Associations of killer cell immunoglobulin like receptors with rheumatoid arthritis among North Indian population

Accepted Manuscript Associations of killer cell immunoglobulin like receptors with rheumatoid arthritis among North Indian population Swayam Prakash, Shahnawaz Alam, Uddalak Bharadwaj, Amita Aggarwal, Ram Nath Mishra, Suraksha Agrawal PII: DOI: Reference:

S0198-8859(14)00153-0 http://dx.doi.org/10.1016/j.humimm.2014.05.014 HIM 9383

To appear in:

Human Immunology

Received Date: Accepted Date:

19 November 2013 29 May 2014

Please cite this article as: Prakash, S., Alam, S., Bharadwaj, U., Aggarwal, A., Mishra, R.N., Agrawal, S., Associations of killer cell immunoglobulin like receptors with rheumatoid arthritis among North Indian population, Human Immunology (2014), doi: http://dx.doi.org/10.1016/j.humimm.2014.05.014

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

1

Associations of killer cell immunoglobulin like receptors with rheumatoid arthritis among North Indian population Swayam Prakash1, Shahnawaz Alam2, Uddalak Bharadwaj3, Amita Aggarwal4, Ram Nath Mishra4, Suraksha Agrawal1, * 1

Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raibareily

Road, Lucknow, Uttar Pradesh, India 2

Department of Nephrology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raibareily

Road, Lucknow, Uttar Pradesh, India 3

Margaret M. and Albert B. Alkek Department of Medicine, Section of Infectious Diseases, Baylor

College of Medicine, Houston, Texas, United States of America 4

Department of Immunology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raibareily

Road, Lucknow, Uttar Pradesh, India

* Corresponding author Suraksha Agrawal, Department of Medical Genetics, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow (UP) 226014, India Phone: 091-522 -668004-8 Ext 4338, 4346, 4347, 4339 Fax No. 091-522 -6680973/6680017 Email: [email protected] Key Words: Killer Immunoglobulin like receptor (KIR); Rheumatoid Arthritis (RA); Autoimmune Disease; Genetic Association; Polymerase Chain Reaction-Sequence Specific Primer (PCR-SSP). Abbreviated title: Genetic association of KIRs with RA

2

Abstract Introduction: Rheumatoid Arthritis (RA) is an autoimmune and chronic inflammatory disease of unknown etiology. Killer cell immunoglobulin-like receptors (KIR) expressed on surface of natural killer cells and CD28 null T-cells which are present in synovial membrane of RA. The present study has evaluated associations of KIR genes with RA among North Indian population from Uttar Pradesh. Material and methods: KIR genotypes were determined in 100 RA cases and 100 healthy controls using sequence specific primer polymerase chain reaction (PCR-SSP) method. Results: RA cases positive for KIR3DS1 (OR=1.17, p-value=0.0498) and KIR2DS2 (OR=2.21, p-value=0.0120) showed risk associations. While, KIR2DL2 (OR=0.40, p-value=0.0026), KIR2DL3 (OR=0.44, p-value=0.0283) and KIR3DL1 (OR=0.32, p-value=0.0012) showed protective associations. Increased incidence of BB genotype (45%) was revealed among cases. Risk association was noted against telomeric region (OR=2.12, p=0.0120) genes for RA. Pairwise linkage disequilibrium (LD) analysis among RA cases revealed KIR2DS1-2DL1 (D'=0.83, r2=0.36), KIR3DL1-3DS1 (D'=1, r2=0.58) and KIR2DL1-2DL2 (D'=1, r2=0.61) to be in significant LD. KIR3DS1 and KIR2DS3 genes showed significant risk associations among RA patients with extra-articular

manifestations

(OR=5.14,

p-value=0.0018; OR=3.79,

p-

value=0.0106) and in limited range of motion in affected joints (OR=14.91, p-value=0.0001; OR=2.95, p-value=0.0126). Conclusion: The KIR activating genes have risk association with RA in the present study.

