Mol Biol Rep DOI 10.1007/s11033-014-3280-9
Human leukocyte antigen (HLA)-C polymorphisms are associated with a decreased risk of rheumatoid arthritis Yiqun Zhang • Hui Zhang • Yong Huang Rongbin Sun • Ruiping Liu • Jie Wei
•
Received: 10 February 2013 / Accepted: 13 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Rheumatoid arthritis (RA) is an autoimmune rheumatological disease thought to have substantial genetic contributions. Several genetic factors involved in the susceptibility to psoriasis and psoriatic arthritis (PsA) have been identified with genome-wide association studies, including human leukocyte antigen (HLA)-C, junction adhesion molecule 2 (JAM2) and REL. Psoriasis and PsA may share many features in common with RA. We hypothesized that this polymorphism may contribute to RA susceptibility in a Chinese population. We studied HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL
Yiqun Zhang and Hui Zhang contributed equally to this study. Y. Zhang Department of Ophthalmology, Affiliated Hospital of Suzhou University, Changzhou No. 4 People’s Hospital, Changzhou 213001, China H. Zhang Y. Huang R. Sun R. Liu (&) Department of Orthopedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou 213003, China e-mail: [email protected] R. Liu Central Laboratory, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou 213003, China J. Wei Department of Orthopaedic Trauma, Beijing Jishuitan Hospital, Beijing 100035, China J. Wei (&) Department of Orthopaedic Trauma, Beijing Jishuitan Hospital, Beijing 100035, China e-mail: [email protected]
rs702873 G/A polymorphisms in 520 patients with RA and 520 controls in a Chinese population. HLA-C rs12191877 C/T polymorphism was in complete linkage disequilibrium (LD) (D0 = 1.0, r2 = 1.0) with HLA-C rs10484554 C/T polymorphism. When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the TT/ CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–0.99, p = 0.044). We found that the HLAC rs12191877 C/T polymorphism was also associated with a decreased risk of RA. HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms were not associated with the risk of RA. These results provide evidence that HLA-C polymorphisms are associated with a decreased risk of RA. Keywords HLA-C Polymorphisms Rheumatoid arthritis Molecular epidemiology Abbreviations CI confidence interval HLA human leukocyte antigen LD linkage disequilibrium OR odds ratio SNP single nucleotide polymorphism
Introduction Rheumatoid arthritis (RA) is characterized by synovitis, progressive erosions, and cartilage destruction. RA is an inflammatory disease and thought to have substantial genetic contributions [1]. Psoriasis and psoriatic arthritis (PsA) are also chronic immune-mediated inflammatory
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diseases associated with appreciable morbidity and disability [2, 3]. PsA shares many common features with RA. Some clinical features between RA and PsA overlap, so genetic susceptibility might also (at least in part) overlap. Several epidemiologic studies have examined the association between psoriasis and inflammatory arthritis to determine if PsA is a distinct entity rather than a chance association of psoriasis with an established inflammatory arthritis [4, 5]. PsA (but not RA) is linked with several major histocompatibility complex (MHC) class-I alleles [6]. Genome-wide association studies (GWAS) are the most popular and powerful methods for identifying susceptibility genes for complex diseases. Several genetic factors involved in susceptibility to psoriasis and PsA have been identified with GWAS [7]. The association of human leukocyte antigen (HLA)-C with PsA was found to be linked with early-onset psoriasis [8]. Within the chromosome 6p21 region codes for several genes important in the immune response (including HLA and non-HLA genes), three single nucleotide polymorphisms (SNPs) have shown strong evidence to be associated with psoriasis or PsA approximately 35 kb upstream of HLA-C (rs12212594, rs12191877 and rs10484554) [7, 9, 10]. One study found that HLA-C encoded variants unique to these haplotypes at the level of translated proteins, which increased the brisk of psoriasis, suggesting that HLA-C is a psoriasis susceptibility gene [11]. Furthermore, a meta-analysis of the PsA subsets of three independent GWAS revealed that the junction adhesion molecule 2 (JAM2) rs2829866 A/T polymorphism is associated with PsA [12]. The REL gene, a key modulator of the nuclear factor kappa B (NFjB) pathway, is known to share shared Crohn’s disease (CD) and psoriasis susceptibility loci [13]. However, although HLA-C, JAM2 and REL play important parts in the immune response, few studies have focused on the influences of HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms in the susceptibilities to RA. We therefore undertook a hospital-based case–control study to genotype 520 patients and 520 controls in a Chinese population.
