**Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to Type1 Diabetes, Vitiligo and Hypoparathyroidism**

Rajni Rani and Archana Singh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/57494

### **1. Introduction**

[55] Petrone A, Giorgi G, Mesturino CA, Capizzi M, Cascino I, Nistico L, Osborn J, Di Mario U, Buzzetti R. Association of DRB1\*04-DQB1\*0301 haplotype and lack of asso‐ ciation of two polymorphic sites at CTLA-4 gene with Hashimoto's thyroiditis in an

[56] Iwama S, Ikezaki A, Kikuoka N, Kim HS, Matsuoka H, Yanagawa T, Sato H, Hoshi M, Sakamaki T, Sugihara S. Association of HLA-DR, -DQ genotype and CTLA-4 gene polymorphism with Graves' disease in Japanese children. Horm Res 2005;63(2) 55-60.

[57] Santamaria P, Barbosa JJ, Lindstrom AL, Lemke TA, Goetz FC, Rich SS. HLA-DQB1 associated susceptibility that distinguishes Hashimoto's thyroiditis from Graves' dis‐

[58] Ikegami H, Awata T, Kawasaki E, Kobayashi T, Maruyama T, Nakanishi K, Shimada A, Amemiya S, Kawabata Y, Kurihara S, Tanaka S, Kanazawa Y, Mochizuki M, Ogi‐ hara T. The association of CTLA4 polymorphism with type 1 diabetes is concentrated in patients complicated with autoimmune thyroid disease: a multicenter collabora‐

[59] Katahira M, Maeda H, Tosaki T, Segawa S. The human leukocyte antigen class II gene has different contributions to autoimmune type 1 diabetes with or without au‐ toimmune thyroid disease in the Japanese population. Diabetes Res Clin Pract

[60] Chuang LM, Wu HP, Chang CC, Tsai WY, Chang HM, Tai TY, Lin BJ. HLA DRB1/ DQA1/DQB1 haplotype determines thyroid autoimmunity in patients with insulin-

[61] Chikuba N, Akazawa S, Yamaguchi Y, Kawasaki E, Takino H, Yoshimoto M, Ohe N, Yamashita K, Yano A, Nagataki S. Immunogenetic heterogeneity in type 1 (insulindependent) diabetes among Japanese--class II antigen and autoimmune thyroid dis‐

[62] Chikuba N, Akazawa S, Yamaguchi Y, Kawasaki E, Takino H, Takao Y, Maeda Y, Okuno S, Yamamoto H, Yokota A, Yoshimoto M, Nagataki S. Type 1 (insulin-de‐ pendent) diabetes mellitus with coexisting autoimmune thyroid disease in Japan. In‐

[63] Sumník Z, Drevínek P, Snajderová M, Kolousková S, Sedláková P, Pechová M, Vavri‐ nec J, Cinek O. HLA-DQ polymorphisms modify the risk of thyroid autoimmunity in children with type 1 diabetes mellitus. J Pediatr Endocrinol Metab 2003;16(6) 851-858.

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Italian population. Thyroid 2001;11(2) 171-175.

2009;85(3) 293-297.

200 HLA and Associated Important Diseases

Immune cells get educated in the thymus during development to recognize self antigens so that there are no immune responses against self antigens. However, whenever they encounter non-self antigens like bacteria or viruses, they are recognized as non-self and immune response takes place against them to save us from infections. While the basic purpose of the immune response is to save us from infections, something goes awry in some cases such that the self antigens are recognized as foreign by one's own immune cells which attack the cells /organs containing those antigens presuming them to be foreign. This results in auto-immune disor‐ ders like Type 1 diabetes, vitiligo or hypoparathyroidism, the focus for the present chapter. All these disorders are complex, multi-factorial, organ specific disorders where auto-immune responses have been implicated due to presence of auto-antibodies and auto-antigen specific T cells in the peripheral circulation of the patients. Although what triggers the autoimmune responses in these disorders is not clearly understood, association with certain major histo‐ compatibility complex (MHC) alleles has been considered a hallmark of autoimmune disorders [1] since they have a role in antigen presentation that orchestrates the antigen specific adaptive immune responses.

Major Histocompatibility Complex (MHC) is a set of glycoprotein molecules present on the cell surface of nucleated cells encoded by several polymorphic genes localized on chromosome 6 of man. In human, MHC is known as Human leukocyte Antigens (HLA). MHC region is spread over 3.84 megabases of chromosome 6p21.3 and is the most gene dense region of the human genome with 224 genes. 128 of these 224 genes are known to be expressed. And 40 % of these genes have immune related functions [2]. HLA constitutes a small part of the MHC

© 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

region. Major function of HLA molecules is to present antigenic peptides to T cells. HLA can be broadly classified into two major classes: class-I and class-II. HLA class-I genes are HLA-A, -B and –C. These are classical class-I genes which are very polymorphic with 2365, 3005 and 1848 alleles respectively.

chains of DQ and DP molecules with 416 and 190 alleles respectively and DQA1 and DPA1 encode the alpha chains of DQ and DP molecules with 51 and 37 alleles respectively [10, 11].

Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to...

http://dx.doi.org/10.5772/57494

203

Besides the classical class-I and class-II genes there are several non-classical HLA genes with specialized functions and also pseudo HLA genes present in the MHC region. However, they will not be discussed here since their roles are not very clearly understood in autoimmunity.

A recurring theme of several autoimmune disorders is the aberrant presentation of self antigens to the immune system that triggers downstream perturbations. Under normal circumstances most of the MHC molecules are occupied by self peptides and the T cells are tolerized against them during thymic education so that auto-immune responses do not take place, however, sometimes there is a break in the tolerance resulting in recognition of self as non-self by the immune system which results in an auto-immune response. This break in tolerance could be due to low expression of some antigens in the thymus which may result in self-reactive T cells to reach the peripheral circulation. Or it could be due to escape of selfreactive T cells from clonal deletion during T cell development. Since associations with MHC alleles have been considered a hallmark of autoimmune disorders due to their role in antigen presentation, we have studied HLA alleles in two common and one rare autoimmune disor‐

DNAs were extracted using standard protocol from 10 ml of blood from type 1 diabetes (N=211), vitiligo (N=1404), hypoparathyroidism patients (N=134) and 902 normal healthy controls from the same ethnic background after obtaining informed consent. All the patients attending diabetes of young clinic were carefully assessed and categorized as type 1, type 2 and fibrocalculous pancreatopathy as per the recent classification by American Diabetes Association expert committee [12, 13]. All of the T1D subjects included in the study required insulin for glycemic control and 51 of them had history of ketosis at presentation. Insulin requiring patients with fibrocalculous pancreatopathy and subjects with diabetes in whom glycemic control was achieved with diet and oral hypoglycemic agent were excluded from the

Vitiligo group consisted of 1404 North Indian patients enrolled at Dr. Ram Manohar Lohia Hospital (RMLH), and All India Institute of Medical Sciences (AIIMS), New Delhi Diagnosis of vitiligo was based on clinical examination done by dermatologists. Clinically, the cases were classified as having Generalized (Vulgaris, Acrofacial, Universalis) or Localized (Focal, Acral, mucosal and segmental) forms of vitiligo. The replication study was done on 355 vitiligo cases

Idiopathic hypoparathyroidism group consisted of 134 unrelated patients who attended the endocrine clinics of All India Institute of Medical Sciences during 1998-2011. The diagnosis of IH was based on presence of hypocalcemia, hyperphosphatemia, low serum intact PTH, normal renal function and serum magnesium levels. Patients with post surgical hypopara‐

ders: Type1 diabetes, vitiligo and hypoparathyroidism.

**2. Patients and controls**

from Gujarat, a state in west of India [15].

study [12, 14].

The MHC class-I molecules are present on the surface of all nucleated cells and present antigenic peptides to the CD8+ cytotoxic T cells. MHC class-I molecule is a hetero-dimer of a heavy and a light chain. The molecular weight of the heavy alpha chain is about 40-45 KDa and that of the light chain, beta 2 microglobulin (β2m) is 12 KDa [3]. The genes for the alpha chains are encoded on chromosome 6. However, the gene for β2m is encoded on human chromosome 15. The heavy chain of the MHC class-I molecule has three domains alpha 1 (α1), alpha 2 (α2) and alpha 3 (α3). Alpha 1 (α1) and alpha 2 (α2) domains are the most polymorphic domains since they constitute the peptide binding groove of the MHC molecule. These polymorphic domains are encoded by exons 2 and 3 of the genes encoding MHC class-I alpha chain which has in total 8 exons. The peptides that are presented by the MHC molecules have allele specific motifs i.e., certain peptides can be presented by certain MHC molecules. The affinity of the peptide to bind to the peptide binding groove is determined by the anchors present on the peptide binding groove where the peptides go and bind through hydrogen bonds. Specific motifs or the amino acid residues present in the pockets of the peptide binding grove which are involved in binding the side chains of complimentary residues of the peptide determine which peptides would bind to which MHC molecule [4, 5]. Pockets B (key residues at positions 9, 45, 63, 67, 70 and 99) and F (key residues at positions 77, 80, 81 and 116) of alpha chain are the main anchors which engage the peptides at their amino acid positions 2 and the C-terminus [6]. However, several alleles of the MHC share peptide binding specificities i.e. similar peptides may be presented by different MHC molecules. These alleles are clustered in supertypes [6].

HLA class-II molecules are expressed on antigen presenting cells like macrophages, dendritic cells, B cells, thymic epithelium and activated T cells [7]. MHC class-II molecules present antigenic peptides to the CD4+ T helper cells (Th cells) which in turn initiate a cascade of immunological events that result in activation of CD8+ cytotoxic T cells and B lymphocytes [2]. CD4+ T helper cells get activated when a non-self antigen is presented to them. T helper cells are of two types T helper 1 (Th1) and Th2. Th1 cells secrete cytokines like Interferon gamma and TNF-alpha which are involved in cell-mediated immune responses as they activate the cytotoxic T cells which have already seen the antigen in the context of MHC class-I. Th2 cells, on the other hand, secrete IL-4, IL-5 and/or IL-6 which are involved in humoral immunity as they activate the B cells to become plasma cells which make the antibodies against antigen they have seen. Thus an immune response takes place which varies in strength depending on the host factors and the peptides being presented.

The classical MHC class-II glycoproteins in humans are HLA-DR, -DP and –DQ. The MHC class-II molecule is a heterodimer of two polypeptide chains: an alpha (25-33 KDa) and a beta chain (24-29KDa) [8, 9]. Unlike MHC class-I, both alpha and beta chains of the class-II molecule are encoded on chromosome 6. DRB1 gene encodes DR beta chain while DRA1 encodes DR alpha chain with 1355 DRB1 alleles and 7 DRA1 alleles. Similarly DQB1 and DPB1 encode beta chains of DQ and DP molecules with 416 and 190 alleles respectively and DQA1 and DPA1 encode the alpha chains of DQ and DP molecules with 51 and 37 alleles respectively [10, 11].

Besides the classical class-I and class-II genes there are several non-classical HLA genes with specialized functions and also pseudo HLA genes present in the MHC region. However, they will not be discussed here since their roles are not very clearly understood in autoimmunity.

A recurring theme of several autoimmune disorders is the aberrant presentation of self antigens to the immune system that triggers downstream perturbations. Under normal circumstances most of the MHC molecules are occupied by self peptides and the T cells are tolerized against them during thymic education so that auto-immune responses do not take place, however, sometimes there is a break in the tolerance resulting in recognition of self as non-self by the immune system which results in an auto-immune response. This break in tolerance could be due to low expression of some antigens in the thymus which may result in self-reactive T cells to reach the peripheral circulation. Or it could be due to escape of selfreactive T cells from clonal deletion during T cell development. Since associations with MHC alleles have been considered a hallmark of autoimmune disorders due to their role in antigen presentation, we have studied HLA alleles in two common and one rare autoimmune disor‐ ders: Type1 diabetes, vitiligo and hypoparathyroidism.