3

1. Introduction Rheumatoid arthritis (RA) is an autoimmune disease affecting 1% of the world population, in which chronic inflammatory response develops [1]. RA is determined by an inflammation of the synovial membrane leading to destruction of cartilage and bone [2]. Several studies [3-5] have investigated the potential involvement of Natural Killer (NK) cells in RA. NK cell-like lymphocytes have been demonstrated in the affected joints in RA patients. It is proposed that NK cells may have a regulatory role through the release of cytokines and cross-talk with dendritic cells [3]. It is also apparent that cytokines play a major role in RA. Reduced NK cell activity has been shown to occur in patients with RA [6-9]. The NK cells display killer cell immunoglobulin-like receptors (KIRs). The KIRs comprise a multigene family of receptors. Highly polymorphic KIR haplotypes are located on chromosome 19q13.4 and are classified into group A or B. The classification is based on the number and type of genes which encode inhibitory and activating KIRs. Group B haplotypes are determined due to the presence of one or more of the KIR2DL5, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS5 and KIR3DS1 genes, where-else , group A haplotypes are characterized by the absence of all these genes [10, 11]. KIRs are expressed on the surface of CD8+ T cells, CD4+ CD28null T-cells, and natural killer (NK) cells. Activatory KIR functions as a co-stimulatory molecule on CD4+ CD28null T-cells, which may lead to the clonal expansion of these cells [4]. RA patients show expanded CD4+ CD28 null T-cells, which play a direct role in acute coronary syndrome [12] like vascular injury. Therefore, KIR may be involved in the pathogenesis of RA. In contrast, CD56 bright KIR-negative cells have been shown to accumulate in the synovial fluid of patients with RA [13]. Previous studies revealing the involvement of KIR receptors in RA have presented a debatable

4

picture. KIR2DS2 is reported to be a risk factor among Caucasian patients with RA [9] and KIR2DS4 was shown to be increased in patients with RA from Taiwan [14]. However, a study among the Japanese population has not found any significant change in KIR gene frequencies in patients with RA as compared to controls [15]. In addition to studies on RA, association of activating KIR receptors KIR2DS1 [16, 17] and KIR2DS2 [16] have been reported in psoriatic arthritis. While higher prevalence of KIR2DL5 [18] and KIR2DS1 [18, 19] have been reported among psoriasis vulgaris cases. The conflicting evidence, in the frequency of KIR receptors, led us to examine the KIR frequency in a cohort of patients and controls with chronic RA from north India. 2. Material and methods 2.1. Genomic DNA Samples A total of 100 North Indian patients with chronic RA (Female = 67 and Male = 33) fulfilling the criteria of the American College of Rheumatology were examined. Age range was 29-65 years, with an average age of 46 years, and all patients were treated with at least one disease-modifying anti-rheumatic drug. We further categorized the RA cases into three broad groups i.e., (i) Cases with all extra articular manifestations (N=21) placing the cases who had systemic manifestations like vasculitis, visceral nodules, sjogren’s syndrome and pulmonary fibrosis , (ii) RA limited to joints (N = 34) and (iii) Other extra articular manifestations (N = 45) placing the cases who had destructive polyarthritis along with extra articular organ involvement and assessed the association of

various KIR gene content in these categories. The normal healthy controls consisted of 100 unrelated north Indian individuals without any history of RA or any other autoimmune diseases (Female = 60 and Male = 40) and were from the same ethnic background. 5ml of whole blood from RA cases and healthy controls was collected in EDTA coated collection vials and DNA

5

was extracted using Quiagen kits (Brand GMbH and Co KG, Cat # 51104). The study was approved by the institute ethics committee and was performed as per the ethical standards laid down by the Declaration of Helsinki. Informed written consent was obtained from all individuals prior to their inclusion in the study.

2.2. KIR Genotyping

KIR typing was performed using a sequence-specific primer (SSP) approach (Supplementary Table 1). PCR-SSP method was implemented to study the KIR complement of human genome. The DNA samples of controls as well as RA patients were typed for the KIR genes responsible for inhibitory signals (2DL1, 2DL2, 2DL3, 3DL1, 3DL2, 3DL3, 2DL4 and 2DL5), those for activating signals (2DS1, 2DS2, 2DS3, 2DS4, 2DS5 and 3DS1), and two pseudo genes (2DP1 and 3DP1), based on the primers described earlier [20, 21] Positive and negative controls were included in every reaction.

2.3. Statistical Analysis Gene and genotype carrier frequency of KIR was determined by direct counting. Frequency differences between the RA patients and control groups for the individual inhibitory and activating KIR genes as well as for the haplogroups was tested for significance at 95% confidence limits using Fisher’s exact test. Bonferroni correction was applied keeping in view the multiple comparisons. The significance levels of the inhibitory or activating KIR genes between RA patients and controls were tested and a linear model with a logistic link was used to test the association between increasing/decreasing extent of activating/inhibiting interactions and the prevalence of RA. p-value ≤ 0.05 were considered significant. The Linkage Disequilibrium

6

has been calculated as per the Lewontin’s principle; strong positive LD has been assigned to the KIR genes having a LD score ranging between 0.8 and 1. Further r2 values for all the KIR genes were calculated according to the Cramer’s principle to strengthen the genetic association No significant differences occurred in the number of females vs. the number of males having each of the KIR genes. Statistical power of the study and the sample size estimation was carried out using

G*Power

version

2

(Heinrich

Heine,

Universitat

Dusseldorf,

Germany).

