir, et al., 2010; Mahfoudh, et al., 2011; Ploski, et al., 1995; Said-Nahal, et al., 2002; Sims, A M, et al., 2007). HLA-DRB1 was found to have a strong association with AS. In British patients the level of this association was increased in DR1 homozygotes, a fact that may suggest a B27-independent association, excluding a possible LD effect (Brown, et al., 1998a; Sims, A M, et al., 2007). On the other hand, in French families the association between DR1 and AS seems to be a consequence of LD with HLA-B27 (Said-Nahal, et al., 2002). Here, it was reported an excess of transmission of HLA-DR4 to patients, independently of its LD with B27. Together with the lack of transmission of DR4 to HLA-B27 positive siblings, this report suggests that the presence of DR4 may contribute to AS simultaneously with B27. The HLA-DR1 is associated to SpA in Mexican patients, however, this finding was not confirmed when only AS patients were analysed (Vargas-Alarcon, et al., 2002). The sample size and the high prevalence of peripheral enthesopathy and arthropathy in the studied group may explain this result, different from the results obtained in the British population.

Consistent results, on the association of DRB1 alleles with AS, have been difficult to ascertain in different ethnic groups. A Finnish report proposed that, in this specific population, the HLA-DRB1 alleles do not seem to play a strong role in AS susceptibility , but may influence the age of symptom onset (Jaakkola, et al., 2006). The results, in this casecontrol study, are limited due to the sample size. Still, a strong association of DRB1\*08, both independent and as part of a HLA-B27 haplotype, was suggested. HLA-DRB1\*08 had been already reported in juvenile-AS B27-positive individuals, in Norwegian population (Ploski, et al., 1995). Two haplotypes were identified in a British case-control study: B\*27positive /DRB1\*07positive and B\*27negative /DRB1\*03positive (Sims, A M, et al., 2007). The frequency of HLA-DRB1\*15 was found to be increased, in Tunisian AS patients, but this allele was in LD with B27 (Mahfoudh, et al., 2011). Within the same population, a casecontrol study reported a significant increase in HLA-DRB1\*11 frequency among patients. It was also found a possible protective effect of HLA-DRB1\*13 (Kchir, et al., 2010).

Recently, in a Spanish/Portuguese cohort, it was reported the association between HLA-DPA1 and HLA-DPB1 alleles and AS (Díaz-Peña, et al., 2011). The study was focused on these two MHC genes that lay on a region unrelated to B27. Both AS patients and controls were HLA-B27 positive. This way it was possible to achieve results from an HLA-B27 neutral point of view. The data showed significant results between AS and DPA1\*01:03,

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 107

The association between Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) and AS was first reported in Caucasians, together with IL23R (Consortium WTCCC/TASC, 2007). Contrarily to the IL23R, the association of *ERAP1* was later confirmed in other ethnic groups (Chen, R., et al., 2011). ERAP1 molecules are encoded by the oxytocinase subfamily, a group of three genes located on the chromosome 5 (5q15) (Brionez & Reveille, 2008), and are

The involvement between *ERAP1* and MHC class I presentation was the first suggested and confirmed function attributed to this aminopeptidase (Saric, et al., 2002; Saveanu, et al., 2005). ERAP1 is MHC class I dependent and play a major role on peptide trimming, processing it to optimal length, for presentation at cell surface (Saveanu, et al., 2005), having an important connection to the immune recognition mechanism related to CD8+ T cells. However, it is proposed that the referred importance varies depending both on the antigen and the cell physiological state. It was shown that, contrarily to other peptidases, ERAP1 cleaves NH2-terminal from peptides longer than ten residues and, consequently, is able to produce peptides containing eight to nine residues from longer precursors (York, et al., 2002). This fact support the relation with MHC class I molecules and indicates a possible coevolution between these 2 groups of molecules, since MHC class I require eight to nine residues peptides for a stable binding (York, et al., 2002). Due to the involvement between ERAP1 and MHC class I, these molecules and their trimming mechanisms are consequently involved with HLA-B27, an MHC class I variant (Campbell, et al., 2011). For some years, it was stated that a deeper understanding about *ERAP1* function and its relation with B27, at peptide production level, would lead to new insights on the paths that support the well known association between HLA-B27 and AS.. This aspiration was recently fulfilled by the latest GWAS on AS (Consortium TASC/WTCCC2, 2011). It was shown that HLA-B27 positive and negative AS cases differ in association with *ERAP1*. This fact provided the first reliable replicated example of a gene-gene interaction in AS, indicating that the mechanism by which HLA-B27 induces AS involves aberrant presentation or handling of peptides

The cleavage of cytokine cell surface receptors is another function documented to *ERAP1*. The shedding of IL-1R2, promoted by ERAP1, was already reported in coimmunoprecipitation experiments (Cui, et al., 2003a). Through the use of cell lines, it was possible to achieve a correspondence between *ERAP1* overexpression, the increase of IL1-R2 shedding and the decrease of membrane–associated IL1-R2. It was also found that ERAP1 is necessary for constitutive IL1-R2 shedding, as basal IL1-R2 shedding is absent from *ERAP1* knockout cell lines (Cui, et al., 2003a). The exact same correlations were already observed in reports regarding TNFR1 (Cui, et al., 2002) and IL6R (Cui, et al., 2003b). These findings were not confirmed when was measured the appearance of those receptors in cell culture supernatants from single-cell suspensions, stimulated with plate-bound anti-CD3 (Clone 145-2C11) and phorbol 12-myristate 13-acetate (PMA), prepared from mouse spleens. No difference in the levels of these receptors was observed over time, indicating that ERAP1 does not have a major influence on cytokine receptor trimming, at least in mice (Consortium TASC/WTCCC2, 2011). Further tests may contribute to the clarification of the mechanisms underlying the association between ERAP1 and AS. An abnormal increase or decrease in the

**2.2 Non-major histocompatibility complex genes** 

located in the endoplasmatic reticulum lumen (Saric, et al., 2002).

**2.2.1 ERAP1/ARTS1** 

(Consortium TASC/WTCCC2, 2011).

DPA1\*02:01, DPB1\*13:01. It was also found an association between two haplotypes and AS, namely: DPA1\*02:01-DPB1\*11:01:01 and DPA1\*02:01-DPB1\*13:01. This was the first study to show an association between this region of MHC and susceptibility to AS. Consequently, further replicates are needed to confirm these results.


Table 1. Genes associated to AS - adapted from (Brown, 2010; Reveille, J D, 2011).

#### **2.2 Non-major histocompatibility complex genes**

#### **2.2.1 ERAP1/ARTS1**

106 Clinical and Molecular Advances in Ankylosing Spondylitis

DPA1\*02:01, DPB1\*13:01. It was also found an association between two haplotypes and AS, namely: DPA1\*02:01-DPB1\*11:01:01 and DPA1\*02:01-DPB1\*13:01. This was the first study to show an association between this region of MHC and susceptibility to AS. Consequently,

Gene Locus Mechanism Population origin

Peptide trimming prior to HLA Class I presentation and cleaving cytokine receptors from cell surface

along axonal and dendritic microtubules Caucasian

to form IL23R Caucasian

helper Th17 T cells Caucasian

remodelling Caucasian (one study)

apoptosis, binds lipopolysaccharide Caucasian (one study)

1 Metabolism of xenobiotics Caucasian (North

Caucasian and non-

Caucasian and non-

Caucasian and Han-

Caucasian and Han-

Caucasian

Caucasian

Chinese

Chinese

Caucasian

European)

Chinese

cohort)

Caucasian and Han-

Caucasian and Japanese

Caucasian (Finnish small

(small cohorts)

Caucasian and Han-Chinese (small cohorts)

further replicates are needed to confirm these results.

Genes with consistent association to ankylosing spondylitis

HLA-B60

ERAP1/ART S1 5q15

CYP2D6 22q13.

ANKH 5p15.2

KIR 19q13.

4

Other genes

HLA-B27 6p21.3 Presents endogenously processed antigens to T cells

(\*4001) 6p21.3 Presents endogenously processed antigens to T cells

IL23R 1p31.3 Differentiation of naive CD4 T cells into helper Th17 T cells

Genes with suggestive association to ankylosing spondylitis

KIF21B 1p32 Transport of essential cellular components

IL12B 5q31 Heterodimerises with the IL23 p19 subunit,

TNFSF15 9q32 Differentiation of naive CD4 T cells into

target cells

TNAP 1p36.1 Receptor on monocytes important in

TGFB1 19q13 Mediates inflammation, fibrosis and bone

CD14 5q31.1 A phosphoethanolamine and pyridoxal-5'-

ANTRX2 4q21 Binds to collagen IV and laminin, possibly


TNFR1 12p13 Influence on TNF signaling Caucasian

involved in extracellular matrix adhesion

Gene Locus Mechanism Population origin

Exports inorganic pyrophosphate from intracellular to extracellular compartments.

Regulates activation of NK cells via recognition of HLA class I molecules on

PO4 actingectophosphatase. Degrades PPi

Table 1. Genes associated to AS - adapted from (Brown, 2010; Reveille, J D, 2011).

Regulates tissue calcification

Gene Locus Mechanism Population origin IL1R2 2q11 Interference in the binding of IL-1 to IL-1R1 Caucasian and nonThe association between Endoplasmic Reticulum Aminopeptidase 1 (ERAP1) and AS was first reported in Caucasians, together with IL23R (Consortium WTCCC/TASC, 2007). Contrarily to the IL23R, the association of *ERAP1* was later confirmed in other ethnic groups (Chen, R., et al., 2011). ERAP1 molecules are encoded by the oxytocinase subfamily, a group of three genes located on the chromosome 5 (5q15) (Brionez & Reveille, 2008), and are located in the endoplasmatic reticulum lumen (Saric, et al., 2002).

The involvement between *ERAP1* and MHC class I presentation was the first suggested and confirmed function attributed to this aminopeptidase (Saric, et al., 2002; Saveanu, et al., 2005). ERAP1 is MHC class I dependent and play a major role on peptide trimming, processing it to optimal length, for presentation at cell surface (Saveanu, et al., 2005), having an important connection to the immune recognition mechanism related to CD8+ T cells. However, it is proposed that the referred importance varies depending both on the antigen and the cell physiological state. It was shown that, contrarily to other peptidases, ERAP1 cleaves NH2-terminal from peptides longer than ten residues and, consequently, is able to produce peptides containing eight to nine residues from longer precursors (York, et al., 2002). This fact support the relation with MHC class I molecules and indicates a possible coevolution between these 2 groups of molecules, since MHC class I require eight to nine residues peptides for a stable binding (York, et al., 2002). Due to the involvement between ERAP1 and MHC class I, these molecules and their trimming mechanisms are consequently involved with HLA-B27, an MHC class I variant (Campbell, et al., 2011). For some years, it was stated that a deeper understanding about *ERAP1* function and its relation with B27, at peptide production level, would lead to new insights on the paths that support the well known association between HLA-B27 and AS.. This aspiration was recently fulfilled by the latest GWAS on AS (Consortium TASC/WTCCC2, 2011). It was shown that HLA-B27 positive and negative AS cases differ in association with *ERAP1*. This fact provided the first reliable replicated example of a gene-gene interaction in AS, indicating that the mechanism by which HLA-B27 induces AS involves aberrant presentation or handling of peptides (Consortium TASC/WTCCC2, 2011).

The cleavage of cytokine cell surface receptors is another function documented to *ERAP1*. The shedding of IL-1R2, promoted by ERAP1, was already reported in coimmunoprecipitation experiments (Cui, et al., 2003a). Through the use of cell lines, it was possible to achieve a correspondence between *ERAP1* overexpression, the increase of IL1-R2 shedding and the decrease of membrane–associated IL1-R2. It was also found that ERAP1 is necessary for constitutive IL1-R2 shedding, as basal IL1-R2 shedding is absent from *ERAP1* knockout cell lines (Cui, et al., 2003a). The exact same correlations were already observed in reports regarding TNFR1 (Cui, et al., 2002) and IL6R (Cui, et al., 2003b). These findings were not confirmed when was measured the appearance of those receptors in cell culture supernatants from single-cell suspensions, stimulated with plate-bound anti-CD3 (Clone 145-2C11) and phorbol 12-myristate 13-acetate (PMA), prepared from mouse spleens. No difference in the levels of these receptors was observed over time, indicating that ERAP1 does not have a major influence on cytokine receptor trimming, at least in mice (Consortium TASC/WTCCC2, 2011). Further tests may contribute to the clarification of the mechanisms underlying the association between ERAP1 and AS. An abnormal increase or decrease in the

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 109

the clinical heterogeneity, ethnic differences or real genetic heterogeneity, the small sample

The Consortium TASC included *ERAP1* in the first GWAS for AS, already mentioned above. This work included a large Caucasian cohort and narrowed down the ERAP1 region, associated to AS, to a block of SNPs lying in a 4.6-kb region between rs27529 (exon 9) and rs469758 (intron 12) (Consortium TASC, 2010). The identified region showed an association 50 times more significant than any other imputed SNP (P<10-11). The overall results showed strong association of two SNPs that were not reported in previous Caucasian studies, rs27037 and rs27434. The data obtained in a Han Chinese group, published before TASC GWAS showed moderate association of rs27037 (P=0.012) and did not observe the association of rs27434 (P=0.14) (Davidson, et al., 2009). The same result was attained by the recent meta-analysis already cited (Chen, R., et al., 2011). On the other hand, the strong association of both markers was recently confirmed in a Korean group (Bang, et al., 2011). Once more, together with ethnic differences, the results seem to be influenced by the effect

The data concerning *ERAP1* haplotypes is limited; however the available results seem to point out to some haplotypes that influence the risk of disease. The rs2044/10060860/30187- CCT haplotypes increased the risk of disease in 3 Canadian case-control cohorts. This haplotype is in one of the two strongly significant haplotypes, out of four, that were identified among Koreans: rs27044/rs17482078/rs10050860/rs30187-GCCT and rs27044/ rs17482078/rs10050860/rs30187-CCCC (Choi, et al., 2010). All these four SNPs formed an LD block with almost complete LD (95<D'<100) (Choi, et al., 2010; Tsui, F. W., et al., 2010). The Canadian study reported a protection effect of rs30187/26618/26653-CTG ERAP1 haplotype (Maksymowych, W. P., et al., 2009). A family-based study was not able to support the significant association of rs2044/10060860/30187 haplotype, but here a relatively small sample was used (Tsui, F. W., et al., 2010). Moreover, the results are probably an underestimate since some of the studied families had more than two affected individuals (3– 5) and the method used by the authors for power estimation took into account only two affected family members. Despite this lack of confirmation, it was possible to identify significant association between rs27044/rs2549782-GT and rs30187/rs2549782-TT haplotypes and AS. In addition, a novel finding showing that rs27044/rs30187/rs2549782- GTT haplotype in the ERAP1-ERAP2 loci was significantly associated with disease

The association between Interleukin 23 receptor (IL23R) and AS has been confirmed throughout the last few years, since the first association evidence, reported by the

IL23R is a member of heamopoietin receptor family, which binds to IL-23 mediating its activity. This hemopoietic cytokine receptor is encoded on chromosome 1 (1p31.3), by a gene located 150kb far from the gene for IL-12Rβ2 (Parham, C., et al., 2002). IL23 is a heterodimeric cytokine consisting of two subunits: p40, which is shared with IL-12, and p19 (Oppmann, et al., 2000). IL-23 has also other biological connections with IL-12; activated human PHA blast T cells, when induced by IL-23, register a better proliferation and IFN-γ

size of the studies or their low statistical power (Chen, R., et al., 2011).

susceptibility for both models used (Tsui, F. W., et al., 2010).

WTCCC/TASC study (Consortium WTCCC/TASC, 2007) .

genetic heterogeneity.

**2.2.2 IL23R** 

amount of those cytokine cell surface receptors is an expected outcome of ERAP1 malfunctioning. Thus, the susceptibility to AS can emerge as a consequence of proinflammatory effects related to that amount variation (Chen, R., et al., 2011; Consortium WTCCC/TASC, 2007).

Since the confirmed association between ERAP1 and AS reported in 2007(Consortium WTCCC/TASC, 2007), some replications occurred in studies involving Caucasian (Consortium TASC, 2010; Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010; Pimentel-Santos, et al., 2009; Tsui, F. W., et al., 2010) and Han Chinese cohorts (Bang, et al., 2011; Choi, et al., 2010; Davidson, et al., 2009). The Wellcome Trust Case-Control consortium and Australo-Anglo-American Spondyloarthritis Consortium first described the association of five *ERAP1* single nucleotide polymorphisms (SNPs) and AS (Consortium WTCCC/TASC, 2007), namely rs27044, rs17482078, rs10050860, rs30187 and rs2287987. Two of these SNPs, rs27044 and rs30187, gathered more consistent results in the subsequent reports and were the only markers that showed significant association both in Caucasian and in Han Chinese groups (Choi, et al., 2010; Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010; Pimentel-Santos, et al., 2009). The significant relation between rs27044 and AS was not confirmed in Canadian, contrarily to all other replicates involving this SNP and Caucasian groups. However, in that same cohort, it was found an *ERAP1* haplotype, containing rs27044, which increased the risk of AS: rs2044/10060860/30187-CCT (Maksymowych, W. P., et al., 2009). The other mentioned SNP, rs30187, did not show a significant association with AS in a group from Hungary (Pazar, et al., 2010). However, this result has low impact compared to the other studies performed, since the obtained P-value was 0.051 and the sample was the smallest of all the mentioned cohorts. The rs30187 (Arg528Lys) is the only coding marker within a SNP block recently identified in GWAS (Consortium TASC, 2010). This variant showed a really strong significant association in another GWAS using a large Caucasian cohort (P=1.8x10-27) (Consortium TASC/WTCCC2, 2011). It was shown that this marker originates a significant decrease in aminopeptidase activity toward a synthetic peptide substrate. Moreover, modeling of ERAP1 protein points out to the presence of Arg528 at the mouth of the putative enzyme substrate pocket. This location can explain the reduction of the aminopeptidase activity of the molecules that contain this variation (Goto, et al., 2008; Kochan, et al., 2011). These results and reported data from family based studies (Tsui, F. W., et al., 2010) support this SNP as one of the *ERAP1* variants with strongest association to AS.

The remaining SNPs found by The Australo-Anglo-American Spondyloarthritis Consortium (TASC) & Wellcome Trust Case Control Consortium (WTCCC) didn't show consistent results in other sudies. The first published study, after WTCCC/TASC report, was not able to identify a significant association between rs2287987, rs17482078 or rs10050860 and AS in a Portuguese group (Pimentel-Santos, et al., 2009). The same result was obtained in a Han Chinese group (Choi, et al., 2010). The rs17482078 significant association (Consortium WTCCC/TASC, 2007) was not confirmed also in already mentioned Hungary group (Pazar, et al., 2010). Other replications using Caucasian cohorts were able to find significant associations between rs2287987, rs10050860 and AS (Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010). Recently, a meta-analysis that included all the *ERAP1* association studies, confirmed the presence of a significant association between the SNPs reported by the Consortium WTCCC/TASC and AS (Chen, R., et al., 2011). Moreover, it was proposed that the contradictory results, obtained in some replications, can be an effect of

amount of those cytokine cell surface receptors is an expected outcome of ERAP1 malfunctioning. Thus, the susceptibility to AS can emerge as a consequence of proinflammatory effects related to that amount variation (Chen, R., et al., 2011; Consortium

Since the confirmed association between ERAP1 and AS reported in 2007(Consortium WTCCC/TASC, 2007), some replications occurred in studies involving Caucasian (Consortium TASC, 2010; Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010; Pimentel-Santos, et al., 2009; Tsui, F. W., et al., 2010) and Han Chinese cohorts (Bang, et al., 2011; Choi, et al., 2010; Davidson, et al., 2009). The Wellcome Trust Case-Control consortium and Australo-Anglo-American Spondyloarthritis Consortium first described the association of five *ERAP1* single nucleotide polymorphisms (SNPs) and AS (Consortium WTCCC/TASC, 2007), namely rs27044, rs17482078, rs10050860, rs30187 and rs2287987. Two of these SNPs, rs27044 and rs30187, gathered more consistent results in the subsequent reports and were the only markers that showed significant association both in Caucasian and in Han Chinese groups (Choi, et al., 2010; Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010; Pimentel-Santos, et al., 2009). The significant relation between rs27044 and AS was not confirmed in Canadian, contrarily to all other replicates involving this SNP and Caucasian groups. However, in that same cohort, it was found an *ERAP1* haplotype, containing rs27044, which increased the risk of AS: rs2044/10060860/30187-CCT (Maksymowych, W. P., et al., 2009). The other mentioned SNP, rs30187, did not show a significant association with AS in a group from Hungary (Pazar, et al., 2010). However, this result has low impact compared to the other studies performed, since the obtained P-value was 0.051 and the sample was the smallest of all the mentioned cohorts. The rs30187 (Arg528Lys) is the only coding marker within a SNP block recently identified in GWAS (Consortium TASC, 2010). This variant showed a really strong significant association in another GWAS using a large Caucasian cohort (P=1.8x10-27) (Consortium TASC/WTCCC2, 2011). It was shown that this marker originates a significant decrease in aminopeptidase activity toward a synthetic peptide substrate. Moreover, modeling of ERAP1 protein points out to the presence of Arg528 at the mouth of the putative enzyme substrate pocket. This location can explain the reduction of the aminopeptidase activity of the molecules that contain this variation (Goto, et al., 2008; Kochan, et al., 2011). These results and reported data from family based studies (Tsui, F. W., et al., 2010) support this SNP as one of the *ERAP1* variants with strongest association to AS. The remaining SNPs found by The Australo-Anglo-American Spondyloarthritis Consortium (TASC) & Wellcome Trust Case Control Consortium (WTCCC) didn't show consistent results in other sudies. The first published study, after WTCCC/TASC report, was not able to identify a significant association between rs2287987, rs17482078 or rs10050860 and AS in a Portuguese group (Pimentel-Santos, et al., 2009). The same result was obtained in a Han Chinese group (Choi, et al., 2010). The rs17482078 significant association (Consortium WTCCC/TASC, 2007) was not confirmed also in already mentioned Hungary group (Pazar, et al., 2010). Other replications using Caucasian cohorts were able to find significant associations between rs2287987, rs10050860 and AS (Harvey, et al., 2009a; Maksymowych, W. P., et al., 2009; Pazar, et al., 2010). Recently, a meta-analysis that included all the *ERAP1* association studies, confirmed the presence of a significant association between the SNPs reported by the Consortium WTCCC/TASC and AS (Chen, R., et al., 2011). Moreover, it was proposed that the contradictory results, obtained in some replications, can be an effect of

WTCCC/TASC, 2007).

the clinical heterogeneity, ethnic differences or real genetic heterogeneity, the small sample size of the studies or their low statistical power (Chen, R., et al., 2011).

The Consortium TASC included *ERAP1* in the first GWAS for AS, already mentioned above. This work included a large Caucasian cohort and narrowed down the ERAP1 region, associated to AS, to a block of SNPs lying in a 4.6-kb region between rs27529 (exon 9) and rs469758 (intron 12) (Consortium TASC, 2010). The identified region showed an association 50 times more significant than any other imputed SNP (P<10-11). The overall results showed strong association of two SNPs that were not reported in previous Caucasian studies, rs27037 and rs27434. The data obtained in a Han Chinese group, published before TASC GWAS showed moderate association of rs27037 (P=0.012) and did not observe the association of rs27434 (P=0.14) (Davidson, et al., 2009). The same result was attained by the recent meta-analysis already cited (Chen, R., et al., 2011). On the other hand, the strong association of both markers was recently confirmed in a Korean group (Bang, et al., 2011). Once more, together with ethnic differences, the results seem to be influenced by the effect genetic heterogeneity.

The data concerning *ERAP1* haplotypes is limited; however the available results seem to point out to some haplotypes that influence the risk of disease. The rs2044/10060860/30187- CCT haplotypes increased the risk of disease in 3 Canadian case-control cohorts. This haplotype is in one of the two strongly significant haplotypes, out of four, that were identified among Koreans: rs27044/rs17482078/rs10050860/rs30187-GCCT and rs27044/ rs17482078/rs10050860/rs30187-CCCC (Choi, et al., 2010). All these four SNPs formed an LD block with almost complete LD (95<D'<100) (Choi, et al., 2010; Tsui, F. W., et al., 2010). The Canadian study reported a protection effect of rs30187/26618/26653-CTG ERAP1 haplotype (Maksymowych, W. P., et al., 2009). A family-based study was not able to support the significant association of rs2044/10060860/30187 haplotype, but here a relatively small sample was used (Tsui, F. W., et al., 2010). Moreover, the results are probably an underestimate since some of the studied families had more than two affected individuals (3– 5) and the method used by the authors for power estimation took into account only two affected family members. Despite this lack of confirmation, it was possible to identify significant association between rs27044/rs2549782-GT and rs30187/rs2549782-TT haplotypes and AS. In addition, a novel finding showing that rs27044/rs30187/rs2549782- GTT haplotype in the ERAP1-ERAP2 loci was significantly associated with disease susceptibility for both models used (Tsui, F. W., et al., 2010).