### **2. Patients and controls**

region. Major function of HLA molecules is to present antigenic peptides to T cells. HLA can be broadly classified into two major classes: class-I and class-II. HLA class-I genes are HLA-A, -B and –C. These are classical class-I genes which are very polymorphic with 2365, 3005 and

The MHC class-I molecules are present on the surface of all nucleated cells and present antigenic peptides to the CD8+ cytotoxic T cells. MHC class-I molecule is a hetero-dimer of a heavy and a light chain. The molecular weight of the heavy alpha chain is about 40-45 KDa and that of the light chain, beta 2 microglobulin (β2m) is 12 KDa [3]. The genes for the alpha chains are encoded on chromosome 6. However, the gene for β2m is encoded on human chromosome 15. The heavy chain of the MHC class-I molecule has three domains alpha 1 (α1), alpha 2 (α2) and alpha 3 (α3). Alpha 1 (α1) and alpha 2 (α2) domains are the most polymorphic domains since they constitute the peptide binding groove of the MHC molecule. These polymorphic domains are encoded by exons 2 and 3 of the genes encoding MHC class-I alpha chain which has in total 8 exons. The peptides that are presented by the MHC molecules have allele specific motifs i.e., certain peptides can be presented by certain MHC molecules. The affinity of the peptide to bind to the peptide binding groove is determined by the anchors present on the peptide binding groove where the peptides go and bind through hydrogen bonds. Specific motifs or the amino acid residues present in the pockets of the peptide binding grove which are involved in binding the side chains of complimentary residues of the peptide determine which peptides would bind to which MHC molecule [4, 5]. Pockets B (key residues at positions 9, 45, 63, 67, 70 and 99) and F (key residues at positions 77, 80, 81 and 116) of alpha chain are the main anchors which engage the peptides at their amino acid positions 2 and the C-terminus [6]. However, several alleles of the MHC share peptide binding specificities i.e. similar peptides may be presented by different MHC molecules. These alleles are clustered in

HLA class-II molecules are expressed on antigen presenting cells like macrophages, dendritic cells, B cells, thymic epithelium and activated T cells [7]. MHC class-II molecules present

CD4+ T helper cells get activated when a non-self antigen is presented to them. T helper cells are of two types T helper 1 (Th1) and Th2. Th1 cells secrete cytokines like Interferon gamma and TNF-alpha which are involved in cell-mediated immune responses as they activate the cytotoxic T cells which have already seen the antigen in the context of MHC class-I. Th2 cells, on the other hand, secrete IL-4, IL-5 and/or IL-6 which are involved in humoral immunity as they activate the B cells to become plasma cells which make the antibodies against antigen they have seen. Thus an immune response takes place which varies in strength depending on the

The classical MHC class-II glycoproteins in humans are HLA-DR, -DP and –DQ. The MHC class-II molecule is a heterodimer of two polypeptide chains: an alpha (25-33 KDa) and a beta chain (24-29KDa) [8, 9]. Unlike MHC class-I, both alpha and beta chains of the class-II molecule are encoded on chromosome 6. DRB1 gene encodes DR beta chain while DRA1 encodes DR alpha chain with 1355 DRB1 alleles and 7 DRA1 alleles. Similarly DQB1 and DPB1 encode beta

T helper cells (Th cells) which in turn initiate a cascade of

cytotoxic T cells and B lymphocytes [2].

1848 alleles respectively.

202 HLA and Associated Important Diseases

supertypes [6].

antigenic peptides to the CD4+

immunological events that result in activation of CD8+

host factors and the peptides being presented.

DNAs were extracted using standard protocol from 10 ml of blood from type 1 diabetes (N=211), vitiligo (N=1404), hypoparathyroidism patients (N=134) and 902 normal healthy controls from the same ethnic background after obtaining informed consent. All the patients attending diabetes of young clinic were carefully assessed and categorized as type 1, type 2 and fibrocalculous pancreatopathy as per the recent classification by American Diabetes Association expert committee [12, 13]. All of the T1D subjects included in the study required insulin for glycemic control and 51 of them had history of ketosis at presentation. Insulin requiring patients with fibrocalculous pancreatopathy and subjects with diabetes in whom glycemic control was achieved with diet and oral hypoglycemic agent were excluded from the study [12, 14].

Vitiligo group consisted of 1404 North Indian patients enrolled at Dr. Ram Manohar Lohia Hospital (RMLH), and All India Institute of Medical Sciences (AIIMS), New Delhi Diagnosis of vitiligo was based on clinical examination done by dermatologists. Clinically, the cases were classified as having Generalized (Vulgaris, Acrofacial, Universalis) or Localized (Focal, Acral, mucosal and segmental) forms of vitiligo. The replication study was done on 355 vitiligo cases from Gujarat, a state in west of India [15].

Idiopathic hypoparathyroidism group consisted of 134 unrelated patients who attended the endocrine clinics of All India Institute of Medical Sciences during 1998-2011. The diagnosis of IH was based on presence of hypocalcemia, hyperphosphatemia, low serum intact PTH, normal renal function and serum magnesium levels. Patients with post surgical hypopara‐ thyroidism were not included. Patients with clinical features of APECED syndrome indicated by mucocutaneous candidiasis or presence of adrenocortical autoantibody were excluded from the study[16].

(26.6/100,000) as compared to rural areas (4.27/100,000) has been reported with a total preva‐ lence of 10.20/100,000. T1D develops as a result of complex interaction of many genetic and environmental factors leading to autoimmune destruction of the insulin producing Pancreatic Beta cells. While 20 genomic intervals have been implicated for the manifestation of the disease [22], role of an intricate network of the products of these genes cannot be ruled out. However, unraveling different factors involved and how they interact in integrated networks is like solving a zig-saw puzzle which is the aim of our studies [12, 14, 17]. However, in this chapter

Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to...

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205

When the patients report to the clinician, he is left with no option but to give daily insulin injections since most of their pancreatic beta cells are already destroyed. So, if we want to stop autoimmune responses in the people who are predisposed to get T1D, we need to identify the prediabetics before the onset of the disease and device ways to inhibit autoimmunity in them.

We have studied the frequencies of MHC class-I alleles for HLA-B locus and MHC class-II alleles for HLA-DRB1 and DQB1 loci in patients with Type 1 diabetes and normal healthy controls using polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes (PCR-SSOP). DNAs were isolated from peripheral blood mononuclear cells (PBMCs) from patients and healthy controls using standard protocol. The second and third exons of the HLA-B gene and second exons of HLA-DRB1 and DQB1 genes were amplified using PCR and hybridized with oligonucleotide probes and the hybridization pattern with different probes was used to identify the alleles present in a sample. The results showed that *HLA-B\*08* (p<7.8X10-13, Odds Ratio (OR) = 3.3)*, HLA-B\*50* (p< 4.2 X 10-21, OR= 7.5)*, HLA-B\*58* (p< 3.3 X 10-6, OR= 2.6)*, HLA- DRB1\*03:01* (p<1.7x10-35, OR= 13.2)*, DRB1\*04:01* (p<0.00001, OR= 5.4)*, and DRB1\*04:05* (p<0.00001, OR=16.01)*,* were significantly increased in the patients (Figure 1a, b) as compared to controls. We had earlier compared the results of the T1D patients with 91 healthy controls [14], however, now we have compared the results of T1D samples with 902 healthy controls, with the same results reinforcing our earlier data. These results were in concordance with earlier studies in North Indians [25-29]. However, we also observed *DRB1\*07:01*(p<8x10-6, OR= 0.19) to be significantly decreased in the patients as compared to controls. In our earlier study when we had less number of controls, we observed *DRB1\*04:03* (p< 0.02, OR=0.25) and *DRB1\*04:04* (p< 0.05, OR= 0.2) also to be significantly reduced in the patients [14], however, when we increased the number of controls to 902, this finding did not remain significant, although *DRB1\*04:03* and *DRB1\*04:04* were reduced in the patients but not significantly. We did not find any significant reduction of HLA-DR2 which has been shown to confer strong protection from T1D in most ethnic groups [30, 31] probably because the DR2 haplotype which has been shown to be protective i.e., *DRB1\*15:01- DQB1\*06:02,* was observed with a low frequency of only 1.06% in North Indians [21]. On the other hand, we observed a marginally increased frequency of *DRB1\*15:01* and reduced frequency of *DRB1\*15:06* in patients as compared to controls, which did not remain significant when *p* was corrected for the number of alleles tested for DRB1 locus. *DRB1\*04:03* has been shown to be associated with protection in a Belgian study on T1D [32]. Our results are in concordance with studies on T1D patients from Sardinia, black population from Zimbabwe,

we will be concentrating on MHC associations and their implications only.

This is possible by using genetic predisposition criteria to get the disease.

Nine hundred and two ethnically matched unaffected controls from North India were used for all three studies and 441 ethnically matched unaffected controls from Gujarat were studied in the replication study for vitiligo only. None of the unaffected controls had personal or family history of T1D, vitiligo, hypoparathyroidism or any other autoimmune or infectious disease.

All Cases and control samples were collected after obtaining informed consent and Institu‐ tional Human Ethics Committee's clearance from all the institutes/Hospitals involved, following declaration of Helsinki protocols.

### **3. Study of** *HLA***-***A, HLA-B* **and** *HLA–DRB1* **alleles**

DNA extraction was carried out by standard procedures from fresh whole blood samples collected in anticoagulant EDTA. Alleles of *HLA*-*A, HLA*-*B* and *HLA*-*DRB1* were studied using Polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes (SSOPs) as described earlier [17] using a bead-based technology (Luminex, Austin, Tx) following Manufacturer's instructions (Labtype SSO kit from One Lambda, Canoga Park, USA). The latest nomenclature for the *HLA* system was used to designate the alleles of the three loci studied [18].

### **4. Statistical analysis**

Frequencies of alleles in the patient samples were compared with their respective unaffected controls using Chi-square analysis and the strength of associations was estimated by odds ratio (OR) and 95% confidence interval using Stata 9.2 statistical program. Fisher's exact test was used when the numbers were five or less in any group i.e. in cases or controls for any allele. In such cases, Odds ratios were calculated using Woolf's method [19] with Haldane's [20] modification as described earlier [21].

