**2. Mediterranean fever (***MEFV***) gene mutations**

#### **2.1 Protein pyrin**

One of the most recognizable diseases of the Mediterranean region is Familial Mediterranean Fever (FMF), a prototype of a monogenic autoinflammatory disease, associated with mutations in the *MEFV* gene that encodes for protein pyrin. Autoinflammatory disorders are characterized by dysregulation of innate immune response, unlike autoimmune diseases that are primarily mediated by adaptive immunity. However, approximately in a third of FMF patients pathogenic *MEFV* mutation is not identified, hence the diagnostic criteria for FMF still rely on clinical manifestations [1, 2]. The *MEFV* gene is composed out of 10 exons and 13 introns, which make 781 amino acids (aa) long, multifunctional, protein pyrin. One of its first described functions is the assembly of an inflammasome. Pyrin acts as a pattern recognition receptor (PRR) that senses intracellular danger signals after which it binds to an adaptor protein and oligomerizes to form a pyrin inflammasome. Subsequently, inflammasome recruits and activates caspase-1, which further cleaves pro-inflammatory molecules, such as interleukin (IL)-1β and IL-18 [1, 3, 4].

As a PRR, pyrin seems to recognize downstream effects of a pathogen-driven modification and/or inactivation of RhoA GTPases - molecules that regulate actin dynamics [3]. By sensing a disturbance in actin signaling, pyrin recognizes common virulence mechanisms and starts an immune response. Several pathogen bacteria employ actin cytoskeleton for their invasion and survival, and by secretion of Rho-inactivating cytotoxins they were shown to activate pyrin inflammasome (e.g. *Clostridium, Vibrio parahaemolyticus, Bordetella pertussis, Yersinia pestis*) [5–7].

Besides, pyrin regulates process of autophagy a highly specific degradation of inflammasomes components. With this process pyrin suppresses IL-1β production, thereby preventing an excessive inflammation. Additionally, autophagy-based secretory pathway enables a group of proteins to exit cytoplasm without entering Golgi apparatus, among which is IL-1β [8, 9]. Hence, *MEFV* mutation-induced alterations affect this pathway and may facilitate interleukins secretion.

The pyrin activation requires at least two independent processes: dephosphorylation and pyrin inflammasome maturation involving microtubule dynamics [2, 7]. In FMF patients with pathogenic *MEFV* variants there is a hyperreactive state of the pyrin inflammasome. It seems that the second control mechanism of pyrin activation is lacking, and that pyrin is maintained inactive only by phosphorylation. Mutations in the exon 10 do not impact pyrin phosphorylation but may affect the control mechanism of microtubule dynamics [4, 7].

#### **2.2 The** *MEFV* **mutations and their effect**

According to the Infevers registry (the registry of hereditary autoinflammatory disorders mutations) there are 377 nucleotide variants identified in the *MEFV* gene so far. Most of them are benign and not involved in pathogenesis of FMF.

The FMF has long been considered an autosomal recessive disease, but with description of cases with heterozygote *MEFV* mutations this definition has changed. The mutations may express their effect in either a recessive or a dominant manner, depending on their location in the gene. Generally, those in the exon 10 are considered recessive, while other manifest their effect in a heterozygous state and are considered dominant (gain-of-function). The most frequently identified FMFcausing *MEFV* variants are in the exon 10 and encompass M694V (c.2080A > G), M680I (c.2040G > C), and V726A (c.2177 T > C) missense mutations, with the carrier frequency of ~10% in the populations of the Mediterranean region [2, 4, 8, 10].

**147**

*Adaptation to Mediterranea*

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

significance (E148Q, K695R, P369S, F479L, and I591T).

immune factors (HLA I gene A), and microbiota [11, 20–22].

**2.3 Potential heterozygote advantage** *of MEFV* **mutations**

positive selection in primates [2, 4, 23, 24].