#### **2.2.2 IL23R**

The association between Interleukin 23 receptor (IL23R) and AS has been confirmed throughout the last few years, since the first association evidence, reported by the WTCCC/TASC study (Consortium WTCCC/TASC, 2007) .

IL23R is a member of heamopoietin receptor family, which binds to IL-23 mediating its activity. This hemopoietic cytokine receptor is encoded on chromosome 1 (1p31.3), by a gene located 150kb far from the gene for IL-12Rβ2 (Parham, C., et al., 2002). IL23 is a heterodimeric cytokine consisting of two subunits: p40, which is shared with IL-12, and p19 (Oppmann, et al., 2000). IL-23 has also other biological connections with IL-12; activated human PHA blast T cells, when induced by IL-23, register a better proliferation and IFN-γ

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 111

these two cohorts and it was in LD with rs11209026. The Toronto population also registered

In the Spanish group, the obtained results were able to corroborate the previous findings for rs1343151, rs11209026 and rs10889677. In this cohort the SNPs revealed a protective effect. The association of the remaining SNPs, reported by the consortium WTCCC/TASC, was not confirmed. The involvement of rs11465804 was not tested, on the other hand, like in the Canadian study, it was included the SNP rs7517847 but no significant result was found for this variation (Rueda, et al., 2008). In this population, rs1343151 showed a stronger association (P=2x10-4). This findings, together with the Canadian study results previously mentioned (Rahman, et al., 2008), points out to a protection effect of the Arg381Gln nonsynonymous polymorphism. The changing of Arg381 for Gln381 may modify the interaction between IL23R and its associated JAK2 kinase (Parham, C., et al., 2002). The authors proposed that this variation can interfere with the IL23R transducing pathway leading to a reduction in cellular response to IL-23, explaining this way the protective effect of Gln381

A Portuguese study was not able to confirm all this findings (Pimentel-Santos, et al., 2009). The modest power of the study can explain the results. However, as it was already mentioned, a significant association was found for rs1004819. The minor allele frequencies observed in this study were similar to those reported in British and North Americans. Furthermore, this association had a similar magnitude of effect to the one already reported in those populations. The attributable risk for rs1004819 in Portuguese cohort, is very similar to the one reported to rs11209032 in the British/North American populations (Consortium WTCCC/TASC, 2007; Pimentel-Santos, et al., 2009). Using the obtained results, the authors performed a meta-analysis study combining the Portuguese data and the previously published Spanish data (Rueda, et al., 2008). Considering fixed effects, three *IL23R* SNPs revealed significant association rs1004819, rs1343151 and rs11209026. When random effects were considered for the analysis of combined data, only rs1004819 was found to have significant association, confirming the result found when only the Portuguese cohort was

The results obtained for rs1004819 in the Iberian population were confirmed in the Hungarian population (Pimentel-Santos, et al., 2009; Safrany, et al., 2009). This study was performed with a small number of samples; even so, it was possible to register some significant associations. It was shown that the presence of rs1004819 allele A increases the risk for AS in more than two-fold. The same increase was registered when rs10889677 was considered. Significant results were also found for rs11209026. Besides the SNPs reported in the initial association study (Consortium WTCCC/TASC, 2007), the authors also included rs11805303, which was significantly increased in patients with AS. The minor allele of this SNP conferred a 1.6-fold risk for the development of the disease in the studied cohort (Safrany, et al., 2009). The presence of rs11209026 revealed no significant difference between patients and controls when only HLA-B27 positive AS patients were included in the statistical analysis. Thus, the association found for rs11805303 and rs10889677 had a marginal significance when comparing controls with the HLA-B27 patients. Nevertheless, all the other variants kept their significant results, showing that HLA-B27 status has no real

significant differences in rs7517847 (Rahman, et al., 2008).

allele (Rahman, et al., 2008; Rueda, et al., 2008).

analyzed (Pimentel-Santos, et al., 2009).

effect on the effect of IL23R (Safrany, et al., 2009).

production, like when they are induced by IL-12. However, IFN-γ levels produced in cells stimulated by IL-23 are always lower than those induced by IL-12. In the presence of IL-23, naive T cells only increase the producing of IFN-γ after a long stimulation. In contrast, memory T cells - human and mouse – and NKL cells register a strong response to IL-23, enhancing IFN-γ production (Oppmann, et al., 2000; Parham, C., et al., 2002). The cells response to either IL-12 or IL-23 is linked to the level of IL-12Rβ2 or IL23R expression, respectively (Parham, C., et al., 2002).

IL23R is one of the two subunits of IL-23 receptor complex, present on IL-23-responsive cells. The other component is IL-12Rβ1 and is shared with IL-12 receptor. Despite the similarities in the structure of both receptors, in the presence of IL23R subunit, cells respond to IL-23 and not to IL-12 (Parham, C., et al., 2002). Human *IL23R* is expressed on T cells, Natural-Killer (NK) cells, monocytes and DCs, all cells that are able to respond to IL-23 (Belladonna, et al., 2002; Parham, C., et al., 2002). The IL23R has an important role in CD4 Tcell differentiation, since it encodes a critical cytokine receptor in the TH17 lymphocyte subset. The TH17 cells were already identified as mediators of inflammatory process in several models of autoimmunity (Cua, et al., 2003; Murphy, et al., 2003). Furthermore, they are considered as a distinct subset of T-cells, expressing high levels of IL-17 in response to stimulation (Park, et al., 2005) and are associated to tissue damage in brain, joints, heart, lungs and intestines (Steinman, 2007). Genetic variants of IL23R have also been related with susceptibility to several autoimmune diseases, namely inflammatory bowel disease (IBD) (Duerr, et al., 2006), psoriasis (Cargill, et al., 2007), multiple sclerosis (Nunez, et al., 2008) and AS (Consortium WTCCC/TASC, 2007). A significant association between *IL-12Rβ1* and AS (rs6556416; P=1.9x10-8), which encodes one of IL23R and IL-12R subunits, was reported recently by the Consortium WTCCC2/TASC (Consortium TASC/WTCCC2, 2011).

The Consortium WTCCC/TASC reported the strong association (p ≤ 0.008) of eight *IL23R* SNPs and AS, rs11209026, rs1004819, rs10489629, rs11465804, rs1343151, rs10889677, rs11209032, rs1495965 (Consortium WTCCC/TASC, 2007). The strongest association reported was with SNP rs11209032 (p= 7.5 × 10-9) with an attributable risk of 9%. The power of this association was consistent, even when only AS cases, without IBD. were considered. It was stated that this fact could be the result of a primary association with AS, and thus, not related to the presence of IBD (Consortium WTCCC/TASC, 2007). These findings were replicated in several studies; however, there are some conflicting results, especially when analyzing different ethnic groups. A recent GWAS used the same British cohort, and included a new sample composed by Australian, British and North American individuals. In this large cohort a strong AS association to SNP rs11209026 (p= 2.3 × 10-9) was identified (Consortium TASC, 2010). This association was soon after confirmed by the most recent GWAS reported by the TASC/WTCCC2 consortia (Consortium TASC/WTCCC2, 2011).

Closely after the WTCCC/TASC report, a Canadian and a Spanish study were able to replicate some of the already studied *IL23R* SNPs (Rahman, et al., 2008; Rueda, et al., 2008). The Canadian study encompassed 3 cohorts and tested the association of 10 *IL23R* SNPs: rs7517847 and rs2201841, plus the SNPs reported by WTCCC/TASC. Significant associations, to the SNPs rs1004819 and rs11209032, were also found in a Portuguese cohort (Pimentel-Santos, et al., 2009), and in the Alberta population, respectively. The SNPs rs11209026 (Arg381Gln) and rs11465804 revealed association with AS in both Newfoundland and Toronto groups. The last SNP, revealed the strongest protective effect in

production, like when they are induced by IL-12. However, IFN-γ levels produced in cells stimulated by IL-23 are always lower than those induced by IL-12. In the presence of IL-23, naive T cells only increase the producing of IFN-γ after a long stimulation. In contrast, memory T cells - human and mouse – and NKL cells register a strong response to IL-23, enhancing IFN-γ production (Oppmann, et al., 2000; Parham, C., et al., 2002). The cells response to either IL-12 or IL-23 is linked to the level of IL-12Rβ2 or IL23R expression,

IL23R is one of the two subunits of IL-23 receptor complex, present on IL-23-responsive cells. The other component is IL-12Rβ1 and is shared with IL-12 receptor. Despite the similarities in the structure of both receptors, in the presence of IL23R subunit, cells respond to IL-23 and not to IL-12 (Parham, C., et al., 2002). Human *IL23R* is expressed on T cells, Natural-Killer (NK) cells, monocytes and DCs, all cells that are able to respond to IL-23 (Belladonna, et al., 2002; Parham, C., et al., 2002). The IL23R has an important role in CD4 Tcell differentiation, since it encodes a critical cytokine receptor in the TH17 lymphocyte subset. The TH17 cells were already identified as mediators of inflammatory process in several models of autoimmunity (Cua, et al., 2003; Murphy, et al., 2003). Furthermore, they are considered as a distinct subset of T-cells, expressing high levels of IL-17 in response to stimulation (Park, et al., 2005) and are associated to tissue damage in brain, joints, heart, lungs and intestines (Steinman, 2007). Genetic variants of IL23R have also been related with susceptibility to several autoimmune diseases, namely inflammatory bowel disease (IBD) (Duerr, et al., 2006), psoriasis (Cargill, et al., 2007), multiple sclerosis (Nunez, et al., 2008) and AS (Consortium WTCCC/TASC, 2007). A significant association between *IL-12Rβ1* and AS (rs6556416; P=1.9x10-8), which encodes one of IL23R and IL-12R subunits, was reported

recently by the Consortium WTCCC2/TASC (Consortium TASC/WTCCC2, 2011).

The Consortium WTCCC/TASC reported the strong association (p ≤ 0.008) of eight *IL23R* SNPs and AS, rs11209026, rs1004819, rs10489629, rs11465804, rs1343151, rs10889677, rs11209032, rs1495965 (Consortium WTCCC/TASC, 2007). The strongest association reported was with SNP rs11209032 (p= 7.5 × 10-9) with an attributable risk of 9%. The power of this association was consistent, even when only AS cases, without IBD. were considered. It was stated that this fact could be the result of a primary association with AS, and thus, not related to the presence of IBD (Consortium WTCCC/TASC, 2007). These findings were replicated in several studies; however, there are some conflicting results, especially when analyzing different ethnic groups. A recent GWAS used the same British cohort, and included a new sample composed by Australian, British and North American individuals. In this large cohort a strong AS association to SNP rs11209026 (p= 2.3 × 10-9) was identified (Consortium TASC, 2010). This association was soon after confirmed by the most recent GWAS reported by the TASC/WTCCC2 consortia (Consortium TASC/WTCCC2, 2011).

Closely after the WTCCC/TASC report, a Canadian and a Spanish study were able to replicate some of the already studied *IL23R* SNPs (Rahman, et al., 2008; Rueda, et al., 2008). The Canadian study encompassed 3 cohorts and tested the association of 10 *IL23R* SNPs: rs7517847 and rs2201841, plus the SNPs reported by WTCCC/TASC. Significant associations, to the SNPs rs1004819 and rs11209032, were also found in a Portuguese cohort (Pimentel-Santos, et al., 2009), and in the Alberta population, respectively. The SNPs rs11209026 (Arg381Gln) and rs11465804 revealed association with AS in both Newfoundland and Toronto groups. The last SNP, revealed the strongest protective effect in

respectively (Parham, C., et al., 2002).

these two cohorts and it was in LD with rs11209026. The Toronto population also registered significant differences in rs7517847 (Rahman, et al., 2008).

In the Spanish group, the obtained results were able to corroborate the previous findings for rs1343151, rs11209026 and rs10889677. In this cohort the SNPs revealed a protective effect. The association of the remaining SNPs, reported by the consortium WTCCC/TASC, was not confirmed. The involvement of rs11465804 was not tested, on the other hand, like in the Canadian study, it was included the SNP rs7517847 but no significant result was found for this variation (Rueda, et al., 2008). In this population, rs1343151 showed a stronger association (P=2x10-4). This findings, together with the Canadian study results previously mentioned (Rahman, et al., 2008), points out to a protection effect of the Arg381Gln nonsynonymous polymorphism. The changing of Arg381 for Gln381 may modify the interaction between IL23R and its associated JAK2 kinase (Parham, C., et al., 2002). The authors proposed that this variation can interfere with the IL23R transducing pathway leading to a reduction in cellular response to IL-23, explaining this way the protective effect of Gln381 allele (Rahman, et al., 2008; Rueda, et al., 2008).

A Portuguese study was not able to confirm all this findings (Pimentel-Santos, et al., 2009). The modest power of the study can explain the results. However, as it was already mentioned, a significant association was found for rs1004819. The minor allele frequencies observed in this study were similar to those reported in British and North Americans. Furthermore, this association had a similar magnitude of effect to the one already reported in those populations. The attributable risk for rs1004819 in Portuguese cohort, is very similar to the one reported to rs11209032 in the British/North American populations (Consortium WTCCC/TASC, 2007; Pimentel-Santos, et al., 2009). Using the obtained results, the authors performed a meta-analysis study combining the Portuguese data and the previously published Spanish data (Rueda, et al., 2008). Considering fixed effects, three *IL23R* SNPs revealed significant association rs1004819, rs1343151 and rs11209026. When random effects were considered for the analysis of combined data, only rs1004819 was found to have significant association, confirming the result found when only the Portuguese cohort was analyzed (Pimentel-Santos, et al., 2009).

The results obtained for rs1004819 in the Iberian population were confirmed in the Hungarian population (Pimentel-Santos, et al., 2009; Safrany, et al., 2009). This study was performed with a small number of samples; even so, it was possible to register some significant associations. It was shown that the presence of rs1004819 allele A increases the risk for AS in more than two-fold. The same increase was registered when rs10889677 was considered. Significant results were also found for rs11209026. Besides the SNPs reported in the initial association study (Consortium WTCCC/TASC, 2007), the authors also included rs11805303, which was significantly increased in patients with AS. The minor allele of this SNP conferred a 1.6-fold risk for the development of the disease in the studied cohort (Safrany, et al., 2009). The presence of rs11209026 revealed no significant difference between patients and controls when only HLA-B27 positive AS patients were included in the statistical analysis. Thus, the association found for rs11805303 and rs10889677 had a marginal significance when comparing controls with the HLA-B27 patients. Nevertheless, all the other variants kept their significant results, showing that HLA-B27 status has no real effect on the effect of IL23R (Safrany, et al., 2009).

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 113

(Kimura-Yoshida, et al., 2004; Nobrega, et al., 2003). Several diseases have been associated with these particular areas of the genome; possible explanations to elucidate the mechanisms underlying these associations include: 1) epigenetic effects, 2) unknown protein-coding transcripts, 3) effects of long range transcriptional regulatory elements and 4)

A GWAS has recently identified the new association of two narrow intergenic regions, in chromosomes 2 (23kb) and 21 (11kb), with AS. This study found, in each intergenic region, a block of SNPs in tight linkage disequilibrium encompassing areas that likely contain the causative variants responsible for the observed association (Consortium TASC, 2010). Another GWAS recent study replicated this association and has shown that SNPs at these intergenic regions are independent of the HLA-B27 presence, since it is associated with AS in both HLA-B27 positive and HLA-B27-negative patients (Consortium TASC/WTCCC2,

The intergenic area 2p15 does not contain any known gene and, until now, this has been the only association to a disease (Reveille, J. D., et al., 2010). The association to AS was already been replicated in a small study in the Spanish population. The SNP rs10865331 was typed in four hundred and fifty six AS patients and 300 healthy donors, and the result was a significant association with AS, while no association was found for rs2242944, in 21q22

The other intergenic AS associated area, at chromosome 21q22, has already been associated with a closely related condition: paediatric-onset inflammatory bowel disease (IBD) (Kugathasan, et al., 2008). Remarkably, the most strongly IBD associated SNP is in strong linkage disequilibrium with the strongest ankylosing spondylitis-associated marker (Consortium TASC, 2010). The nearest gene, to this region, lies at 82kb distance (PSMG1) and encodes for a proteasome assembly chaperone 1 (Consortium TASC, 2010). According to the authors' opinion, it is unlikely that *PSMG1* is a candidate gene directly involved in AS susceptibility since 1) this gene was not differentially expressed in peripheral blood mononuclear cells from cases with active AS when compared with healthy controls, 2) the large distance to the associated locus and 3) the lack of evidence of a relevant biological

It is suggested that both these regions at chromosomes 2p15 and 21q22, contain long mRNA-like noncoding RNA species or until now unreported protein-coding genes that may

Interleukin-1 (IL-1) and its related family members are key cytokines in autoimmune and inflammatory diseases produced by monocytes, macrophages, and dendritic cells. They are cell surface associated proteins and stimulate the expression of several genes, affecting both the innate and acquired immune systems (Dinarello, 2002). IL1-F1, IL1-F2, IL1-F3, and IL1- F4 are the primary members of the family. IL1-F1, IL1-F2, and IL1-F4 are each agonist while IL1-F3 is a receptor antagonist for IL1-F1, IL1-F2 (Dinarello, 2002). When the antagonist occupies the receptor, there is no signal transduction as the IL1-F1 and IL1-F2 can not bind

be involved in AS susceptibility (Consortium TASC, 2010).

**3. Genes suggestively associated with ankylosing spondylitis** 

effects of non-coding RNA that can influence gene expression (Brown, 2010).

2011).

function.

**3.1 IL1 gene cluster** 

(Consortium TASC/WTCCC2, 2011).

A meta-analysis, reported at the same year, included the first United Kingdom and United States cohort published (Consortium WTCCC/TASC, 2007), the Canadian groups (Alberta, Newfoundland and Toronto) (Rahman, et al., 2008), the Spanish results (Rueda, et al., 2008) plus a new British group (Karaderi, et al., 2009). Just like stated by WTCCC/TASC, the results of this study confirmed the association between AS and all the eight IL23R SPNs analyzed. Once more, the strongest associations were seen with rs11209026 (P<10-10) and rs11209032 (P=4.06x10-9) (Karaderi, et al., 2009). The last variant had the same order of magnitude reported by the WTCCC/TASC study (Consortium WTCCC/TASC, 2007). This meta-analysis confirmed the connection of IL23R and AS susceptibility in Caucasians.

The first replication, performed in a group with a different ethnic background, did not confirm the association of IL23R and AS in Han Chinese (Davidson, et al., 2009; Sung, et al., 2009). These findings may indicate a difference in the mechanism of disease pathogenesis between Caucasian and Han Chinese populations. The authors proposed that the development of AS developing Chinese can result from a mechanism independent of any *IL23R* polymorphism. This difference could be the result of the association with a different gene also involved in the *IL23R* signaling pathway (Davidson, et al., 2009). Other explanation to this lack of association is the absence of polymorphism of rs11209026 in Chinese. This variant, as described above, is pointed out as one of the causative SNP for disease susceptibility in Europeans. However, recent published data reported association of some *IL23R* SNPs and AS in a Chinese cohort (Wang, et al., 2010). In this group it was found, for the first time, a significant association between rs6677188, located in the intergenic region, and AS susceptibility. The previously reported association of rs11209032 in Caucasians was confirmed also in Chinese. Through the analysis of paiwised LD, it was found that rs11209032 and rs6677188 were in strong LD, in this population (Wang, et al., 2010).

#### **2.2.3 KIF21B**

The gene *KIF21B* (Kinesin Family 21B) is one of the most recent loci with confirmed association to AS. This gene belongs to a family of kinesin motor proteins that are involved in the transport of essential components along axonal and denditric microtubules by neurons. A GWS found strong association for rs11584383 (1q32) (P = 1.6x10-10), a SNP located downstream of and flanked by *KIF21B* (Danoy, et al., 2010). This strong association was corroborated by WTCCC2/TASC. Here, the SNP rs2297909 showed the strongest association with AS (P=5.2 × 10−12) (Consortium TASC/WTCCC2, 2011). Both studies were conducted with Caucasian cohorts. Despite the confirmed association in this ethnic group, further replications in other ethnic groups are mandatory.

#### **2.3 Intergenic regions - 2p15 and 21q22**

It has been estimated that approximately 25% of the human genome consists of gene deserts defined as generally long regions, ranging from a few base pairs to 5.1 Mb, containing no protein-coding sequences and with no obvious biological functions (Venter, et al., 2001). Ovcharenko *et al*. found that conservation clearly separates two distinct categories of gene deserts: weakly conserved variable gene deserts and more conserved stable gene deserts (Ovcharenko, et al., 2005). Moreover, it has already been shown that some human gene deserts harbour distant regulatory elements that are deeply conserved in vertebrate species

A meta-analysis, reported at the same year, included the first United Kingdom and United States cohort published (Consortium WTCCC/TASC, 2007), the Canadian groups (Alberta, Newfoundland and Toronto) (Rahman, et al., 2008), the Spanish results (Rueda, et al., 2008) plus a new British group (Karaderi, et al., 2009). Just like stated by WTCCC/TASC, the results of this study confirmed the association between AS and all the eight IL23R SPNs analyzed. Once more, the strongest associations were seen with rs11209026 (P<10-10) and rs11209032 (P=4.06x10-9) (Karaderi, et al., 2009). The last variant had the same order of magnitude reported by the WTCCC/TASC study (Consortium WTCCC/TASC, 2007). This meta-analysis confirmed the connection of IL23R and AS susceptibility in Caucasians.

The first replication, performed in a group with a different ethnic background, did not confirm the association of IL23R and AS in Han Chinese (Davidson, et al., 2009; Sung, et al., 2009). These findings may indicate a difference in the mechanism of disease pathogenesis between Caucasian and Han Chinese populations. The authors proposed that the development of AS developing Chinese can result from a mechanism independent of any *IL23R* polymorphism. This difference could be the result of the association with a different gene also involved in the *IL23R* signaling pathway (Davidson, et al., 2009). Other explanation to this lack of association is the absence of polymorphism of rs11209026 in Chinese. This variant, as described above, is pointed out as one of the causative SNP for disease susceptibility in Europeans. However, recent published data reported association of some *IL23R* SNPs and AS in a Chinese cohort (Wang, et al., 2010). In this group it was found, for the first time, a significant association between rs6677188, located in the intergenic region, and AS susceptibility. The previously reported association of rs11209032 in Caucasians was confirmed also in Chinese. Through the analysis of paiwised LD, it was found that rs11209032 and rs6677188 were in strong LD, in this population (Wang, et al.,

The gene *KIF21B* (Kinesin Family 21B) is one of the most recent loci with confirmed association to AS. This gene belongs to a family of kinesin motor proteins that are involved in the transport of essential components along axonal and denditric microtubules by neurons. A GWS found strong association for rs11584383 (1q32) (P = 1.6x10-10), a SNP located downstream of and flanked by *KIF21B* (Danoy, et al., 2010). This strong association was corroborated by WTCCC2/TASC. Here, the SNP rs2297909 showed the strongest association with AS (P=5.2 × 10−12) (Consortium TASC/WTCCC2, 2011). Both studies were conducted with Caucasian cohorts. Despite the confirmed association in this ethnic group,

It has been estimated that approximately 25% of the human genome consists of gene deserts defined as generally long regions, ranging from a few base pairs to 5.1 Mb, containing no protein-coding sequences and with no obvious biological functions (Venter, et al., 2001). Ovcharenko *et al*. found that conservation clearly separates two distinct categories of gene deserts: weakly conserved variable gene deserts and more conserved stable gene deserts (Ovcharenko, et al., 2005). Moreover, it has already been shown that some human gene deserts harbour distant regulatory elements that are deeply conserved in vertebrate species

further replications in other ethnic groups are mandatory.

**2.3 Intergenic regions - 2p15 and 21q22** 

2010).

**2.2.3 KIF21B** 

(Kimura-Yoshida, et al., 2004; Nobrega, et al., 2003). Several diseases have been associated with these particular areas of the genome; possible explanations to elucidate the mechanisms underlying these associations include: 1) epigenetic effects, 2) unknown protein-coding transcripts, 3) effects of long range transcriptional regulatory elements and 4) effects of non-coding RNA that can influence gene expression (Brown, 2010).

A GWAS has recently identified the new association of two narrow intergenic regions, in chromosomes 2 (23kb) and 21 (11kb), with AS. This study found, in each intergenic region, a block of SNPs in tight linkage disequilibrium encompassing areas that likely contain the causative variants responsible for the observed association (Consortium TASC, 2010). Another GWAS recent study replicated this association and has shown that SNPs at these intergenic regions are independent of the HLA-B27 presence, since it is associated with AS in both HLA-B27 positive and HLA-B27-negative patients (Consortium TASC/WTCCC2, 2011).

The intergenic area 2p15 does not contain any known gene and, until now, this has been the only association to a disease (Reveille, J. D., et al., 2010). The association to AS was already been replicated in a small study in the Spanish population. The SNP rs10865331 was typed in four hundred and fifty six AS patients and 300 healthy donors, and the result was a significant association with AS, while no association was found for rs2242944, in 21q22 (Consortium TASC/WTCCC2, 2011).