### **5. Type 1 diabetes**

Type 1 diabetes (T1D) is an incurable, multi-factorial and complex autoimmune disorder characterized by the loss of insulin producing beta cells of the pancreas. This results in abnormal metabolism of glucose that may be detrimental for several other complications like ketoacidosis, retinopathy, nephropathy and even cardio-vascular diseases and pre-mature deaths [22]. World-wide disease affects 1 in 300-400 children [23]. The incidence in South India has been reported to be 10.5/100,000/year [24]. In North India, a higher prevalence in urban (26.6/100,000) as compared to rural areas (4.27/100,000) has been reported with a total preva‐ lence of 10.20/100,000. T1D develops as a result of complex interaction of many genetic and environmental factors leading to autoimmune destruction of the insulin producing Pancreatic Beta cells. While 20 genomic intervals have been implicated for the manifestation of the disease [22], role of an intricate network of the products of these genes cannot be ruled out. However, unraveling different factors involved and how they interact in integrated networks is like solving a zig-saw puzzle which is the aim of our studies [12, 14, 17]. However, in this chapter we will be concentrating on MHC associations and their implications only.

thyroidism were not included. Patients with clinical features of APECED syndrome indicated by mucocutaneous candidiasis or presence of adrenocortical autoantibody were excluded from

Nine hundred and two ethnically matched unaffected controls from North India were used for all three studies and 441 ethnically matched unaffected controls from Gujarat were studied in the replication study for vitiligo only. None of the unaffected controls had personal or family history of T1D, vitiligo, hypoparathyroidism or any other autoimmune or infectious disease. All Cases and control samples were collected after obtaining informed consent and Institu‐ tional Human Ethics Committee's clearance from all the institutes/Hospitals involved,

DNA extraction was carried out by standard procedures from fresh whole blood samples collected in anticoagulant EDTA. Alleles of *HLA*-*A, HLA*-*B* and *HLA*-*DRB1* were studied using Polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes (SSOPs) as described earlier [17] using a bead-based technology (Luminex, Austin, Tx) following Manufacturer's instructions (Labtype SSO kit from One Lambda, Canoga Park, USA). The latest nomenclature for the *HLA* system was used to designate the alleles of the

Frequencies of alleles in the patient samples were compared with their respective unaffected controls using Chi-square analysis and the strength of associations was estimated by odds ratio (OR) and 95% confidence interval using Stata 9.2 statistical program. Fisher's exact test was used when the numbers were five or less in any group i.e. in cases or controls for any allele. In such cases, Odds ratios were calculated using Woolf's method [19] with Haldane's [20]

Type 1 diabetes (T1D) is an incurable, multi-factorial and complex autoimmune disorder characterized by the loss of insulin producing beta cells of the pancreas. This results in abnormal metabolism of glucose that may be detrimental for several other complications like ketoacidosis, retinopathy, nephropathy and even cardio-vascular diseases and pre-mature deaths [22]. World-wide disease affects 1 in 300-400 children [23]. The incidence in South India has been reported to be 10.5/100,000/year [24]. In North India, a higher prevalence in urban

the study[16].

204 HLA and Associated Important Diseases

three loci studied [18].

**4. Statistical analysis**

**5. Type 1 diabetes**

modification as described earlier [21].

following declaration of Helsinki protocols.

**3. Study of** *HLA***-***A, HLA-B* **and** *HLA–DRB1* **alleles**

When the patients report to the clinician, he is left with no option but to give daily insulin injections since most of their pancreatic beta cells are already destroyed. So, if we want to stop autoimmune responses in the people who are predisposed to get T1D, we need to identify the prediabetics before the onset of the disease and device ways to inhibit autoimmunity in them. This is possible by using genetic predisposition criteria to get the disease.

We have studied the frequencies of MHC class-I alleles for HLA-B locus and MHC class-II alleles for HLA-DRB1 and DQB1 loci in patients with Type 1 diabetes and normal healthy controls using polymerase chain reaction followed by hybridization with sequence specific oligonucleotide probes (PCR-SSOP). DNAs were isolated from peripheral blood mononuclear cells (PBMCs) from patients and healthy controls using standard protocol. The second and third exons of the HLA-B gene and second exons of HLA-DRB1 and DQB1 genes were amplified using PCR and hybridized with oligonucleotide probes and the hybridization pattern with different probes was used to identify the alleles present in a sample. The results showed that *HLA-B\*08* (p<7.8X10-13, Odds Ratio (OR) = 3.3)*, HLA-B\*50* (p< 4.2 X 10-21, OR= 7.5)*, HLA-B\*58* (p< 3.3 X 10-6, OR= 2.6)*, HLA- DRB1\*03:01* (p<1.7x10-35, OR= 13.2)*, DRB1\*04:01* (p<0.00001, OR= 5.4)*, and DRB1\*04:05* (p<0.00001, OR=16.01)*,* were significantly increased in the patients (Figure 1a, b) as compared to controls. We had earlier compared the results of the T1D patients with 91 healthy controls [14], however, now we have compared the results of T1D samples with 902 healthy controls, with the same results reinforcing our earlier data. These results were in concordance with earlier studies in North Indians [25-29]. However, we also observed *DRB1\*07:01*(p<8x10-6, OR= 0.19) to be significantly decreased in the patients as compared to controls. In our earlier study when we had less number of controls, we observed *DRB1\*04:03* (p< 0.02, OR=0.25) and *DRB1\*04:04* (p< 0.05, OR= 0.2) also to be significantly reduced in the patients [14], however, when we increased the number of controls to 902, this finding did not remain significant, although *DRB1\*04:03* and *DRB1\*04:04* were reduced in the patients but not significantly. We did not find any significant reduction of HLA-DR2 which has been shown to confer strong protection from T1D in most ethnic groups [30, 31] probably because the DR2 haplotype which has been shown to be protective i.e., *DRB1\*15:01- DQB1\*06:02,* was observed with a low frequency of only 1.06% in North Indians [21]. On the other hand, we observed a marginally increased frequency of *DRB1\*15:01* and reduced frequency of *DRB1\*15:06* in patients as compared to controls, which did not remain significant when *p* was corrected for the number of alleles tested for DRB1 locus. *DRB1\*04:03* has been shown to be associated with protection in a Belgian study on T1D [32]. Our results are in concordance with studies on T1D patients from Sardinia, black population from Zimbabwe, Lithuania, Czecks, Lebanese, Brazilians and African Americans where *DRB1\*03:01, DRB1\*04:01* and *DRB1\*04:05* have been shown to be associated with the disease [33-40]. T1D [32]. Our results are in concordance with studies on T1D patients from Sardinia, black population from Zimbabwe, Lithuania, Czecks, Lebanese, Brazilians and African Americans where *DRB1\*03:01, DRB1\*04:01* and *DRB1\*04:05* have been shown to be associated with the disease [33-40].

**(\*0301,X) are significantly increased in T1D as compared to controls. e.** *HLA‐B‐DRB1\*03* **haplotypes in T1D patients and controls. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles.**  HLA-DRB1 and DQB1 are in strong linkage disequilibrium. We also studied the alleles of *DQB1* locus*. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 1c). However *DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were significantly reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and *DQB1\*05:03* (6x10-4, OR=0.28) were reduced in the patients [14]. **Figure 1.** Distribution of *HLA-DRB1*, *HLA-B* and *DQB1* alleles in Type 1 diabetes and healthy controls. a. *HLA-DRB1* alleles significantly increased or decreased in T1D are shown. *DRB1\*03:01*, *\*04:01* and *\*04:05* are significantly in‐ creased and *DRB1\*07:01*is significantly reduced in T1D as compared to controls. b. *HLA-B\*08*, *B\*50:01* and *B\*58* are significantly increased in T1D as compared to controls c. *DQB1\*02:01* is significantly increased and *DQB1\*03:01* and *DQB1\*05:03* are significantly reduced in T1D as compared to controls. d. Homozygous *DRB1\*03:01* and heterozygous *DRB1\*03:01* with *\*04:01*, *\*04:05* or any other allele (\*0301,X) are significantly increased in T1D as compared to con‐ trols. e. *HLA-B-DRB1\*03* haplotypes in T1D patients and controls. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles.

**Figure 1. Distribution of** *HLA‐DRB1***,** *HLA‐B* **and** *DQB1* **alleles in Type 1 diabetes and healthy controls. a.** *HLA‐DRB1* **alleles significantly increased or decreased in T1D are shown.** *DRB1\*03:01***,** *\*04:01* **and** *\*04:05* **are significantly increased and** *DRB1\*07:01***is significantly reduced in T1D as compared to controls. b.** *HLA‐ B\*08***,** *B\*50:01* **and** *B\*58* **are significantly increased in T1D as compared to controls c.** *DQB1\*02:01* **is significantly increased and** *DQB1\*03:01* **and** *DQB1\*05:03* **are significantly reduced in T1D as compared to controls. d. Homozygous** *DRB1\*03:01* **and heterozygous** *DRB1\*03:01* **with** *\*04:01***,** *\*04:05* **or any other allele**

Figure 1d shows the homozygosity and heterozygosity of *DRB1\*03:01* and *DRB1\*04* alleles significantly increased in T1D. Homozygous *DRB1\*03:01* (p<4.6X10-39, OR=23.1), heterozygous *DRB1\*03:01/\*04:05* (p<5.5X10-6, OR =124.2) and *DRB1\*03:01/\*04:01* (p<0.0001, OR =17.6) were significantly increased in the patients as compared to controls. Heterozygous *03:01*/X (i.e. any other allele) (p<5.9X10-6, OR = 2.8) was also significantly increased in the patients as compared to controls. Heterozygous *DRB1\*07:01/X* (p < 1.3X10-7, OR = 0.09) were significantly reduced in the T1D patients as compared to controls suggesting their protective role. Thirty two percent of the patients studied were homozygous for *DRB1\*0301* as compared to 2% of the controls and this difference was highly significant. Homozygosity of *DQB1\*02:01* was significantly (p<1x10-5, OR=5.4) increased in the patients. *DQB1\*03:02* which was not significantly increased in the patients, showed a significant increase in heterozygous combination with *DQB1\*0201* (p<3x10-5, OR=34.16) [14]. In fact none of the controls had *DQB1\*0201/\*0302* heterozygous combination [14]. In a Swedish study, *DQA1\*0301/DQB1\*0302* and heterozygous combinations of *DQA1\*0301/DQB1\*0302* and *DQA1\*0201/DQB1\*0501* have been shown to confer the highest

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Figure 1 e shows the *HLA-B-DRB1* haplotypes that were significantly increased in T1D patients. HLA alleles are very closely linked to each other in such a way that the haplotypes are inherited en-bloc. Three *HLA-DRB1\*03* haplotypes, *B\*08-DRB1\*03:01*(p<1x10-14, OR=3.7), *B\*50- DRB1\*03:01*(p<8x10-24, OR=11.2) and *B\*58-DRB1\*03:01* (p<1.4x10-12, OR=5.1) and *DRB1\*03:01* haplotypes with non *B\*8/ B\*50/ B\*58* alleles (p<2.8x10-8, OR=3.4) were significantly increased in the patients as compared to controls. On the other hand non- *B\*8/ B\*50/ B\*58-non DRB1\*03:01* haplotypes were significantly reduced (p<1.5x10-44, OR=0.09) in T1D patients as

Thus our studies show a significant increase of HLA class-I and II alleles which may have a

and CD4+

Vitiligo is a depigmenting disorder of the skin caused by autoimmune destruction of pigment producing cells called melanocytes. It effects 0.5-1 % of the world population [42], while in India the incidence varies from 0.25 to 2.5% [43, 44] in most ethnic groups, however, it has been reported to be 8.8% in populations from western states of Gujarat and Rajasthan [45]. Vitiligo manifests in several forms which can be broadly classified in two clinical subtypes: generalized and localized [42]. While precise etiology of vitiligo is not very well understood, several hypotheses have been proposed which include autoimmune [46], neural [47], auto-cytotoxic [48] and genetic hypotheses [49]. However, autoimmune hypothesis gets credence due to cooccurrence of other autoimmune diseases in vitiligo patients in some cases, presence of auto reactive T cells and circulating auto-antibodies in the patients [46, 50-55]. The role of genetic factors has been suggested due to the fact that 7% of first-degree relatives of vitiligo develop

T cells respectively.

susceptibility [41]

compared to controls [12].

**6. Vitiligo**

the disease. [56, 57].

role in auto-antigen presentation to CD8+

HLA-DRB1 and DQB1 are in strong linkage disequilibrium. We also studied the alleles of *DQB1* locus*. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 1c). However *DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and *DQB1\*05:03* (6x10-4, OR=0.28) were reduced in the patients [14].