3. Results Significant statistical power was calculated against patient and control groups (RA-Control: 0.96, RA limited to joints-control: 0.80, other extra articular manifestations-control: 0.87) which justified the sample size for the present study (Supplementary Table 2). However, for all extra articular manifestations the sample size was inadequate as it was evident from the insignificant statistical power of the study.

3.1. Variation in KIR gene content

Decreased incidence of KIR2DL2, 2DL3 and 3DL1 genes were observed among RA patients. Odds of occurrence of RA in patient group were linearly related to the increased incidence of activating KIR2DS2 (p = 0.0120, OR = 2.21, 95%CI = 1.22-3.99) and KIR3DS1 (p = 0.0039, OR = 1.53, 95%CI = 0.29-1.96). On the other hand there was a significant protective effect of inhibitory KIR2DL2 (p = 0.0026, OR = 0.40, 95%CI = 0.22-0.71), 2DL3 (p = 0.0283, OR = 0.44, 95%CI = 0.22-0.88) and 3DL1 (p=0.0012, OR=0.32, 95%CI=0.16-0.63) gene among patients and controls. Other KIR inhibitory (KIR2DL1 and 2DL5) and activating (KIR2DS2, 2DS3, 2DS4 and 2DS5) genes revealed no statistical significance against case and control

7

groups.

3.2. Genotype and haplo-group variation Genotypic data obtained for the RA patients and controls was classified into group A and Bx haplogroups (Table-1). The A haplogroup carries a fixed set of nine genes including a single activating gene (2DS4), and various inhibitory genes, and is designated as inhibitory haplogroup. On the other hand haplogroup-Bx is characterized by the presence of additional activating genes and absence of group-A specific variable inhibitory KIR genes (KIR2DL2, 2DL3 and 3DL1). The total KIR profiles among patients and controls are shown in Table-2. We have observed significant difference for the KIR BB and AB genotypes. However, an increased incidence of BB genotype among patients (45.0%) as compared to controls (34.0%) was found. AB genotype was found in higher frequency among the controls (54.0%) in comparison to RA (42.0%) cases. The telomeric region (T4) comprising of KIR2DL5, KIR3DS1, KIR2DS5 and KIR2DS1 genes showed susceptibility (p=0.0120, OR=2.12, 95%CI=1.22-3.99) to RA. Among the four KIR Bx Subgroups namely C4T4, C4Tx, CxT4, CxTx; C4Tx showed protective association (p=0.0001, OR=0.24, 95%CI=0.12-0.48) for RA. 3.3. Linkage disequilibrium (LD) analysis LD results showed same trend as obtained on the basis of allele frequency and KIR profiles. Strong positive LD was observed between 2DS1-2DL1 (D'=0.83, r2=0.36), 3DL1-3DS1 (D'=1, r2=0.58), 2DL1-2DL2 (D'=1, r2=0.41) and 2DL2-2DL3 (D'=1, r2=0.61) among RA cases. Along with these few more positive LD’s were observed against 2DL2-2DS2 (D'=1, r2=0.67), 2DS12DL5 (D'=0.95, r2=0.77), 2DS3-2DL5 (D'=0.98, r2=0.70), 2DS5-2DL5 (D'=0.95, r2=0.64) and 2DL1-2DL3 (D'=0.98, r2=0.81) (Table-3).

8

3.4.Gene content distribution in different clinical parameters of RA While comparing the all extra articular cases with control significant risk associations were found against KIR2DS3 (p-value=0.0106, O.R. = 3.79) and KIR3DS1 (p-value=0.0018, O.R. = 5.14) genes. KIR2DS5 revealed susceptible association (p-value = 0.0115, O.R = 5.16) when comparison was drawn among all extra articular cases with RA cases limited to joints. Comparison between RA limited to joints cases and healthy controls showed susceptibility for KIR3DS1 (p-value = 0.0001, O.R = 14.9) and KIR2DS3 (p-value = 0.0126, O.R = 2.95) genes. Protective associations against all genes were concurred while comparison was made for controls with cases with other extra articular manifestations (Table 4).