The other intergenic AS associated area, at chromosome 21q22, has already been associated with a closely related condition: paediatric-onset inflammatory bowel disease (IBD) (Kugathasan, et al., 2008). Remarkably, the most strongly IBD associated SNP is in strong linkage disequilibrium with the strongest ankylosing spondylitis-associated marker (Consortium TASC, 2010). The nearest gene, to this region, lies at 82kb distance (PSMG1) and encodes for a proteasome assembly chaperone 1 (Consortium TASC, 2010). According to the authors' opinion, it is unlikely that *PSMG1* is a candidate gene directly involved in AS susceptibility since 1) this gene was not differentially expressed in peripheral blood mononuclear cells from cases with active AS when compared with healthy controls, 2) the large distance to the associated locus and 3) the lack of evidence of a relevant biological function.

It is suggested that both these regions at chromosomes 2p15 and 21q22, contain long mRNA-like noncoding RNA species or until now unreported protein-coding genes that may be involved in AS susceptibility (Consortium TASC, 2010).

#### **3. Genes suggestively associated with ankylosing spondylitis**

#### **3.1 IL1 gene cluster**

Interleukin-1 (IL-1) and its related family members are key cytokines in autoimmune and inflammatory diseases produced by monocytes, macrophages, and dendritic cells. They are cell surface associated proteins and stimulate the expression of several genes, affecting both the innate and acquired immune systems (Dinarello, 2002). IL1-F1, IL1-F2, IL1-F3, and IL1- F4 are the primary members of the family. IL1-F1, IL1-F2, and IL1-F4 are each agonist while IL1-F3 is a receptor antagonist for IL1-F1, IL1-F2 (Dinarello, 2002). When the antagonist occupies the receptor, there is no signal transduction as the IL1-F1 and IL1-F2 can not bind

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 115

In a recent study on unrelated cases of AS among Australian, British, and North American individuals of European descent, carried out by TASC, a strong association between polymorphism in *ANTXR2* gene and AS was found (Consortium TASC, 2010). In the same year, investigating on AS susceptibility in 1164 Korean patients, Bang et al. confirmed the differentially expression of *ANTXR2* gene in AS (Bang, et al., 2011). In contrast no association was observed between *ANTXR2* polymorphism and AS in Chinese Han population (Chen, C., et al., 2010). With these divergent results, it is still not clear if ANTXR2 influences the disease and how it is involved in the pathogenesis. IT has been suggested that it can act at intestinal permeability level given its function in epithelial barriers (Thomas & Brown, 2010). The previous results reported by TASC were confirmed by another GWS that also found significant association in the ANTXR2 area (P = 9.4 × 10−8)

The Tumor Necrosis Factor Ligand Superfamily, Member 15 (TNFSF15, also known as TNF superfamily ligand A, TL1A or vascular endothelial cell growth inhibitor, VEGI) is a TNFlike factor encoded on the chromosome 9 (9q32). The molecules are primarily expressed in endothelial cells and its expression is highly inducible by TNF and IL-1A (Migone, et al., 2002; Yue, et al., 1999). Strong evidences point to a significant genetic association between the TNFSF15 gene and inflammatory bowel disease (IBD). The data was obtained in populations from different ethnic backgrounds, namely Caucasians and Japanese (Yamazaki, et al., 2005). A WGAS for AS in Caucasians reported the existence of linkage on chromosome 9q (Laval, et al., 2001). This was supported by another WGS for spondyloarthropathies (SpA) susceptibility genes, performed on multiplex families, which detected a significant linkage on chromosome 9q31-34, including the TNFSF15 encoding region (Miceli-Richard, et al., 2004). However, this region was not identified in a recent

Recently, comprehensive linkage and association analyses reported, for the first time, an association between several SNPs near the *TNFSF15* gene and SpA (Zinovieva, et al., 2009). The authors aimed to narrow down the susceptibility region for SpA found on the first GWAS reported by the same authors (Miceli-Richard, et al., 2004). The obtained data showed two areas of statistically significance linkage. The highest linkage peak was located on the marker D9S1824 at 115.9 Mb from the p-telomere. The second significant area was located near D9S1682, a suggestive linkage peak reported in WGS for AS (Laval, et al., 2001).

The analysis of combined data from family-based and case/control studies showed a strong association (P<0.001) of 7 SNPs: rs4979459, rs7849556, rs10817669, rs10759734, rs6478105, rs10982396 and rs10733612 (Zinovieva, et al., 2009). The SNP rs4246905 also showed a significant result but with a lower level (P=0.01). Six of this SNPs compose a 40.3 Kb block with a high degree of LD, found in the same study. This LD block included two genes, *LOC389786* and *TNFSF15* (Zinovieva, et al., 2009). The rs6478105 revealed to be the strongest individual associated SNP in the overall dataset (P=3x10-5). The SNPs associated with Crohn's disease were not associated with SpA in this study. Haplotypes research also confirmed this tendency and showed that haplotypes composed of markers of this block were significantly associated with the disease. Two significant individual haplotypes were

This finding supports the validity of linkage between this region and SpA.

(Consortium TASC/WTCCC2, 2011).

GWAS (Consortium TASC, 2010).

**3.3 TNFSF15** 

the specific receptor. All *IL-1* genes are located on the long arm of chromosome 2 except IL-18 and IL-18 binding protein (IL-18BP) which are located on chromosome 11. Some member of the family as IL1-F5, IL1-F7, and IL1-F9 are gene duplications and they are very closely related to IL1-F3 (Mulero, et al., 1999). The exact function of these genes is still unclear.

The IL-1 receptor family is composed of nine genes. IL1-R1, IL1-R2, and IL1-R3 are the bona fide receptors while the rest are called 'orphan' receptors as they lack of specific ligand Anyway, some gene regulation studies found non-specific proteins binding to orphan receptors (Gayle, et al., 1996; Moritz, et al., 1998; Parnet, et al., 1996; Torigoe, et al., 1997). The most studied receptor of the family is the IL1-R2 (Symons, et al., 1991). IL1-R2 receptor acts as a decoy receptor. It has a high affinity for IL1-F1 and IL1-F2 and a lower affinity for IL1-F3. After the cleavage from cell membrane by ERAP1, IL1-R2 binds IL1-F1 interfering with IL1-F1 / IL1-R1 binding. Its extracellular domain is homologue of the IL1-R1 but the intracellular domain is shorter and lacks of TIR domain (Dunne & O'Neill, 2003). Therefore when activated, IL-R2 is unable to initiate any biological response.

Positive association between AS and *IL1-F3* gene was observed in earlier studies with high frequency of the allele 2 of *IL1-F3* variable nucleotide tandem repeat (VNTR) in AS patients (Dunne & O'Neill, 2003; van der Paardt, et al., 2002). The result was further confirmed through a recent study on meta-analysis of *IL-1* gene cluster (Wu & Gu, 2007). A significant association between two *IL1-F3* intronic SNPs and AS susceptibility was also found at position 30735 and 30017 in exon 6 (Chou, et al., 2006; Maksymowych, W. P., et al., 2003). A significant difference of the distribution of haplotypes was found between the AS affected and healthy individuals. On the contrary, in earlier studies no association between AS and *IL1-F3* gene was found (Djouadi, et al., 2001; Jin, et al., 2004; Kim, et al., 2005; Maksymowych, W. P., et al., 2006; Timms, A. E., et al., 2004). These findings were also observed in the genome scan carried out by North American Spondylitis Consortium (NASC) (Jin, et al., 2004). In this work, six exons and introns in 102 white patients and 50 controls were sequenced and no association was revealed. Moreover, other genes in the *IL-1* cluster have been analyzed. In nine *IL-1* genes, were identified SNPs showing significant association with AS (Timms, A. E., et al., 2004). Maksymowych et al. identified 14 SNPs with high association in at least one cohort (Maksymowych, W. P., et al., 2006). The most significant cohorts were in IL1-F1 and IL1-F2. In a more recent meta-analysis nine SNPs were analyzed and a strong association was observed in three IL1-F1 loci (Sims, A. M., et al., 2008).

#### **3.2 ANTXR2**

The *ANTXR2* gene encodes for a transmembrane protein which serves as receptor of anthrax toxin (Thomas & Brown, 2010). Also known as capillary morphogenesis protein 2 (CMP2), the receptor recognises and binds the toxin, allowing anthrax to attach the cells and triggering the disease process. It is widely expressed in human tissue including in the hearth, lung, liver, placenta, small intestine, kidney, colon, and skeletal muscles. Expressed primarily in macrofages, it is involved in capillary formation and extracellular matrix adhesion (Reveille, J D, 2011). Recessive mutations in ANTXR2 gene are associated to two autosomal diseases, infantile systemic hyalinosis (ISH; MIM#237490) and juvenile hyaline fibromatosis (JHF; MIM#228600) (Consortium TASC, 2010).

In a recent study on unrelated cases of AS among Australian, British, and North American individuals of European descent, carried out by TASC, a strong association between polymorphism in *ANTXR2* gene and AS was found (Consortium TASC, 2010). In the same year, investigating on AS susceptibility in 1164 Korean patients, Bang et al. confirmed the differentially expression of *ANTXR2* gene in AS (Bang, et al., 2011). In contrast no association was observed between *ANTXR2* polymorphism and AS in Chinese Han population (Chen, C., et al., 2010). With these divergent results, it is still not clear if ANTXR2 influences the disease and how it is involved in the pathogenesis. IT has been suggested that it can act at intestinal permeability level given its function in epithelial barriers (Thomas & Brown, 2010). The previous results reported by TASC were confirmed by another GWS that also found significant association in the ANTXR2 area (P = 9.4 × 10−8) (Consortium TASC/WTCCC2, 2011).

#### **3.3 TNFSF15**

114 Clinical and Molecular Advances in Ankylosing Spondylitis

the specific receptor. All *IL-1* genes are located on the long arm of chromosome 2 except IL-18 and IL-18 binding protein (IL-18BP) which are located on chromosome 11. Some member of the family as IL1-F5, IL1-F7, and IL1-F9 are gene duplications and they are very closely related to IL1-F3 (Mulero, et al., 1999). The exact function of these genes is still unclear.

The IL-1 receptor family is composed of nine genes. IL1-R1, IL1-R2, and IL1-R3 are the bona fide receptors while the rest are called 'orphan' receptors as they lack of specific ligand Anyway, some gene regulation studies found non-specific proteins binding to orphan receptors (Gayle, et al., 1996; Moritz, et al., 1998; Parnet, et al., 1996; Torigoe, et al., 1997). The most studied receptor of the family is the IL1-R2 (Symons, et al., 1991). IL1-R2 receptor acts as a decoy receptor. It has a high affinity for IL1-F1 and IL1-F2 and a lower affinity for IL1-F3. After the cleavage from cell membrane by ERAP1, IL1-R2 binds IL1-F1 interfering with IL1-F1 / IL1-R1 binding. Its extracellular domain is homologue of the IL1-R1 but the intracellular domain is shorter and lacks of TIR domain (Dunne & O'Neill, 2003). Therefore

Positive association between AS and *IL1-F3* gene was observed in earlier studies with high frequency of the allele 2 of *IL1-F3* variable nucleotide tandem repeat (VNTR) in AS patients (Dunne & O'Neill, 2003; van der Paardt, et al., 2002). The result was further confirmed through a recent study on meta-analysis of *IL-1* gene cluster (Wu & Gu, 2007). A significant association between two *IL1-F3* intronic SNPs and AS susceptibility was also found at position 30735 and 30017 in exon 6 (Chou, et al., 2006; Maksymowych, W. P., et al., 2003). A significant difference of the distribution of haplotypes was found between the AS affected and healthy individuals. On the contrary, in earlier studies no association between AS and *IL1-F3* gene was found (Djouadi, et al., 2001; Jin, et al., 2004; Kim, et al., 2005; Maksymowych, W. P., et al., 2006; Timms, A. E., et al., 2004). These findings were also observed in the genome scan carried out by North American Spondylitis Consortium (NASC) (Jin, et al., 2004). In this work, six exons and introns in 102 white patients and 50 controls were sequenced and no association was revealed. Moreover, other genes in the *IL-1* cluster have been analyzed. In nine *IL-1* genes, were identified SNPs showing significant association with AS (Timms, A. E., et al., 2004). Maksymowych et al. identified 14 SNPs with high association in at least one cohort (Maksymowych, W. P., et al., 2006). The most significant cohorts were in IL1-F1 and IL1-F2. In a more recent meta-analysis nine SNPs were analyzed and a strong association was observed in three IL1-F1 loci (Sims, A. M., et al.,

The *ANTXR2* gene encodes for a transmembrane protein which serves as receptor of anthrax toxin (Thomas & Brown, 2010). Also known as capillary morphogenesis protein 2 (CMP2), the receptor recognises and binds the toxin, allowing anthrax to attach the cells and triggering the disease process. It is widely expressed in human tissue including in the hearth, lung, liver, placenta, small intestine, kidney, colon, and skeletal muscles. Expressed primarily in macrofages, it is involved in capillary formation and extracellular matrix adhesion (Reveille, J D, 2011). Recessive mutations in ANTXR2 gene are associated to two autosomal diseases, infantile systemic hyalinosis (ISH; MIM#237490) and juvenile hyaline

fibromatosis (JHF; MIM#228600) (Consortium TASC, 2010).

when activated, IL-R2 is unable to initiate any biological response.

2008).

**3.2 ANTXR2** 

The Tumor Necrosis Factor Ligand Superfamily, Member 15 (TNFSF15, also known as TNF superfamily ligand A, TL1A or vascular endothelial cell growth inhibitor, VEGI) is a TNFlike factor encoded on the chromosome 9 (9q32). The molecules are primarily expressed in endothelial cells and its expression is highly inducible by TNF and IL-1A (Migone, et al., 2002; Yue, et al., 1999). Strong evidences point to a significant genetic association between the TNFSF15 gene and inflammatory bowel disease (IBD). The data was obtained in populations from different ethnic backgrounds, namely Caucasians and Japanese (Yamazaki, et al., 2005). A WGAS for AS in Caucasians reported the existence of linkage on chromosome 9q (Laval, et al., 2001). This was supported by another WGS for spondyloarthropathies (SpA) susceptibility genes, performed on multiplex families, which detected a significant linkage on chromosome 9q31-34, including the TNFSF15 encoding region (Miceli-Richard, et al., 2004). However, this region was not identified in a recent GWAS (Consortium TASC, 2010).

Recently, comprehensive linkage and association analyses reported, for the first time, an association between several SNPs near the *TNFSF15* gene and SpA (Zinovieva, et al., 2009). The authors aimed to narrow down the susceptibility region for SpA found on the first GWAS reported by the same authors (Miceli-Richard, et al., 2004). The obtained data showed two areas of statistically significance linkage. The highest linkage peak was located on the marker D9S1824 at 115.9 Mb from the p-telomere. The second significant area was located near D9S1682, a suggestive linkage peak reported in WGS for AS (Laval, et al., 2001). This finding supports the validity of linkage between this region and SpA.

The analysis of combined data from family-based and case/control studies showed a strong association (P<0.001) of 7 SNPs: rs4979459, rs7849556, rs10817669, rs10759734, rs6478105, rs10982396 and rs10733612 (Zinovieva, et al., 2009). The SNP rs4246905 also showed a significant result but with a lower level (P=0.01). Six of this SNPs compose a 40.3 Kb block with a high degree of LD, found in the same study. This LD block included two genes, *LOC389786* and *TNFSF15* (Zinovieva, et al., 2009). The rs6478105 revealed to be the strongest individual associated SNP in the overall dataset (P=3x10-5). The SNPs associated with Crohn's disease were not associated with SpA in this study. Haplotypes research also confirmed this tendency and showed that haplotypes composed of markers of this block were significantly associated with the disease. Two significant individual haplotypes were

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 117

The cytochrome P450 enzymes are responsible for the majority of oxidative (phase I) drug metabolism (1)(Gonzalez, 1992); they are polymorphic although 5-10% of Europeans lack this activity (described as poor metabolisers) (Cholerton, et al., 1992). At least 15 allelic variants of *CYP2D6*, that is inherited as an autosomal recessive trait, can cause poor metaboliser phenotype, but 75% of these are CYP2D6\*4 (Brown, et al., 2000). The *CYP2D6* genotype was found to be associated with other chronic inflammatory disease and with

Beyeler et al. (Beyeler, et al., 1996) first reported a relationship between this gene and the susceptibility to AS, investigating 54 patients and 662 healthy volunteers. The association was modest between AS and CYP2D6 genotype and the effect was greatest for the *CYP2D6B*

Brown et al. (Brown, et al., 2000) studied linkage of the *CYP2D6* gene and association of the main poor metabolizer genotype in 617 unrelated AS patients, 402 healthy controls, and in 361 families with AS. Significant association was observed between homozygosity for CYP2D6\*4 and AS, but heterozygosity for allele 4 was not disease associated, and weak linkage of the *CYP2D6* polymorphism and AS was found with a LOD score of 0.9. The authors suggested that dysfunction of the *CYP2D6* gene increases the risk of AS, although

A more recent Turkish study, investigating 100 unrelated AS patients and 52 healthy controls, found no significant risk of AS development for patients with one or two

In conclusion, the cytochrome P450 gene debrisoquine 4-hydroxilase (*CYP2D6*), encoded at 22q13.1, may have a moderate support for involvement in susceptibility to AS. The mechanisms for susceptibility are unknown but it is possible that the ubiquitous environmental trigger of AS is a natural toxin and reducing its metabolism could increase

The Killer immunoglobulin-like receptor (KIR) genes are a polymorphic group of genes located on chromosome 19q13.4, and they span 150Kb of the leucocyte receptor complex (LCR) (Hsu, K. C., et al., 2002). They express a family of proteins which are activating and inhibitory receptors expressed on natural killer (NK) cells and on a subset of T cells (CD8+). They have been classified in two types: Activating KIRs (KIR2DS and KIR3DS), which have a short (S) cytoplasmic tail with the capacity to interact with activating adaptor proteins such as DAP12 (Lanier, et al., 1998), and inhibitory *KIR* (KIR2DL and KIR3DL), which has one or two immunoreceptor tyrosine-based inhibition motifs in their long (L) cytoplasmic tail. Further to the allelic polymorphism, haplotypic variability is also described and according to gene content, haplotypes were divided in two basic groups: Haplotype A contains only one activating KIR gene, 2DS4, whereas Haplotype B contains various combinations of activating *KIR* genes, KIR2DS1, -2DS2, -2DS3, -2DS5, - 3DS1, and -2DS4,

only contributing a small proportion of the overall risk of the disease.

**3.5 CYP2D6** 

allele.

cancer (Baer, et al., 1986; Daly, et al., 1994).

CYP2D6\*4 alleles (Erden, et al., 2009).

susceptibility to disease (Brown, 2006).

**4. Other genes** 

**4.1 KIR** 

found in family-based study: rs7849556/ rs10817669/ rs10759734/ rs6478105/ rs10982396/ rs10733612 - AAAACC and rs7849556/ rs10817669/ rs10759734/ rs6478105/ rs10982396/ rs10733612 - CGGACT. The pooled case/control investigation revealed a significant strong association of the individual haplotype rs7849556/ rs10817669/ rs10759734/ rs6478105/ rs10982396/ rs10733612 - CGGGGT (Zinovieva, et al., 2009).

TNFSF15 is a ligand for the receptors DR3 (death domain receptor 3, also known as TNFRSF25 (tumor necrosis factor receptor super-family, member 25) and TR6/DcR3 (decoy receptor 3, also called TNFRSF6B - tumor necrosis factor receptor super-family, member 6b) (Migone, et al., 2002). The ligand-receptor pairing of TNFSF15-DR3 was already pointed as a regulator of Th17 differentiation and activation (Pappu, et al., 2008; Takedatsu, et al., 2008). An increased level of Th17 cells in AS patients, when compared to healthy controls, was already reported (Jandus, et al., 2008). Therefore, targeting the TNFSF15-DR3 pathway could provide new insights in the role of TNFS15 in AS development.

#### **3.4 TNFRSF1A and TRADD**

The TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A, also known as tumor necrosis factor receptor 1 - TNFR1) association with AS was reported by the TASC GWAS. Several *TNFRSF1A* SNPs showed moderate levels of association in the discovery set and, among them, rs1800693 showed the strongest association (P=6.9x10-5) (Consortium TASC, 2010). It was hypothasized that TNF1, encoded by *TNFRSF1A*, was cleaved by ERAP1. The data suggested that ERAP1 extracellular domain binds to the TNFR1 extracellular domain and acts as an extracellular TNFR1 regulatory protein that would promote TNFR1 shedding (Cui, et al., 2002). However, as already mentioned in ERAP1 section, no correlation between presence of ERAP1 and decrease of TNFR1 levels was observed in cultured cells from mice (Consortium TASC/WTCCC2, 2011).

TNF antagonists are highly effective in suppressing inflammation in AS. Thus, some data obtained from studies with mice showed that mesenchymal cells are common primary targets for TNF in the development of AS, and that selective expression of *TNFRSF1A* on those cells is enough to cause the complete development of AS, as well as inflammatory polyarthritis and inflammatory bowel disease (IBD) (Armaka, et al., 2008). The determination of the polymorphisms involved on that action would allow a better understanding of the relation between *TNFRSF1A* gene and AS. TRADD (TNF receptor type 1-associated death domain) is located on chromosome 16q, a region already reported in linkage studies (Laval, et al., 2001). TRADD registered moderate levels of association and lies between the SNPs rs9033 and rs868213, which already showed strong association with AS (Pointon, et al., 2010). This gene is a key component of the TNFR1-signaling cascade and is involved in TLR3, TLR4 and D3 signaling (Chen, N. J., et al., 2008; Chinnaiyan, et al., 1996; Hsu, H., et al., 1995).

Recently it was found an AS associated SNP at chromosome 12p13 between LTBR (lymphotoxin beta receptor) and TNFRSF1A (rs11616188; P = 4.1 × 10−12). The authors also found an association at chromosome 17q21 near *TBKBP1* (encoding TBK binding protein 1), a component of the TNFR signaling pathway (rs8070463, P = 5.3 × 10−8). Here, TRADD also showed suggestive association with AS in this study, namely SNP rs9033 (P = 4.9 × 10−5) (Consortium TASC/WTCCC2, 2011). Once more, it is mandatory to continue research at this level to determine the precise mechanism underlying these associations.

#### **3.5 CYP2D6**

116 Clinical and Molecular Advances in Ankylosing Spondylitis

found in family-based study: rs7849556/ rs10817669/ rs10759734/ rs6478105/ rs10982396/ rs10733612 - AAAACC and rs7849556/ rs10817669/ rs10759734/ rs6478105/ rs10982396/ rs10733612 - CGGACT. The pooled case/control investigation revealed a significant strong association of the individual haplotype rs7849556/ rs10817669/ rs10759734/ rs6478105/

TNFSF15 is a ligand for the receptors DR3 (death domain receptor 3, also known as TNFRSF25 (tumor necrosis factor receptor super-family, member 25) and TR6/DcR3 (decoy receptor 3, also called TNFRSF6B - tumor necrosis factor receptor super-family, member 6b) (Migone, et al., 2002). The ligand-receptor pairing of TNFSF15-DR3 was already pointed as a regulator of Th17 differentiation and activation (Pappu, et al., 2008; Takedatsu, et al., 2008). An increased level of Th17 cells in AS patients, when compared to healthy controls, was already reported (Jandus, et al., 2008). Therefore, targeting the TNFSF15-DR3 pathway could

The TNFRSF1A (Tumor necrosis factor receptor superfamily member 1A, also known as tumor necrosis factor receptor 1 - TNFR1) association with AS was reported by the TASC GWAS. Several *TNFRSF1A* SNPs showed moderate levels of association in the discovery set and, among them, rs1800693 showed the strongest association (P=6.9x10-5) (Consortium TASC, 2010). It was hypothasized that TNF1, encoded by *TNFRSF1A*, was cleaved by ERAP1. The data suggested that ERAP1 extracellular domain binds to the TNFR1 extracellular domain and acts as an extracellular TNFR1 regulatory protein that would promote TNFR1 shedding (Cui, et al., 2002). However, as already mentioned in ERAP1 section, no correlation between presence of ERAP1 and decrease of TNFR1 levels was

TNF antagonists are highly effective in suppressing inflammation in AS. Thus, some data obtained from studies with mice showed that mesenchymal cells are common primary targets for TNF in the development of AS, and that selective expression of *TNFRSF1A* on those cells is enough to cause the complete development of AS, as well as inflammatory polyarthritis and inflammatory bowel disease (IBD) (Armaka, et al., 2008). The determination of the polymorphisms involved on that action would allow a better understanding of the relation between *TNFRSF1A* gene and AS. TRADD (TNF receptor type 1-associated death domain) is located on chromosome 16q, a region already reported in linkage studies (Laval, et al., 2001). TRADD registered moderate levels of association and lies between the SNPs rs9033 and rs868213, which already showed strong association with AS (Pointon, et al., 2010). This gene is a key component of the TNFR1-signaling cascade and is involved in TLR3, TLR4 and D3 signaling (Chen, N. J., et al., 2008; Chinnaiyan, et al., 1996;

Recently it was found an AS associated SNP at chromosome 12p13 between LTBR (lymphotoxin beta receptor) and TNFRSF1A (rs11616188; P = 4.1 × 10−12). The authors also found an association at chromosome 17q21 near *TBKBP1* (encoding TBK binding protein 1), a component of the TNFR signaling pathway (rs8070463, P = 5.3 × 10−8). Here, TRADD also showed suggestive association with AS in this study, namely SNP rs9033 (P = 4.9 × 10−5) (Consortium TASC/WTCCC2, 2011). Once more, it is mandatory to continue research at this

rs10982396/ rs10733612 - CGGGGT (Zinovieva, et al., 2009).

provide new insights in the role of TNFS15 in AS development.

observed in cultured cells from mice (Consortium TASC/WTCCC2, 2011).

level to determine the precise mechanism underlying these associations.