*DRB1\*04:01* and *DRB1\*04:05* have been shown to be associated with the disease [33-40]. T1D [32]. Our results are in concordance with studies on T1D patients from Sardinia, black population from Zimbabwe, Lithuania, Czecks, Lebanese, Brazilians and African Americans where *DRB1\*03:01, DRB1\*04:01* and Figure 1d shows the homozygosity and heterozygosity of *DRB1\*03:01* and *DRB1\*04* alleles significantly increased in T1D. Homozygous *DRB1\*03:01* (p<4.6X10-39, OR=23.1), heterozygous *DRB1\*03:01/\*04:05* (p<5.5X10-6, OR =124.2) and *DRB1\*03:01/\*04:01* (p<0.0001, OR =17.6) were significantly increased in the patients as compared to controls. Heterozygous *03:01*/X (i.e. any other allele) (p<5.9X10-6, OR = 2.8) was also significantly increased in the patients as compared to controls. Heterozygous *DRB1\*07:01/X* (p < 1.3X10-7, OR = 0.09) were significantly reduced in the T1D patients as compared to controls suggesting their protective role. Thirty two percent of the patients studied were homozygous for *DRB1\*0301* as compared to 2% of the controls and this difference was highly significant. Homozygosity of *DQB1\*02:01* was significantly (p<1x10-5, OR=5.4) increased in the patients. *DQB1\*03:02* which was not significantly increased in the patients, showed a significant increase in heterozygous combination with *DQB1\*0201* (p<3x10-5, OR=34.16) [14]. In fact none of the controls had *DQB1\*0201/\*0302* heterozygous combination [14]. In a Swedish study, *DQA1\*0301/DQB1\*0302* and heterozygous combinations of *DQA1\*0301/DQB1\*0302* and *DQA1\*0201/DQB1\*0501* have been shown to confer the highest susceptibility [41]

> Figure 1 e shows the *HLA-B-DRB1* haplotypes that were significantly increased in T1D patients. HLA alleles are very closely linked to each other in such a way that the haplotypes are inherited en-bloc. Three *HLA-DRB1\*03* haplotypes, *B\*08-DRB1\*03:01*(p<1x10-14, OR=3.7), *B\*50- DRB1\*03:01*(p<8x10-24, OR=11.2) and *B\*58-DRB1\*03:01* (p<1.4x10-12, OR=5.1) and *DRB1\*03:01* haplotypes with non *B\*8/ B\*50/ B\*58* alleles (p<2.8x10-8, OR=3.4) were significantly increased in the patients as compared to controls. On the other hand non- *B\*8/ B\*50/ B\*58-non DRB1\*03:01* haplotypes were significantly reduced (p<1.5x10-44, OR=0.09) in T1D patients as compared to controls [12].

> Thus our studies show a significant increase of HLA class-I and II alleles which may have a role in auto-antigen presentation to CD8+ and CD4+ T cells respectively.

### **6. Vitiligo**

Lithuania, Czecks, Lebanese, Brazilians and African Americans where *DRB1\*03:01,*

**\***

**\***

**Figure 1.** Distribution of *HLA-DRB1*, *HLA-B* and *DQB1* alleles in Type 1 diabetes and healthy controls. a. *HLA-DRB1* alleles significantly increased or decreased in T1D are shown. *DRB1\*03:01*, *\*04:01* and *\*04:05* are significantly in‐ creased and *DRB1\*07:01*is significantly reduced in T1D as compared to controls. b. *HLA-B\*08*, *B\*50:01* and *B\*58* are significantly increased in T1D as compared to controls c. *DQB1\*02:01* is significantly increased and *DQB1\*03:01* and *DQB1\*05:03* are significantly reduced in T1D as compared to controls. d. Homozygous *DRB1\*03:01* and heterozygous *DRB1\*03:01* with *\*04:01*, *\*04:05* or any other allele (\*0301,X) are significantly increased in T1D as compared to con‐ trols. e. *HLA-B-DRB1\*03* haplotypes in T1D patients and controls. \* denotes alleles that show statistically significant

HLA-DRB1 and DQB1 are in strong linkage disequilibrium. We also studied the alleles of *DQB1* locus*. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 1c). However *DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and *DQB1\*05:03* (6x10-4, OR=0.28)

a b c

**Figure 1. Distribution of** *HLA‐DRB1***,** *HLA‐B* **and** *DQB1* **alleles in Type 1 diabetes and healthy controls. a.** *HLA‐DRB1* **alleles significantly increased or decreased in T1D are shown.** *DRB1\*03:01***,** *\*04:01* **and** *\*04:05* **are significantly increased and** *DRB1\*07:01***is significantly reduced in T1D as compared to controls. b.** *HLA‐ B\*08***,** *B\*50:01* **and** *B\*58* **are significantly increased in T1D as compared to controls c.** *DQB1\*02:01* **is significantly increased and** *DQB1\*03:01* **and** *DQB1\*05:03* **are significantly reduced in T1D as compared to controls. d. Homozygous** *DRB1\*03:01* **and heterozygous** *DRB1\*03:01* **with** *\*04:01***,** *\*04:05* **or any other allele**

**\* \* \* \***

**\***

**\* \***

HLA-DRB1 and DQB1 are in strong linkage disequilibrium. We also studied the alleles of *DQB1* locus*. DQB1\*02:01* which is linked to *DRB1\*03:01* was significantly increased (p<1x10-8, OR=5.08) in patients (Figure 1c). However

**(\*0301,X) are significantly increased in T1D as compared to controls. e.** *HLA‐B‐DRB1\*03* **haplotypes in T1D patients and controls. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles.** 

Rani et al., Tissue Antigens, 2004, Kumar et al., Tissue Antigens , 2007

**\***

e

**\* \***

*DRB1\*04:05* have been shown to be associated with the disease [33-40].

 **\* \* \***

**\***

**\* \***

increase or decrease in the frequencies of the depicted alleles.

were reduced in the patients [14].

d

**\***

**\***

**\***

206 HLA and Associated Important Diseases

*DQB1\*03:02* and *DQB1\*03:07*, alleles linked with *DRB1\*04:01*, *DRB1\*04:03*, *DRB1\*04:04* and *DRB1\*04:05* were not significantly increased in the patients because two of these alleles *DRB1\*04:01* and *DRB1\*04:05* were increased in the patients and the other two DR4 alleles *DRB1\*04:03* and *DRB1\*04:04* were significantly reduced in the patients. *DQB1\*03:01* (p<6x10-4, OR=0.27) and *DQB1\*05:03* (6x10-4, OR=0.28) were reduced in the patients [14]. Vitiligo is a depigmenting disorder of the skin caused by autoimmune destruction of pigment producing cells called melanocytes. It effects 0.5-1 % of the world population [42], while in India the incidence varies from 0.25 to 2.5% [43, 44] in most ethnic groups, however, it has been reported to be 8.8% in populations from western states of Gujarat and Rajasthan [45]. Vitiligo manifests in several forms which can be broadly classified in two clinical subtypes: generalized and localized [42]. While precise etiology of vitiligo is not very well understood, several hypotheses have been proposed which include autoimmune [46], neural [47], auto-cytotoxic [48] and genetic hypotheses [49]. However, autoimmune hypothesis gets credence due to cooccurrence of other autoimmune diseases in vitiligo patients in some cases, presence of auto reactive T cells and circulating auto-antibodies in the patients [46, 50-55]. The role of genetic factors has been suggested due to the fact that 7% of first-degree relatives of vitiligo develop the disease. [56, 57].

Recent genome wide studies [58, 59] have shown that several *single nucleotide polymor‐ phisms* (*SNPs*) in the *MHC* region were significantly associated with the disease. Using imputation based on the previous studies done on associations of HLA, the authors showed that certain *HLA-class-I* alleles were associated with vitiligo but they could not impute *HLA class-II* alleles. [58]. As mentioned before MHC is the most polymorphic system of the human genome. With such a great diversity at each locus, linkage disequilibrium (LD) of *HLA* alleles with di-allelic *SNPs* may not be appropriate since several *HLA* alleles may be in LD with one or the alternate *SNP*. Thus the derivation based on these LDs may not be accurate especially in the populations from where no previous *HLA* data is available i.e. if there is no HLA data available it would be impossible to determine the linkage disequilibrium of HLA alleles with di-allelic SNPs. So, we conducted a very robust study on a sample size of 1404 vitiligo cases consisting of 1097 generalized and 307 localized vitiligo cases and 902 unaffected controls from North India for the alleles at *HLA*-*A, -B,*(class-I) and -*DRB1* (Class-II) loci using molecular methods and also replicated the study on 355 cases (250 general‐ ized and 105 localized) and 441 controls from Gujarat, a state in west of India [15]. We also reported differences in the amino acid signatures of peptide binding pockets of the HLA molecules in vitiligo patients as compared to controls [15].

Our study showed a lot of diversity in number and frequencies of HLA alleles in patients and controls, with 78, 160 and 106 alleles in cases and 68, 111 and 94 alleles in controls for *HLA-A, -B* and *-DRB1* loci respectively from North India. Similar diversity was observed in the replication study as well. However, *HLA-A\*33:01*(p<1.21x10-15, OR=2.57 in North Indians and p<0.008, OR=1.69 in Gujaratis), *B\*44:03* (p<7.05x10-27, OR=3.53 in North Indians and p<1.54x10-8, OR=3.13 in Gujaratis) and *DRB1\*07:01* (p<3.16x10-30, OR=2.8 in North Indians and p<9.81x10-15, OR=3.19 in Gujaratis) were significantly increased in both initial and replication study (Figure 2), suggesting these alleles are the markers for Vitiligo in both North India and Gujarat. These associations were significant irrespective of the age at onset or the gender of the cases. So, to determine which *HLA* alleles have primary association, we sequentially deleted samples having *DRB1\*07:01, A\*33:01*and *B\*44:03* from both the case and control groups and then analysed the data for remainder alleles. The analysis showed that association of *DRB1\*07:01* with vitiligo seemed to be primary because in the absence of *DRB1\*07:01,* the class-I alleles *A\*33:01* and *B\*44:03* did not remain significantly increased. However, in the absence of *A\*33:01*and *B\*44:03, DRB1\*07:01* still remained significantly increased in both the popula‐ tions studied. The basic predisposing alleles in both localized and generalized vitiligo are same; however, similarities with the controls in terms of allele frequencies of some alleles and amino acid signature of the DR beta chain (figure 3) seem to be protective from generalized distri‐ bution of the lesions in localized vitiligo[15].

haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03-DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01- B\*08:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01- DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and *A\*26:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo cases in

**Figure 2.** HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show statis‐

\* \* \* \* \*

\* \* \*

Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to...

\* \* \*

\* \* \*

\* \* \*

OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo cases in both initial and replication study[15].

tically significant increase or decrease in the frequencies of the depicted alleles.

**Figure 2. HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show statistically significant increase or** 

Singh et al., Journal of Investigative Dermatology,2012

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\* \* \*

\*\* \*

\* \*\*

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\*\* \*\* \*

Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles may be on the same chromosome making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03- DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01-B\*080:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01-DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and *A\*26:01-DRB1\*03:01* was significantly reduced (p<0.00005,

We further studied the amino acid signatures of the peptide binding pockets of DRB1 in vitiligo cases and unaffected controls Eleven residues representing integral parts of peptide binding pockets of DR beta chain were analysed at positions 26, 28, 30, 37, 47, 67, 70, 71, 74, 77 and 86 [62]. The protein sequences were downloaded for all the alleles observed from the HLA database (http://www.ebi.ac.uk/cgi-bin/imgt/HLA/align.cgi) and the amino acids present at

both initial and replication study[15].