4. Discussion

Most of the genetic association studies dealing with KIRs are on autoimmune diseases and viral infections. Polygenic and multi-allelic diversity of KIR has made the KIR gene locus a promising target for disease association studies. In relation to autoimmune diseases involvements of specific KIRs have been suggested. Signaling pathways involved with the aberrant expression of KIRs on T cells have elucidated the importance of pathogenesis of rheumatoid arthritis [22]. Our results revealed significance for KIR2DL2, KIR2DL3, KIR3DL1, KIR2DS2 and KIR3DS1 genes with RA. KIR2DL2, KIR2DL3 and KIR2DS2 share the HLA class-I ligand with group alleles located on HLA-C locus. These KIRs are further associated with CD28 null T-cells [23]. While, KIR3DL1 and KIR3DS1 genes have been associated with HLA-Bw4 ligand. The present study was designed to clarify the relevance of KIR gene polymorphism in RA. We have investigated cases and controls drawn from a homogeneous population of Uttar Pradesh,

9

India. Thus, the bias because of population stratification is limited. The results are similar between cases and controls barring few KIR loci and the concurrence of some of the KIR gene frequencies with that in the literature as provided in the allelefrequencies.net database [24]. KIR2DS2 (OR=2.21) and KIR3DS1 (OR=1.53) genes showed genetic susceptibility in our study. Upon comparing our results with other populations we found that KIR2DS2 is common in western European and Polish RA populations studied by Yen et al [25]. On the contrary no association of these genes was revealed among the Caucasian population with RA from Northern Ireland [26].

The results of the present study are confiscatory with a previous finding of KIRs association (Table 5) with RA. This may be due to racial differences which contribute to KIR gene frequencies. The KIR2DS4 frequency in the Taiwan [14] control populations was not very high as compared to other world populations [24]. One previous study on psoriatic arthritis reported the associations of KIR genes with RA on the basis of strength of inhibition/activation [16]. The results were explained as a continuum moving from high inhibition (absence of activating genes KIR2DS1/S2) to high activation (presence of both KIR2DS1 and KIR2DS2).

We have observed that the KIR2DL3 gene frequency was decreased (OR=0.44) in RA. It has been earlier reported that IFN-γ plays a pivotal role in RA auto-reactivity [27]. KIR2DL3 gene encodes an inhibitory receptor for KIR2DL3. The inhibitory receptor modulates intracellular signaling which in turn results in the blockade of cytotoxic secretion of IFN-γ like cytokines [28]. Keeping these facts in view the protective role of KIR2DL3 cannot be denied.

KIR2DS2 gene revealed two fold increased risk (OR=2.21), suggesting that it may act as a

10

susceptibility factor for RA. KIR2DS2 also reportedly play a prominent role in the increased IFN-γ secretion through two biological mechanisms. These two mechanisms result in enhanced secretion of perforin and granzyme which in turn lead to destruction of joints in RA patients [9]. Firstly, intracellular signaling in NK cell has been activated by KIR2DS2 upon its non-covalent association with DAP-12 adapter molecule. DAP-12 adapter molecule possesses domains for immune tyrosine like activating motifs. Secondly, in the CD28null T-cells KIR2DS2 gene has been reported to act as a co-stimulatory molecule. This phenomenon causes increased T-cell receptor activation [4]. KIR2DL2 gene was found with low incidence among RA patients from north India in the present study. Its role as inhibitor of intracellular signaling could be blocked in RA patients by two hypothetical mechanisms. The first mechanism suggests that differences in the sequence of peptides contents in HLA class I molecules can regulate the activity of NK cell antagonizing inhibition mediated by KIR2DL2 favoring activation by its counterpart KIR2DS2 [29]. While the second mechanism proposes that antibodies to KIR2DL2 are found in 30% of RA patients and probably are involved in the breakdown of self tolerance [30]. null

CD4- CD28

T cells have been found to express NKG2D in the blood and synovial tissue of

patients with RA [23]. NKG2D is absent on normal T cells but is induced by TNF-α and IL-15, cytokines which are present in synovial and sera of patients with RA. The ligands for NKGD are the major histocompatibility complex class I chain-related genes MICA and MICB, and alleles of these genes have been found to be associated with RA [3]. Interestingly the expression of KIRs, notably KIR2DS2, on a subset of T cells and not NK cells, has previously been shown to play an important role in RA, in particular towards vascular complications of the disease [31]. To conclude, initial studies which have shown significant associations of RA with KIR gene

11

content needs further confirmation in independent cohorts. However, the major drawback of studies related to KIRs is that it is difficult to replicate such studies with conflicting results. This has lead to uncertainty over what might be real associations. In this instance, we have not able to confirm certain previous studies related to the association of KIR genes in context of RA. To overcome such inconsistencies in results, there is a need to increase the sample size and to carry out such studies in different ethnic groups. The role of other non-KIR like receptors on the NK cells also cannot be ignored which are involved in T-cell auto-reactivity hence leading to the pathogenesis of RA.

Acknowledgements: We are thankful to the Department of Biotechnology, New Delhi for carrying out this work.