**2.4 Diversity of** *MEFV* **mutations**

In order to achieve a better classification of pathogenic *MEFV* mutations, and to set a guidelines for genetic diagnostic testing of hereditary recurrent fevers, Shinar et al. [11], adopted the final consensus document that proposed a group of bialelic mutations to be used for definition of FMF. This group comprises 14 mutations, 9 of which are clearly pathogenic (M694V, M694I, M680I, V726A, R761H, A744S, I692del, E167D, and T267I), while 5 mutations are designated as of unknown

Depending on the type of mutations, permissive environmental factors and genetic background, clinical picture of FMF may vary from typical recurrent inflammatory attacks to mild symptoms or asymptomatic cases. The most frequent symptoms are recurrent episodes of fever with serosal inflammation, arthralgia or arthritis, abdominal pain, and localized erythematous skin rash. Episodes are self-limited and usually resolve within 48-72 h. Heterozygous patients usually have milder symptoms and shorter and less frequent attacks. Some asymptomatic carriers may have elevated inflammatory and oxidative stress biomarkers [4, 10, 12, 13]. The most concerning long-term complication is renal amyloidosis, and renal transplantation is the choice in most end-stage renal disease [14, 15]. Standard treatment is a prolonged use of colchicine, although there are resistant cases. One potential cause of resistance is the vitamin D deficiency in these patients [16–18]. The M694V homozygote mutation is mostly associated with early onset of disease and severe course. It seems that environmental factors have stronger influence on this mutation [19, 20]. It is interesting to note the impact of environmental factors, since patients of the same ethnicity have different phenotype depending on the country they live in, *i.e.* the Eastern or Western Europe. Besides, a set of additional factors influence the phenotype, such as patient's age and sex, micro-RNAs,

The higher frequency of *MEFV* mutations among multiple populations in the Mediterranean region suggests an existence of a heterozygote advantage. Mostly accepted theories explaining this assumption are those that recognize mutations as an adaptation to yet undetermined endemic infectious agent or, more probably, a group of agents. It seems that *MEFV's* exon 10 had been exposed to the episodic

Several bacteria secrete invasive factors (toxins) that covalently modify RhoA or its regulators. Other may inhibit pyrin inflammasome assembly by keeping it in the phosphorylated state, such as a protein YopM produced by bacteria *Yersinia enterocolitica*. Pyrin activation occurs when RhoA GTPases are disabled to promote their downstream signaling. In that sense, bacteria *Yersinia pestis* (bubonic plaguebacterium) is proposed as a possible agent that had led to the selection of gain-offunction *MEFV* mutations [25]. Other hypotheses imply that mutated pyrin may confer a protection against tuberculosis or brucellosis, but without direct evidence [2]. Potential association between mutated pyrin and defense against tuberculosis is within the processes of autophagy and inflammasome activation. *Mycobacterium tuberculosis* is capable of arresting phagolysosome biogenesis in macrophages and prevents inflammasome activation by its Zn-metalloprotease, while mutated pyrin mediated stimulation of autophagic pathways may overcome this block [2, 26, 27].

Higher frequency of pathogenic *MEFV* mutations in the Mediterranean basin is mainly explained by a founder effect and balancing selection. The M694V and

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

**2.1 Protein pyrin**

**2. Mediterranean fever (***MEFV***) gene mutations**

One of the most recognizable diseases of the Mediterranean region is Familial

Mediterranean Fever (FMF), a prototype of a monogenic autoinflammatory disease, associated with mutations in the *MEFV* gene that encodes for protein pyrin. Autoinflammatory disorders are characterized by dysregulation of innate immune response, unlike autoimmune diseases that are primarily mediated by adaptive immunity. However, approximately in a third of FMF patients pathogenic *MEFV* mutation is not identified, hence the diagnostic criteria for FMF still rely on clinical manifestations [1, 2]. The *MEFV* gene is composed out of 10 exons and 13 introns, which make 781 amino acids (aa) long, multifunctional, protein pyrin. One of its first described functions is the assembly of an inflammasome. Pyrin acts as a pattern recognition receptor (PRR) that senses intracellular danger signals after which it binds to an adaptor protein and oligomerizes to form a pyrin inflammasome. Subsequently, inflammasome recruits and activates caspase-1, which further cleaves pro-inflammatory molecules, such as interleukin (IL)-1β and IL-18 [1, 3, 4]. As a PRR, pyrin seems to recognize downstream effects of a pathogen-driven modification and/or inactivation of RhoA GTPases - molecules that regulate actin dynamics [3]. By sensing a disturbance in actin signaling, pyrin recognizes common virulence mechanisms and starts an immune response. Several pathogen bacteria employ actin cytoskeleton for their invasion and survival, and by secretion of Rho-inactivating cytotoxins they were shown to activate pyrin inflammasome (e.g. *Clostridium, Vibrio parahaemolyticus, Bordetella pertussis, Yersinia pestis*) [5–7]. Besides, pyrin regulates process of autophagy a highly specific degradation of inflammasomes components. With this process pyrin suppresses IL-1β production, thereby preventing an excessive inflammation. Additionally, autophagy-based secretory pathway enables a group of proteins to exit cytoplasm without entering Golgi apparatus, among which is IL-1β [8, 9]. Hence, *MEFV* mutation-induced