**3.4 TNFRSF1A and TRADD** 

Hsu, H., et al., 1995).

The cytochrome P450 enzymes are responsible for the majority of oxidative (phase I) drug metabolism (1)(Gonzalez, 1992); they are polymorphic although 5-10% of Europeans lack this activity (described as poor metabolisers) (Cholerton, et al., 1992). At least 15 allelic variants of *CYP2D6*, that is inherited as an autosomal recessive trait, can cause poor metaboliser phenotype, but 75% of these are CYP2D6\*4 (Brown, et al., 2000). The *CYP2D6* genotype was found to be associated with other chronic inflammatory disease and with cancer (Baer, et al., 1986; Daly, et al., 1994).

Beyeler et al. (Beyeler, et al., 1996) first reported a relationship between this gene and the susceptibility to AS, investigating 54 patients and 662 healthy volunteers. The association was modest between AS and CYP2D6 genotype and the effect was greatest for the *CYP2D6B* allele.

Brown et al. (Brown, et al., 2000) studied linkage of the *CYP2D6* gene and association of the main poor metabolizer genotype in 617 unrelated AS patients, 402 healthy controls, and in 361 families with AS. Significant association was observed between homozygosity for CYP2D6\*4 and AS, but heterozygosity for allele 4 was not disease associated, and weak linkage of the *CYP2D6* polymorphism and AS was found with a LOD score of 0.9. The authors suggested that dysfunction of the *CYP2D6* gene increases the risk of AS, although only contributing a small proportion of the overall risk of the disease.

A more recent Turkish study, investigating 100 unrelated AS patients and 52 healthy controls, found no significant risk of AS development for patients with one or two CYP2D6\*4 alleles (Erden, et al., 2009).

In conclusion, the cytochrome P450 gene debrisoquine 4-hydroxilase (*CYP2D6*), encoded at 22q13.1, may have a moderate support for involvement in susceptibility to AS. The mechanisms for susceptibility are unknown but it is possible that the ubiquitous environmental trigger of AS is a natural toxin and reducing its metabolism could increase susceptibility to disease (Brown, 2006).

#### **4. Other genes**

#### **4.1 KIR**

The Killer immunoglobulin-like receptor (KIR) genes are a polymorphic group of genes located on chromosome 19q13.4, and they span 150Kb of the leucocyte receptor complex (LCR) (Hsu, K. C., et al., 2002). They express a family of proteins which are activating and inhibitory receptors expressed on natural killer (NK) cells and on a subset of T cells (CD8+). They have been classified in two types: Activating KIRs (KIR2DS and KIR3DS), which have a short (S) cytoplasmic tail with the capacity to interact with activating adaptor proteins such as DAP12 (Lanier, et al., 1998), and inhibitory *KIR* (KIR2DL and KIR3DL), which has one or two immunoreceptor tyrosine-based inhibition motifs in their long (L) cytoplasmic tail. Further to the allelic polymorphism, haplotypic variability is also described and according to gene content, haplotypes were divided in two basic groups: Haplotype A contains only one activating KIR gene, 2DS4, whereas Haplotype B contains various combinations of activating *KIR* genes, KIR2DS1, -2DS2, -2DS3, -2DS5, - 3DS1, and -2DS4,

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 119

The *ANKH* gene maps to human chromosome 5 (5p15.1) and encodes a 492 amino acid multiple-pass transmembrane protein (ANK) which transports the inorganic pyrophosphate (PPi) across the plasma membrane into the extracellular compartment (Gurley, K.A., et al., 2006b). ANK function is essential in joints to inhibit mineral formation in joints and maintain mobility (Gurley, K. A., et al., 2006a). Mutations in the *ANKH* gene have been consistently associated with two autosomal dominant skeletal disorders: familial

In the last decade, a very small number of reports suggested an association of AN*KH* with AS. In 2003, two polymorphisms - ANKH-OR and ANKH-TR - in complete linkage disequilibrium, located in the 5'-noncoding region and in the promoter region of this gene, respectively, were found to be significantly associated with AS. After linkage analysis and family-based association studies the authors concluded that *ANKH* could be among the most important non-MHC loci for AS susceptibility (Tsui, F W, et al., 2003). In a follow-up study, with 201 multiplex AS families, it was reported that the region associated with AS in women only showed significance in the test of interaction among the subset of families with affected individuals of both genders. These findings supported the concept that *ANKH* plays a role in genetic susceptibility to AS revealing a gender-genotype specificity in this interaction (Tsui, H W, et al., 2005). Contradicting these results, a small study performed in a cohort of 233 patients and 478 controls, revealed no association between *ANKH* locus and

chondrocalcinosis (MIM #118600) and craniometaphyseal dysplasia (MIM #123000).

either susceptibility to AS or its clinical manifestations (Timms, A E, et al., 2003).

GWAS studies with large cohorts.

by chronic inflammation of the sacroiliac joints.

**4.3 TGFB1** 

In another study, the authors examined a total of 45 SNPs in 15 genes by a sequential screening. 170 Japanese AS patients and 896 controls for the SNPs were first genotyped. Then, eight SNPs with P < 0.05 in the first screen were genotyped for 108 additional Japanese patients. The replication of the association of the most significant SNP was checked by genotyping 219 Taiwanese AS patients and 185 controls. After combining the first and second screens, four SNPs showed nominal significance of P < 0.05. One synonymous SNP in *ANKH*, c.963T > G, showed a marginal association in the Japanese population (P = 0.045) (Furuichi, et al., 2008). This association is not consistent and was not replicated in recent

The *TGFB1* gene codifies for the human transforming growth factor β1 (TGFβ1) (van der Paardt, et al., 2005b) located on chromosome 19q21.1. It is a multifunctional cytokine involved in inflammation, fibrosis and bone remodelling (Reveille, J D, 2011). The concentration of TGFβ1 in cartilage and bone is 100 times superior to other tissues (Centrella, et al., 1991). It was demonstrate that injections of TGFβ1 into young rat bone induce formation of cartilaginous mass and subsequently bone tissue (Joyce, et al., 1990). Whether the effect is positive with growing bones or negative with damaged bones depends on the concentration of TGFβ1 and the presence of other hormones (Archer & Keat, 1999). Therefore, TGFβ1 represent a good candidate for a key cytokine in a disease characterized

A marginal association between TGFB1 and AS was observed in a study of Finnish and British families (Jaakkola, et al., 2004) and in a Scottish case study (McGarry, et al., 2002).

**4.2 ANKH** 

exhibiting extreme diversity, resulting in different signaling potentials to NK and T cells (Hsu, K. C., et al., 2002).

The precise function of some KIRs is controversial but it was demonstrated that mature class I complexes act as ligands for immunomodulatary receptors, and it is known that KIR proteins recognize subsets of HLA-A, -B, or –C alleles (Parham, P., 2005). It is possible that KIRs may synergise with HLAs to generate activating or inhibitory compound genotypes that provide different levels of activation and inhibition for NK or T cells, which may be associated with differing susceptibility to or protection against a range of diseases.

A study in Caucasian populations demonstrated genetic evidence for the implication of KIR3DL1 and the activating counterpart, KIR3DS1 in AS (Lopez-Larrea, C., et al., 2006). In this study the inhibitory allele was decreased in AS patients compared with B27-positive healthy controls, whereas KIR3DS1 was increased in AS patients. Another study by the same group in two Asian B27-positive populations (China and Thailand), reported some KIR associations with AS susceptibility (Diaz-Pena, et al., 2008). The authors hypothesized that AS patients could possess more activating *KIR* genes than the healthy control subjects, which could create a genetic imbalance between inhibitory and activating *KIR* genes that could have influence on the AS pathogenesis.

Another study genotyped 200 UK AS patients and 405 healthy controls for 14 *KIR* genes. Additionally, sequence-specific oligonucleotide probes were used to subtype 368 cases with AS and 366 controls for 12 KIR3DL2 alleles. The authors concluded that neither the *KIR* gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to AS (Harvey, et al., 2009b).

Two more recent studies, one from China and another one from Spain investigated the association of KIR and AS susceptibility. The Chinese study investigated 115 unrelated HLA-B27-positive AS patients and 119 HLA-B27-positive healthy controls, and concluded that the frequencies of KIR2DL1 and KIR2DL5 were significantly higher in the AS patient group although they did not reach statistical significance. Furthermore, the investigators also concluded that HLA-Cw\*08 was present more frequently in AS patients than in healthy B27 controls, raising the possibility that HLA-Cw\*08 recognized KIRs through the Asp80, thereby contributing to the immune regulation in AS (Jiao, et al., 2010). In the Spanish study 270 AS patients and 435 healthy HLA-B27-positive controls from Spain were genotyped for KIR3DL1/S1 alleles. The authors found that the KIR3DS1\*013 allele frequency was increased in patients with AS, and that the null allele KIR3DL1\*004 was a unique inhibitory KIR3DL1 allele that showed a negative association with AS (Diaz-Pena, et al., 2010).

Genome-wide scans have implicated regions on chromosomes 2q, 6q, 10q, 11q, 16q, 17q, and 19q in AS. The *KIR* genes are located on chromosome 19q13.4 in the LCR, and obviously are good candidates for AS susceptibility (Carter, et al., 2007). More recent investigations using different approaches like the report of the TASC/WTCCC2, did not identify this chromosome region as involved in AS susceptibility (Consortium TASC/WTCCC2, 2011). The influence of KIR/HLA genotypes in AS susceptibility may be mediated by a general imbalance between the protective/inhibitory and the risk/activating allotypes. Further studies in other populations are needed to confirm the role of KIR genes in AS susceptibility.

#### **4.2 ANKH**

118 Clinical and Molecular Advances in Ankylosing Spondylitis

exhibiting extreme diversity, resulting in different signaling potentials to NK and T cells

The precise function of some KIRs is controversial but it was demonstrated that mature class I complexes act as ligands for immunomodulatary receptors, and it is known that KIR proteins recognize subsets of HLA-A, -B, or –C alleles (Parham, P., 2005). It is possible that KIRs may synergise with HLAs to generate activating or inhibitory compound genotypes that provide different levels of activation and inhibition for NK or T cells, which may be

A study in Caucasian populations demonstrated genetic evidence for the implication of KIR3DL1 and the activating counterpart, KIR3DS1 in AS (Lopez-Larrea, C., et al., 2006). In this study the inhibitory allele was decreased in AS patients compared with B27-positive healthy controls, whereas KIR3DS1 was increased in AS patients. Another study by the same group in two Asian B27-positive populations (China and Thailand), reported some KIR associations with AS susceptibility (Diaz-Pena, et al., 2008). The authors hypothesized that AS patients could possess more activating *KIR* genes than the healthy control subjects, which could create a genetic imbalance between inhibitory and activating *KIR* genes that

Another study genotyped 200 UK AS patients and 405 healthy controls for 14 *KIR* genes. Additionally, sequence-specific oligonucleotide probes were used to subtype 368 cases with AS and 366 controls for 12 KIR3DL2 alleles. The authors concluded that neither the *KIR* gene content of particular KIR haplotypes nor KIR3DL2 polymorphisms contribute to AS

Two more recent studies, one from China and another one from Spain investigated the association of KIR and AS susceptibility. The Chinese study investigated 115 unrelated HLA-B27-positive AS patients and 119 HLA-B27-positive healthy controls, and concluded that the frequencies of KIR2DL1 and KIR2DL5 were significantly higher in the AS patient group although they did not reach statistical significance. Furthermore, the investigators also concluded that HLA-Cw\*08 was present more frequently in AS patients than in healthy B27 controls, raising the possibility that HLA-Cw\*08 recognized KIRs through the Asp80, thereby contributing to the immune regulation in AS (Jiao, et al., 2010). In the Spanish study 270 AS patients and 435 healthy HLA-B27-positive controls from Spain were genotyped for KIR3DL1/S1 alleles. The authors found that the KIR3DS1\*013 allele frequency was increased in patients with AS, and that the null allele KIR3DL1\*004 was a unique inhibitory KIR3DL1 allele that showed a negative association with AS (Diaz-Pena,

Genome-wide scans have implicated regions on chromosomes 2q, 6q, 10q, 11q, 16q, 17q, and 19q in AS. The *KIR* genes are located on chromosome 19q13.4 in the LCR, and obviously are good candidates for AS susceptibility (Carter, et al., 2007). More recent investigations using different approaches like the report of the TASC/WTCCC2, did not identify this chromosome region as involved in AS susceptibility (Consortium TASC/WTCCC2, 2011). The influence of KIR/HLA genotypes in AS susceptibility may be mediated by a general imbalance between the protective/inhibitory and the risk/activating allotypes. Further studies in other populations are needed to confirm the

associated with differing susceptibility to or protection against a range of diseases.

(Hsu, K. C., et al., 2002).

(Harvey, et al., 2009b).

et al., 2010).

role of KIR genes in AS susceptibility.

could have influence on the AS pathogenesis.

The *ANKH* gene maps to human chromosome 5 (5p15.1) and encodes a 492 amino acid multiple-pass transmembrane protein (ANK) which transports the inorganic pyrophosphate (PPi) across the plasma membrane into the extracellular compartment (Gurley, K.A., et al., 2006b). ANK function is essential in joints to inhibit mineral formation in joints and maintain mobility (Gurley, K. A., et al., 2006a). Mutations in the *ANKH* gene have been consistently associated with two autosomal dominant skeletal disorders: familial chondrocalcinosis (MIM #118600) and craniometaphyseal dysplasia (MIM #123000).

In the last decade, a very small number of reports suggested an association of AN*KH* with AS. In 2003, two polymorphisms - ANKH-OR and ANKH-TR - in complete linkage disequilibrium, located in the 5'-noncoding region and in the promoter region of this gene, respectively, were found to be significantly associated with AS. After linkage analysis and family-based association studies the authors concluded that *ANKH* could be among the most important non-MHC loci for AS susceptibility (Tsui, F W, et al., 2003). In a follow-up study, with 201 multiplex AS families, it was reported that the region associated with AS in women only showed significance in the test of interaction among the subset of families with affected individuals of both genders. These findings supported the concept that *ANKH* plays a role in genetic susceptibility to AS revealing a gender-genotype specificity in this interaction (Tsui, H W, et al., 2005). Contradicting these results, a small study performed in a cohort of 233 patients and 478 controls, revealed no association between *ANKH* locus and either susceptibility to AS or its clinical manifestations (Timms, A E, et al., 2003).

In another study, the authors examined a total of 45 SNPs in 15 genes by a sequential screening. 170 Japanese AS patients and 896 controls for the SNPs were first genotyped. Then, eight SNPs with P < 0.05 in the first screen were genotyped for 108 additional Japanese patients. The replication of the association of the most significant SNP was checked by genotyping 219 Taiwanese AS patients and 185 controls. After combining the first and second screens, four SNPs showed nominal significance of P < 0.05. One synonymous SNP in *ANKH*, c.963T > G, showed a marginal association in the Japanese population (P = 0.045) (Furuichi, et al., 2008). This association is not consistent and was not replicated in recent GWAS studies with large cohorts.

#### **4.3 TGFB1**

The *TGFB1* gene codifies for the human transforming growth factor β1 (TGFβ1) (van der Paardt, et al., 2005b) located on chromosome 19q21.1. It is a multifunctional cytokine involved in inflammation, fibrosis and bone remodelling (Reveille, J D, 2011). The concentration of TGFβ1 in cartilage and bone is 100 times superior to other tissues (Centrella, et al., 1991). It was demonstrate that injections of TGFβ1 into young rat bone induce formation of cartilaginous mass and subsequently bone tissue (Joyce, et al., 1990). Whether the effect is positive with growing bones or negative with damaged bones depends on the concentration of TGFβ1 and the presence of other hormones (Archer & Keat, 1999). Therefore, TGFβ1 represent a good candidate for a key cytokine in a disease characterized by chronic inflammation of the sacroiliac joints.

A marginal association between TGFB1 and AS was observed in a study of Finnish and British families (Jaakkola, et al., 2004) and in a Scottish case study (McGarry, et al., 2002).

Genetics in Ankylosing Spondylitis – Beyond HLA-B\*27 121

AS susceptibility is no longer an HLA-B27 exclusive. The association of AS and ERAP1, IL23R and the intergenic regions 2p15 and 21q22 has been confirmed in large cohort studies. This knowledge increased the complexity of genes involved in AS susceptibility; however, it

The identification of associated genetic regions outside MHC will require a large number of replicates in populations with different ethnic backgrounds. The consistent data already obtained, and the results that will come up from new replicates, should provide a solid basis to new research that may unravel the mechanisms underlying all these associations. Consequently, the AS pathway will start to present an even more conclusive and consistent shape, providing new tools to clinicians and allowing an improvement in the disease diagnosis and treatment. More, the identified and confirmed markers could be used to

Archer, J. R. & A. C. Keat. (1999). Ankylosing spondylitis: time to focus on ankylosis. *J* 

Armaka, M., M. Apostolaki, P. Jacques, D. L. Kontoyiannis, D. Elewaut & G. Kollias. (2008).

Armas, J. B., S. Gonzalez, J. Martinez-Borra, F. Laranjeira, E. Ribeiro, J. Correia, M. L.

Baer, A. N., C. B. McAllister, G. R. Wilkinson, R. L. Woosley & T. Pincus. (1986). Altered

Ball, E. J. & M. A. Khan. (2001). HLA-B27 polymorphism. *Joint Bone Spine*,Vol. 68, No. 5,

Bang, S. Y., T. H. Kim, B. Lee, E. Kwon, S. H. Choi, K. S. Lee, S. C. Shim, A. Pope, P. Rahman,

Belladonna, M. L., J. C. Renauld, R. Bianchi, C. Vacca, F. Fallarino, C. Orabona, M. C.

Beyeler, C., M. Armstrong, H. A. Bird, J. R. Idle & A. K. Daly. (1996). Relationship between

Mesenchymal cell targeting by TNF as a common pathogenic principle in chronic inflammatory joint and intestinal diseases. *J Exp Med*,Vol. 205, No. 2, (Feb 18), pp.

Ferreira, M. Toste, A. Lopez-Vazquez & C. Lopez-Larrea. (1999). Susceptibility to ankylosing spondylitis is independent of the Bw4 and Bw6 epitopes of HLA-B27

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J. D. Reveille & R. D. Inman. (2011). Genetic studies of ankylosing spondylitis in Koreans confirm associations with ERAP1 and 2p15 reported in white patients. *J* 

Fioretti, U. Grohmann & P. Puccetti. (2002). IL-23 and IL-12 have overlapping, but distinct, effects on murine dendritic cells. *J Immunol*,Vol. 168, No. 11, (Jun 1), pp.

genotype for the cytochrome P450 CYP2D6 and susceptibility to ankylosing spondylitis and rheumatoid arthritis. *Ann Rheum Dis*,Vol. 55, No. 1, (Jan), pp. (66-

is shortening the way to a better understanding about AS immunopathogenesis.

create a complete diagnosis testing panel, along with HLA-B27.

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**5. Conclusion** 

**6. References** 

The Scottish genotype showed a strong correlation with high concentration of TGFβ1. On the other side, no significant association with AS susceptibility was reported in two following studies in Dutch and Southern Chinese populations (Howe, et al., 2005; van der Paardt, et al., 2005b); although, in the Dutch study, the frequency of mutated allele was greater in AS patients than in the healthy individuals. The TGFB1 polymorphism might be implicated to the AS disease at bone formation level but further studies are necessary. Understanding the role of TGFβ1 in AS could be interesting especially to develop new pharmacological approaches in order to prevent the disorder (van der Paardt, et al., 2005b).

#### **4.4 CD14**

The Cluster of differentiation 14 (CD14) gene maps to chromosome 5 (5q22-q3215q31.1) and encodes 2 protein forms: a 50 to 55 kD glycosylphosphatidylinositol-anchored membrane protein (mCD14) and a monocyte or liver-derived soluble serum protein (sCD14) that lacks the anchor. Both molecules are essential for lipopolysaccharide (LPS)-dependent signal transduction. Increased sCD14 levels are associated with inflammatory infectious diseases and high mortality in gram-negative shock (LeVan, et al., 2001).

A putative role of *CD14* in the pathogenesis of AS was investigated due to its significant role on the innate immune system. The polymorphism C-260T was studied in genomic DNA from 113 unrelated Dutch AS patients and 170 healthy controls. No significant differences were found between the frequency of this allele in patients and controls suggesting that this polymorphism is not involved in the susceptibility to AS (van der Paardt, et al., 2005a). On the other hand, evidence of association was identified between this allele and AS in a small cohort of Finnish families (Pointon, et al., 2008). No other studies about CD14 and its association with AS have been published suggesting that this association is not established.

#### **4.5 TNAP**

Tissue-Nonspecific Alkaline Phosphatase (TNAP) is an isozyme of a family of four homologous human alkaline phosphatase genes. It is present in the matrix vesicles and has the ability of hydrolyze PPi. In humans, this enzyme is enconded by the gene *TNAP*, that maps in chromosome 1, containing 12 exons. Deactivating mutations in the *TNAP* gene cause hypophosphatasia [MIM#241500 (Infantile form), MIM#146300 (Adult type)], characterized by poorly mineralized cartilage and bones, spontaneous bone fractures, chondrocalcinosis by calcium pyrophosphate deposition and elevated concentrations of pyrophosphate (PPi) (Mornet, et al., 1998).

One study has shown the significant association of the *TNAP* haplotype rs3767155(G) / rs3738099 (G) / rs1780329 (T) with AS but in men only (Tsui, H. W., et al., 2007). This association was later investigated in a case control study involving a cohort of 353 AS patients and 514 unrelated healthy controls, and a family-based association study with 57 pedigrees, of the Chinese Han population. Two intronic SNPs (rs3767155 and rs1780329) were genotyped; the results showed no significant difference in allele, genotype or haplotype frequencies between AS patients and controls (Cheng, et al., 2009). No other studies have replicated the association of TNAP to AS.

#### **5. Conclusion**

120 Clinical and Molecular Advances in Ankylosing Spondylitis

The Scottish genotype showed a strong correlation with high concentration of TGFβ1. On the other side, no significant association with AS susceptibility was reported in two following studies in Dutch and Southern Chinese populations (Howe, et al., 2005; van der Paardt, et al., 2005b); although, in the Dutch study, the frequency of mutated allele was greater in AS patients than in the healthy individuals. The TGFB1 polymorphism might be implicated to the AS disease at bone formation level but further studies are necessary. Understanding the role of TGFβ1 in AS could be interesting especially to develop new pharmacological approaches in order to prevent the disorder (van der Paardt, et al.,

The Cluster of differentiation 14 (CD14) gene maps to chromosome 5 (5q22-q3215q31.1) and encodes 2 protein forms: a 50 to 55 kD glycosylphosphatidylinositol-anchored membrane protein (mCD14) and a monocyte or liver-derived soluble serum protein (sCD14) that lacks the anchor. Both molecules are essential for lipopolysaccharide (LPS)-dependent signal transduction. Increased sCD14 levels are associated with inflammatory infectious diseases

A putative role of *CD14* in the pathogenesis of AS was investigated due to its significant role on the innate immune system. The polymorphism C-260T was studied in genomic DNA from 113 unrelated Dutch AS patients and 170 healthy controls. No significant differences were found between the frequency of this allele in patients and controls suggesting that this polymorphism is not involved in the susceptibility to AS (van der Paardt, et al., 2005a). On the other hand, evidence of association was identified between this allele and AS in a small cohort of Finnish families (Pointon, et al., 2008). No other studies about CD14 and its association with AS have been published suggesting that this

Tissue-Nonspecific Alkaline Phosphatase (TNAP) is an isozyme of a family of four homologous human alkaline phosphatase genes. It is present in the matrix vesicles and has the ability of hydrolyze PPi. In humans, this enzyme is enconded by the gene *TNAP*, that maps in chromosome 1, containing 12 exons. Deactivating mutations in the *TNAP* gene cause hypophosphatasia [MIM#241500 (Infantile form), MIM#146300 (Adult type)], characterized by poorly mineralized cartilage and bones, spontaneous bone fractures, chondrocalcinosis by calcium pyrophosphate deposition and elevated concentrations of

One study has shown the significant association of the *TNAP* haplotype rs3767155(G) / rs3738099 (G) / rs1780329 (T) with AS but in men only (Tsui, H. W., et al., 2007). This association was later investigated in a case control study involving a cohort of 353 AS patients and 514 unrelated healthy controls, and a family-based association study with 57 pedigrees, of the Chinese Han population. Two intronic SNPs (rs3767155 and rs1780329) were genotyped; the results showed no significant difference in allele, genotype or haplotype frequencies between AS patients and controls (Cheng, et al., 2009). No other

and high mortality in gram-negative shock (LeVan, et al., 2001).