12

References 1. McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011; 365(23):2205-2219. 2. Ritchlin, C. T. Mechanisms of erosion in rheumatoid arthritis. J. Rheumatol. 2004; 31: 1229-1237. 3. Pridgeon C, Lennon GP, Pazmany L, Thompson RN, Christmas SE, Moots RJ. Natural killer cells in the synovial fluid of rheumatoid arthritis patients exhibit a CD56bright, CD94 bright,CD158 negative phenotype. Rheumatology 2003; 42(7): 870-8. 4. Namekawa T, Snyder MR, Yen JH Goehring BE, Leibson PJ, Weyand CM et al. Killer Cell activating receptors function as costimulatory molecules on CD4+CD8null T cells clonally expanded in Rheumatoid Arthritis. J Immunol 2000; 165(2):1138-45. 5. Brennan FM, McInnes IB. Evidence that cytokines play a role in rheumatoid arthritis. J Clin Invest 2008; 118(11):3537-45. 6. Karsh J, Dorval G, Osterland CK. Natural cytotoxicity in rheumatoid arthritis and systemic lupus erythematosus. Clin Immunol Immunopathol 1981; 19:437–46. 7. Combe B, Pope R, Darnell B, Talal N. Modulation of natural killer cell activity in the rheumatoid joint and peripheral blood. Scand J Immunol 1984; 20:551–8. 8. Lipsky PE. Natural killer cell function in rheumatoid arthritis. Clin Exp Rheumatol 1986; 4: 303–5. 9. Yen J-H, Moore BE, Nakajima T, Scholl D, Schaid DJ, Weyand CM et al. Major histocompatibility complex class I-recognising receptors are disease risk genes in rheumatoid arthritis. J Exp Med 2001; 193:1159–67.

13

10. Uhrberg M, Valiante NM, Shum BP, Shilling HG, Lienert-Weidenbach K, Arnett KL et al. Human diversity in killer cell inhibitory receptor genes. Immunity 1997 7:753-63 11. Hsu KC, Chida S, Geraghty DE, Dupont B. The killer cell immunoglobulin-like receptor (KIR) genomic region: gene-order, haplotypes and allelic polymorphism. Immunological Review 2002 190:40-52 12. Weyand CM, Goronzy JJ, Liuzzo G, Kopecky SL, Holmes DR Jr, Frye RL. T-cell immunity in acute coronary syndromes. Mayo Clin Proc 2001; 76:1011–20. 13. Dalbeth N, Cullan MF. A subset of natural killer cells is greatly expanded within inflamed joints. Arthritis Rheum 2002; 46:1763–72. 14. Yen JH, Lin CH, Tsai WC, Wu CC, Ou TT, Hu CJ et al. Killer cell immunoglobulin-like receptor genes repertoire in rheumatoid arthritis. Scand J Rheumatol 2006; 35: 124–7. 15. Miyashita R, Tsuchiya N, Matsuta K, Yabe T, Tokunaga K. An association study: KIR and HLA genotypes in the Japanese patients with rheumatoid arthritis. In:Abstract from 8th Annual Meeting of Society for Natural Immunity. Basel: Karger AG, 2004; 56. 16. Nelson GW, Martin MP, Gladman D, Wade J, Trowsdale J, Carrington M. Cutting edge: heterozygote advantage in autoimmune disease: hierarchy of protection/susceptibility conferred by HLA and killer Ig-like receptor combinations in psoriatic arthritis. J Immunol 2004; 173: 4273–6. 17. Williams F, Meenagh A, Sleator C, Cook D, Fernandez-Vina M, Bowcock AM et al. Activating killer cell immunoglobulin-like receptor gene KIR2DS1 is associated with psoriatic arthritis. Hum Immunol 2005; 66: 836–41. 18. Suzuki Y, Hamamoto Y, Ogasawara Y, Ishikawa K, Yoshikawa Y, Sasazuki T et al. Genetic polymorphisms of killer cell immunoglobulin-like receptors are associated with

14

susceptibility to psoriasis vulgaris. J Invest Dermatol 2004; 122:1133–6. 19. Łuszczek W, Mańczak M, Cisło M, Nockowski P, Wiśniewski A, Jasek M et al. Gene for the activating natural killer cell receptor, KIR2DS1, is associated with susceptibility to psoriasis vulgaris. Hum Immunol 2004; 65:758–66. 20. Vilches C, Castano J, Gomez-Lozano N, Estefanıa E. Facilitation of KIR genotyping by a PCR-SSP method that amplifies short DNA fragments. Tissue Antigens 2007; 70(5):415– 22. 21. Murdoch S, Soed M, Kircheisen R, Mazhar B, Slim R. Detailed gene and allele content analysis of three homozygous KIR haplotypes. Tissue Antigens 2006; 68(1):72–7. 22.

Mavropoulos A, Rigopoulou EI, Liaskos C, Bogdanos DP, Sakkas LI. The role of p38 MAPK in the aetiopathogenesis of psoriasis and psoriatic arthritis. Clin Dev Immunol 2013;2013:569751.