Patients and methods Subjects We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study.
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Five hundred and twenty RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 [14] were consecutively recruited from the Changzhou Second Hospital-Affiliated Hospital of Nanjing Medical University (Changzhou, China), the Changzhou First Hospital (Changzhou, China), and the Changzhou Traditional Chinese Medical Hospital (Changzhou, China), between September 2010 and May 2012. The controls were patients without RA, matched RA for age (±5 years) and sex, and recruited from the same institutions during the same time period; most of the controls were admitted to the hospitals for the treatment of trauma. Each patient was interviewed by trained personnel using a pre-tested questionnaire to obtain information on demographic data and related risk factors for RA. After the interview, 2 mL of peripheral blood was collected from each subject. Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetraacetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was done by MALDI-TOF MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [15]. Cases and controls at a proportion of &1:1 were assayed. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom), and analyzed by MALDI-TOF MS. Genotype calling was done in real time with MassARRAY RT software (version 3.1; Sequenom), and analyzed using MassARRAY Typer software (version 4.0; Sequenom). For quality control, repeated analyses were undertaken on 10 % of randomly selected samples. Statistical analysis Differences in demographics, variables, and genotypes of the HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphism variants were evaluated using a v2 test. The associations between HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A genotypes and risk of RA was estimated by computing odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–
Mol Biol Rep Table 1 Patient demographics and risk factors in rheumatoid arthritis and controls Variable
RA cases (n = 520)
Controls (n = 520)
p
Age (years)
54.72 (± 15.27)
54.17 (± 10.50)
0.496
Female/male
390/130
385/135
0.722
Age at onset, years, mean ± SD
46.43 (± 13.28)
–
–
Disease duration, years, mean ± SD
8.34 (± 9.40)
–
–
Treatment duration, years, mean ± SD
7.03 (± 7.93)
–
–
RF-positive, no. (%)
414 (79.6 %)
–
–
ACPA-positive, no. (%)
269 (51.7 %)
–
–
CRP-positive, no. (%)
220 (42.3 %)
–
–
ESR, mm/h
36.93 (± 29.41)
–
–
DAS28
4.55 (± 1.53)
–
–
I
68 (13.1 %)
–
–
II III
236 (45.4 %) 183 (35.2 %)
– –
– –
IV
33 (6.3 %)
–
–
Functional class, no. (%)
RF Rheumatoid factor, ACPA Anti-cyclic citrullinated peptide antibody, CRP C-reactive protein, ESR Erythrocyte sedimentation rate, DAS28 RA disease activity score
Weinberg equilibrium (HWE) was tested by a goodness-offit v2 test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).
Results Characteristics of the study population The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (p = 0.496 and 0.722, respectively) for RA patients and controls. Among 1,040 DNA samples (520 RA patients and 520 controls), we successfully genotyped 1,018 (97.9 %) samples in the first step, then in the second step we genotyping the rest 22 samples and 104 (10 % of all samples) randomly selected samples. At last, the HLA-C rs10484554 C/T polymorphism was successful in 518 (99.6 %) RA patients and 520 (100.0 %) controls. Genotyping for HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A was successful ranging from 99.4 to 99.7 % (Table 2). The concordance rates of repeated analyses were