2005b).

**4.4 CD14** 

**4.5 TNAP** 

association is not established.

pyrophosphate (PPi) (Mornet, et al., 1998).

studies have replicated the association of TNAP to AS.

AS susceptibility is no longer an HLA-B27 exclusive. The association of AS and ERAP1, IL23R and the intergenic regions 2p15 and 21q22 has been confirmed in large cohort studies. This knowledge increased the complexity of genes involved in AS susceptibility; however, it is shortening the way to a better understanding about AS immunopathogenesis.

The identification of associated genetic regions outside MHC will require a large number of replicates in populations with different ethnic backgrounds. The consistent data already obtained, and the results that will come up from new replicates, should provide a solid basis to new research that may unravel the mechanisms underlying all these associations. Consequently, the AS pathway will start to present an even more conclusive and consistent shape, providing new tools to clinicians and allowing an improvement in the disease diagnosis and treatment. More, the identified and confirmed markers could be used to create a complete diagnosis testing panel, along with HLA-B27.

#### **6. References**


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**8** 

*1Portugal 2Australia* 

**Lessons from Genomic Profiling in AS** 

*1Universidade Nova de Lisboa, Faculdade de Ciências Médicas,* 

*Chronic Diseases Research Center (CEDOC), Lisboa, 2University of Queensland Diamantina Institute,* 

*Princess Alexandra Hospital, Brisbane,* 

Fernando M. Pimentel-Santos1, Jaime C. Branco1 and Gethin Thomas2

Ankylosing Spondylitis (AS) is a common cause of chronic inflammatory arthritis worldwide, with a prevalence of 0.2-0.9% in white European populations (Braun *et al.*, 1998), with unknown etiology. The progressive ankylosis of affected joints is currently irreversible and it is, therefore, logical that early diagnosis and treatment offers the best opportunity to improve its prognosis. Several studies have shown a delay of more than 8 years between the onset of symptoms and diagnosis, with consequent delay in starting an effective therapy (Feldtkeller *et al.*, 2003; Hamilton *et al.*, 2011). This is a critical period clinically, with diagnosis frequently occurring after significant irreversible radiological damage has already occurred. Currently, diagnosis of AS relies on a combination of clinical and imaging parameters (van der Linden *et al.*, 1984 and Boonen *et al.*, 2010) with no single blood derived biomarker that by itself is sufficiently sensitive and specific to identify AS cases or to be

In this context, recent advances in molecular biology, in particular, the completion of the genome human sequence, the improvement in computational tools and the rapid access to large databases, allow an integrated understanding of biological systems, through "omic" approaches. The main challenge, however, is to extract relevant knowledge from the huge amount of data provided by these technologies for the development of biomarkers for diagnosis, prognosis, therapy monitoring and both prediction and monitoring of treatment response. Such technological advances represent the beginning of patient-specific

In contrast to traditional DNA-based diagnostic tests that largely focus on single genes associated with rare conditions, microarray-based genotyping and expression assays are ideal for the study of diseases with underlying complex genetic causes (Li *et al.*, 2008). Microarray gene expression technology can be used for the detection and quantification of differentially expressed genes. Its ability to study expression of several thousand genes or even all of the genes of the entire genome in a single experiment has changed biomedical research. Gene-expression profiling confers a "snapshot" of cellular activity providing information on the mechanisms mediating stress responses of human cells (Belcher *et al.*, 2000; Guillemin *et al.*, 2002), identification of signaling cascades (Shaffer *et al.*, 2000; Diehn *et* 

**1. Introduction** 

useful in disease management.

personalized medicine (Kandpal *et al.*, 2009).

M. Wei, R. Wides, C. Xiao, C. Yan, et al. (2001). The sequence of the human genome. *Science*,Vol. 291, No. 5507, (Feb 16), pp. (1304-51), 0036-8075 (Print), 0036- 8075 (Linking)


### **Lessons from Genomic Profiling in AS**

Fernando M. Pimentel-Santos1, Jaime C. Branco1 and Gethin Thomas2

*1Universidade Nova de Lisboa, Faculdade de Ciências Médicas, Chronic Diseases Research Center (CEDOC), Lisboa, 2University of Queensland Diamantina Institute, Princess Alexandra Hospital, Brisbane, 1Portugal 2Australia* 

#### **1. Introduction**

134 Clinical and Molecular Advances in Ankylosing Spondylitis

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Zinovieva, E., C. Bourgain, A. Kadi, F. Letourneur, B. Izac, R. Said-Nahal, N. Lebrun, N.

Chinese patients. *Rheumatology*,Vol. 43, No. 7, (Jun 2004), pp. (839-42), Wu, Z. & J. R. Gu. (2007). A meta-analysis on interleukin-1 gene cluster polymorphism and

No. 7, (Feb 13), pp. (433-7), 0376-2491 (Print), 0376-2491 (Linking)

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1529-2908 (Print), 1529-2908 (Linking)

No. 3, (Jan 15), pp. (1479-86), 0021-9258

No. 6, (Jun), 1553-7390

M. Wei, R. Wides, C. Xiao, C. Yan, et al. (2001). The sequence of the human genome. *Science*,Vol. 291, No. 5507, (Feb 16), pp. (1304-51), 0036-8075 (Print), 0036-

nucleotide polymorphisms and expression of IL23R in Chinese ankylosing spondylitis patients. *Rheumatol Int*,Vol. 30, No. 7, (May), pp. (955-9), 1437-160X

strongly associated with ankylosing spondylitis in HLA-B27-negative Taiwan

genetic susceptibility for ankylosing spondylitis. *Zhonghua Yi Xue Za Zhi*,Vol. 87,

Juji & K. Ito. (1995). Association of HLA-B39 with HLA-B27-negative ankylosing spondylitis and pauciarticular juvenile rheumatoid arthritis in Japanese patients. Evidence for a role of the peptide-anchoring B pocket. *Arthritis Rheum*,Vol. 38, No.

Takazoe, T. Tanaka, T. Ichimori, S. Saito, A. Sekine, A. Iida, A. Takahashi, T. Tsunoda, M. Lathrop & Y. Nakamura. (2005). Single nucleotide polymorphisms in TNFSF15 confer susceptibility to Crohn's disease. *Hum Mol Genet*,Vol. 14, No. 22,

(2002). The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues. *Nat Immunol*,Vol. 3, No. 12, (Dec), pp. (1177-84),

X. Wang, Z. Xia, W. E. DeWolf, Jr. & G. Z. Feuerstein. (1999). TL1, a novel tumor necrosis factor-like cytokine, induces apoptosis in endothelial cells. Involvement of activation of stress protein kinases (stress-activated protein kinase and p38 mitogen-activated protein kinase) and caspase-3-like protease. *J Biol Chem*,Vol. 274,

Cagnard, A. Vigier, S. Jacques, C. Miceli-Richard, H. J. Garchon, S. Heath, C. Charon, D. Bacq, A. Boland, D. Zelenika, G. Chiocchia & M. Breban. (2009). Comprehensive linkage and association analyses identify haplotype, near to the TNFSF15 gene, significantly associated with spondyloarthritis. *PLoS Genet*,Vol. 5, Ankylosing Spondylitis (AS) is a common cause of chronic inflammatory arthritis worldwide, with a prevalence of 0.2-0.9% in white European populations (Braun *et al.*, 1998), with unknown etiology. The progressive ankylosis of affected joints is currently irreversible and it is, therefore, logical that early diagnosis and treatment offers the best opportunity to improve its prognosis. Several studies have shown a delay of more than 8 years between the onset of symptoms and diagnosis, with consequent delay in starting an effective therapy (Feldtkeller *et al.*, 2003; Hamilton *et al.*, 2011). This is a critical period clinically, with diagnosis frequently occurring after significant irreversible radiological damage has already occurred. Currently, diagnosis of AS relies on a combination of clinical and imaging parameters (van der Linden *et al.*, 1984 and Boonen *et al.*, 2010) with no single blood derived biomarker that by itself is sufficiently sensitive and specific to identify AS cases or to be useful in disease management.

In this context, recent advances in molecular biology, in particular, the completion of the genome human sequence, the improvement in computational tools and the rapid access to large databases, allow an integrated understanding of biological systems, through "omic" approaches. The main challenge, however, is to extract relevant knowledge from the huge amount of data provided by these technologies for the development of biomarkers for diagnosis, prognosis, therapy monitoring and both prediction and monitoring of treatment response. Such technological advances represent the beginning of patient-specific personalized medicine (Kandpal *et al.*, 2009).

In contrast to traditional DNA-based diagnostic tests that largely focus on single genes associated with rare conditions, microarray-based genotyping and expression assays are ideal for the study of diseases with underlying complex genetic causes (Li *et al.*, 2008). Microarray gene expression technology can be used for the detection and quantification of differentially expressed genes. Its ability to study expression of several thousand genes or even all of the genes of the entire genome in a single experiment has changed biomedical research. Gene-expression profiling confers a "snapshot" of cellular activity providing information on the mechanisms mediating stress responses of human cells (Belcher *et al.*, 2000; Guillemin *et al.*, 2002), identification of signaling cascades (Shaffer *et al.*, 2000; Diehn *et* 

Lessons from Genomic Profiling in AS 137

Despite minor differences between platforms, the basic steps involved in a microarrays experiment are similar (Fig. 1) (Repsilber *et al.*, 2005). Key points in undertaking an

2. Selection of the tissue/cell most relevant to the question and the selection of the control

3. Total mRNA is extracted from the chosen tissue/cell, and reverse transcribed

5. Bound probes are detected and quantified by imaging tools and every gene/probe

6. Signals are corrected for common bias i.e. normalized. For each mRNA, the signal intensity difference between the disease and the control sample correlates to the change in gene expression (genes up- or down-regulated) that might be associated with the studied condition. Several methods have been implemented to reduce variability in DNA microarray experiments (Workman *et al.*, 2002). A critical step in the whole procedure is an appropriate analysis of the large volumes of data generated using sophisticated software. Bioconductor (www.bioconductor.org) or BRB ArrayTools (Simon *et al.*, 2007), examples of bioinformatic platforms, provide tools for analysis and

7. Candidate genes are validated through another technology. Usually quantitative

Large-scale gene expression analysis, is in fact, a flourishing technology with potential applications in several fields of Biology and Medicine as indicated by the large number of peer-reviewed articles (n=35502) containing the words "gene" and "microarray" found in

Microarray profiling of gene expression is a powerful tool for discovery, but the ability to manage and compare the resulting data can be problematic. Biological, experimental, and technical variations between studies of the same phenotype/phenomena create substantial

a) The success of the microarrays experience greatly depends on whether the hypothesis and rationale have been appropriately formulated through a clearly delineated question. It influences the study design as a whole, from sample collection, to experimental design, and

b) While most of the early studies used primary tissues involved in the disease, such as tumor biopsies, more recently a number of gene expression profiling studies have focused on peripheral blood to identify systemic markers of disease. However, gene expression patterns in peripheral blood cells greatly depend on inter-individual variations and technical aspects such as blood sampling techniques, cell and RNA isolation as well as storage temperature or delays in processing. However although significant inter-individual variations in gene expression patterns in peripheral blood cells can be seen, these differences

generating cDNA which is labelled with radioactive or fluorescent markers.

4. Labeled transcripts are hybridized onto the microarray.

reverse-transcription PCR (qPCR) is the preferred method.

differences in results. Some of these issues will be discussed in detail.

finally, the strategies for data analysis (Smith & Rosa, 2007).

8. Data is integrated and applied to the initial question.

expression profiling study are:

group.

1. Establish your research question.

assigned a signal intensity.

comprehension of genomic data.

**1.2 Microarray challenges and concerns** 

Pubmed upto June 2011.

*al.*, 2002), disease changes, or mechanisms underlying therapy responses (Raetz & Moos, 2004). It represents an advance to the traditional molecular genomic techniques that have been previously applied in a large broad of clinical research as cancer, infections, metabolic, genetics and more recently, in rheumatic diseases.

#### **1.1 Microarray fundamentals**

Gene expression techniques, based on measuring mRNA levels, have greatly evolved since the development of the Northern Blot, in 1975 (Southern, 1975) to microarrays, in the mid 1990s (Shalon *et al.*, 1996). From a single labeled mRNA (probe), hybridized on a membrane (Northern Blot), to multiple probes hybridized on a membrane (macroarrays) or on glass (microarrays), the improvement was tremendous. Today several platforms, with predesigned and custom arrays are available in the market (Hardiman, 2004) from Affymetrix, Agilent and Illumina. Table 1 summarizes similarities and differences between the most widely used platforms.


Table 1. Microarray platform comparison.

*al.*, 2002), disease changes, or mechanisms underlying therapy responses (Raetz & Moos, 2004). It represents an advance to the traditional molecular genomic techniques that have been previously applied in a large broad of clinical research as cancer, infections, metabolic,

Gene expression techniques, based on measuring mRNA levels, have greatly evolved since the development of the Northern Blot, in 1975 (Southern, 1975) to microarrays, in the mid 1990s (Shalon *et al.*, 1996). From a single labeled mRNA (probe), hybridized on a membrane (Northern Blot), to multiple probes hybridized on a membrane (macroarrays) or on glass (microarrays), the improvement was tremendous. Today several platforms, with predesigned and custom arrays are available in the market (Hardiman, 2004) from Affymetrix, Agilent and Illumina. Table 1 summarizes similarities and differences between the most

Platforms

**Array format** 25-mer 60-mer 50-mer

**temperature** 45ºC 60ºC 55ºC

**Affymetrix Agilent Illumina** 

**Fluorescent Direct Label Kit (cDNA labeling):** 10μg total RNA, or200ng polyA+ RNA **Low input RNA Fluorescent Linear Amplification kit (Amplified cDNA labeling):** 50ng total RNA **Low input RNA Fluorescent Linear Amplification kit (Amplified cRNA labeling):** 50ng total RNA

**Fluorescent Direct Label Kit:**  3-4 hours **Low input RNA Fluorescent Linear Amplification kit Amplified cDNA labeling**: 10 hours **Amplified cRNA labeling:** 6 hours

Cyanine 3 (Cy3) and cyanine 5 (Cy5) fluorescent labeling

Reproducibility; content; mature platform; sensitivity; customization

Two-color dye bias and ozonerelated degradation

50-500ng total RNA

16h

Streptavidin-Cy3

Reproducibility; Full genome coverage; Sensitivity; Low background; Mature platform; Low cost/sample; Low starting material required

Currently only available for human, rat and mouse studies; Less probes per gene; not so sensitive to detect splice variants.

genetics and more recently, in rheumatic diseases.

**1.1 Microarray fundamentals** 

widely used platforms.

**Starting RNA** 

**Hybridization** 

**Advantages** 

**Disadvantages** 

**requirement** <sup>5</sup>μg total RNA

**Hybridization time** 16h

**Detection method** Streptavidin-

Table 1. Microarray platform comparison.

phycoerythrin

Reproducibility; Full genome coverage; Mature platform; Customization; More probes per gene.

Short oligonucleotides; Less sensitive; High cost/sample.

Despite minor differences between platforms, the basic steps involved in a microarrays experiment are similar (Fig. 1) (Repsilber *et al.*, 2005). Key points in undertaking an expression profiling study are:


#### **1.2 Microarray challenges and concerns**

Large-scale gene expression analysis, is in fact, a flourishing technology with potential applications in several fields of Biology and Medicine as indicated by the large number of peer-reviewed articles (n=35502) containing the words "gene" and "microarray" found in Pubmed upto June 2011.

Microarray profiling of gene expression is a powerful tool for discovery, but the ability to manage and compare the resulting data can be problematic. Biological, experimental, and technical variations between studies of the same phenotype/phenomena create substantial differences in results. Some of these issues will be discussed in detail.

a) The success of the microarrays experience greatly depends on whether the hypothesis and rationale have been appropriately formulated through a clearly delineated question. It influences the study design as a whole, from sample collection, to experimental design, and finally, the strategies for data analysis (Smith & Rosa, 2007).

b) While most of the early studies used primary tissues involved in the disease, such as tumor biopsies, more recently a number of gene expression profiling studies have focused on peripheral blood to identify systemic markers of disease. However, gene expression patterns in peripheral blood cells greatly depend on inter-individual variations and technical aspects such as blood sampling techniques, cell and RNA isolation as well as storage temperature or delays in processing. However although significant inter-individual variations in gene expression patterns in peripheral blood cells can be seen, these differences

Lessons from Genomic Profiling in AS 139

is required. Over the last few years a number of papers have reviewed in detail how to analyze typical microarray data experiments (Allison *et al.*, 2006; Reimers, 2010), to interpret them (Michiels *et al.*, 2007) and to report the results (Dupuy & Simon, 2007). The multidimensionality of microarrays and possible solutions to deal with this issue are well

e) Confirmation and validation studies are another crucial step. For confirmation studies the initial results must be reproduced using another assay technology, usually qPCR. Validation studies require an independent study in a new sample cohort to confirm that the gene signatures defined previously replicate satisfactorily in a similar clinical setting. It may be performed by the same research team or ideally by others. These aditional steps reduce false

Establishing a consensus to optimize each step of the procedure would therefore generate more reproducibility in results from different studies. Evidence-based guidelines to perform meta-analysis of array data are in progress (Ramasamy *et al.*, 2008) but establishing consensus in experimental design and protocols is still the most likely method to minimize variation. Clinical trials to confirm the gene signature's clinical utility on diagnosis and

Several microarrays studies have been published looking at spondyloarthritis (SpA). A number of early studies used different tissue sources and smaller microarrays with wholegenome arrays prohibitively expensive (Reviewed in Thomas & Brown MA, 2010a, 2010b). The first study in 2002 identified genes more highly expressed in peripheral blood mononuclear cells (PBMC) of patients with SpA, rheumatoid arthritis (RA) and psoriatic arthritis (PsA), in comparison to normal subjects (Gu *et al.*, 2002a). A 588-gene microarray was used as a screening tool and the results were validated by reverse transcriptionpolymerase chain reaction (RT-PCR). A total of 16 genes were identified encoding differentiation markers, cytokines, cytokine/chemokine receptors and signalling and adhesion molecules. An increased expression of C-X-C chemokine receptor type 4 (*CXCR4*) and its ligand Stromal cell-derived factor-1 (SDF-1), in synovial fluid cells, were seen in all three arthritis groups. The conclusion was that the CXCR4/SDF-1 is a potential proinflammatory axis for SpA, PsA and RA. However no genes were identified that could

In another study gene expression profiles of synovial fluid mononuclear cells (SFMC) from SpA and RA patients were compared with PBMC of healthy controls to evaluate the unfolded protein response (UPR) hypothesis and identify which cytokines/chemokines were being expressed and which cell fractions were involved. An 1176-gene microarray was used and the results were validated by RT-PCR. There was an increase in transcripts encoding Monocyte chemotactic protein-1 (MCP-1), proteasome subunit C2 and Binding immunoglobulin protein (BiP), which suggest the existence of an UPR. BiP was higher in SpA SFMC compared to RA SFMC and macrophages were potentially identified as the cell

A third study identified a gene expression profile in gut biopsies that could differentiate SpA patients with sub-clinical gut inflammation from SpA patients without gut disease.

treatment decisions are mandatory, after the identification of reliable biomarkers.

discussed in a recent review (Michiels *et al.*, 2011).

positives and the potential for biases (Michiels *et al.*, 2007, 2011).

**1.3 Microarray applications in rheumatology/spondyloarthritis** 

discriminate between the different diseases.

type involved (Gu *et al.*, 2002b).

are often much less than the differences between blood samples from healthy donors and from patients. These observations and the accessibility of peripheral blood, strongly suggests that gene expression analysis of peripheral blood is probably the best source for the assessment of systemic differences or changes in gene expression associated with disease or drug response. (Debey *et al.*, 2004).

Fig. 1. Design, experimental and data analysis steps in a typical microarray gene expression experiment. Adapted from Repsilber *et al.,* 2005.

c) Appropriate experimental design is another critical step for the success of a microarray experiment. It's important to control and exclude as many biases as possible (Ransohoff, 2007). Integrity and purity of RNA extracted, cDNA labeling and hybridization procedures may affect reproducibility, thus these steps need to be standardized and optimized. However, several key issues regarding appropriate replication remains in discussion: the minimum sample size, the necessity of running multiple arrays with the same samples or the potential benefits and risks associated with pooling samples (Smith & Rosa, 2007). Increasing the sample size will lower the false discovery and false negative rates but it represents an expensive option (Pawitan *et al.*, 2005). Given the well-established reproducible commercially available platforms, technical replication is not required currently. Finally, pooling samples can reduce the variation between arrays but potential outliers may get masked or may compromise the entire pool (Smith & Rosa, 2007). To guaranty an improvement of data quality, replication studies in independent patient series must be performed, but these analyses are often lacking (Ionnidis *et al.*, 2009).

d) Data analysis currently represents a major challenge for researchers. A closer look at the literature reveals many conflicting results. A consensus regarding strategies in data analysis

are often much less than the differences between blood samples from healthy donors and from patients. These observations and the accessibility of peripheral blood, strongly suggests that gene expression analysis of peripheral blood is probably the best source for the assessment of systemic differences or changes in gene expression associated with disease or

Fig. 1. Design, experimental and data analysis steps in a typical microarray gene expression

c) Appropriate experimental design is another critical step for the success of a microarray experiment. It's important to control and exclude as many biases as possible (Ransohoff, 2007). Integrity and purity of RNA extracted, cDNA labeling and hybridization procedures may affect reproducibility, thus these steps need to be standardized and optimized. However, several key issues regarding appropriate replication remains in discussion: the minimum sample size, the necessity of running multiple arrays with the same samples or the potential benefits and risks associated with pooling samples (Smith & Rosa, 2007). Increasing the sample size will lower the false discovery and false negative rates but it represents an expensive option (Pawitan *et al.*, 2005). Given the well-established reproducible commercially available platforms, technical replication is not required currently. Finally, pooling samples can reduce the variation between arrays but potential outliers may get masked or may compromise the entire pool (Smith & Rosa, 2007). To guaranty an improvement of data quality, replication studies in independent patient series

must be performed, but these analyses are often lacking (Ionnidis *et al.*, 2009).

d) Data analysis currently represents a major challenge for researchers. A closer look at the literature reveals many conflicting results. A consensus regarding strategies in data analysis

drug response. (Debey *et al.*, 2004).

experiment. Adapted from Repsilber *et al.,* 2005.

is required. Over the last few years a number of papers have reviewed in detail how to analyze typical microarray data experiments (Allison *et al.*, 2006; Reimers, 2010), to interpret them (Michiels *et al.*, 2007) and to report the results (Dupuy & Simon, 2007). The multidimensionality of microarrays and possible solutions to deal with this issue are well discussed in a recent review (Michiels *et al.*, 2011).

e) Confirmation and validation studies are another crucial step. For confirmation studies the initial results must be reproduced using another assay technology, usually qPCR. Validation studies require an independent study in a new sample cohort to confirm that the gene signatures defined previously replicate satisfactorily in a similar clinical setting. It may be performed by the same research team or ideally by others. These aditional steps reduce false positives and the potential for biases (Michiels *et al.*, 2007, 2011).

Establishing a consensus to optimize each step of the procedure would therefore generate more reproducibility in results from different studies. Evidence-based guidelines to perform meta-analysis of array data are in progress (Ramasamy *et al.*, 2008) but establishing consensus in experimental design and protocols is still the most likely method to minimize variation. Clinical trials to confirm the gene signature's clinical utility on diagnosis and treatment decisions are mandatory, after the identification of reliable biomarkers.

#### **1.3 Microarray applications in rheumatology/spondyloarthritis**

Several microarrays studies have been published looking at spondyloarthritis (SpA). A number of early studies used different tissue sources and smaller microarrays with wholegenome arrays prohibitively expensive (Reviewed in Thomas & Brown MA, 2010a, 2010b). The first study in 2002 identified genes more highly expressed in peripheral blood mononuclear cells (PBMC) of patients with SpA, rheumatoid arthritis (RA) and psoriatic arthritis (PsA), in comparison to normal subjects (Gu *et al.*, 2002a). A 588-gene microarray was used as a screening tool and the results were validated by reverse transcriptionpolymerase chain reaction (RT-PCR). A total of 16 genes were identified encoding differentiation markers, cytokines, cytokine/chemokine receptors and signalling and adhesion molecules. An increased expression of C-X-C chemokine receptor type 4 (*CXCR4*) and its ligand Stromal cell-derived factor-1 (SDF-1), in synovial fluid cells, were seen in all three arthritis groups. The conclusion was that the CXCR4/SDF-1 is a potential proinflammatory axis for SpA, PsA and RA. However no genes were identified that could discriminate between the different diseases.