**decrease in the frequencies of the depicted alleles.** 

North Indians

\* \*

\*\* \* \* \*

Gujaratis (replication study)

\* \*\*

\*

\*

\* \*

Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles being on the same chro‐ mosome may be making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed

North Indians

Recent genome wide studies [58, 59] have shown that several *single nucleotide polymor‐ phisms* (*SNPs*) in the *MHC* region were significantly associated with the disease. Using imputation based on the previous studies done on associations of HLA, the authors showed that certain *HLA-class-I* alleles were associated with vitiligo but they could not impute *HLA class-II* alleles. [58]. As mentioned before MHC is the most polymorphic system of the human genome. With such a great diversity at each locus, linkage disequilibrium (LD) of *HLA* alleles with di-allelic *SNPs* may not be appropriate since several *HLA* alleles may be in LD with one or the alternate *SNP*. Thus the derivation based on these LDs may not be accurate especially in the populations from where no previous *HLA* data is available i.e. if there is no HLA data available it would be impossible to determine the linkage disequilibrium of HLA alleles with di-allelic SNPs. So, we conducted a very robust study on a sample size of 1404 vitiligo cases consisting of 1097 generalized and 307 localized vitiligo cases and 902 unaffected controls from North India for the alleles at *HLA*-*A, -B,*(class-I) and -*DRB1* (Class-II) loci using molecular methods and also replicated the study on 355 cases (250 general‐ ized and 105 localized) and 441 controls from Gujarat, a state in west of India [15]. We also reported differences in the amino acid signatures of peptide binding pockets of the HLA

Our study showed a lot of diversity in number and frequencies of HLA alleles in patients and controls, with 78, 160 and 106 alleles in cases and 68, 111 and 94 alleles in controls for *HLA-A, -B* and *-DRB1* loci respectively from North India. Similar diversity was observed in the replication study as well. However, *HLA-A\*33:01*(p<1.21x10-15, OR=2.57 in North Indians and p<0.008, OR=1.69 in Gujaratis), *B\*44:03* (p<7.05x10-27, OR=3.53 in North Indians and p<1.54x10-8, OR=3.13 in Gujaratis) and *DRB1\*07:01* (p<3.16x10-30, OR=2.8 in North Indians and p<9.81x10-15, OR=3.19 in Gujaratis) were significantly increased in both initial and replication study (Figure 2), suggesting these alleles are the markers for Vitiligo in both North India and Gujarat. These associations were significant irrespective of the age at onset or the gender of the cases. So, to determine which *HLA* alleles have primary association, we sequentially deleted samples having *DRB1\*07:01, A\*33:01*and *B\*44:03* from both the case and control groups and then analysed the data for remainder alleles. The analysis showed that association of *DRB1\*07:01* with vitiligo seemed to be primary because in the absence of *DRB1\*07:01,* the class-I alleles *A\*33:01* and *B\*44:03* did not remain significantly increased. However, in the absence of *A\*33:01*and *B\*44:03, DRB1\*07:01* still remained significantly increased in both the popula‐ tions studied. The basic predisposing alleles in both localized and generalized vitiligo are same; however, similarities with the controls in terms of allele frequencies of some alleles and amino acid signature of the DR beta chain (figure 3) seem to be protective from generalized distri‐

Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles being on the same chro‐ mosome may be making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed

molecules in vitiligo patients as compared to controls [15].

208 HLA and Associated Important Diseases

bution of the lesions in localized vitiligo[15].

Singh et al., Journal of Investigative Dermatology,2012

*DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01-B\*080:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007,

**Figure 2. HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles.**  Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles may be on the same chromosome making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03-* **Figure 2.** HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show statis‐ tically significant increase or decrease in the frequencies of the depicted alleles.

haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03-DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01- B\*08:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01- DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and *A\*26:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo cases in both initial and replication study[15]. OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01-DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and *A\*26:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo cases in both initial and replication study[15].

We further studied the amino acid signatures of the peptide binding pockets of DRB1 in vitiligo cases and unaffected controls Eleven residues representing integral parts of peptide binding pockets of DR beta chain were analysed at positions 26, 28, 30, 37, 47, 67, 70, 71, 74, 77 and 86 [62]. The protein sequences were downloaded for all the alleles observed from the HLA database (http://www.ebi.ac.uk/cgi-bin/imgt/HLA/align.cgi) and the amino acids present at


OR=0.15 in Gujaratis) in vitiligo cases in both initial and replication study[15].

**Figure 2. HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show** 

Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles may be on the same chromosome making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03-DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01-B\*080:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01-DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and

> generalized vitiligo in both the initial and replication study. However, the amino acid signature similar to healthy controls were observed at positions Arg30β/Cys30β, Ser37β, Ile67β/Leu67β Asp70β, Ala74β in North Indians and Cys30β, Ser37β, Asp70β, Arg71β/Lys Ser71β, Ala74β/ Arg74β and Thr77β /

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Most of the studies previously have been done on Generalized vitiligo which has been considered to be an autoimmune disease. We showed for the first time that both generalized and localized vitiligo have the same predisposing MHC alleles i.e., *B\*44:03 and DRB1\*07:01* in both the populations studied*.* Association of *MHC* alleles with localized vitiligo clearly suggests an autoimmune etiology of the disease due to antigen presenting function of the MHC molecules. Thus there seems to be a need to re-look at the aetiopathogenesis of localized vitiligo in light of our results. While it may be an autoimmune disorder, similarities with unaffected controls in terms of *HLA* alleles and amino acid signature of the peptide binding pockets of

Idiopathic hypoparathyroidism (IH) is a rare endocrine disease where parathyroid gland is functionally impaired resulting in subnormal PTH secretion, hypocalcemia, hyperphospha‐ temia and associated complications [63]. While it may be an autoimmune disorder [63], there is hardly any evidence to confirm the autoimmune etiology of the disease. Since there was no comprehensive study on the immunogenetic basis of IH, we studied the association of *HLA class-I* and *II* alleles in a large group of North Indian patients (N=134) with IH [16, 64-70] and compared them to 902 healthy controls from the same ethnic background. We also reported significant differences in the amino acid signatures of peptide binding pockets of the HLA

Just like vitiligo, we observed diversity in number and frequencies of HLA alleles with 23, 40 and 44 alleles in patients for *HLA-A, -B, and -DRB1* loci, respectively. However, specific alleles at these loci were found to be predisposing. *HLA-A\*26:01* (*p*<1.71x10-34, OR = 9.29), *HLA-B\*08:01* (*p*<8.19x10-6*,* OR = 2.59), and *HLA-DRB1\*03:01* (*p*<0.013, OR = 1.67) and *HLA-DRB1\*12:02* (*p<*0.0046, OR = 3.52) were significantly increased in IH when compared with healthy controls. However, alleles *HLA-A\*01:01 (p<*0.039, OR = 0.60), *HLA-A\*03:01* (*p*<0.004*,* OR=0.33), *HLA-B\*15:01* (*p*<0.0038, OR=0.17) and *HLA-B\*57:01* (*p*<0.0074*,* OR=0.3) were signif‐ icantly reduced in IH as compared with controls (Figure 4a). Since *HLA-A\*26:01* showed the highest significance, we studied whether any other allele was significantly increased in the absence of *HLA-A\*26:01*. So, all the samples with *A\*26:01* were removed from both cases and controls and then analysis was done for significant differences in frequencies of the remainder alleles. In the absence of *HLA-A\*26:01,* no other allele showed any significant differences [16]. Associations of *A\*26:01* and *B\*08:01* with IH were significant irrespective of the gender and age at onset of the disease. To investigate whether there was any association of HLA alleles with associated clinical autoimmunity in IH, patients were categorized into two groups, those with associated clinical autoimmunity and those without. While *HLA-A\*26:01* was signifi‐

DR beta chain may be contributing to the localized distribution of the lesions [15].

Asn77β in Gujaratis [15].

**7. Idiopathic hypoparathyroidism**

molecules in patients with IH as compared with controls [16].

**statistically significant increase or decrease in the frequencies of the depicted alleles.** 


**Figure 3. Amino acid signatures for the peptide binding pockets of HLA molecules for DR beta1 chain in all vitiligo,** 

**generalized vitiligo and localized vitiligo patients from North India and Gujarat. Amino acids shown in red are significantly increased in vitiligo patients while amino acids shown in green are significantly reduced in vitiligo patients as compared to controls and those in black are not significantly different from controls (Singh et al., JID, 2012). Figure 3.** Amino acid signatures for the peptide binding pockets of HLA molecules for DR beta1 chain in all vitiligo, generalized vitiligo and localized vitiligo patients from North India and Gujarat. Amino acids shown in red are signifi‐ cantly increased in vitiligo patients while amino acids shown in green are significantly reduced in vitiligo patients as compared to controls and those in black are not significantly different from controls (Singh et al., JID, 2012).

the aforesaid positions were compared in patients with controls. Figure 3 shows the amino acid residues at the aforesaid positions that were either significantly increased or decreased in all vitiligo patients, generalized and localzed vitiligo patients when compared with controls. The results show that Glu28β, Leu30β/Arg30β Phe37β, Tyr47β, Ile67β, Asp70β, Arg71β, Gln74β, Thr77β and Gly86β make the molecular signature of the peptide binding pockets of DRB1 for predisposition to develop vitiligo in both the initial as well as in the replication study (Figure 3). Asp28β, Tyr30β/ Cys30β, Ser37β, Phe47β, Leu67β, Gln70β, Lys71β, Ala/Arg74β, Asn77β and Val86β make the molecular signature for the protection from Vitiligo in both North India and Gujarat. [15].

We observed subtle differences in the molecular signatures of the peptide binding pockets of DR beta chain in localized and generalized vitiligo. Localized vitiligo patients show similarities in the amino acid signatures with not only generalized vitiligo but also with healthy controls probably responsible for localized distribution of the lesions in them. Amino acid signature at positions Glu28β, Leu30β, Phe37β, Tyr47β and Gln74β in the localized vitiligo patients is similar to generalized vitiligo in both the initial and replication study. However, the amino acid signature similar to healthy controls were observed at positions Arg30β/Cys30β, Ser37β, Ile67β/Leu67β Asp70β, Ala74β in North Indians and Cys30β, Ser37β, Asp70β, Arg71β/Lys Ser71β, Ala74β/ Arg74β and Thr77β / Asn77β in Gujaratis [15].

Most of the studies previously have been done on Generalized vitiligo which has been considered to be an autoimmune disease. We showed for the first time that both generalized and localized vitiligo have the same predisposing MHC alleles i.e., *B\*44:03 and DRB1\*07:01* in both the populations studied*.* Association of *MHC* alleles with localized vitiligo clearly suggests an autoimmune etiology of the disease due to antigen presenting function of the MHC molecules. Thus there seems to be a need to re-look at the aetiopathogenesis of localized vitiligo in light of our results. While it may be an autoimmune disorder, similarities with unaffected controls in terms of *HLA* alleles and amino acid signature of the peptide binding pockets of DR beta chain may be contributing to the localized distribution of the lesions [15].