alterations affect this pathway and may facilitate interleukins secretion.

so far. Most of them are benign and not involved in pathogenesis of FMF.

control mechanism of microtubule dynamics [4, 7].

**2.2 The** *MEFV* **mutations and their effect**

The pyrin activation requires at least two independent processes: dephosphorylation and pyrin inflammasome maturation involving microtubule dynamics [2, 7]. In FMF patients with pathogenic *MEFV* variants there is a hyperreactive state of the pyrin inflammasome. It seems that the second control mechanism of pyrin activation is lacking, and that pyrin is maintained inactive only by phosphorylation. Mutations in the exon 10 do not impact pyrin phosphorylation but may affect the

According to the Infevers registry (the registry of hereditary autoinflammatory disorders mutations) there are 377 nucleotide variants identified in the *MEFV* gene

The FMF has long been considered an autosomal recessive disease, but with description of cases with heterozygote *MEFV* mutations this definition has changed. The mutations may express their effect in either a recessive or a dominant manner, depending on their location in the gene. Generally, those in the exon 10 are considered recessive, while other manifest their effect in a heterozygous state and are considered dominant (gain-of-function). The most frequently identified FMFcausing *MEFV* variants are in the exon 10 and encompass M694V (c.2080A > G), M680I (c.2040G > C), and V726A (c.2177 T > C) missense mutations, with the carrier frequency of ~10% in the populations of the Mediterranean region [2, 4, 8, 10].

**146**

In order to achieve a better classification of pathogenic *MEFV* mutations, and to set a guidelines for genetic diagnostic testing of hereditary recurrent fevers, Shinar et al. [11], adopted the final consensus document that proposed a group of bialelic mutations to be used for definition of FMF. This group comprises 14 mutations, 9 of which are clearly pathogenic (M694V, M694I, M680I, V726A, R761H, A744S, I692del, E167D, and T267I), while 5 mutations are designated as of unknown significance (E148Q, K695R, P369S, F479L, and I591T).

Depending on the type of mutations, permissive environmental factors and genetic background, clinical picture of FMF may vary from typical recurrent inflammatory attacks to mild symptoms or asymptomatic cases. The most frequent symptoms are recurrent episodes of fever with serosal inflammation, arthralgia or arthritis, abdominal pain, and localized erythematous skin rash. Episodes are self-limited and usually resolve within 48-72 h. Heterozygous patients usually have milder symptoms and shorter and less frequent attacks. Some asymptomatic carriers may have elevated inflammatory and oxidative stress biomarkers [4, 10, 12, 13]. The most concerning long-term complication is renal amyloidosis, and renal transplantation is the choice in most end-stage renal disease [14, 15]. Standard treatment is a prolonged use of colchicine, although there are resistant cases. One potential cause of resistance is the vitamin D deficiency in these patients [16–18].

The M694V homozygote mutation is mostly associated with early onset of disease and severe course. It seems that environmental factors have stronger influence on this mutation [19, 20]. It is interesting to note the impact of environmental factors, since patients of the same ethnicity have different phenotype depending on the country they live in, *i.e.* the Eastern or Western Europe. Besides, a set of additional factors influence the phenotype, such as patient's age and sex, micro-RNAs, immune factors (HLA I gene A), and microbiota [11, 20–22].