23. Fasth AE, Björkström NK, Anthoni M, Malmberg KJ, Malmström V. Activating NK-cell receptors co-stimulate CD4+CD28- T cells in patients with rheumatoid arthritis. Eur J Immunol 2010; 40(2):378-87. 24.

Gonzalez-Galarza FF, Christmas S, Middleton D, Jones AR. Allele frequency net: a database and online repository for immune gene frequencies in worldwide populations. Nucleic Acid Research 2011; 39, D913-D919.

25.

Yen JH, Moore BE, Nakajima T, Scholl D, Schaid DJ, Weyand CM et al. Major Histocompatibility Complex Class I-recognizing Receptors Are Disease Risk Genes in Rheumatoid Arthritis. J Exp Med 2001; 193(10):1159-67.

15

26. Middleton D, Meenagh A, Wright GD. No association in frequency of KIR receptors in patients with rheumatoid arthritis from Northern Ireland. Tissue Antigens 2007; 69(6):577-82. 27. Flodström-Tullberg M, Bryceson YT, Shi FD, Höglund P, Ljunggren HG. Natural killer cells in human autoimmunity. Curr Opin Immunol 2009; 21(6):634-40. 28. Rajagopalan S, Long EO. Understanding how combinations of HLA and KIR genes influence disease. J Exp Med 2005; 201(7):1025-9. 29. Fadda L, Borhis G, Ahmed P, Cheent K, Pageon SV, Cazaly A et al. Peptide antagonismas a mechanism for NK cell activation. Proc Natl Acad Sci U S A. 2010; 107(22):10160-5. 30. Matsui T, Otsuka M, Maenaka K, Furukawa H, Yabe T, Yamamoto K et al. Detection of Autoantibodies to Killer Immunoglobulin Like Receptors Using Recombinant Fusion Proteins for Two Killer Immunoglobulin-Like Receptors in Patients With Systemic Autoimmune Diseases. Arthritis Rheum 2001; 44(2):384-8. 31. Ramírez-De los Santos S, Sánchez-Hernández PE, Muñoz-Valle JF, Palafox-Sánchez CA, Rosales-Rivera LY, García-Iglesias T et al. Associations of killer cell immunoglobulin like receptor genes with rheumatoid arthritis. Disease Markers 2012; 33:201-206.

Table 1 Distribution of KIR Genes, Genotypes, Haplogroups and Bx linkage Groups among Rheumatoid Arthritis Patients and Controls in North Indian Population Sl.No

Gene

Patients [N=100(%)]

Controls [N=100(%)]

p-value

OR

95% CI

KIR Gene Carrier Frequencies 1

KIR2DL1

84 (84.0%)

92(92.0%)

0.1264

0.45

0.18-1.22

2

KIR2DL3

70 (70.0%)

84(84.0%)

0.0283*

0.44

0.22-0.88

3

KIR2DL2

46 (46.0%)

68(68.0%)

0.0026*

0.40

0.22-0.71

4

KIR2DL5

53 (53.0%)

66(66.0%)

0.0836

0.58

0.32-1.02

5

KIR3DL1

63 (63.0%)

84(84.0%)

0.0012*

0.32

0.16-0.63

6

KIR3DL3

100(100%)

100(100%)

-

-

-

7

KIR2DL4

100(100%)

100(100%)

-

-

-

8

KIR3DL2

100(100%)

100(100%)

-

-

-

9

KIR2DS2

45 (45.0%)

27 (27.0%)

0.0120*

2.21

1.22-3.99

10

KIR2DS3

23 (23.0%)

30(30.0%)

0.3364

0.69

0.37-1.31

11

KIR3DS1

52 (52.0%)

28(28.0%)

0.0498*

1.17

0.67-2.04

12

KIR2DS5

44 (44.0%)

53(53.0%)

0.2576

0.69

0.39-1.21

13

KIR2DS1

28 (28.0%)

42(42.0%)

0.0536

0.53

0.29-0.96

14

KIR2DS4

61 (61.0%)

69(69.0%)

0.2994

0.70

0.39-1.26

15

KIR2DP1

72 (72.0%)

89(89.0%)

0.0039*

0.31

0.14-0.68

KIR Genotype and Haplogroup frequencies 1

AA

8(8.0%)

10(10.0%)

0.8056

0.78

0.29-2.07

2

AB

42(42.0%)

54(54.0%)

0.1193

0.61

0.35-1.07

3

BB

45(45.0%)

34(34.0%)

0.1478

1.58

0.89-2.81

4

A

8(8.0%)

10(10.0%)

0.8056

0.78

0.29-2.07

5

Bx

87(87.0%)