### **7. Idiopathic hypoparathyroidism**

the aforesaid positions were compared in patients with controls. Figure 3 shows the amino acid residues at the aforesaid positions that were either significantly increased or decreased in all vitiligo patients, generalized and localzed vitiligo patients when compared with controls. The results show that Glu28β, Leu30β/Arg30β Phe37β, Tyr47β, Ile67β, Asp70β, Arg71β, Gln74β, Thr77β and Gly86β make the molecular signature of the peptide binding pockets of DRB1 for predisposition to develop vitiligo in both the initial as well as in the replication study (Figure 3). Asp28β, Tyr30β/ Cys30β, Ser37β, Phe47β, Leu67β, Gln70β, Lys71β, Ala/Arg74β, Asn77β and Val86β make the molecular

**compared to controls and those in black are not significantly different from controls (Singh et al., JID, 2012). Figure 3.** Amino acid signatures for the peptide binding pockets of HLA molecules for DR beta1 chain in all vitiligo, generalized vitiligo and localized vitiligo patients from North India and Gujarat. Amino acids shown in red are signifi‐ cantly increased in vitiligo patients while amino acids shown in green are significantly reduced in vitiligo patients as

**Figure 2. HLA-A, B and DRB1 loci alleles significantly increased in all vitiligo, generalized vitiligo and localized vitiligo patients when compared with healthy controls in North Indian and Gujarat patients. \* denotes alleles that show** 

Removal of *DRB1\*07:01* from both patients and control groups resulted in removal of all the samples that had *A\*33:01* and *B\*44:03,* suggesting that these alleles may be on the same chromosome making a haplotype, such that when samples with *DRB1\*07:01* are removed they also get removed simultaneously. To prove that we estimated the *HLA* haplotype frequencies using the expectation-maximization (EM) algorithm [60, 61] using Arlequin Ver 3.5 (http://cmpg.unibe.ch/software/arlequin35/). Haplotype analysis for three loci showed haplotypes *A\*33:01-B\*44:03-DRB1\*07:01* (p<8.75x10-23, OR=6.21 in North Indians and p<0.000016, OR=3.65 in Gujaratis) and *A\*24:02-B\*44:03-DRB1\*07:01* (p<0.0019, OR=3.83 in North Indians and p<0.0034, OR=7.73 in Gujaratis) were significantly increased and *A\*26:01-B\*080:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007, OR=0.15 in Gujaratis) in vitiligo patients in both the initial study as well as in the replication study. Analysis of two-locus haplotypes revealed haplotypes *A\*33:01-DRB1\*07:01* (p<6.97x10-26, OR=6.55 in North Indians and p<0.00001, OR=3.59 in Gujaratis) and *B\*44:03-DRB1\*07:01* (p < 2.4 x 10-29, OR=4.01 in North Indians and p<1.84x10-14, OR=6.34 in Gujaratis) was significantly increased and *A\*26:01-DRB1\*03:01* was significantly reduced (p<0.00005, OR=0.33 in North Indians and p<0.0007,

**statistically significant increase or decrease in the frequencies of the depicted alleles.** 

OR=0.15 in Gujaratis) in vitiligo cases in both initial and replication study[15].

**North Indians All Vitiligo Gujaratis All Vitiligo North Indian Generalized Gujaratid Generalized North Indian Localized Gujarati Localized** 

210 HLA and Associated Important Diseases

**North Indians All Vitiligo Gujaratis All Vitiligo North Indian Generalized Gujaratid Generalized North Indian Localized Gujarati Localized** 

 **Position 28 30 30 37 47 67 70 71 74 77 86 Amino Acid E L R F Y I D R Q T G** 

**Position 28 30 30 37 47 67 70 71 74 74 77 86 Amino Acid D Y C S F L Q K A R N V** 

> **Significantly increased <0.009 to 10-6 Significantly increased p<0.01 to 0.05**

**Significantly reduced p<0.01 to 0.05 Significantly reduced p<0.009 to 10-6**

**Figure 3. Amino acid signatures for the peptide binding pockets of HLA molecules for DR beta1 chain in all vitiligo, generalized vitiligo and localized vitiligo patients from North India and Gujarat. Amino acids shown in red are significantly increased in vitiligo patients while amino acids shown in green are significantly reduced in vitiligo patients as**

**Non significant** 

compared to controls and those in black are not significantly different from controls (Singh et al., JID, 2012).

We observed subtle differences in the molecular signatures of the peptide binding pockets of DR beta chain in localized and generalized vitiligo. Localized vitiligo patients show similarities in the amino acid signatures with not only generalized vitiligo but also with healthy controls probably responsible for localized distribution of the lesions in them. Amino acid signature at positions Glu28β, Leu30β, Phe37β, Tyr47β and Gln74β in the localized vitiligo patients is similar to

signature for the protection from Vitiligo in both North India and Gujarat. [15].

Idiopathic hypoparathyroidism (IH) is a rare endocrine disease where parathyroid gland is functionally impaired resulting in subnormal PTH secretion, hypocalcemia, hyperphospha‐ temia and associated complications [63]. While it may be an autoimmune disorder [63], there is hardly any evidence to confirm the autoimmune etiology of the disease. Since there was no comprehensive study on the immunogenetic basis of IH, we studied the association of *HLA class-I* and *II* alleles in a large group of North Indian patients (N=134) with IH [16, 64-70] and compared them to 902 healthy controls from the same ethnic background. We also reported significant differences in the amino acid signatures of peptide binding pockets of the HLA molecules in patients with IH as compared with controls [16].

Just like vitiligo, we observed diversity in number and frequencies of HLA alleles with 23, 40 and 44 alleles in patients for *HLA-A, -B, and -DRB1* loci, respectively. However, specific alleles at these loci were found to be predisposing. *HLA-A\*26:01* (*p*<1.71x10-34, OR = 9.29), *HLA-B\*08:01* (*p*<8.19x10-6*,* OR = 2.59), and *HLA-DRB1\*03:01* (*p*<0.013, OR = 1.67) and *HLA-DRB1\*12:02* (*p<*0.0046, OR = 3.52) were significantly increased in IH when compared with healthy controls. However, alleles *HLA-A\*01:01 (p<*0.039, OR = 0.60), *HLA-A\*03:01* (*p*<0.004*,* OR=0.33), *HLA-B\*15:01* (*p*<0.0038, OR=0.17) and *HLA-B\*57:01* (*p*<0.0074*,* OR=0.3) were signif‐ icantly reduced in IH as compared with controls (Figure 4a). Since *HLA-A\*26:01* showed the highest significance, we studied whether any other allele was significantly increased in the absence of *HLA-A\*26:01*. So, all the samples with *A\*26:01* were removed from both cases and controls and then analysis was done for significant differences in frequencies of the remainder alleles. In the absence of *HLA-A\*26:01,* no other allele showed any significant differences [16].

Associations of *A\*26:01* and *B\*08:01* with IH were significant irrespective of the gender and age at onset of the disease. To investigate whether there was any association of HLA alleles with associated clinical autoimmunity in IH, patients were categorized into two groups, those with associated clinical autoimmunity and those without. While *HLA-A\*26:01* was signifi‐ cantly increased in both the groups as compared with controls, *HLA-B\*08:01* and *DRB1\*12:02* were significantly increased in patients without any associated clinical autoimmunity when compared with controls [16]. of the disease. To investigate whether there was any association of HLA alleles with associated clinical autoimmunity in IH, patients were categorized into two groups, those with associated clinical autoimmunity and those without. While *HLA-A\*26:01* was significantly increased in both the groups as compared with controls, *HLA-B\*08:01* and *DRB1\*12:02* were significantly increased in patients without

any associated clinical autoimmunity when compared with controls [16].

Associations of *A\*26:01* and *B\*08:01* with IH were significant irrespective of the gender and age at onset

MHC molecules are extremely polymorphic, however, alleles with similar peptide binding pockets may be clustered in one group [6], called supertypes, such that HLA molecules in one supertype may present similar peptides[16]. Assignment of alleles in the supertypes have been done based on published motifs, binding data, shared repertoires of binding peptides and the amino acid sequences of different alleles at the peptide binding pockets [6]. Supertype A01 (which includes *A\*26:01*) was significantly increased in IH with 65.67% of the patients as compared to 39.36% of the controls having this supertype (figure 4 c). However, *A\*26:01* was observed only in 50.75% of the patients as compared to 9.98% of the controls. About 15% of the IH cases had different alleles of A01 supertype (other than *A\*26:01*)*,* which did not show statistically significant differences due to small numbers*.* Significant increase of A01 supertype suggests that similar auto-antigenic peptides may be getting presented by different MHC

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While the amino acid residue at a particular position in different alleles in a supertype may not be the same, they would be similar in nature such that they would bind similar peptides. For instance, all the alleles falling in supertype A01 will bind small and aliphatic residues in the B pocket and aromatic and large hydrophobic residues in F pocket [6]. Thus, the nature of the shared amino acids and not amino acid per se constitutes the molecular signatures of the

The peptide binding groove of the MHC class-I molecules contain small pockets where the side chains of complimentary residues of the peptide bind [4]. Pockets B (key residues at positions 9, 45, 63, 67, 70 and 99) and F (key residues at positions 77, 80, 81 and 116) of alpha chain are the main anchors which engage the peptides at its amino acid position 2 and the Cterminus [6]. Since we observed a significant increase in an HLA-A locus allele (*HLA-A\*26:01*), we analysed the amino acid signatures of the peptide binding groove of the alleles of A-locus to check for shared peptide binding specificity. For this purpose, the protein sequences of the *HLA-A* locus alleles were downloaded from the HLA database as mentioned for the vitiligo study and the amino acids present at the aforesaid positions were compared in patients with controls. We observed that Tyr9α, Asn63α, Val67α, His70α, Asn77α, Ile80α and Ala81α make the molecular signature for HLA-A peptide binding pockets for predisposition to IH, however, Phe9α, Glu63α, Met67α, Gln70α, Asp77α, Thr80α and Leu81α make the molecular signature for negative association with the disease (Figure 4d). While the most significant association was derived from *HLA-A\*26:01*, peptide binding grooves of other alleles shared some of the

MHC is the most polymorphic system of the man with more than 6000 alleles for different loci. With such a great diversity, association of particular *MHC* alleles with a disease has functional implications due to the antigen presenting function of the MHC. The peptides presented by the MHC molecules have allele specific motifs [4]. The affinity of the peptide to a particular MHC molecule is determined by the amino acid residues present in peptide binding pockets of the peptide binding groove. Shared amino acids in the peptide binding pockets have been demonstrated in autoimmune diseases like Type 1 diabetes [71], rheumatoid arthritis [72] and thyroiditis [62]. Investigation of amino acid signatures for the peptide binding pockets of HLA-A and HLA-B alpha chain and HLA-DR beta chain revealed specific molecular signatures for

molecules which belong to the supertype [6, 16].

peptide binding groove of different alleles in a supertype.

residues with the peptide binding grove of *HLA-A\*26:01* (Figure 5)[16].

Metabolism, 2012

**Figure 4.** *HLA-A, B* **and** *DRB1* **loci alleles significantly increased in idiopathic hypoparathyroidism patients when compared with healthy controls in North Indian patients. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles . Figure 4.** *HLA-A, B* and *DRB1* loci alleles significantly increased in idiopathic hypoparathyroidism patients when com‐ pared with healthy controls in North Indian patients. \* denotes alleles that show statistically significant increase or decrease in the frequencies of the depicted alleles.