## **2.3 Potential heterozygote advantage** *of MEFV* **mutations**

The higher frequency of *MEFV* mutations among multiple populations in the Mediterranean region suggests an existence of a heterozygote advantage. Mostly accepted theories explaining this assumption are those that recognize mutations as an adaptation to yet undetermined endemic infectious agent or, more probably, a group of agents. It seems that *MEFV's* exon 10 had been exposed to the episodic positive selection in primates [2, 4, 23, 24].

Several bacteria secrete invasive factors (toxins) that covalently modify RhoA or its regulators. Other may inhibit pyrin inflammasome assembly by keeping it in the phosphorylated state, such as a protein YopM produced by bacteria *Yersinia enterocolitica*. Pyrin activation occurs when RhoA GTPases are disabled to promote their downstream signaling. In that sense, bacteria *Yersinia pestis* (bubonic plaguebacterium) is proposed as a possible agent that had led to the selection of gain-offunction *MEFV* mutations [25]. Other hypotheses imply that mutated pyrin may confer a protection against tuberculosis or brucellosis, but without direct evidence [2]. Potential association between mutated pyrin and defense against tuberculosis is within the processes of autophagy and inflammasome activation. *Mycobacterium tuberculosis* is capable of arresting phagolysosome biogenesis in macrophages and prevents inflammasome activation by its Zn-metalloprotease, while mutated pyrin mediated stimulation of autophagic pathways may overcome this block [2, 26, 27].

#### **2.4 Diversity of** *MEFV* **mutations**

Higher frequency of pathogenic *MEFV* mutations in the Mediterranean basin is mainly explained by a founder effect and balancing selection. The M694V and

V726A mutations seem to emerge in human genome about 2000 years ago, according to their association with specific microsatellite haplotypes in different populations [21, 28]. Presence of the E148Q (c.442G > C) mutation in different ethnic groups in the region supports the hypothesis of its recurrent nature or founder effect, probably stemming from the Asian countries, such as China and India, where E148Q is also frequent [20, 29]. The M694I (c.2082 G > A) mutation is merely present in North Africa and is estimated that occurred in an indigenous population of Berbers before colonizations in the 7th century BC [4, 20].

The M694V, M694I, M680I, and V726A mutations are most common in the Eastern Mediterranean countries, that is in Turkish, Armenian, Arab, and Jewish populations (**Figure 1**). The carrier rate of the mutations in these populations is estimated to be 1:5 to 1:7. Consequently, FMF mainly affects people of these ethnicities [2, 5, 19, 30–32]. The prevalence of *MEFV* mutations and FMF is much lower in the Western Mediterranean countries (France and Spain). Actually, the ethnic origin of these patients is usually from populations with higher mutation frequencies. Also, higher prevalence of homozygous mutations in the East might reflect the consequence of a local custom of consanguinity marriages [20, 33, 34].

Beside differences in *MEFV* mutations distribution between countries, there are variations within countries as well. For example, in Turkey, 94% of diagnosed FMF patients were from central-western parts of the country. However, more than a half of them had a family origin from the eastern provinces, pointing to the migration routes of mutations and disease [19, 35, 36]. The similarity between *MEFV* mutations present in Turks and Jordanians can also be explained by the local migrations, during the Ottoman Empire [20, 37]. The M694V mutation is the most common in Arab FMF patients. Unlike others, Arabs in North Africa have higher rate of M694I mutation that is probably acquired through the intermarriages with the local autochthonous population [20, 31, 38–43].

There is a dissimilar pattern of *MEFV* mutations among Jewish population, as there is a number of distinct Jewish ethnic groups in Europe. Although the aforementioned mutations are mostly present in Jewish FMF patients, their exact frequencies differ depending on a country and ethnic group [16, 23]. For example, high carrier rate of M694V is identified in Jewish FMF patients living in North Africa (Morocco) (11.1%) and Iraq (2.9%), but is rarely observed among Ashkenazim [20], while the V726A is prevalent among Ashkenazi (7.4%) and Iraqi Jews (12.8%) [44]. In the study of *MEFV* mutation prevalence in the Israeli society, M694V was common mutation among non-Ashkenazi Jews, E148Q was observed in

**149**

it spread further [51].

of inflammatory and autoimmune diseases [52].