100 %. The genotype distributions of HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A in all subjects are illustrated in Table 3. The observed genotype frequencies for the polymorphism in controls were in HWE for HLA-C rs12212594 T/C (p = 0.895), HLAC rs12191877 C/T (p = 0.431), JAM2 rs2829866 A/T (p = 0.413) and REL rs702873 G/A (p = 0.993). However, the observed genotype frequencies for the polymorphism in controls were not in HWE for HLA-C rs10484554 C/T (p = 0.009). Associations between HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and the risk of RA The genotype frequencies of the HLA-C rs10484554 C/T polymorphism were 84.6 % (CC), 14.5 % (CT) and 1.0 % (TT) in RA patients, and 79.4 % (CC), 18.1 % (CT) and 2.5 % (TT) in controls (p = 0.040) (Table 3). When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the TT/CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–0.99, p = 0.044). When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the CT or TT genotype was not associated with the risk for RA (CT vs. CC, adjusted OR = 0.77, 95 % CI = 0.55–1.07, p = 0.116; TT vs. CC, adjusted OR = 0.38, 95 % CI = 0.13–1.08, p = 0.068). In the recessive model, when the HLA-C rs10484554 CC/CT genotypes were used as the reference group, the TT homozygote genotype was not associated with susceptibility to RA (adjusted OR = 0.40, 95 % CI = 0.14–1.12, p = 0.082). HLA-C rs12191877 C/T polymorphism was in complete linkage disequilibrium (LD) (D0 = 1.0, r2 = 1.0) with HLA-C rs10484554 C/T polymorphism. The genotype frequencies of the HLA-C rs12191877 C/T polymorphism were 84.5 % (CC), 14.5 % (CT) and 1.0 % (TT) in RA patients, and 79.4 % (CC), 18.1 % (CT) and 2.5 % (TT) in controls (p = 0.043) (Table 3). When the HLAC rs12191877 CC homozygote genotype was used as the reference group, the TT/CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–1.00, p = 0.047). When the HLA-C rs12191877 CC homozygote genotype was used as the reference group, the CT or TT genotype was not associated with the risk for RA (CT vs. CC, adjusted OR = 0.77, 95 % CI = 0.55–1.07, p = 0.123; TT vs. CC, adjusted OR = 0.38, 95 % CI = 0.13–1.08, p = 0.069). In the recessive model, when the HLA-C rs12191877 CC/CT genotypes were used as the reference group, the TT
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Mol Biol Rep Table 2 Primary information for HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms Genotyped SNPs
Chr Pos
Regulome DB scorea
Location
MAF for Chinese in database
MAF in our controls (n = 520)
p value for HWE test
% genotyping value
HLA-C rs10484554 C/T
6p21.3
No data
Intergenic region
0.102
0.115
0.655
99.8
HLA-C rs12212594 T/C
6p21.3
No data
Intergenic region
0.065
0.090
1.000
99.7
HLA-C rs12191877 C/T JAM2 rs2829866 A/T
6p21.3 21q21.2
6 6
Intergenic region Intron
0.094 0.375
0.115 0.369
0.752 0.403
99.6 99.4
REL rs702873 G/A
2p16.1
No Data
Intron
0.131
0.132
0.752
99.7
a
http://www.regulomedb.org/, HLA-C rs10484554 C/T polymorphism was in complete linkage disequilibrium (LD) (D = 1.0, r2 = 1.0) with HLA-C rs12191877 C/T polymorphism 0
MAF minor allele frequency HWE Hardy–Weinberg equilibrium
homozygote genotype was not associated with susceptibility to RA (adjusted OR = 0.40, 95 % CI = 0.14–1.132, p = 0.083). None of the HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms achieved a significant difference in the genotype distributions between cases and controls. Logistic regression analyses revealed that HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms were not associated with the risk of RA (Table 3).