In another study gene expression profiles of synovial fluid mononuclear cells (SFMC) from SpA and RA patients were compared with PBMC of healthy controls to evaluate the unfolded protein response (UPR) hypothesis and identify which cytokines/chemokines were being expressed and which cell fractions were involved. An 1176-gene microarray was used and the results were validated by RT-PCR. There was an increase in transcripts encoding Monocyte chemotactic protein-1 (MCP-1), proteasome subunit C2 and Binding immunoglobulin protein (BiP), which suggest the existence of an UPR. BiP was higher in SpA SFMC compared to RA SFMC and macrophages were potentially identified as the cell type involved (Gu *et al.*, 2002b).

A third study identified a gene expression profile in gut biopsies that could differentiate SpA patients with sub-clinical gut inflammation from SpA patients without gut disease.

Lessons from Genomic Profiling in AS 141

One of the most intriguing aspects regarding AS pathogenesis is the possible link between pathogens and disease onset. There are several pieces of evidence that an abnormal host response against pathogens is implicated in AS and/or SpA pathogenesis. Sixty percent of patients with SpA without diagnosed Crohn's disease evidenced endoscopic or histological signs of gut inflammation (Mielants *et al.*, 1995). Moreover, studies showing HLA-B27 transgenic rats do not develop inflammatory intestinal or peripheral joint disease in a germfree environment support a role of commensal gut flora in the shared pathogenesis of gut

Pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of the host defense system. These receptors are transmembrane receptors such as Tolllike receptors (TLRs) or C-type lectin receptors (CLRs) and cytosolic receptors RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) (Jeong & Lee, 2011). Interestingly, expression changes in genes involved in innate immune response such as *TLRs* (Assassi *et al.*, 2011), *NLRP2* (Sharma *et al.*, 2009) and *CLEC4D* (Pimentel-Santos *et al.*, 2011) were

Fig. 2. Possible functional interactions between innate immune receptors and AS candidate

TLRs are characterized by an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain and a cytoplasmic Toll/IL-1R (TIR) domain. As many as 13 TLR

consistently observed in several different studies using microarray technology.

**2.1 The link between an abnormal innate immune response and AS** 

**2. Lessons from genomic profiling in AS** 

and joint manifestations (Taurog *et al.*, 1994).

genes (Adapted from Thomas & Brown, 2010a).

2625 differentially expressed sequence tags were initially identified through macroarrays in colon biopsies from Crohn's and SpA patients which were then used to construct a microarray which was used to screen a further sample cohort. Ninety five expressed sequence tags clustered patients with Crohn's and those with SpA and chronic gut inflammation (Laukens *et al.*, 2006).

This chapter, Lessons from Genomic Profiling in AS will be focused on studies using peripheral blood and microarray platforms covering the whole genome. The results seem to be quite heterogeneous reflecting the different methodologies involved, as commented above. Several aspects, summarized in Figure 1, may introduce variability and bias in the results, specifically;


Based on seven papers published since 2007, several pathways relevant to potential SpA pathological processes have been identified. Moreover, potential biomarkers with applications to diagnosis and treatment response prediction in clinical practice were also flagged. Table 2, summarizes the similarities and methodological differences between the studies and reinforces the caution that should be observed when translating these findings to clinical practice. All the knowledge obtained must be interpreted as hypotheses which need validation in future studies.


**AS:** Ankylosing spondylitis; **SPA:** Spondyloarthritis; **HC:** Healthy controls; **RA:** Rheumatoid arthritis; **LBP:** Lumbar back pain; **SLE:** Systemic lupus erythematosus; **mNYC**: modified New York criteria; **ESSG:** European Spondyloarthropathy Study Group; **PBMC:** Peripheral blood mononuclear cells; **qPCR**: Quantitative reverse transcription polymerase chain reaction.

Table 2. Comparison between published microarrays studies in SpA.

2625 differentially expressed sequence tags were initially identified through macroarrays in colon biopsies from Crohn's and SpA patients which were then used to construct a microarray which was used to screen a further sample cohort. Ninety five expressed sequence tags clustered patients with Crohn's and those with SpA and chronic gut

This chapter, Lessons from Genomic Profiling in AS will be focused on studies using peripheral blood and microarray platforms covering the whole genome. The results seem to be quite heterogeneous reflecting the different methodologies involved, as commented above. Several aspects, summarized in Figure 1, may introduce variability and bias in the

a. Patient selection: numbers of patients, the criteria used to classify and include the patients, different degrees of activity/severity of the disease and patients receiving different therapies are examples of heterogeneity that might influence the final results.

Based on seven papers published since 2007, several pathways relevant to potential SpA pathological processes have been identified. Moreover, potential biomarkers with applications to diagnosis and treatment response prediction in clinical practice were also flagged. Table 2, summarizes the similarities and methodological differences between the studies and reinforces the caution that should be observed when translating these findings to clinical practice. All the knowledge obtained must be interpreted as hypotheses which

**Subjects Criteria Samples Microarray Validation** 

35AS+18HC mNYC PBMC Illumina qPCR

78AS+78HC mNYC Whole blood Illumina qPCR

ESSG, Amor Macrophage Affymetrix qPCR

Score Whole blood Affymetrix Microarrays

Calin PBMC Illumina qPCR

mNYC Whole bood Illumina qPCR

(2nd set)

b. Cell Source used for analysis: PBMC vs. whole blood or a specific cell subset. c. Differences in microarray platform technology and data analysis tools.

mNYC

Likelihood

**AS:** Ankylosing spondylitis; **SPA:** Spondyloarthritis; **HC:** Healthy controls; **RA:** Rheumatoid arthritis; **LBP:** Lumbar back pain; **SLE:** Systemic lupus erythematosus; **mNYC**: modified New York criteria; **ESSG:** European Spondyloarthropathy Study Group; **PBMC:** Peripheral blood mononuclear

cells; **qPCR**: Quantitative reverse transcription polymerase chain reaction. Table 2. Comparison between published microarrays studies in SpA.

*al.* **2010** 16AS mNYC PBMC Affymetrix qPCR

d. Differences in methodology used regarding validation of candidate biomarkers.

inflammation (Laukens *et al.*, 2006).

need validation in future studies.

6AS+2uSPA 9HC

11uSPA+7uSPA 25HC

18AS+18HC

21AS+28uSPA 23AS+18uSPA 26HC+12RA+5LBP

> 16AS + 14HC+ SLE+SSC 27AS+27HC

18AS+18HC

**Smith** *et al.* **2008** 

**Haroon** *et* 

**Sharma**  *et al.* **2009** 

**Duan** *et al.* **2010** 

**Gu** *et al. 2***009** 

**Assassi** *et al.* **2011** 

**Santos** *et al.* **2011** 

results, specifically;

#### **2. Lessons from genomic profiling in AS**

#### **2.1 The link between an abnormal innate immune response and AS**

One of the most intriguing aspects regarding AS pathogenesis is the possible link between pathogens and disease onset. There are several pieces of evidence that an abnormal host response against pathogens is implicated in AS and/or SpA pathogenesis. Sixty percent of patients with SpA without diagnosed Crohn's disease evidenced endoscopic or histological signs of gut inflammation (Mielants *et al.*, 1995). Moreover, studies showing HLA-B27 transgenic rats do not develop inflammatory intestinal or peripheral joint disease in a germfree environment support a role of commensal gut flora in the shared pathogenesis of gut and joint manifestations (Taurog *et al.*, 1994).

Pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of the host defense system. These receptors are transmembrane receptors such as Tolllike receptors (TLRs) or C-type lectin receptors (CLRs) and cytosolic receptors RIG-I-like receptors (RLRs) and NOD-like receptors (NLRs) (Jeong & Lee, 2011). Interestingly, expression changes in genes involved in innate immune response such as *TLRs* (Assassi *et al.*, 2011), *NLRP2* (Sharma *et al.*, 2009) and *CLEC4D* (Pimentel-Santos *et al.*, 2011) were consistently observed in several different studies using microarray technology.

Fig. 2. Possible functional interactions between innate immune receptors and AS candidate genes (Adapted from Thomas & Brown, 2010a).

TLRs are characterized by an extracellular leucine-rich repeat (LRR) domain, a transmembrane domain and a cytoplasmic Toll/IL-1R (TIR) domain. As many as 13 TLR

Lessons from Genomic Profiling in AS 143

As such, the reduced expression of Nod-like receptor family, pyrin domain containing 2 *(NLRP2)* in AS was a very interesting observation (Sharma *et al.*, 2009). NLRP2, as with other NLRs, induces an inhibition of the NF*k*B signaling pathway, leading to regulation of IL1*β*, a relevant cytokine in the disease process. The downregulation of *NLRP2* may therefore lead to upregulation of IL-1*β.* Supporting this, polymorphisms in *NLR* genes have also been implicated in Behçet's disease and Crohn's disease which share some clinical features with AS (Cummings *et al.*, 2010; Kappen *et al.*, 2009). Another interesting point is the association of *CARD9* with Crohn's disease and AS (Pointon *et al.*, 2010) which has a pivotal role in

Another family of receptors of particular interest are the C-type lectins which display a distinct protein domain, the carbohydrate recognition domain (CRD). Based on the organization of their CRDs, 17 distinct groups have been defined (Drickamer & Fadden, 2002; Zelensky & Gready, 2005). While some recognize DAMPs which facilitate adhesion between cells, adhesion of cells to extracellular matrix and other non-enzymatic functions, others may act as PRRs (Graham & Brown, 2009) after PAMP recognition. Upon ligand biding, C-type lectin receptors can induce a variety of cellular responses, and can be functionally divided into those that inhibit or those that induce cellular activation. In general, inhibitory receptors contain a consensus immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domains, while activation receptors either contain an immunoreceptor tyrosine-based activation motif (ITAM), or associate with signalling adaptor molecules. Depending on whether signalling is through ITAM or ITIM , either activation of Src homology 2 (SH2) domain-containing protein tyrosine kinases (SyK, ZAP 10) or SH2 containing-phosphatases (SHP-1, SHP-2) are recruited, thereby up or

downmodulating cellular activation, respectively (Majeed *et al.*, 2001; Long, 1999).

cooperation with MyD88-mediated TLR signaling (Kanazawa, 2007).

MyD88-mediated TLR signaling (Graham & Brown GD, 2009).

Genes encoding for each family are distinctly clustered in the telomeric Natural Killer-gene complex (NKC), on chromosome 12. The Dectin-1 cluster of receptors, includes Dectin-1, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), C-type lectin-like receptor-1 (CLEC-1), CLEC-2, CLEC12B, CLEC9A and myeloid inhibitory C-type lectin-like receptor (MICL). The Dectin-2 cluster of receptors, includes Dectin-2, DCIR, DCAR, BDCA-2, Mincle

Dectin-1, is expressed in dendritic cells, monocytes, macrophages, neutrophils and weakly in a subset of T cells, B cells and eosinophils. It recognizes fungal β-glucan, working as an activating receptor uniquely possessing an ITAM in the cytoplasmic domain. The induction of phagocytosis, production of reactive oxygen species and cytokine production is mediated by NF-kB and spleen tyrosine kinase (Syk). In addition, some of these effects require

Dectin-2 and Mincle are expressed in macrophages, dendritic cells and weakly in Langerhans cells and monocytes. The receptors recognize several pathogens (*Candida albicans, Saccharomyces cerevisiae, Mycoplasma tuberculosis, Histoplasma capsulatum*) but also endogenous ligands. Both have characteristic short cytoplasmic domains and are associated with FcRγ domains. Their activation, inducing the production of proinflammatory cytokines, is mediated by Syk- and CARD9-dependent pathways but independently of

CLEC4D has been found to be expressed in a monocyte/macrophage restricted manner, and although no ligand or biological function has as yet been described, the receptor has been

NOD2 signaling.

and CLEC4D (Graham & Brown, 2009).

family members have been identified in mammalian systems with TLRs 1 to 10 expressed in humans. They can be divided into 2 groups according to cellular localization and respective ligands. TLRs 1, 2, 4, 5, and 6, are expressed on the cell surface and recognize microbial components in the outer membrane of bacteria. TLRs 3, 7, 8 and 9 are found in intracellular vesicles and recognize microbial nucleic acids (Sirisinha, 2011). TLRs are expressed in various immune (monocytes, macrophages, dendritic cells, B cells) and non-immune (epithelial cells, endothelial cells, fibroblasts) cells. *TLR4* was overexpressed in SpA patients in peripheral whole blood cells, assessed by microarray (Assassi *et al.*, 2011; Pimentel-Santos *et al.*, 2011), in PBMCs, measured by flow cytometry (De Rycke *et al.*, 2005) and in lymphocytes, monocytes and neutrophils by qPCR (Yang *et al.*, 2007). The main ligand for TLR4 is lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, however, it also recognizes other exogenous pathogens such as mannan from *Candida albicans*, glycoinositolphospholipid from *Trypanosoma*, and the envelope proteins from mouse mammary tumor virus (MMTV) and respiratory syncytial virus (RSV). It also recognizes some endogenous molecules, including heat-shock proteins (HSP60, HSP70, and HSP gp96), fibrinogen, oligosaccharides of hyaluronic acid, extracellular domain A of fibronectin, heparan sulfate, myeloid-related proteins (Mrp8 and Mrp14), oxidized LDL, saturated fatty acid and amyloid-β (Jeong & Lee, 2011). Microarray analysis also showed overexpression of TLR5 in peripheral whole blood cells from SpA patients (Assassi S *et al.,* 2011; Pimentel-Santos *et al.*, 2011). Flagellin, a primary component of Gram negative bacteria flagella, is the main ligand for TLR5 (Hayashi *et al.*, 2001), which is mainly expressed on the luminar surface of epithelial cells in the mucosal tissues and respiratory tract (Gewirtz *et al.*, 2001).

The wide responsiveness of TLRs to a wide variety of external and internal signals, and the link that these receptors establish between the innate and adaptative immune systems, reinforces the theory that TLRs are strongly implicated in the development of chronic inflammatory diseases. However, mechanistic studies are needed in order to clarify the role of specific receptor subtypes in AS development.

Members of the NOD-like receptor (NLR) family consist of a central nucleotide-binding and oligomerization (NACHT) domain, which is commonly flanked by C-terminal leucine-rich repeat (LRRs) domain and N-terminal caspase recruitment (CARD) or pyrin (PYD) domains (Schroder & Tschopp, 2010). So far, 20 NLR family members have been identified in humans. Two main subgroups have been described. One, including NODs (NOD 1-5 and CIITA), detects pathogen-associated molecular patterns (PAMPs) existing in Gram-negative bacteria cell walls and elicit responses that are distinct from those of the TLRs. The other NLR subgroup involves a large family of molecular complexes known as the "inflammasomes", the NLRPs (NLRP1-14) and the IPAF subfamily, consisting of IPAF and NAIP (Fitzgerald, 2010; Schroder & Tschopp, 2010). The inflammasomes are macromolecular cytosolic complexes composed of several proteins, some of which are found in all inflammasomes (pro-caspase-1, Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain-ASC), and others which are present depending on the inflammasome type (cardinal, pro-caspase-5, domain with function to find-FIIND). These complexes are involved in the innate immune response recognizing both endogenous signals (adenosine triphosphate, urate, and calcium pyrophosphate crystals) as well as external pathogen-derived products (bacterial RNA, bacterial toxins) (Drenth & van der Meer, 2006).

family members have been identified in mammalian systems with TLRs 1 to 10 expressed in humans. They can be divided into 2 groups according to cellular localization and respective ligands. TLRs 1, 2, 4, 5, and 6, are expressed on the cell surface and recognize microbial components in the outer membrane of bacteria. TLRs 3, 7, 8 and 9 are found in intracellular vesicles and recognize microbial nucleic acids (Sirisinha, 2011). TLRs are expressed in various immune (monocytes, macrophages, dendritic cells, B cells) and non-immune (epithelial cells, endothelial cells, fibroblasts) cells. *TLR4* was overexpressed in SpA patients in peripheral whole blood cells, assessed by microarray (Assassi *et al.*, 2011; Pimentel-Santos *et al.*, 2011), in PBMCs, measured by flow cytometry (De Rycke *et al.*, 2005) and in lymphocytes, monocytes and neutrophils by qPCR (Yang *et al.*, 2007). The main ligand for TLR4 is lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, however, it also recognizes other exogenous pathogens such as mannan from *Candida albicans*, glycoinositolphospholipid from *Trypanosoma*, and the envelope proteins from mouse mammary tumor virus (MMTV) and respiratory syncytial virus (RSV). It also recognizes some endogenous molecules, including heat-shock proteins (HSP60, HSP70, and HSP gp96), fibrinogen, oligosaccharides of hyaluronic acid, extracellular domain A of fibronectin, heparan sulfate, myeloid-related proteins (Mrp8 and Mrp14), oxidized LDL, saturated fatty acid and amyloid-β (Jeong & Lee, 2011). Microarray analysis also showed overexpression of TLR5 in peripheral whole blood cells from SpA patients (Assassi S *et al.,* 2011; Pimentel-Santos *et al.*, 2011). Flagellin, a primary component of Gram negative bacteria flagella, is the main ligand for TLR5 (Hayashi *et al.*, 2001), which is mainly expressed on the luminar surface of epithelial cells in the mucosal tissues and respiratory

The wide responsiveness of TLRs to a wide variety of external and internal signals, and the link that these receptors establish between the innate and adaptative immune systems, reinforces the theory that TLRs are strongly implicated in the development of chronic inflammatory diseases. However, mechanistic studies are needed in order to clarify the role

Members of the NOD-like receptor (NLR) family consist of a central nucleotide-binding and oligomerization (NACHT) domain, which is commonly flanked by C-terminal leucine-rich repeat (LRRs) domain and N-terminal caspase recruitment (CARD) or pyrin (PYD) domains (Schroder & Tschopp, 2010). So far, 20 NLR family members have been identified in humans. Two main subgroups have been described. One, including NODs (NOD 1-5 and CIITA), detects pathogen-associated molecular patterns (PAMPs) existing in Gram-negative bacteria cell walls and elicit responses that are distinct from those of the TLRs. The other NLR subgroup involves a large family of molecular complexes known as the "inflammasomes", the NLRPs (NLRP1-14) and the IPAF subfamily, consisting of IPAF and NAIP (Fitzgerald, 2010; Schroder & Tschopp, 2010). The inflammasomes are macromolecular cytosolic complexes composed of several proteins, some of which are found in all inflammasomes (pro-caspase-1, Apoptosis-associated Speck-like Protein Containing a Caspase Recruitment Domain-ASC), and others which are present depending on the inflammasome type (cardinal, pro-caspase-5, domain with function to find-FIIND). These complexes are involved in the innate immune response recognizing both endogenous signals (adenosine triphosphate, urate, and calcium pyrophosphate crystals) as well as external pathogen-derived products (bacterial RNA, bacterial toxins) (Drenth & van der

tract (Gewirtz *et al.*, 2001).

Meer, 2006).

of specific receptor subtypes in AS development.

As such, the reduced expression of Nod-like receptor family, pyrin domain containing 2 *(NLRP2)* in AS was a very interesting observation (Sharma *et al.*, 2009). NLRP2, as with other NLRs, induces an inhibition of the NF*k*B signaling pathway, leading to regulation of IL1*β*, a relevant cytokine in the disease process. The downregulation of *NLRP2* may therefore lead to upregulation of IL-1*β.* Supporting this, polymorphisms in *NLR* genes have also been implicated in Behçet's disease and Crohn's disease which share some clinical features with AS (Cummings *et al.*, 2010; Kappen *et al.*, 2009). Another interesting point is the association of *CARD9* with Crohn's disease and AS (Pointon *et al.*, 2010) which has a pivotal role in NOD2 signaling.

Another family of receptors of particular interest are the C-type lectins which display a distinct protein domain, the carbohydrate recognition domain (CRD). Based on the organization of their CRDs, 17 distinct groups have been defined (Drickamer & Fadden, 2002; Zelensky & Gready, 2005). While some recognize DAMPs which facilitate adhesion between cells, adhesion of cells to extracellular matrix and other non-enzymatic functions, others may act as PRRs (Graham & Brown, 2009) after PAMP recognition. Upon ligand biding, C-type lectin receptors can induce a variety of cellular responses, and can be functionally divided into those that inhibit or those that induce cellular activation. In general, inhibitory receptors contain a consensus immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic domains, while activation receptors either contain an immunoreceptor tyrosine-based activation motif (ITAM), or associate with signalling adaptor molecules. Depending on whether signalling is through ITAM or ITIM , either activation of Src homology 2 (SH2) domain-containing protein tyrosine kinases (SyK, ZAP 10) or SH2 containing-phosphatases (SHP-1, SHP-2) are recruited, thereby up or downmodulating cellular activation, respectively (Majeed *et al.*, 2001; Long, 1999).

Genes encoding for each family are distinctly clustered in the telomeric Natural Killer-gene complex (NKC), on chromosome 12. The Dectin-1 cluster of receptors, includes Dectin-1, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), C-type lectin-like receptor-1 (CLEC-1), CLEC-2, CLEC12B, CLEC9A and myeloid inhibitory C-type lectin-like receptor (MICL). The Dectin-2 cluster of receptors, includes Dectin-2, DCIR, DCAR, BDCA-2, Mincle and CLEC4D (Graham & Brown, 2009).

Dectin-1, is expressed in dendritic cells, monocytes, macrophages, neutrophils and weakly in a subset of T cells, B cells and eosinophils. It recognizes fungal β-glucan, working as an activating receptor uniquely possessing an ITAM in the cytoplasmic domain. The induction of phagocytosis, production of reactive oxygen species and cytokine production is mediated by NF-kB and spleen tyrosine kinase (Syk). In addition, some of these effects require cooperation with MyD88-mediated TLR signaling (Kanazawa, 2007).

Dectin-2 and Mincle are expressed in macrophages, dendritic cells and weakly in Langerhans cells and monocytes. The receptors recognize several pathogens (*Candida albicans, Saccharomyces cerevisiae, Mycoplasma tuberculosis, Histoplasma capsulatum*) but also endogenous ligands. Both have characteristic short cytoplasmic domains and are associated with FcRγ domains. Their activation, inducing the production of proinflammatory cytokines, is mediated by Syk- and CARD9-dependent pathways but independently of MyD88-mediated TLR signaling (Graham & Brown GD, 2009).

CLEC4D has been found to be expressed in a monocyte/macrophage restricted manner, and although no ligand or biological function has as yet been described, the receptor has been

Lessons from Genomic Profiling in AS 145

In the second study downregulation of several pro-inflammatory genes were described highlighting another aspect of AS pathogenesis (Pimentel-Santos *et al.*, 2011). Protein tyrosine phosphatase, non-receptor type 1 *(PTPN1)* and Dedicator of cytokinesis 10 *(DOCK10)*, which are both involved in mediating IL4 actions (Paul & Ohara., 1987) were downregulated. Protein tyrosine phosphatase 1B (PTP1B), the *PTPN1* protein product, is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signalling pathways, including the downstream processes involved in C-type lectin receptor activation (Majeed *et al.*, 2001; Long, 1999) and IL4 signalling (Lu *et al.*, 2008). Dock10 is also regulated by IL4 in B cells (Yelo *et al.*, 2008). This is of particular interest as IL4 may play a role in AS pathogenesis. Interleukin 4 (IL4), has a variety of stimulatory and inhibitory actions on B and T cells (O'Garra *et al.*, 1988; Jelinek & Lipsky 1988; Rousset *et al.*, 1988). Recent studies have also indicated a potential role for IL4 producing CD8+ T cells in the pathogenesis of AS. Although CD8+ T cells are predominately associated with the production of 'TH1' cytokines, such as IFN*γ*, there is now good evidence that some subsets of these cells can also produce 'TH2' cytokines such as IL4, IL5 and IL10 (Baek *et al.*, 2008). The potential functions associated with IL4-producing CD8+ T cells are as yet unclear but the subtype CD8+/TCR αβ+ T cells, with a regulatory phenotype and function (expressing CD25+, CTLA4+, Foxp3+, but negative for IFNγ and perforin), were previously described in peripheral blood of AS patients (Jarvis *et al.*, 2005). These results were confirmed in a recent study suggesting an altered pattern of CD8+ T cell differentiation in AS and in HLAB27+ healthy individuals. This predisposition to generate IL4+CD8+ T cells may play a role in pathogenesis of SpA (Zhang *et al.*, 2009). Further supporting this theory, *RUNX3* was identified as a candidate gene in a GWAS (Australo-Anglo-American Spondyloarthritis Consortium (TASC), 2010). The association of *RUNX3* with AS provides additional evidence of a role for CD8+ T cells in the disease. It's expression in immature lymphocytes is triggered by IL7R signalling, leading to suppression

Although there are some differences between the different expression profiling studies, their findings do contribute to a greater understanding of the pathogenesis of AS, particularly in

Bone formation and bone loss take place at sites closely located to each other presenting an "apparent paradox", which is reflected in the changes in bone and cartilage metabolism occurring in the AS disease process (Carter & Lories, 2011). Ossification is the hallmark of AS and has been linked to aberrant activation of bone morphogenic protein (BMP) and wingless (WNT) signaling. Bone resorption, driven by the impact of inflammation on the bone remodeling cycle, occurs simultaneously, with up to 56% of patients developing systemic osteopenia and some of them systemic osteoporosis (Lange

Biomarkers, reflecting structural damage and disease activity, constitute a high priority for the understanding of the pathogenesis of AS and for the new therapy discovery. Two microarray-based studies have contributed to the improvement of knowledge in this field. A bone remodeling signature was described associated with an overexpression of *BMP6*, Proprotein convertase subtilisin/kexin type 6 *(PCSK6)*, Kringle containing transmembrane

of CD4 and upregulation of CD8 expression (Park *et al.*, 2010).