*Adaptation to Mediterranea*

*DOI: http://dx.doi.org/10.5772/intechopen.94081*

support the diagnosis of FMF [2, 5, 10, 11].

common mutation for this region [13, 48].

patients of all ethnic groups, while K695R (c.2084A > G) seem to be characteristic mutation present in Jews [45]. In Sephardic Jews, the overall *MEFV* mutation carrier rate is between 1:8 and 1:16 [46], M694V is predominant, while other mutations,

The M680I mutation is common in Armenians and is associated with milder phenotype of the disease. Nevertheless, Armenian patients with FMF have common pathogenic mutations as the previous populations, such as M694V (~50%), fol-

The E148Q mutation is the most frequent sequence alteration in the general population, but its clinical significance is still debatable. It is mostly encountered in a heterozygous state in asymptomatic individuals. When in homozygous state it is associated with FMF-like disease, with mild symptoms and later onset of disease. Thus, with regard to the SHARE recommendations heterozygous E148Q does not

The P369S and K695R are rare mutations with reduced penetrance, often found in asymptomatic carriers or in complex alleles in FMF patients [23, 30, 47]. They were relatively common in general Ashkenazi Jewish sample in the USA, with the carrier frequency of ~1:5 in FMF patients [16]. The P369S was the most frequent mutation in healthy Armenians and it might ameliorate the phenotypic presentation of the co-existing exon 10 mutations in patients. P369S homozygotes were even

Unexpectedly high K695R mutation rate was determined in the countries of Central and South-Eastern Europe. This region is characterized with limited heterogeneity of *MEFV* mutations, with only eight different mutations determined in healthy subjects and FMF patients (K695R, E148Q, V726A, M694V, F756C, I591T, S730F and A744S). The K695R mutation was most common mutation, found in 40% of healthy and 32% of FMF patients, which supports the idea that this is a

One another *MEFV* variation of unknown significance is the R202Q (c.605G>A), often considered a polymorphism due to its high heterozygous frequency, among healthy individuals. Due to its poor conservation during evolution it is assumed it appeared later compared to other common mutations [49]. The R202Q alteration in homozygous state was associated with FMF-like symptoms in some cases [47, 50]. In our study of *MEFV* distribution in Serbia, 45% of healthy individuals had heterozygous R202Q, while 10% were homozygotes. Although considered healthy, the homozygotes reported self-limited episodes of fever of unknown origin and unspecific abdominal pain [13]. The results indicate a pathogenic role of R202Q homozygosity, perhaps along with other permissive environmental and genetic factors of a patient. In isolated populations there is a greater chance for arising of specific genotypes. One example of specific *MEFV* mutation distribution is an island of Cyprus, due to its distinct ancestry and relative isolation in the Mediterranean. The *MEFV* mutations carrier rate in the Greek-Cypriot patients suspected for FMF is 1:25, with V726A, M694V, E167D (c.501G > C) and F479L (c.1437C > G) being the most common mutations. F479L is very rare elsewhere but in Greek-Cypriots its frequency is 20.6%. Interestingly, F479L was always co-inherited in *cis* with E167D mutation. It is hypothesized that F479L originated in Cyprus as a founder mutation, from where

It would be ideal when every population would perform a genetic testing for *MEFV* mutations and accordingly establish a set of the most frequent, which could be used in a screening for suspected FMF patients, as well as other inflammatory conditions, since *MEFV* mutations are found to be a modifying factor in a number

such as E148Q, P369S (c.459G > T), K695R and V726A, are rare [16].

lowed by V726A, M680I, and R761H (c.2282G > A) [10, 30, 32].

observed among the asymptomatic Ashkenazi Jews [16, 30].