88(88.0%)

1.0000

0.91

0.39-2.11

KIR Bx Subgroup (Linkage Groups) Frequencies 1

C4T4

19(19.0%)

13(13.0%)

0.3350

1.57

0.72-3.38

2

C4Tx

14(14.0%)

40(40.0%)

<0.0001*

0.24

0.12-0.48

3

CxT4

54(54.0%)

42(42.0%)

0.1193

1.62

0.92-2.83

4

CxTx

10(10.0%)

11(11.0%)

1.0000

0.89

0.36-2.22

5

C4

33(33.0%)

53(53.0%)

0.0065*

0.43

0.24-0.77

6

T4

73(73.0%)

55(55.0%)

0.0120*

2.12

1.22-3.99

* = statistically significant (p<0.05).

Genotype ID’s have been given as per the Allelefrequencies.net format.

Genotype ID

Genotype

Bx Bx Bx Bx Bx Bx AA Bx Bx Bx AA Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx Bx

33 234 55 38 9 233 195 337 27 400 1 433 20 19 202 2 93 21 11 18 79 17 413 13 4 64 336 438

AB BB AB AB AB AB BB AB AB AB AA AB AB AB AB AB BB AB AB AB BB AB BB AB AB AB AB BB

Linkage Group

Haplotype Group

KIR3DP1

KIR2DP1

KIR3DL3

KIR3DL2

KIR2DL4

KIR2DS5

KIR2DS3

KIR2DS2

KIR2DS1

KIR3DS1

KIR2DL5

KIR2DL2

KIR2DS4

KIR2DL3

KIR2DL1

KIR3DL1

Table 2 KIR genotyping profiles of rheumatoid arthritis cases

CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxTx CxT4 C4T4 CxTx C4Tx CxT4 CxT4 CxTx CxTx C4Tx CxTx CxTx CxTx CxTx

Table 3 Linkage Disequilibrium analysis for 12 KIR genes among rheumatoid arthritis cases Gene

KIR3DL1

KIR2DL1

KIR2DL3

KIR2DS4

KIR2DL2

KIR2DL5

KIR3DS1

KIR2DS1

KIR2DS2

KIR2DS3

KIR2DS5

KIR2DP1

KIR3DL1

*

0.0359

0.8120

0.7027

0.0411

0.0609

0.5802

0.1674

0.0119

0.0006

0.0818

0.0225

KIR2DL1

1

*

0.3391

0.0026

0.4117

0.0021

0.00009

0.3669

0.1118

0.0134

0.0333

0.1697

KIR2DL3

0.98

1

*

0.0039

0.6118

0.0020

0.0032

0.0007

0.0107

0.0110

0.0015

0.2095

KIR2DS4

0.93

0.24

0.17

*

0.0450

0.0757

0.3088

0.2043

0.0168

0.0018

0.1016

0.0924

KIR2DL2

1

1

1

0.94

*

0.1811

0.0011

0.0137

0.6753

0.0827

0.0531

0.0253

KIR2DL5

1

0.19

0.06

1

0.52

*

0.1904

0.7764

0.1801

0.7017

0.6432

0.00001

KIR3DS1

1

0.02

0.09

0.98

0.08

0.90

*

0.3724

0.0092

0.0721

0.2416

0.0185

KIR2DS1

0.92

0.83

0.04

0.91

0.26

0.95

0.70

*

0.0188

0.0171

0.4812

0.0078

KIR2DS2

0.33

1

0.18

0.35

1

0.58

0.15

0.18

*

0.1675

0.0143

0.0004

KIR2DS3

0.07

0.22

0.17

0.07

0.76

0.98

0.28

0.16

0.66

*

0.0016

0.0006

KIR2DS5

0.71

0.46

0.05

0.71

0.46

0.95

0.62

0.77

0.14

0.06

*

0.0029

KIR2DP1

0.19

0.52

1

0.43

1

0.01

0.34

0.25

0.05

0.09

0.17

*

The pair-wise LD values have been assigned as per the Mattiuz’s principle and the r2 value is calculated as per Cramer’s V statistics principle. The r2 values are showed above the diagonal. The D’* values are showed below the diagonal.