**2.3 Bone ossification and resorption processes** 

*et al.*, 2005).

the delineation of the roles of the innate and adaptive immune responses.

shown to be upregulated at the transcript level in a number of disease settings, similarly to two other members of the family, Mincle and Dectin-2. They are able to recognize and promote pathogen clearance and induce inflammatory signals. This process seems to follow the Syk and CARD9 pathway which was recently implicated in a mouse model of SpA (Ruutu *et al.*, 2010). The upregulation of *CLEC4D*, observed for the first time in an expression profiling study of AS patients (Pimentel-Santos *et al.*, 2011), supports the importance of innate immune mechanisms in AS pathology. However, further studies are required to confirm this hypothesis.

#### **2.2 Proinflammatory** *vs.* **immunosuppressive signatures**

Transcriptional profiling studies have demonstrated that transcripts involved in the inflammatory response were differentially expressed in AS patients and controls, but reports on the nature of these changes seem to vary. A proinflammatory profile in peripheral blood monocyte cells (PBMCs), from undifferentiated spondyloarthritis (uSpA) and AS, is indicated by an increased expression of *RGS1, NR4A2, HBEGF* and *SOCS3,* in both groups (Gu *et al.*, 2009). However, other reports suggest decreased immune responsiveness such as a "reverse *IFNγ* signature" (Smith *et al.*, 2008), and immunosuppressive phenotypes (Duan *et al.*, 2010, Pimentel-Santos *et al.*, 2011*).* The main reason for these differences in the transcriptomic profiles, between the first study and the 3 later studies, is unknown but differences in patients and methodologies may contribute.

IFN*γ* dysregulation in AS is supported by previous studies of cytokines expression. A lower frequency of IFN*γ* positive T cells has been reported in AS patients (Rudwaleit *et al.*, 2001) and gut biopsy samples show a reduced TH1 profile in lymphocytes from SpA patients (Van Damme *et al.*, 2001). Moreover, IFN*γ* is expressed at lower levels in synovium from SpA compared to rheumatoid arthritis patients (Canete *et al.*, 2000). This knowledge may contribute to understanding AS pathogenesis as decresead IFN*γ* production by macrophages could impair the host's ability to clear pathogenic organisms. Recent studies support this theory (Rothfuchs *et al.*, 2001; Inman *et al.*, 2006), and may implicate arthritogenic organisms in AS susceptibility. In addition, IFN*γ* reduction, can contribute to activation of the IL-23/IL-17 axis a major axis in AS pathogenesis.

Complementary to the report in macrophages from peripheral blood of AS patients (Smith *et al.*, 2008), two different studies, from PBMCs and whole blood, have shown an immunosuppressive phenotype (Duan *et al.*, 2010, Pimentel-Santos *et al.*, 2011*)*. The first one validated three downregulated genes, Nuclear receptor subfamily 4, group A, member 2 (*NR4A2)*, Tumor necrosis factor, alpha-induced protein 3 *(TNFAIP3)* and CD69 molecule *(CD69)*. NR4A2 has been associated with T-cell subset communication and the macrophage inflammatory response. TNFAIP3 serves as negative feedback system for the TNFα induced by NFkB, acting as an anti-inflammatory molecule to control prolonged inflammation. CD69 is an early leukocyte activation molecule expressed at sites of active inflammation. Of further interest were the results of Ingenuity Pathways Analysis using the differentially expressed geneset showing altered activity of the JAK/STAT signaling pathway in AS patients (Duan *et al.*, 2010). Both *STAT3* and *JAK2* have been shown to be genetically associated with IBD and AS (Barrett *et al.*, 2008; Danoy *et al.*, 2010; The Australo-Anglo-American-Spondyloarthritis-Consortium (TASC), 2011), and represent key downstream molecules of the IL-23/IL-17 pathway (Ma *et al.*, 2008).

shown to be upregulated at the transcript level in a number of disease settings, similarly to two other members of the family, Mincle and Dectin-2. They are able to recognize and promote pathogen clearance and induce inflammatory signals. This process seems to follow the Syk and CARD9 pathway which was recently implicated in a mouse model of SpA (Ruutu *et al.*, 2010). The upregulation of *CLEC4D*, observed for the first time in an expression profiling study of AS patients (Pimentel-Santos *et al.*, 2011), supports the importance of innate immune mechanisms in AS pathology. However, further studies are required to

Transcriptional profiling studies have demonstrated that transcripts involved in the inflammatory response were differentially expressed in AS patients and controls, but reports on the nature of these changes seem to vary. A proinflammatory profile in peripheral blood monocyte cells (PBMCs), from undifferentiated spondyloarthritis (uSpA) and AS, is indicated by an increased expression of *RGS1, NR4A2, HBEGF* and *SOCS3,* in both groups (Gu *et al.*, 2009). However, other reports suggest decreased immune responsiveness such as a "reverse *IFNγ* signature" (Smith *et al.*, 2008), and immunosuppressive phenotypes (Duan *et al.*, 2010, Pimentel-Santos *et al.*, 2011*).* The main reason for these differences in the transcriptomic profiles, between the first study and the 3 later studies, is unknown but differences in patients and methodologies may contribute.

IFN*γ* dysregulation in AS is supported by previous studies of cytokines expression. A lower frequency of IFN*γ* positive T cells has been reported in AS patients (Rudwaleit *et al.*, 2001) and gut biopsy samples show a reduced TH1 profile in lymphocytes from SpA patients (Van Damme *et al.*, 2001). Moreover, IFN*γ* is expressed at lower levels in synovium from SpA compared to rheumatoid arthritis patients (Canete *et al.*, 2000). This knowledge may contribute to understanding AS pathogenesis as decresead IFN*γ* production by macrophages could impair the host's ability to clear pathogenic organisms. Recent studies support this theory (Rothfuchs *et al.*, 2001; Inman *et al.*, 2006), and may implicate arthritogenic organisms in AS susceptibility. In addition, IFN*γ* reduction, can contribute to

Complementary to the report in macrophages from peripheral blood of AS patients (Smith *et al.*, 2008), two different studies, from PBMCs and whole blood, have shown an immunosuppressive phenotype (Duan *et al.*, 2010, Pimentel-Santos *et al.*, 2011*)*. The first one validated three downregulated genes, Nuclear receptor subfamily 4, group A, member 2 (*NR4A2)*, Tumor necrosis factor, alpha-induced protein 3 *(TNFAIP3)* and CD69 molecule *(CD69)*. NR4A2 has been associated with T-cell subset communication and the macrophage inflammatory response. TNFAIP3 serves as negative feedback system for the TNFα induced by NFkB, acting as an anti-inflammatory molecule to control prolonged inflammation. CD69 is an early leukocyte activation molecule expressed at sites of active inflammation. Of further interest were the results of Ingenuity Pathways Analysis using the differentially expressed geneset showing altered activity of the JAK/STAT signaling pathway in AS patients (Duan *et al.*, 2010). Both *STAT3* and *JAK2* have been shown to be genetically associated with IBD and AS (Barrett *et al.*, 2008; Danoy *et al.*, 2010; The Australo-Anglo-American-Spondyloarthritis-Consortium (TASC), 2011), and represent key downstream

confirm this hypothesis.

**2.2 Proinflammatory** *vs.* **immunosuppressive signatures** 

activation of the IL-23/IL-17 axis a major axis in AS pathogenesis.

molecules of the IL-23/IL-17 pathway (Ma *et al.*, 2008).

In the second study downregulation of several pro-inflammatory genes were described highlighting another aspect of AS pathogenesis (Pimentel-Santos *et al.*, 2011). Protein tyrosine phosphatase, non-receptor type 1 *(PTPN1)* and Dedicator of cytokinesis 10 *(DOCK10)*, which are both involved in mediating IL4 actions (Paul & Ohara., 1987) were downregulated. Protein tyrosine phosphatase 1B (PTP1B), the *PTPN1* protein product, is a ubiquitously expressed enzyme shown to negatively regulate multiple tyrosine phosphorylation-dependent signalling pathways, including the downstream processes involved in C-type lectin receptor activation (Majeed *et al.*, 2001; Long, 1999) and IL4 signalling (Lu *et al.*, 2008). Dock10 is also regulated by IL4 in B cells (Yelo *et al.*, 2008). This is of particular interest as IL4 may play a role in AS pathogenesis. Interleukin 4 (IL4), has a variety of stimulatory and inhibitory actions on B and T cells (O'Garra *et al.*, 1988; Jelinek & Lipsky 1988; Rousset *et al.*, 1988). Recent studies have also indicated a potential role for IL4 producing CD8+ T cells in the pathogenesis of AS. Although CD8+ T cells are predominately associated with the production of 'TH1' cytokines, such as IFN*γ*, there is now good evidence that some subsets of these cells can also produce 'TH2' cytokines such as IL4, IL5 and IL10 (Baek *et al.*, 2008). The potential functions associated with IL4-producing CD8+ T cells are as yet unclear but the subtype CD8+/TCR αβ+ T cells, with a regulatory phenotype and function (expressing CD25+, CTLA4+, Foxp3+, but negative for IFNγ and perforin), were previously described in peripheral blood of AS patients (Jarvis *et al.*, 2005). These results were confirmed in a recent study suggesting an altered pattern of CD8+ T cell differentiation in AS and in HLAB27+ healthy individuals. This predisposition to generate IL4+CD8+ T cells may play a role in pathogenesis of SpA (Zhang *et al.*, 2009). Further supporting this theory, *RUNX3* was identified as a candidate gene in a GWAS (Australo-Anglo-American Spondyloarthritis Consortium (TASC), 2010). The association of *RUNX3* with AS provides additional evidence of a role for CD8+ T cells in the disease. It's expression in immature lymphocytes is triggered by IL7R signalling, leading to suppression of CD4 and upregulation of CD8 expression (Park *et al.*, 2010).

Although there are some differences between the different expression profiling studies, their findings do contribute to a greater understanding of the pathogenesis of AS, particularly in the delineation of the roles of the innate and adaptive immune responses.

#### **2.3 Bone ossification and resorption processes**

Bone formation and bone loss take place at sites closely located to each other presenting an "apparent paradox", which is reflected in the changes in bone and cartilage metabolism occurring in the AS disease process (Carter & Lories, 2011). Ossification is the hallmark of AS and has been linked to aberrant activation of bone morphogenic protein (BMP) and wingless (WNT) signaling. Bone resorption, driven by the impact of inflammation on the bone remodeling cycle, occurs simultaneously, with up to 56% of patients developing systemic osteopenia and some of them systemic osteoporosis (Lange *et al.*, 2005).

Biomarkers, reflecting structural damage and disease activity, constitute a high priority for the understanding of the pathogenesis of AS and for the new therapy discovery. Two microarray-based studies have contributed to the improvement of knowledge in this field. A bone remodeling signature was described associated with an overexpression of *BMP6*, Proprotein convertase subtilisin/kexin type 6 *(PCSK6)*, Kringle containing transmembrane

Lessons from Genomic Profiling in AS 147

related DKK2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. In this context, the transmembrane proteins KREMEN1 and -2 were recently identified as additional DKK receptors, which bind to both DKK1 and DKK2 with high affinity (Mao & Niehrs, 2003). It was shown that DKK1 was able to simultaneously bind to LRP5/6 and KREMEN and that the ternary complex was rapidly endocytosed, thus preventing the WNT-LRP interaction. The interaction with KREMEN seems to be not essential but it plays a role in facilitating DKK-mediated antagonism if the level of LRP5/6 is high (Wang *et al.*, 2008). The upregulation of *KREMEN1* and *CTNNAL1* genes by these mechanisms can compromise bone formation. In contrast, upregulation of *BMP6* and its regulator *PCSK6* can contribute to the AS ossification process. BMPs, members of the transforming growth factorβ (TGF β) superfamily, play a crucial role in embryonic development, cell lineage determination, and osteoblastic differentiation and function. Enthesitis, a distinctive feature of SpA, is associated with heterotopic cartilage and bone formation (enthesophyte) (Benjamin & McGonagle, 2001). Different BMPs are expressed in distinct stages of ankylosing enthesitis shown in the DBA/1 mouse model. BMP2 is found in proliferating cells and entheseal cells committing their differentiation fate to chondrogenesis. BMP7 is recognized in prehypertrophic chondrocytes and BMP6 in hypertrophic chondrocytes (Lories *et al.*, 2005). Several regulators of endochondral bone formation with different effects in different stages were described (Kronenberg, 2003). It is therefore possible that the presence of progenitor cells at the entheseal site promotes bone formation in SpA patients. Activation of the BMP signaling pathway (phosphorylated Smad1/5) was found in cells at

the sites of entheseal inflammation in patients with AS (Lories *et al.*, 2005).

Fig. 4. Model representing the effects of SPARC on marrow mesenchymal progenitors

(adapated from Delany & Hankenson, 2009).

protein 1 *(KREMEN1)* and Catenin (cadherin-associated protein) alpha-like 1 *(CTNNAL1)* genes in SpA patients (Sharma *et al.*, 2009).

Fig. 3. The canonical WNT signaling pathway (adapted from Carter & Lories, 2011).

*KREMEN1* and *CTNNAL1* are negative regulators of WNT/catenin pathway via dickkopf homolog 1 (DKK1), or by direct inhibition of β-catenin, respectively. Although four different intracellular pathways can be triggered upon WNT receptor interaction, the WNT/β-catenin or "canonical" pathway is of particular interest in bone and cartilage biology. This pathway involves the interaction of WNT ligands with frizzled (FZD) receptors and low-density lipoprotein receptor-related protein 4, 5 or 6 (LRP 4, 5 or 6) co-receptors. In the absence of a WNT-FZD-LRP 4/5/6 interaction, cytoplasmic β-catenin is captured within a destruction complex comprising adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3β (GSK-3β), and casein kinase 1α (CK1 α). The kinases phosphorylate β-catenin, which leads to ubiquitinylation and subsequent destruction in a proteasome complex. When WNT does complex with FZD and LRP 4/5/6, axin binds to the cytoplasmic tail of LRP5 or 6, thereby phosphorylating and inhibiting GSK-3β (Gordon & Nusse, 2006). This process enables cytoplasmic β-catenin accumulation which then translocates to the nucleus, where it interacts with transcription factor (TCF)/lymphoid enhancer factor (LEF) family members and modulates WNT target gene expression (Gordon & Nusse, 2006). Several proteins that are not involved in β-catenin stability can also regulate β-catenin signaling. One example is the direct association of α-catenin with β-catenin in the nucleus which interferes with protein-DNA interactions required for TCF-mediated transcription (Giannini *et al.*, 2000). In addition, different endogenous antagonists inhibit WNT signalling; DKK1 and sclerostin (SOST). DKK1 acts by direct binding to and inhibiting the WNT co-receptor LRP6. The

protein 1 *(KREMEN1)* and Catenin (cadherin-associated protein) alpha-like 1 *(CTNNAL1)*

Fig. 3. The canonical WNT signaling pathway (adapted from Carter & Lories, 2011).

*KREMEN1* and *CTNNAL1* are negative regulators of WNT/catenin pathway via dickkopf homolog 1 (DKK1), or by direct inhibition of β-catenin, respectively. Although four different intracellular pathways can be triggered upon WNT receptor interaction, the WNT/β-catenin or "canonical" pathway is of particular interest in bone and cartilage biology. This pathway involves the interaction of WNT ligands with frizzled (FZD) receptors and low-density lipoprotein receptor-related protein 4, 5 or 6 (LRP 4, 5 or 6) co-receptors. In the absence of a WNT-FZD-LRP 4/5/6 interaction, cytoplasmic β-catenin is captured within a destruction complex comprising adenomatous polyposis coli (APC), axin, glycogen synthase kinase 3β (GSK-3β), and casein kinase 1α (CK1 α). The kinases phosphorylate β-catenin, which leads to ubiquitinylation and subsequent destruction in a proteasome complex. When WNT does complex with FZD and LRP 4/5/6, axin binds to the cytoplasmic tail of LRP5 or 6, thereby phosphorylating and inhibiting GSK-3β (Gordon & Nusse, 2006). This process enables cytoplasmic β-catenin accumulation which then translocates to the nucleus, where it interacts with transcription factor (TCF)/lymphoid enhancer factor (LEF) family members and modulates WNT target gene expression (Gordon & Nusse, 2006). Several proteins that are not involved in β-catenin stability can also regulate β-catenin signaling. One example is the direct association of α-catenin with β-catenin in the nucleus which interferes with protein-DNA interactions required for TCF-mediated transcription (Giannini *et al.*, 2000). In addition, different endogenous antagonists inhibit WNT signalling; DKK1 and sclerostin (SOST). DKK1 acts by direct binding to and inhibiting the WNT co-receptor LRP6. The

genes in SpA patients (Sharma *et al.*, 2009).

related DKK2, however, can function either as LRP6 agonist or antagonist, depending on the cellular context, suggesting that its activity is modulated by unknown co-factors. In this context, the transmembrane proteins KREMEN1 and -2 were recently identified as additional DKK receptors, which bind to both DKK1 and DKK2 with high affinity (Mao & Niehrs, 2003). It was shown that DKK1 was able to simultaneously bind to LRP5/6 and KREMEN and that the ternary complex was rapidly endocytosed, thus preventing the WNT-LRP interaction. The interaction with KREMEN seems to be not essential but it plays a role in facilitating DKK-mediated antagonism if the level of LRP5/6 is high (Wang *et al.*, 2008). The upregulation of *KREMEN1* and *CTNNAL1* genes by these mechanisms can compromise bone formation. In contrast, upregulation of *BMP6* and its regulator *PCSK6* can contribute to the AS ossification process. BMPs, members of the transforming growth factorβ (TGF β) superfamily, play a crucial role in embryonic development, cell lineage determination, and osteoblastic differentiation and function. Enthesitis, a distinctive feature of SpA, is associated with heterotopic cartilage and bone formation (enthesophyte) (Benjamin & McGonagle, 2001). Different BMPs are expressed in distinct stages of ankylosing enthesitis shown in the DBA/1 mouse model. BMP2 is found in proliferating cells and entheseal cells committing their differentiation fate to chondrogenesis. BMP7 is recognized in prehypertrophic chondrocytes and BMP6 in hypertrophic chondrocytes (Lories *et al.*, 2005). Several regulators of endochondral bone formation with different effects in different stages were described (Kronenberg, 2003). It is therefore possible that the presence of progenitor cells at the entheseal site promotes bone formation in SpA patients. Activation of the BMP signaling pathway (phosphorylated Smad1/5) was found in cells at the sites of entheseal inflammation in patients with AS (Lories *et al.*, 2005).

Fig. 4. Model representing the effects of SPARC on marrow mesenchymal progenitors (adapated from Delany & Hankenson, 2009).

Lessons from Genomic Profiling in AS 149

Low back pain (LBP) is a very common symptom, responsible for 3% of annual medical visits in the USA (Licciardone, 2008). However only 5% of the chronic back pain seen in general practice designated as "inflammatory", is associated with SpA (Underwood & Dawes, 1995). To classify patients with AS or SpA, various criteria sets can be used. The modified New York Criteria (van der Linden *et al.*, 1984) for AS, the Amor criteria (Amor *et al.*, 1990) and the European Spondyloarthropathy Study Group (ESSG) criteria (Amor *et al.*, 1991), developed in the 1990s, before magnetic resonance imaging (MRI) was available, addressed all SpA subtypes. Recently, it has been proposed to divide SpA patients into subgroups according to clinical presentation. The Assessment of SpondyloArthritis International Society (ASAS) group has developed criteria to classify patients with axial SpA with or without radiographic sacroiliitis, and patients with predominant peripheral SpA (Rudwaleit *et al.*, 2009b; Rudwaleit, 2010). With a sensitivity of 82.9% and a specificity of 84.4% , these axial SpA criteria perform better than the ESSG and Amor criteria, even after adding "sacroiliitis on MRI" to the latter. The peripheral criteria with sensitivity of 77.8% and specificity of 82.8% are also promising for use in clinical practice (Rudwaleit, 2010). The ASAS criteria have been developed as classification criteria but they are likely be useful as diagnostic criteria, especially in patients with non-radiographic axial SpA at an outpatient rheumatology clinic (van den Berg & van der Heijde, 2010). This may help to make an early diagnosis and prevent the current diagnostic delay, described as 5 to 10 years between the first occurrence of symptoms and an AS diagnosis (Feldtkeller *et al.*, 2003; Haibel *et al.*, 2007). It prevents unnecessary diagnostic tests and more importantly makes it possible to commence more effective therapies earlier. This is crucial as at early disease stages, even those without definite radiologic sacroiliitis, can suffer as much pain and have as high a disease activity as patients with established AS (Rudwaleit *et al.*, 2009a). Therefore, it's important to consider all patients with SpA with predominantly axial involvement irrespective of the presence or absence of radiographic changes as belonging to one disease continuum (Rudwaleit, 2005). Despite all these advantages with the new ASAS criteria, one of the major reasons for diagnosis delay is a low awareness of AS among physicians in primary care (Sieper, 2009). In this particular setting, several concerns have been raised regarding the use of ASAS criteria for diagnostic purposes (van den Berg &. van der Heijde, 2010). Thus current diagnosis of AS and SpA still relies on clinical and imaging parameters that may be relatively complex for general use in primary care. Screening parameters for an early referral of AS patients, easy to apply by the non-specialist, sensitive, specific and not too expensive, should be identified. For the rheumatology community this represents a great challenge. Expression studies can identify a small number of genes whose expression profile

**2.4 Biomarkers for early diagnostic purposes** 

might serve as cost effective set of surrogate biomarkers for AS.

One study has identified a small number of genes whose expression profile might serve as a cost-effective set of surrogate biomarkers for AS and uSpA (Gu *et al.*, 2009). In this PBMCbased microarray study, all included patients fulfilled Calin criteria for inflammatory back pain and were taking non-steroidal anti-inflammatory drugs (NSAID's) and/or sulfasalazine. They concluded that the overall gene expression was higher in uSpA than in AS patients suggesting that early axial SpA is associated with a more systemic inflammatory process. This may represent an interesting point as biomarkers are more helpfull in the early stage of SpA rather than the late stage. (Gu et al., 2009). Alternatively, it may reflect the less accurate diagnosis involved in uSpA and might be due some uSpA patients being

Another bone remodeling signature was identified in association with a downregulation of *SPOCK2, EP300* and *PPP2R1A* in AS, which are possible mediators in the ossification process (Pimentel-Santos *et al.*, 2011).

SPOCK2, also known as Sparc/osteonectin, is a non-collagenous bone protein. It is a member of the matricellular class of glycoproteins which includes periostin, tenascin C, osteopontin, bone sialoprotein, thrombospondin-1 and thrombospondin-2 (Alford & Hankenson, 2006). It has been hypothesized to play a role in the regulation, production, assembly and maintenance of the matrix turnover in cartilage (Hausser *et al.*, 2004; Gruber *et al.*, 2005). In this process TGFβ and IFNγ exert antagonistic effects, and play important roles in the physiologic regulation of extracellular matrix turnover. In fact, *TGFβ* positively regulates collagen type 1 (COL1A2) through the Smad signal transduction pathway, whereas *IFNγ* inhibits COL1A2 through Stat1. Additionally, protein phosphatase 2, regulatory subunit A (*PPP2R1A),* also downregulated in AS (Pimentel-Santos *et al.*, 2011), is thought to mediate *TGFβ* regulation through Smad (Heikkinen *et al.*, 2010). Animal models using SPARC-null mice have provided excellent information on the function of this protein in bone. SPARC-null mice develop profound low-turnover osteopenia (bone loss), associated with decreased numbers of osteoblasts and osteoclasts, and a markedly decreased bone-formation rate (Delany *et al.*, 2000; Boskey, 2003). Moreover SPARC-null mice have decreased trabecular bone volume due to decreased trabecular number (Machado dos Reis *et al.*, 2008) and an increase in extra-skeletal adipose deposits (Mansergh *et al.*, 2007). *In vitro* studies showed accumulation of SPARC during early osteoblastic differentiation, likely in association with collagen matrix, which decreases as the cells acquire more osteoblastic characteristics. This expression pattern seems appropriate because SPARC regulates collagen fibril assembly, and matrix is abundantly deposited in the earlier stages of differentiating cultures. SPARC has a positive effect on maintaining and expanding the mesenchymal progenitor pool, and promotes osteoblastogenesis/osteoblast function and decreases adipogenesis (Delany & Hankenson, 2009.). Expression of SPARC by osteoclasts has not been reported. Therefore, the mechanisms by which SPARC limits osteoclast formation may involve the direct interaction with osteoclasts or osteoclast precursors through the bone matrix, and/or the effect of SPARC on immune cells, marrow stromal cells, and osteoblasts supporting osteoclast development (Machado do Reis *et al.* 2008). In summary, recent findings supports the idea that SPARC play a critical role in regulating bone remodeling and maintaining bone mass. Thus its dysregulated expression may contribute to the aberrant matrix formation in AS.