We constructed HLA haplotypes for HLA-A-B-DRB1 and HLA-A-B and HLA-A-DRB1 for 133 cases

and 902 controls using Arlequin 3.5 program (http://cmpg.unibe.ch/software/arlequin35/). Haplotype *HLA-A\*26:01-B\*08:01-DRB1\*03:01* (*p*<4.47x10-8, OR = 4.38), was significantly increased in cases as compared with controls. Two locus haplotype analysis (figure 4b) showed haplotype *A\*26:01- B\*08:01*(*p*<1.48x10-11, OR = 4.72), *A\*26:01-B\*51:01* (*p*<7.59x10-6, OR = 17.26), *A\*26:01-B\*35:03*  (*p*<0.0028, OR = 8.48), and *A\*11:01-B\*40:06* (*p*<0.0009, OR = 4.46), were significantly increased in IH as compared to controls. However, the association of *A\*26:01* with IH seems to be primary since in the absence of *A\*26:01*, none of the other alleles like *B\*08:01, B\*51:01* or *DRB1\*03:01* remained We constructed HLA haplotypes for HLA-A-B-DRB1 and HLA-A-B and HLA-A-DRB1 for 133 cases and 902 controls using Arlequin 3.5 program (http://cmpg.unibe.ch/software/arle‐ quin35/). Haplotype *HLA-A\*26:01-B\*08:01-DRB1\*03:01* (*p*<4.47x10-8, OR = 4.38), was signifi‐ cantly increased in cases as compared with controls. Two locus haplotype analysis (figure 4b) showed haplotypes *A\*26:01-B\*08:01*(*p*<1.48x10-11, OR = 4.72), *A\*26:01-B\*51:01* (*p*<7.59x10-6, OR = 17.26), *A\*26:01-B\*35:03* (*p*<0.0028, OR = 8.48), and *A\*11:01-B\*40:06* (*p*<0.0009, OR = 4.46), were significantly increased in IH as compared to controls. However, the association of *A\*26:01* with IH seems to be primary since in the absence of *A\*26:01*, none of the other alleles like *B\*08:01, B\*51:01* or *DRB1\*03:01* remained significantly different suggesting that the significant increase in these alleles is due to their being in linkage disequilibrium with the *A\*26:01*[16].

MHC molecules are extremely polymorphic, however, alleles with similar peptide binding pockets may be clustered in one group [6], called supertypes, such that HLA molecules in one supertype may present similar peptides[16]. Assignment of alleles in the supertypes have been done based on published motifs, binding data, shared repertoires of binding peptides and the amino acid sequences of different alleles at the peptide binding pockets [6]. Supertype A01 (which includes *A\*26:01*) was significantly increased in IH with 65.67% of the patients as compared to 39.36% of the controls having this supertype (figure 4 c). However, *A\*26:01* was observed only in 50.75% of the patients as compared to 9.98% of the controls. About 15% of the IH cases had different alleles of A01 supertype (other than *A\*26:01*)*,* which did not show statistically significant differences due to small numbers*.* Significant increase of A01 supertype suggests that similar auto-antigenic peptides may be getting presented by different MHC molecules which belong to the supertype [6, 16].

cantly increased in both the groups as compared with controls, *HLA-B\*08:01* and *DRB1\*12:02* were significantly increased in patients without any associated clinical autoimmunity when

any associated clinical autoimmunity when compared with controls [16].

\*

\*

remainder alleles. In the absence of *HLA-A\*26:01,* no other allele showed any significant differences

Associations of *A\*26:01* and *B\*08:01* with IH were significant irrespective of the gender and age at onset of the disease. To investigate whether there was any association of HLA alleles with associated clinical autoimmunity in IH, patients were categorized into two groups, those with associated clinical autoimmunity and those without. While *HLA-A\*26:01* was significantly increased in both the groups as compared with controls, *HLA-B\*08:01* and *DRB1\*12:02* were significantly increased in patients without

b

\*

\* \*

Goswami et al., Journal of clinical Endocrinology and

 \*

> \*

\*

**Figure 4.** *HLA-A, B* **and** *DRB1* **loci alleles significantly increased in idiopathic hypoparathyroidism patients when compared with healthy controls in North Indian patients. \* denotes alleles that show statistically significant increase or** 

**Figure 4.** *HLA-A, B* and *DRB1* loci alleles significantly increased in idiopathic hypoparathyroidism patients when com‐ pared with healthy controls in North Indian patients. \* denotes alleles that show statistically significant increase or

We constructed HLA haplotypes for HLA-A-B-DRB1 and HLA-A-B and HLA-A-DRB1 for 133 cases and 902 controls using Arlequin 3.5 program (http://cmpg.unibe.ch/software/arle‐ quin35/). Haplotype *HLA-A\*26:01-B\*08:01-DRB1\*03:01* (*p*<4.47x10-8, OR = 4.38), was signifi‐ cantly increased in cases as compared with controls. Two locus haplotype analysis (figure 4b) showed haplotypes *A\*26:01-B\*08:01*(*p*<1.48x10-11, OR = 4.72), *A\*26:01-B\*51:01* (*p*<7.59x10-6, OR = 17.26), *A\*26:01-B\*35:03* (*p*<0.0028, OR = 8.48), and *A\*11:01-B\*40:06* (*p*<0.0009, OR = 4.46), were significantly increased in IH as compared to controls. However, the association of *A\*26:01* with IH seems to be primary since in the absence of *A\*26:01*, none of the other alleles like *B\*08:01, B\*51:01* or *DRB1\*03:01* remained significantly different suggesting that the significant increase in these alleles is due to their being in linkage disequilibrium with the *A\*26:01*[16].

Metabolism, 2012

We constructed HLA haplotypes for HLA-A-B-DRB1 and HLA-A-B and HLA-A-DRB1 for 133 cases and 902 controls using Arlequin 3.5 program (http://cmpg.unibe.ch/software/arlequin35/). Haplotype *HLA-A\*26:01-B\*08:01-DRB1\*03:01* (*p*<4.47x10-8, OR = 4.38), was significantly increased in cases as compared with controls. Two locus haplotype analysis (figure 4b) showed haplotype *A\*26:01- B\*08:01*(*p*<1.48x10-11, OR = 4.72), *A\*26:01-B\*51:01* (*p*<7.59x10-6, OR = 17.26), *A\*26:01-B\*35:03*  (*p*<0.0028, OR = 8.48), and *A\*11:01-B\*40:06* (*p*<0.0009, OR = 4.46), were significantly increased in IH as compared to controls. However, the association of *A\*26:01* with IH seems to be primary since in the absence of *A\*26:01*, none of the other alleles like *B\*08:01, B\*51:01* or *DRB1\*03:01* remained

compared with controls [16].

212 HLA and Associated Important Diseases

\*

\*

\* \*

\*

**decrease in the frequencies of the depicted alleles .** 

decrease in the frequencies of the depicted alleles.

<sup>d</sup> <sup>c</sup>

\*

\* \*

[16].

a

While the amino acid residue at a particular position in different alleles in a supertype may not be the same, they would be similar in nature such that they would bind similar peptides. For instance, all the alleles falling in supertype A01 will bind small and aliphatic residues in the B pocket and aromatic and large hydrophobic residues in F pocket [6]. Thus, the nature of the shared amino acids and not amino acid per se constitutes the molecular signatures of the peptide binding groove of different alleles in a supertype.

The peptide binding groove of the MHC class-I molecules contain small pockets where the side chains of complimentary residues of the peptide bind [4]. Pockets B (key residues at positions 9, 45, 63, 67, 70 and 99) and F (key residues at positions 77, 80, 81 and 116) of alpha chain are the main anchors which engage the peptides at its amino acid position 2 and the Cterminus [6]. Since we observed a significant increase in an HLA-A locus allele (*HLA-A\*26:01*), we analysed the amino acid signatures of the peptide binding groove of the alleles of A-locus to check for shared peptide binding specificity. For this purpose, the protein sequences of the *HLA-A* locus alleles were downloaded from the HLA database as mentioned for the vitiligo study and the amino acids present at the aforesaid positions were compared in patients with controls. We observed that Tyr9α, Asn63α, Val67α, His70α, Asn77α, Ile80α and Ala81α make the molecular signature for HLA-A peptide binding pockets for predisposition to IH, however, Phe9α, Glu63α, Met67α, Gln70α, Asp77α, Thr80α and Leu81α make the molecular signature for negative association with the disease (Figure 4d). While the most significant association was derived from *HLA-A\*26:01*, peptide binding grooves of other alleles shared some of the residues with the peptide binding grove of *HLA-A\*26:01* (Figure 5)[16].

MHC is the most polymorphic system of the man with more than 6000 alleles for different loci. With such a great diversity, association of particular *MHC* alleles with a disease has functional implications due to the antigen presenting function of the MHC. The peptides presented by the MHC molecules have allele specific motifs [4]. The affinity of the peptide to a particular MHC molecule is determined by the amino acid residues present in peptide binding pockets of the peptide binding groove. Shared amino acids in the peptide binding pockets have been demonstrated in autoimmune diseases like Type 1 diabetes [71], rheumatoid arthritis [72] and thyroiditis [62]. Investigation of amino acid signatures for the peptide binding pockets of HLA-A and HLA-B alpha chain and HLA-DR beta chain revealed specific molecular signatures for linkage disequilibrium with the *A\*26:01*[16].


significantly different suggesting that the significant increase in these alleles is due to their being in

 MHC molecules are extremely polymorphic, however, alleles with similar peptide binding pockets may be clustered in one group [6], called supertypes, such that HLA molecules in one supertype may present similar peptides[16]. Assignment of alleles in the supertypes have been done based on published motifs, binding data, shared repertoires of binding peptides and the amino acid sequences of different alleles at the peptide binding pockets [6]. Supertype A01 (which includes *A\*26:01*) was significantly increased in IH with 65.67% of the patients as compared to 39.36% of the controls having this supertype (figure 4 c). However, *A\*26:01* was observed only in 50.75% of the patients as compared to 9.98% of the controls. About 15% of the IH cases had different alleles of A01 supertype (other than *A\*26:01*)*,* which did not show statistically significant differences due to small numbers*.* Significant increase of A01 supertype

> These data suggest the autoantigenic peptides presented by different MHC molecules are restricted by these common alleles in a population which are associated with the autoimmune disorders. Of about 350 alleles that were observed in the three patient groups and healthy controls, *A\*26:01* (9.98%), *A\*33:01* (11.53%), *B\*08:01* (12.42%), *B\*44:03* (10.75%), DRB1*\*03:01* (19.29%) and *DRB1\*07:01* (27.49%), were quite common alleles in the healthy North Indians

Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to...

http://dx.doi.org/10.5772/57494

215

Our results suggest affinity of auto-antigenic peptides for predisposing MHC class-II and class-I molecules which may be involved in orchestrating (through CD4+ T cells) and implementing the autoimmune responses (through CD8+ T cells) in Type 1 diabetes, vitiligo and hypopara‐

We are thankful to the study participants: cases and the controls that provided blood samples for the study. Authors are thankful to Dr. Mitali Mukerji, IGIB, Delhi, for providing access to the Indian Genome Variation Consortium samples that have been used as unaffected controls in these studies. The projects were funded in part by grants from Department of Science and Technology (DST), Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India and partly by Core funds of National Institute of Immunology, New Delhi, India. Diabetes work was supported by Department of Science & Technology, New Delhi, India, grant No. SP/SO/B54/98. Vitiligo work was supported by grants for 'Genetic and autoimmune factors associated with vitiligo' (Grant number BT/PR6653/Med/12/258/2005) and the 'Program support for Skin pigmentation and melanocyte-keratinocyte biology' (grant number BT/01/COE/07/07) from the Department of Biotechnology, India. The hypoparathyr‐ oidism work was sponsored by Department of Biotechnology, grant number BT/ PR15022/Med/30/589/2010. The patient sample for this work came from All India Institute of Medical Sciences (AIIMS), New Delhi and Dr. Ram Manohar Lohia Hospital (RMLH), New Delhi, India. We would like to thank Dr. R. Goswami, Department of Endocrinology and Metabolism, (AIIMS) for T1D and hypoparathyroidism samples. We would like to acknowl‐ edge Dr. H. Kar (RMLH), Dr. V. K. Sharma (AIIMS) and Dr. Somesh Gupta (AIIMS), the dermatologists for providing the vitiligo samples. We thankfully acknowledge Dr. Rashee‐ dunnisa Begum and her team for providing vitiligo samples for the replication study from Gujarat. We would like to acknowledge the students and project fellows who have been involved in doing this work. Mr. Kapoor Chand's technical support is acknowledged.