#### **Figure 1.**

*Allele frequencies of common MEFV mutations in FMF patients (%).*

#### *Adaptation to Mediterranea DOI: http://dx.doi.org/10.5772/intechopen.94081*

*Genetic Variation*

V726A mutations seem to emerge in human genome about 2000 years ago, according to their association with specific microsatellite haplotypes in different populations [21, 28]. Presence of the E148Q (c.442G > C) mutation in different ethnic groups in the region supports the hypothesis of its recurrent nature or founder effect, probably stemming from the Asian countries, such as China and India, where E148Q is also frequent [20, 29]. The M694I (c.2082 G > A) mutation is merely present in North Africa and is estimated that occurred in an indigenous population

The M694V, M694I, M680I, and V726A mutations are most common in the Eastern Mediterranean countries, that is in Turkish, Armenian, Arab, and Jewish populations (**Figure 1**). The carrier rate of the mutations in these populations is estimated to be 1:5 to 1:7. Consequently, FMF mainly affects people of these ethnicities [2, 5, 19, 30–32]. The prevalence of *MEFV* mutations and FMF is much lower in the Western Mediterranean countries (France and Spain). Actually, the ethnic origin of these patients is usually from populations with higher mutation frequencies. Also, higher prevalence of homozygous mutations in the East might reflect the

Beside differences in *MEFV* mutations distribution between countries, there are variations within countries as well. For example, in Turkey, 94% of diagnosed FMF patients were from central-western parts of the country. However, more than a half of them had a family origin from the eastern provinces, pointing to the migration routes of mutations and disease [19, 35, 36]. The similarity between *MEFV* mutations present in Turks and Jordanians can also be explained by the local migrations, during the Ottoman Empire [20, 37]. The M694V mutation is the most common in Arab FMF patients. Unlike others, Arabs in North Africa have higher rate of M694I mutation that is probably acquired through the intermarriages with the local

There is a dissimilar pattern of *MEFV* mutations among Jewish population, as there is a number of distinct Jewish ethnic groups in Europe. Although the aforementioned mutations are mostly present in Jewish FMF patients, their exact frequencies differ depending on a country and ethnic group [16, 23]. For example, high carrier rate of M694V is identified in Jewish FMF patients living in North Africa (Morocco) (11.1%) and Iraq (2.9%), but is rarely observed among Ashkenazim [20], while the V726A is prevalent among Ashkenazi (7.4%) and Iraqi Jews (12.8%) [44]. In the study of *MEFV* mutation prevalence in the Israeli society, M694V was common mutation among non-Ashkenazi Jews, E148Q was observed in

consequence of a local custom of consanguinity marriages [20, 33, 34].

autochthonous population [20, 31, 38–43].

*Allele frequencies of common MEFV mutations in FMF patients (%).*

of Berbers before colonizations in the 7th century BC [4, 20].

**148**

**Figure 1.**

patients of all ethnic groups, while K695R (c.2084A > G) seem to be characteristic mutation present in Jews [45]. In Sephardic Jews, the overall *MEFV* mutation carrier rate is between 1:8 and 1:16 [46], M694V is predominant, while other mutations, such as E148Q, P369S (c.459G > T), K695R and V726A, are rare [16].

The M680I mutation is common in Armenians and is associated with milder phenotype of the disease. Nevertheless, Armenian patients with FMF have common pathogenic mutations as the previous populations, such as M694V (~50%), followed by V726A, M680I, and R761H (c.2282G > A) [10, 30, 32].

The E148Q mutation is the most frequent sequence alteration in the general population, but its clinical significance is still debatable. It is mostly encountered in a heterozygous state in asymptomatic individuals. When in homozygous state it is associated with FMF-like disease, with mild symptoms and later onset of disease. Thus, with regard to the SHARE recommendations heterozygous E148Q does not support the diagnosis of FMF [2, 5, 10, 11].

The P369S and K695R are rare mutations with reduced penetrance, often found in asymptomatic carriers or in complex alleles in FMF patients [23, 30, 47]. They were relatively common in general Ashkenazi Jewish sample in the USA, with the carrier frequency of ~1:5 in FMF patients [16]. The P369S was the most frequent mutation in healthy Armenians and it might ameliorate the phenotypic presentation of the co-existing exon 10 mutations in patients. P369S homozygotes were even observed among the asymptomatic Ashkenazi Jews [16, 30].