Table 4 Association of KIR gene cluster with clinical features of rheumatoid arthritis Frequency (%)

2DL1

2DL3

2DL2

2DL5

3DL1

3DL3

2DL4

3DL2

2DS2

2DS3

3DS1

2DS5

2DS1

2DS4

2DP1

Control (N=100)

92

84

68

66

84

100

100

100

39

30

28

53

42

69

89

Patients (N=100)

84

70

46

53

63

100

100

100

27

23

52

44

28

61

72

All Extra Articular Manifestations

13(61)

12(57)

9(47)

8(42)

11(56)

10(52)

4(21)

7(33)

10(50)

13(62)

14(66)

16(76)

11(52)

18(85)

7(33)

16(35)

16(35)

11(24)

27(60)

14(31)

19(42)

22(48)

23(51)

12(26)

12(54)

5(11)

4(9)

6(13)

13(29)

10(22)

6(17)

11(32)

17(50)

18(52)

18(53)

23(67)

8(23)

14(41)

17(50)

19(55)

29(85)

13(38)

16(47)

24(70)

11(32)

(N=21) Other Extra Articular Manifestations (N=45) RA limited to joints (N=34) p-value RA vs Control

p-value

0.1264

0.0283

0.0026

0.0836

0.0012

-

-

-

0.0120

0.3364

0.0498

0.2576

0.0432

0.2994

0.0039

All Extra Articular vs Control

p-value

0.0012

0.0142

0.0445

0.0256

0.0030

0.0001

0.0001

0.0001

0.4745

0.0106

0.0018

0.0567

0.4700

0.1813

0.0001

O.R.

0.14

0.25

0.35

0.31

0.20

0.004

0.001

0.002

1.42

3.79

5.14

2.83

1.51

2.69

0.06

p-value

0.0013

0.0943

0.7817

0.4052

1.0000

0.1661

0.7505

0.7757

1.0000

0.7808

0.1775

0.0115

0.7848

0.3279

1.0000

O.R.

7.58

2.78

0.75

0.54

0.97

0.43

0.76

0.71

0.90

1.28

0.34

5.16

1.23

2.50

1.04

p-value

0.0001

0.0001

0.0001

0.5750

0.0001

0.0001

0.0001

0.0001

0.1890

0.8434

0.0315

0.0001

0.0006

0.0001

0.0001

O.R.

0.04

0.10

0.15

0.77

0.08

0.003

0.004

0.005

0.56

0.84

0.32

0.08

0.21

0.18

0.03

p-value

0.0001

0.0001

0.0699

0.2186

0.0008

0.0001

0.0001

0.0001

0.3156

0.0126

0.0001

0.1662

0.6898

1.0000

0.0001

O.R.

0.01

0.09

0.47

0.57

0.21

0.01

0.001

0.003

1.56

2.95

14.91

0.54

1.22

1.07

0.05

p-value

0.1273

0.8143

0.0317

0.6472

0.0654

0.0398

0.0344

0.4952

0.0378

0.0110

0.0001

0.0023

0.0019

0.0003

0.4409

O.R.

2.57

1.15

0.32

1.33

0.40

0.34

3.10

1.49

0.36

0.28

0.02

0.15

0.17

0.16

0.59

All Extra Articular vs Limited to joints

Other Extra Articular vs Control

Limited to joints vs Control

Other Extra Articular vs Limited to joints

p<0.05=Statistically Significant; Frequency given in percentages (%); O.R.: Odds Ratio

5

Table 5 Distribution of KIR Genes among rheumatoid arthritis cases in different populations Sl.No

Gene

North India (N=100)

Caucasian (N=177)

Taiwan (N=122)

Mexico (N=100)

KIR gene Carrier frequency 1

KIR2DL1

84 (84.0%)

177 (99.4%)

98 (80.3%)

96 (96.0%)

2

KIR2DL3

70 (70.0%)

163 (92.1%)

81 (66.4%)

79 (79.0%)

3

KIR2DL2

46 (46.0%)

97 (54.8%)

70 (57.4%)

70 (70.0%)

4

KIR2DL5

53 (53.0%)

-

-

47 (47.0%)

5

KIR3DL1

63 (63.0%)

166 (93.8%)

105 (86.1%)

98 (98.0%)

6

KIR3DL3

100(100%)

-

-

100 (100.0%)

7

KIR2DL4

100(100%)

-

-

100 (100.0%)

8

KIR3DL2

100(100%)

-

109 (89.3%)

100 (100.0%)

9

KIR2DS2

27 (27.0%)

100 (56.6%)

41 (33.6%)

55 (55.0%)

10

KIR2DS3

23 (23.0%)

51 (28.8%)

19 (15.6%)

19 (19.0%)

11

KIR3DS1

52 (52.0%)

59 (33.3%)

45 (36.9%)

37 (37.0%)

12

KIR2DS5

44 (44.0%)

41 (23.2%)

-

36 (36.0%)

13

KIR2DS1

28 (28.0%)

64 (36.2%)

72 (59.0%)

41 (41.0%)

14

KIR2DS4

61 (61.0%)

151 (85.3%)

90 (73.8%)

96 (96.0%)

15

KIR2DP1

72 (72.0%)

-

-

97 (97.0%)