Molecular Immunogenetics Group, National Institute of Immunology, New Delhi, India

that were associated with the three autoimmune disorders discussed above.

thyroidism.

**Acknowledgements**

**Author details**

Rajni Rani and Archana Singh

*p* **< 0.017 to 1.06x10-10, Significantly increased in IH as compared to controls**

*p* **< 0.017 to 4.79x10-10, Significantly reduced in IH as compared to controls**

**A locus alleles observed in IH cases showing significant increase or decrease in the number of amino acids. N=259 Figure 5.** Amino acid signatures (single letter codes) of the peptide binding pockets and HLA supertypes of 23 HLA-A locus alleles observed in IH cases showing significant increase or decrease in the number of amino acids. N=259 indi‐ cates 259 alleles were observed in 134 patients which were compared with amino acid signatures of 1698 alleles from 902 controls.

**Figure 5. Amino acid signatures (single letter codes) of the peptide binding pockets and HLA supertypes of 23 HLA-**

predisposition and protection from these diseases. Our data shows that haplotypes *B\*8- DRB1\*03:01*, *B\*50-DRB1\*03:01* and *B\*58-DRB1\*03:01* were significantly increased in T1D patients [12]. *A\*26:01-B\*08:01-DRB1\*03:01* and *A\*26:01* with other B-locus alleles were signif‐ icantly increased in hypoparathyroidism [16]. And in yet another autoimmune disorder, vitiligo, *A\*33:01-B\*44:03-DRB1\*07:01* is the predisposing haplotype and *A\*26:01-B\*08:01- DRB1\*03:01* seems to be protective haplotype, with *DRB1\*07:01* having primary association with the disease [15]. While in T1D and vitiligo it is the MHC class-II alleles*DRB1\*03:01* and*DRB1\*07:01* respectively, which had primary association, in hypoparathyroidism it is the MHC class-I allele *HLA-A\*26:01* which has primary association. Interestingly, in T1D *DRB1\*03:01* is predisposing and *DRB1\*07:01* is protective from the disease. However, in vitiligo it is exactly the opposite i.e., *DRB1\*07:01* is predisposing and DRB1\*03:01 is protective. These data suggest the autoantigenic peptides presented by different MHC molecules are restricted by these common alleles in a population which are associated with the autoimmune disorders. Of about 350 alleles that were observed in the three patient groups and healthy controls, *A\*26:01* (9.98%), *A\*33:01* (11.53%), *B\*08:01* (12.42%), *B\*44:03* (10.75%), DRB1*\*03:01* (19.29%) and *DRB1\*07:01* (27.49%), were quite common alleles in the healthy North Indians that were associated with the three autoimmune disorders discussed above.

Our results suggest affinity of auto-antigenic peptides for predisposing MHC class-II and class-I molecules which may be involved in orchestrating (through CD4+ T cells) and implementing the autoimmune responses (through CD8+ T cells) in Type 1 diabetes, vitiligo and hypopara‐ thyroidism.

### **Acknowledgements**

We are thankful to the study participants: cases and the controls that provided blood samples for the study. Authors are thankful to Dr. Mitali Mukerji, IGIB, Delhi, for providing access to the Indian Genome Variation Consortium samples that have been used as unaffected controls in these studies. The projects were funded in part by grants from Department of Science and Technology (DST), Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India and partly by Core funds of National Institute of Immunology, New Delhi, India. Diabetes work was supported by Department of Science & Technology, New Delhi, India, grant No. SP/SO/B54/98. Vitiligo work was supported by grants for 'Genetic and autoimmune factors associated with vitiligo' (Grant number BT/PR6653/Med/12/258/2005) and the 'Program support for Skin pigmentation and melanocyte-keratinocyte biology' (grant number BT/01/COE/07/07) from the Department of Biotechnology, India. The hypoparathyr‐ oidism work was sponsored by Department of Biotechnology, grant number BT/ PR15022/Med/30/589/2010. The patient sample for this work came from All India Institute of Medical Sciences (AIIMS), New Delhi and Dr. Ram Manohar Lohia Hospital (RMLH), New Delhi, India. We would like to thank Dr. R. Goswami, Department of Endocrinology and Metabolism, (AIIMS) for T1D and hypoparathyroidism samples. We would like to acknowl‐ edge Dr. H. Kar (RMLH), Dr. V. K. Sharma (AIIMS) and Dr. Somesh Gupta (AIIMS), the dermatologists for providing the vitiligo samples. We thankfully acknowledge Dr. Rashee‐ dunnisa Begum and her team for providing vitiligo samples for the replication study from Gujarat. We would like to acknowledge the students and project fellows who have been involved in doing this work. Mr. Kapoor Chand's technical support is acknowledged.

### **Author details**

predisposition and protection from these diseases. Our data shows that haplotypes *B\*8- DRB1\*03:01*, *B\*50-DRB1\*03:01* and *B\*58-DRB1\*03:01* were significantly increased in T1D patients [12]. *A\*26:01-B\*08:01-DRB1\*03:01* and *A\*26:01* with other B-locus alleles were signif‐ icantly increased in hypoparathyroidism [16]. And in yet another autoimmune disorder, vitiligo, *A\*33:01-B\*44:03-DRB1\*07:01* is the predisposing haplotype and *A\*26:01-B\*08:01- DRB1\*03:01* seems to be protective haplotype, with *DRB1\*07:01* having primary association with the disease [15]. While in T1D and vitiligo it is the MHC class-II alleles*DRB1\*03:01* and*DRB1\*07:01* respectively, which had primary association, in hypoparathyroidism it is the MHC class-I allele *HLA-A\*26:01* which has primary association. Interestingly, in T1D *DRB1\*03:01* is predisposing and *DRB1\*07:01* is protective from the disease. However, in vitiligo it is exactly the opposite i.e., *DRB1\*07:01* is predisposing and DRB1\*03:01 is protective.

**Figure 5. Amino acid signatures (single letter codes) of the peptide binding pockets and HLA supertypes of 23 HLA-A locus alleles observed in IH cases showing significant increase or decrease in the number of amino acids. N=259** 

**Figure 5.** Amino acid signatures (single letter codes) of the peptide binding pockets and HLA supertypes of 23 HLA-A locus alleles observed in IH cases showing significant increase or decrease in the number of amino acids. N=259 indi‐ cates 259 alleles were observed in 134 patients which were compared with amino acid signatures of 1698 alleles from

*p* **< 0.017 to 1.06x10-10, Significantly increased in IH as compared to controls**

*p* **< 0.017 to 4.79x10-10, Significantly reduced in IH as compared to controls**

significantly different suggesting that the significant increase in these alleles is due to their being in

 MHC molecules are extremely polymorphic, however, alleles with similar peptide binding pockets may be clustered in one group [6], called supertypes, such that HLA molecules in one supertype may present similar peptides[16]. Assignment of alleles in the supertypes have been done based on published motifs, binding data, shared repertoires of binding peptides and the amino acid sequences of different alleles at the peptide binding pockets [6]. Supertype A01 (which includes *A\*26:01*) was significantly increased in IH with 65.67% of the patients as compared to 39.36% of the controls having this supertype (figure 4 c). However, *A\*26:01* was observed only in 50.75% of the patients as compared to 9.98% of the controls. About 15% of the IH cases had different alleles of A01 supertype (other than *A\*26:01*)*,* which did not show statistically significant differences due to small numbers*.* Significant increase of A01 supertype suggests that similar auto-antigenic peptides may be getting presented by different MHC molecules which

**HLA-A N=259 Position Position Position Position Position Position Position supertypes** 

**A\*26:01 68 Y N V H N I A** A01 **A\*0101 22 F E M H N T L** A01 **A\*29:01 5 T Q V Q N T L** A01 **A\*30:01 4 S E V Q D T L** A01 **A\*32:01 3 F E V H S I A** A01 **A\*26:25N 1 Y N V P N T L** A01 **A\*11:01 42 Y E V Q D T L** A03 **A\*68:01 18 Y N V Q D T L** A03 **A\*24:02 28 S E V H N I A** A24 **A\*23:01 1 S E V H N I A** A24 **A\*23:15 1 S E V H N I A A24 A\*24:07 1 S E V Q N I A** A24 **A\*02:06 4 Y E V H D T L** A02 **A\*02:05 3 Y E V H D T L** A02 **A\*33:01 10 T N V H D T L** A03 **A\*33:03 2 T N V H D T L** A03 **A\*30:36 1 S E V Q D T L** A03 **A\*02:01 13 F E V H D T L** A02 **A\*02:11 12 F E V H D T L** A02 **A\*02:03 6 F E V H D T L** A02 **A\*03:01 7 F E V Q D T L** A03 **A\*31:01 6 T E V H D T L** A03 **A\*03:02 1 F E V Q D T L** A03

 **\*Alleles No. 9 63 67 70 77 80 81** 

linkage disequilibrium with the *A\*26:01*[16].

belong to the supertype [6, 16].

214 HLA and Associated Important Diseases

902 controls.

Rajni Rani and Archana Singh

Molecular Immunogenetics Group, National Institute of Immunology, New Delhi, India

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Functional Implications of MHC Associations in Autoimmune Diseases with Special Reference to...

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**Chapter 8**

**HLA in Gastrointestinal Inflammatory Disorders**

The human leukocyte antigen (HLA) region is located on chromosome 6p21.3 and encodes genes for the major histocompatibility complex (MHC). MHC genes are among the most polymorphic genes in the vertebrate genome. HLA region contains hundreds of genes with immunological function that is characterized by a high gene density, variability and extensive linkage disequilibrium [1]. HLA encodes proteins with critical role in immunity, including antigen processing and presentation, and self-recognition by immune cells, as ligands receptors, cytokines, signaling factors, heat shock proteins, and transcription regulators. The HLA region is involved in many biological processes such as histocompatibility, inflammation,

HLA genes have many possible variations, allowing each person's immune system to react to a wide range of foreign invaders. Some HLA genes have hundreds of identified versions (alleles); more than 100 diseases involving abnormal immune function and some forms of cancer have been associated with different alleles of HLA genes [3,4]. Is paradoxical that genes evolved to protect the host against infectious diseases are cause of many genetic disorders themselves. It is often unclear what role HLA genes play in the risk of developing these

The MHC is the most gene-dense region of the mammalian genome playing a key role in immune defense and autoimmunity [5]. The complex is divided into three subgroups (MHC classes I, II, and III). The class I region is located at the telomeric end of the MHC and encodes heterodimeric peptide-binding proteins as well as antigen processing molecule. The class I family is divided into classical (HLA-A, -B, -C) and non-classical (HLA-E, -F and –G) genes. The classical genes are highly polymorphic and constitutively expressed by most somatic cells, and are co-dominantly expressed on the cell surface and responsible for presenting intracell‐

> © 2014 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ligands for immune cell receptors, and the complement cascade [2].

M.I. Torres, T. Palomeque and P. Lorite

http://dx.doi.org/10.5772/57497

**1. Introduction**

**1.1. HLA GENE**

diseases.

Additional information is available at the end of the chapter