Interestingly, the protein produced by *EP300* belongs to the group of nuclear p300/CBP transcriptional coactivators for both Smad3 and Stat1a that integrate signals that positively or negatively regulate *COL1A2* transcription (Ghosh *et al.*, 2001). Transactivated p300, controlled by phosphoinositide-3 kinase (PI3K)/AKT, is also an important transcriptional co-activator of Sox9, which modulates the expression of the major extracellular matrix component, aggrecan (Cheng *et al.*, 2009). Moreover, there is some evidence supporting a p300 interaction with the Wnt pathway as it is a β-catenin transcriptional coactivator. Downregulation of these genes might lead to a loss of matrix integrity thereby accelerating tissue damage. This may be reinforced by a pro-inflammatory status associated with downregulation of *EP300* (Ahmad *et al.*, 2007).

Another bone remodeling signature was identified in association with a downregulation of *SPOCK2, EP300* and *PPP2R1A* in AS, which are possible mediators in the ossification

SPOCK2, also known as Sparc/osteonectin, is a non-collagenous bone protein. It is a member of the matricellular class of glycoproteins which includes periostin, tenascin C, osteopontin, bone sialoprotein, thrombospondin-1 and thrombospondin-2 (Alford & Hankenson, 2006). It has been hypothesized to play a role in the regulation, production, assembly and maintenance of the matrix turnover in cartilage (Hausser *et al.*, 2004; Gruber *et al.*, 2005). In this process TGFβ and IFNγ exert antagonistic effects, and play important roles in the physiologic regulation of extracellular matrix turnover. In fact, *TGFβ* positively regulates collagen type 1 (COL1A2) through the Smad signal transduction pathway, whereas *IFNγ* inhibits COL1A2 through Stat1. Additionally, protein phosphatase 2, regulatory subunit A (*PPP2R1A),* also downregulated in AS (Pimentel-Santos *et al.*, 2011), is thought to mediate *TGFβ* regulation through Smad (Heikkinen *et al.*, 2010). Animal models using SPARC-null mice have provided excellent information on the function of this protein in bone. SPARC-null mice develop profound low-turnover osteopenia (bone loss), associated with decreased numbers of osteoblasts and osteoclasts, and a markedly decreased bone-formation rate (Delany *et al.*, 2000; Boskey, 2003). Moreover SPARC-null mice have decreased trabecular bone volume due to decreased trabecular number (Machado dos Reis *et al.*, 2008) and an increase in extra-skeletal adipose deposits (Mansergh *et al.*, 2007). *In vitro* studies showed accumulation of SPARC during early osteoblastic differentiation, likely in association with collagen matrix, which decreases as the cells acquire more osteoblastic characteristics. This expression pattern seems appropriate because SPARC regulates collagen fibril assembly, and matrix is abundantly deposited in the earlier stages of differentiating cultures. SPARC has a positive effect on maintaining and expanding the mesenchymal progenitor pool, and promotes osteoblastogenesis/osteoblast function and decreases adipogenesis (Delany & Hankenson, 2009.). Expression of SPARC by osteoclasts has not been reported. Therefore, the mechanisms by which SPARC limits osteoclast formation may involve the direct interaction with osteoclasts or osteoclast precursors through the bone matrix, and/or the effect of SPARC on immune cells, marrow stromal cells, and osteoblasts supporting osteoclast development (Machado do Reis *et al.* 2008). In summary, recent findings supports the idea that SPARC play a critical role in regulating bone remodeling and maintaining bone mass. Thus its dysregulated expression may

Interestingly, the protein produced by *EP300* belongs to the group of nuclear p300/CBP transcriptional coactivators for both Smad3 and Stat1a that integrate signals that positively or negatively regulate *COL1A2* transcription (Ghosh *et al.*, 2001). Transactivated p300, controlled by phosphoinositide-3 kinase (PI3K)/AKT, is also an important transcriptional co-activator of Sox9, which modulates the expression of the major extracellular matrix component, aggrecan (Cheng *et al.*, 2009). Moreover, there is some evidence supporting a p300 interaction with the Wnt pathway as it is a β-catenin transcriptional coactivator. Downregulation of these genes might lead to a loss of matrix integrity thereby accelerating tissue damage. This may be reinforced by a pro-inflammatory status associated with

process (Pimentel-Santos *et al.*, 2011).

contribute to the aberrant matrix formation in AS.

downregulation of *EP300* (Ahmad *et al.*, 2007).

#### **2.4 Biomarkers for early diagnostic purposes**

Low back pain (LBP) is a very common symptom, responsible for 3% of annual medical visits in the USA (Licciardone, 2008). However only 5% of the chronic back pain seen in general practice designated as "inflammatory", is associated with SpA (Underwood & Dawes, 1995). To classify patients with AS or SpA, various criteria sets can be used. The modified New York Criteria (van der Linden *et al.*, 1984) for AS, the Amor criteria (Amor *et al.*, 1990) and the European Spondyloarthropathy Study Group (ESSG) criteria (Amor *et al.*, 1991), developed in the 1990s, before magnetic resonance imaging (MRI) was available, addressed all SpA subtypes. Recently, it has been proposed to divide SpA patients into subgroups according to clinical presentation. The Assessment of SpondyloArthritis International Society (ASAS) group has developed criteria to classify patients with axial SpA with or without radiographic sacroiliitis, and patients with predominant peripheral SpA (Rudwaleit *et al.*, 2009b; Rudwaleit, 2010). With a sensitivity of 82.9% and a specificity of 84.4% , these axial SpA criteria perform better than the ESSG and Amor criteria, even after adding "sacroiliitis on MRI" to the latter. The peripheral criteria with sensitivity of 77.8% and specificity of 82.8% are also promising for use in clinical practice (Rudwaleit, 2010). The ASAS criteria have been developed as classification criteria but they are likely be useful as diagnostic criteria, especially in patients with non-radiographic axial SpA at an outpatient rheumatology clinic (van den Berg & van der Heijde, 2010). This may help to make an early diagnosis and prevent the current diagnostic delay, described as 5 to 10 years between the first occurrence of symptoms and an AS diagnosis (Feldtkeller *et al.*, 2003; Haibel *et al.*, 2007). It prevents unnecessary diagnostic tests and more importantly makes it possible to commence more effective therapies earlier. This is crucial as at early disease stages, even those without definite radiologic sacroiliitis, can suffer as much pain and have as high a disease activity as patients with established AS (Rudwaleit *et al.*, 2009a). Therefore, it's important to consider all patients with SpA with predominantly axial involvement irrespective of the presence or absence of radiographic changes as belonging to one disease continuum (Rudwaleit, 2005). Despite all these advantages with the new ASAS criteria, one of the major reasons for diagnosis delay is a low awareness of AS among physicians in primary care (Sieper, 2009). In this particular setting, several concerns have been raised regarding the use of ASAS criteria for diagnostic purposes (van den Berg &. van der Heijde, 2010). Thus current diagnosis of AS and SpA still relies on clinical and imaging parameters that may be relatively complex for general use in primary care. Screening parameters for an early referral of AS patients, easy to apply by the non-specialist, sensitive, specific and not too expensive, should be identified. For the rheumatology community this represents a great challenge. Expression studies can identify a small number of genes whose expression profile might serve as cost effective set of surrogate biomarkers for AS.

One study has identified a small number of genes whose expression profile might serve as a cost-effective set of surrogate biomarkers for AS and uSpA (Gu *et al.*, 2009). In this PBMCbased microarray study, all included patients fulfilled Calin criteria for inflammatory back pain and were taking non-steroidal anti-inflammatory drugs (NSAID's) and/or sulfasalazine. They concluded that the overall gene expression was higher in uSpA than in AS patients suggesting that early axial SpA is associated with a more systemic inflammatory process. This may represent an interesting point as biomarkers are more helpfull in the early stage of SpA rather than the late stage. (Gu et al., 2009). Alternatively, it may reflect the less accurate diagnosis involved in uSpA and might be due some uSpA patients being

Lessons from Genomic Profiling in AS 151

inflammatory markers at the start of therapy predicted a greater improvement in disease activity, (Lord *et al.*, 2010). Predictors of improvement in function, measured using the BASFI, have shown a strong association with gender (significantly greater improvement in women) and concurrent DMARDs therapy (Lord et al, 2010). Finally, prevention of damage is another important outcome of therapy. Slow radiographic progression of the disease and the relatively small fraction of patients progressing over a period of 2-3 years makes radiographic evaluation less sensitive for damage evaluation. However, the major predictor of progression is previous existing radiographic damage. While it is clear that anti-TNFα agents have a structural benefit in inflammation-mediated resorptive damage as indicated by changes in bone and cartilage metabolism, an effect on radiographic progression remains to be demonstrated in AS (de Vlam, 2010). A study of the relationship of biomarker levels, disease activity and the spinal inflammation detected by MRI was performed in patients with ankylosing spondylitis (AS) receiving Infliximab over a 24 week period. Early reductions in IL-6 (by week 2) but not CRP or vascular endothelial growth factor (VEGF), were significantly associated with reductions in MRI activity and BASDAI scores by week 24 in the infliximab group (Visvanathan *et al.*, 2008). However the structural changes of this

Gene expression profiling constitutes a widely available and promising technology to identify treatment-associated changes. In two recent studies it was demonstrated that anti-TNF alpha treatment leads to significant alteration of gene expression and protein profiles, supporting the use of systematic gene expression and proteomic analysis to shed new light on pathogenic pathways with importance in the chronic inflammation of AS (Haroon *et al.*, 2010; Grcevic *et al.*, 2010). Anti-TNFα therapy induced a rapid change in the expression profile within 2 weeks in AS patients with down-regulation of lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes *(LIGHT*), interferon α receptor 1 (*IFNAR1),* interleukin 17 receptor *(IL17R)* and erythropoietin receptor *(EPOR)*  genes. *LIGHT*, a member of the TNF superfamily, was the most significantly downregulated gene and serum soluble LIGHT levels correlate well with other inflammatory markers such as, CRP and ESR. However, no significant differences between responders and non-responders were observed in either *LIGHT* mRNA expression or LIGHT serum levels. A time gap between changes in inflammatory mediators and improvements in subjective disease severity scoring metrics may explain these findings (Haroon *et al.*, 2010). Although these results are interesting more studies are needed for validation. Another study using peripheral blood expression profiles based on PBMCs cells assessed several bone-regulatory factors as potential discriminators of different forms of arthritis, disease activity and therapy responsiveness (Grcevic *et al.*, 2010). ROC curve analysis suggested higher expression of Runx2 was a potential molecular marker for AS. Although no increased gene expression of *BMP-4* or *LIGHT* in AS patients compared with healthy controls were seen, higher expression was evident in AS patients resistant to conventional therapy. Thus *LIGHT* might

be considered an interesting biomarker to consider in future studies.

Another marker which must be considered for a treatment-response marker is the CX3CL1- CC3CR1 complex. In RA, CX3CL1 levels decline in patients showing a clinical response to infliximab treatment. Moreover, patients with active RA who did not show a clinical response to infliximab showed higher basal CX3CL1 levels than those who did (Odai *et al.*, 2009). These results suggest that the CX3CL1-CX3CR1 in patients with active RA may be

effect are not known.

misdiagnosed and actually suffering from a different inflammatory condition. A member of the family of regulators of G protein signaling *(RGS1,)* was identified as the most promising biomarker for uSpA and AS, with this gene more highly expressed in uSpA than in AS. They demonstrated a receiver operating characteristic (ROC) area under the curve (AUC) range between 0.93-0.99. Biomarkers with ROC AUC 0.8-1.0 are usually considered to be useful in clinical practice (Rao, 2003). To evaluate arthritis related factors that might enhance *RGS1* expression, a panel of 25 cytokines and chemokines on a monocyte derived human cell line were used. The 2 strongest activators of *RGS1* expression were TNFα and IL-17. However, in order to be implemented in clinical practice further studies are clearly needed. It requires a multicenter, multi-ethnic validation but also comparison with results obtained through MRI and the new ASAS classification criteria. There are several other concerns. This gene was differentially expressed between AS patients and healthy controls, in another microarray study PBMC based (Duan *et al.*, 2010), but contrary to the first study it was underexpressed. Finally, it wasn't identified as differentially expressed in a recent published study from a well defined population of Portuguese ethnicity background (Pimentel-Santos *et al.*, 2011). These distinct results reinforce the need for larger studies involving different ethnic groups.

#### **2.5 Gene expression changes after anti-TNFα therapy**

Biomarkers that allow quantitative assessment of treatment response have great potential in clinical practice. They enable appropriate choice of therapy, drug dosage to maximize effect and minimize toxicity, and monitor disease outcomes representing the foundation of evidence-based medicine (de Vlam, 2010). The introduction of biologic therapies targeting TNFα (infliximab, etanercept, adalimumab, golimumab) has changed clinical practice with several benefits regarding clinical management and prognosis. Additionally, the scientific community is waiting for the market introduction of new biological treatments with new targets in the near future. Identification of markers of treatment response would be of great clinical benefit by facilitating better targeting of these treatments to those most likely to respond, and potentially significantly reduce treatment costs by minimizing use of these expensive agents in patients unlikely to respond.

Until now the Visual Analogue Scale (VAS) pain, VAS general health, BASDAI, inflammatory parameters and composite response criteria are used to evaluate treatment effect in AS. ASAS defined and validated three levels of response: ASAS20, ASAS40, and ASAS partial remission, for patients treated with non-steroidal anti-inflammatory drugs and TNFα blockade (Anderson *et al.*, 2001). The recent introduction of the ASDAS criteria (van der Heijde *et al.*, 2009) seems to be a highly discriminatory instrument for assessing AS disease activity and monitoring changes in disease and is finding good use in clinical practice. However all these criteria aren't predictors of response to therapy and greatly rely on subjective self-evaluation and are not free from disease-unrelated influences, so biomarkers with high sensitivity and specificity for treatment response are highly desirable.

Current markers of response such as younger age, HLA-B27 carriage, elevation of acute phase reactants (CRP), and marked spinal inflammation, as shown by MRI, may be predictors of good response; conversely, older age, structural damage and poor function may be predictors of poor- or non-response (Rudwaleit *et al.*, 2004; Rudwaleit *et al.*, 2008). Data from the British Society of Rheumatology Biologics Register has shown raised

misdiagnosed and actually suffering from a different inflammatory condition. A member of the family of regulators of G protein signaling *(RGS1,)* was identified as the most promising biomarker for uSpA and AS, with this gene more highly expressed in uSpA than in AS. They demonstrated a receiver operating characteristic (ROC) area under the curve (AUC) range between 0.93-0.99. Biomarkers with ROC AUC 0.8-1.0 are usually considered to be useful in clinical practice (Rao, 2003). To evaluate arthritis related factors that might enhance *RGS1* expression, a panel of 25 cytokines and chemokines on a monocyte derived human cell line were used. The 2 strongest activators of *RGS1* expression were TNFα and IL-17. However, in order to be implemented in clinical practice further studies are clearly needed. It requires a multicenter, multi-ethnic validation but also comparison with results obtained through MRI and the new ASAS classification criteria. There are several other concerns. This gene was differentially expressed between AS patients and healthy controls, in another microarray study PBMC based (Duan *et al.*, 2010), but contrary to the first study it was underexpressed. Finally, it wasn't identified as differentially expressed in a recent published study from a well defined population of Portuguese ethnicity background (Pimentel-Santos *et al.*, 2011). These distinct results reinforce the need for larger studies involving different

Biomarkers that allow quantitative assessment of treatment response have great potential in clinical practice. They enable appropriate choice of therapy, drug dosage to maximize effect and minimize toxicity, and monitor disease outcomes representing the foundation of evidence-based medicine (de Vlam, 2010). The introduction of biologic therapies targeting TNFα (infliximab, etanercept, adalimumab, golimumab) has changed clinical practice with several benefits regarding clinical management and prognosis. Additionally, the scientific community is waiting for the market introduction of new biological treatments with new targets in the near future. Identification of markers of treatment response would be of great clinical benefit by facilitating better targeting of these treatments to those most likely to respond, and potentially significantly reduce treatment costs by minimizing use of these

Until now the Visual Analogue Scale (VAS) pain, VAS general health, BASDAI, inflammatory parameters and composite response criteria are used to evaluate treatment effect in AS. ASAS defined and validated three levels of response: ASAS20, ASAS40, and ASAS partial remission, for patients treated with non-steroidal anti-inflammatory drugs and TNFα blockade (Anderson *et al.*, 2001). The recent introduction of the ASDAS criteria (van der Heijde *et al.*, 2009) seems to be a highly discriminatory instrument for assessing AS disease activity and monitoring changes in disease and is finding good use in clinical practice. However all these criteria aren't predictors of response to therapy and greatly rely on subjective self-evaluation and are not free from disease-unrelated influences, so biomarkers with high sensitivity and specificity for treatment response are highly desirable. Current markers of response such as younger age, HLA-B27 carriage, elevation of acute phase reactants (CRP), and marked spinal inflammation, as shown by MRI, may be predictors of good response; conversely, older age, structural damage and poor function may be predictors of poor- or non-response (Rudwaleit *et al.*, 2004; Rudwaleit *et al.*, 2008). Data from the British Society of Rheumatology Biologics Register has shown raised

ethnic groups.

**2.5 Gene expression changes after anti-TNFα therapy** 

expensive agents in patients unlikely to respond.

inflammatory markers at the start of therapy predicted a greater improvement in disease activity, (Lord *et al.*, 2010). Predictors of improvement in function, measured using the BASFI, have shown a strong association with gender (significantly greater improvement in women) and concurrent DMARDs therapy (Lord et al, 2010). Finally, prevention of damage is another important outcome of therapy. Slow radiographic progression of the disease and the relatively small fraction of patients progressing over a period of 2-3 years makes radiographic evaluation less sensitive for damage evaluation. However, the major predictor of progression is previous existing radiographic damage. While it is clear that anti-TNFα agents have a structural benefit in inflammation-mediated resorptive damage as indicated by changes in bone and cartilage metabolism, an effect on radiographic progression remains to be demonstrated in AS (de Vlam, 2010). A study of the relationship of biomarker levels, disease activity and the spinal inflammation detected by MRI was performed in patients with ankylosing spondylitis (AS) receiving Infliximab over a 24 week period. Early reductions in IL-6 (by week 2) but not CRP or vascular endothelial growth factor (VEGF), were significantly associated with reductions in MRI activity and BASDAI scores by week 24 in the infliximab group (Visvanathan *et al.*, 2008). However the structural changes of this effect are not known.

Gene expression profiling constitutes a widely available and promising technology to identify treatment-associated changes. In two recent studies it was demonstrated that anti-TNF alpha treatment leads to significant alteration of gene expression and protein profiles, supporting the use of systematic gene expression and proteomic analysis to shed new light on pathogenic pathways with importance in the chronic inflammation of AS (Haroon *et al.*, 2010; Grcevic *et al.*, 2010). Anti-TNFα therapy induced a rapid change in the expression profile within 2 weeks in AS patients with down-regulation of lymphotoxins exhibiting inducible expression and competing with herpes simplex virus glycoprotein D for herpesvirus entry mediator, a receptor expressed by T lymphocytes *(LIGHT*), interferon α receptor 1 (*IFNAR1),* interleukin 17 receptor *(IL17R)* and erythropoietin receptor *(EPOR)*  genes. *LIGHT*, a member of the TNF superfamily, was the most significantly downregulated gene and serum soluble LIGHT levels correlate well with other inflammatory markers such as, CRP and ESR. However, no significant differences between responders and non-responders were observed in either *LIGHT* mRNA expression or LIGHT serum levels. A time gap between changes in inflammatory mediators and improvements in subjective disease severity scoring metrics may explain these findings (Haroon *et al.*, 2010). Although these results are interesting more studies are needed for validation. Another study using peripheral blood expression profiles based on PBMCs cells assessed several bone-regulatory factors as potential discriminators of different forms of arthritis, disease activity and therapy responsiveness (Grcevic *et al.*, 2010). ROC curve analysis suggested higher expression of Runx2 was a potential molecular marker for AS. Although no increased gene expression of *BMP-4* or *LIGHT* in AS patients compared with healthy controls were seen, higher expression was evident in AS patients resistant to conventional therapy. Thus *LIGHT* might be considered an interesting biomarker to consider in future studies.

Another marker which must be considered for a treatment-response marker is the CX3CL1- CC3CR1 complex. In RA, CX3CL1 levels decline in patients showing a clinical response to infliximab treatment. Moreover, patients with active RA who did not show a clinical response to infliximab showed higher basal CX3CL1 levels than those who did (Odai *et al.*, 2009). These results suggest that the CX3CL1-CX3CR1 in patients with active RA may be

Lessons from Genomic Profiling in AS 153

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sensitive to anti-TNFα therapy and confirm that CX3CL1 plays a crucial role in the pathogenesis of RA, although further investigations are required. These results suggest that CX3CL1-CX3CR1 may be also relevant in AS process. This is further supported with the underexpression of this gene in AS patients (Pimentel-Santos *et al.*, 2011).


Table 3. Potential clinical applications of microarray findings.

#### **3. Conclusion**

All the studies described above have contributed to increased knowledge of the physiopathological processes involved in AS and have identified potential disease relevant biomarkers with significance for clinical practice (see Table 3). The integration of the expression profiling data with information obtained from "omic" approaches such as proteomic and metabolomic analyses as well as with clinical and imaging data, may further elucidate disease processes and therapeutic responses in AS.

#### **4. Acknowledgment**

We thank Mafalda Matos for her help in figures and tables production.

#### **5. References**

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sensitive to anti-TNFα therapy and confirm that CX3CL1 plays a crucial role in the pathogenesis of RA, although further investigations are required. These results suggest that CX3CL1-CX3CR1 may be also relevant in AS process. This is further supported with the

> Bone remodelling and cartilage matrix turnover

signaling 1 Diagnosis of early AS/uSPA

mediator Response to anti-TNF alpha

underexpression of this gene in AS patients (Pimentel-Santos *et al.*, 2011).

*BMP6* Bone morphogenic protein 6

*CTNNAL1* Catenin (cadherin-associated

*PCSK6* Proprotein convertase

*KREMEN1* Kringle containing

*SPOCK2* Sparc/osteonectin *EP300* Nuclear p300

*PPP2R1A* Protein phosphatase 2,

*RGS1* Regulators of G protein

**3. Conclusion** 

**4. Acknowledgment** 

319-325, ISSN, 1503-0775.

**5. References** 

*LIGHT* Ligand for herpesvirus entry

Table 3. Potential clinical applications of microarray findings.

elucidate disease processes and therapeutic responses in AS.

We thank Mafalda Matos for her help in figures and tables production.

**Gene symbol Designation Potential role** 

subtilisin/kexin type 6

protein) alpha-like 1

regulatory subunit A

treatment *CX3CL1-CX3CR1* 

motif) receptor 1

Chemokine (C-X3-C motif) ligand 1 - chemokine (C-X3-C

All the studies described above have contributed to increased knowledge of the physiopathological processes involved in AS and have identified potential disease relevant biomarkers with significance for clinical practice (see Table 3). The integration of the expression profiling data with information obtained from "omic" approaches such as proteomic and metabolomic analyses as well as with clinical and imaging data, may further

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NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. *Immunity*, Vol.20, No.3 (March 2004) pp.

transmembrane protein 1


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### *Edited by Jacome Bruges-Armas*

The first section of the book entitled Clinical and Molecular Advances in Ankylosing Spondylitis is a review of the clinical manifestations of Ankylosing Spondylitis (AS) and Spondyloarthritis (SpA). The book includes chapters on Bone Mineral Density measurements, two chapters on the temporomandibular joints, axial fractures, clinical manifestations, diagnosis, and treatment. Molecular genetics and immune response are analyzed in the second section of the book; information on HLA-B\*27, other MHC genes and the immune response of AS patients to bacteria is reviewed and updated. Two chapters are dedicated to recent information on non-MHC genes in AS susceptibility, and to new data on disease pathways generated from gene expression studies on peripheral blood.

Photo by Dr\_Microbe / iStock

Clinical and Molecular Advances in Ankylosing Spondylitis

Clinical and Molecular

Advances in Ankylosing

Spondylitis

*Edited by Jacome Bruges-Armas*