Unexpectedly high K695R mutation rate was determined in the countries of Central and South-Eastern Europe. This region is characterized with limited heterogeneity of *MEFV* mutations, with only eight different mutations determined in healthy subjects and FMF patients (K695R, E148Q, V726A, M694V, F756C, I591T, S730F and A744S). The K695R mutation was most common mutation, found in 40% of healthy and 32% of FMF patients, which supports the idea that this is a common mutation for this region [13, 48].

One another *MEFV* variation of unknown significance is the R202Q (c.605G>A), often considered a polymorphism due to its high heterozygous frequency, among healthy individuals. Due to its poor conservation during evolution it is assumed it appeared later compared to other common mutations [49]. The R202Q alteration in homozygous state was associated with FMF-like symptoms in some cases [47, 50]. In our study of *MEFV* distribution in Serbia, 45% of healthy individuals had heterozygous R202Q, while 10% were homozygotes. Although considered healthy, the homozygotes reported self-limited episodes of fever of unknown origin and unspecific abdominal pain [13]. The results indicate a pathogenic role of R202Q homozygosity, perhaps along with other permissive environmental and genetic factors of a patient.

In isolated populations there is a greater chance for arising of specific genotypes. One example of specific *MEFV* mutation distribution is an island of Cyprus, due to its distinct ancestry and relative isolation in the Mediterranean. The *MEFV* mutations carrier rate in the Greek-Cypriot patients suspected for FMF is 1:25, with V726A, M694V, E167D (c.501G > C) and F479L (c.1437C > G) being the most common mutations. F479L is very rare elsewhere but in Greek-Cypriots its frequency is 20.6%. Interestingly, F479L was always co-inherited in *cis* with E167D mutation. It is hypothesized that F479L originated in Cyprus as a founder mutation, from where it spread further [51].

It would be ideal when every population would perform a genetic testing for *MEFV* mutations and accordingly establish a set of the most frequent, which could be used in a screening for suspected FMF patients, as well as other inflammatory conditions, since *MEFV* mutations are found to be a modifying factor in a number of inflammatory and autoimmune diseases [52].

## **3. Behcet's disease**

Behcet's disease (BD) is an autoinflammatory and polygenic disease, more frequent in Mediterranean countries than in rest of Europe. Most cases are identified in countries of the Middle East and along the ancient Silk Route. The highest prevalence among Mediterranean countries is probably in Turkey, with estimated prevalence of 4.2/1000 in Istanbul [53]. This is a rare, sporadic, multi-systemic disease with undetermined cause. The main clinical features are constitutional symptoms and recurrent fever, oral aphthous, genital ulcers, with gastrointestinal, musculoskeletal, neurological, and vascular involvement [54].

Several host genetic factors are implicated in the pathogenesis of BD. The strongest is the association with the major histocompatibility complex HLA–B51 allele, which increases the risk of disease for about 6-fold. Approximately 50% of BD patients possess this HLA variant. Besides, HLA-B51 contributes to the specific clinical features in BD such as less severe disease course, but a higher frequency of ocular manifestations [55, 56].

Behçet's disease can be a comorbidity of FMF, and vice versa, *MEFV* mutations are common finding in BD patients. Some *MEFV* alterations are detected more often in BD patients than healthy subjects, such as P706 polymorphism. In a cohort of Turkish patients, clinical association was found between heterozygous *MEFV* mutation, principally M694V, and vascular involvement [51, 55, 57].

Interestingly, arthritis in BD is self-limiting and nondestructive in nature, pointing to the existence of an inherited protective factor/s. Such a role has been observed for plasminogen activator inhibitor 1 (PAI-1), which levels were higher in synovial fluid of BD patients than healthy. PAI-1 acted protective against destructive arthritis but had promoting effect towards hyperfibrinolysis in BD vasculopathy. However, PAI-1 common polymorphism 4G/5G was not associated with pathogenesis nor development of thrombosis in these patients [58–60].

Besides, several other alterations are described to influence BD occurrence and course, including MHC class 1 polypeptide-related sequence, T cell mediated cytokine dysregulation (of IL-6, IL-8, IL-10), DNA methylation, etc. [55, 61].