Discussion We determined the association between the HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and the risk of RA in a Chinese population. We found that the HLA-C rs10484554 C/T and rs12191877 C/T polymorphisms may be associated with a decreased risk of RA. We have identified DNA features and regulatory elements that contain the coordinate of the SNP (http://www. regulomedb.org/), we find that HLA-C rs12191877 C/T and JAM2 rs2829866 A/T SNPs are functional (Table 2). While HLA-C rs10484554 C/T polymorphism was in complete LD with HLA-C rs12191877 C/T polymorphism. HLA-C plays a key part in the presentation of antigens to CD8 ? T cells, which predominate in the epidermis of psoriatic lesions. Several other studies have also identified oligoclonal T cell receptor rearrangements in psoriasis [16]. HLA-C also has a role in binding to killer immunoglobulin-like receptors (KIRs) upon natural killer (NK) cells, and KIRs genes have been associated with PsA [17, 18]. However, it could also be argued that inflammatory arthritis may be an outcome of different immune responses
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in joints caused by different triggers with different underlying genetic susceptibilities. Indeed, enthesitis is thought by many researchers to be the primary abnormality in PsA, with synovitis being a secondary phenomenon. This is in contrast to RA, in which synovitis takes primacy. The results of the present genetic study support the notion of the opposite allele between RA and psoriasis/PsA. In a recently conducted psoriasis GWAS, evidence for interaction between the HLA-C and endoplasmic reticulum aminopeptidase 1 (ERAP1) loci was reported [9]. ERAP1 variants influenced psoriasis susceptibility in individuals carrying only the HLA-C risk allele [19]. Association of different autoimmune diseases with opposite alleles of the same susceptibility variant has been reported. For example, the minor T allele of the protein tyrosine phosphatase (PTPN22) rs2476601 SNP confers susceptibility to RA, type-1 diabetes and autoimmune thyroid disease, whereas the major C allele confers susceptibility to Crohn’s disease [20]. Similarly, association at the REL locus SNP, rs13017599, has been reported in psoriasis but the opposite allele has also been associated with RA [9, 21]. These findings may suggest that RA clusters with the classical autoantibody-associated autoimmune diseases, whereas PsA shares greater genetic similarity to psoriasis and sero-negative diseases such as Crohn’s disease. These SNPs could be markers for susceptibility to psoriasis and may not be specific to PsA. Testing these markers in psoriasis patients screened to exclude cases with evidence of inflammatory arthritis would be very interesting. The REL locus encoding the NFjB inflammatory pathway member c-REL has been reported in a large GWAS to be associated with type-I psoriasis but with a different SNP, i.e., rs702873 (OR 1.12). Most RA risk loci are identified using patients who have anti-cyclic citrullinated peptide antibodies (ACPA). Given the importance of seronegativity in the classification of PsA, evaluating the
Mol Biol Rep Table 3 Logistic regression analysis of associations between HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and risk of rheumatoid arthritis Genotype
RA (n = 520)
Controls (n = 520)
No. (%)
No. (%)
Adjusted OR* (95 % CI)
CC
438 (84.6)
413 (79.4)
1.00
CT TT
75 (14.5) 5 (1.0)
94 (18.1) 13 (2.5)
0.77 (0.55–1.07) 0.38 (0.13–1.08)
0.116 0.068
CT?TT
80 (15.4)
107 (20.6)
0.72 (0.52–0.99)
0.044
CC?CT
513 (99.0)
507 (97.5)
1.00
5 (1.0)
13 (2.5)
85 (8.2)
120 (11.5)
TT
450 (87.0)
430 (82.7)
TC
66 (12.8)
86 (16.5)
0.75 (0.53–1.06)
0.098
p
HLA-C rs10484554 C/T
TT T allele
0.40 (0.14–1.12)
0.082
HLA-C rs12212594 T/C
CC
1.00
1 (0.2)
4 (0.8)
0.26 (0.03–2.31)
0.225
TC?CC
67 (13.0)
90 (17.3)
0.73 (0.51–1.02)
0.067
TT ? TC
516 (99.8)
516 (99.2)
CC
1.00
1 (0.2)
4 (0.8)
68 (6.6)
94 (9.0)
CC CT
436 (84.5) 75 (14.5)
413 (79.4) 94 (18.1)
1.00 0.77 (0.55–1.07)
0.123
TT
5 (1.0)
13 (2.5)
0.38 (0.13–1.08)
0.069
CT?TT
80 (15.5)
107 (20.6)
0.72 (0.52–1.00)
0.047
CC?CT
511 (99.0)
507 (97.5)
1.00
5 (1.0)
13 (2.5)
85 (8.2)
120 (11.5)
TT
220 (42.7)
211 (40.7)
1.00
TA
234 (45.4)
233 (44.9)
0.96 (0.74–1.25)
0.754
C allele
0.27 (0.03–2.42)
0.241
HLA-C rs12191877 C/T
TT T allele
0.40 (0.14–1.13)
0.083
JAM2 rs2829866 A/T
AA
61 (11.8)
75 (14.5)
0.79 (0.53–1.16)
0.223
TA?AA
295 (57.3)
308 (59.3)
0.92 (0.72–1.18)
0.493
TT?TA
454 (88.2)
444 (85.5)
1.00
AA
61 (11.8)
75 (14.5)
366 (35.5)
383 (36.9)
GG GA
378 (73.1) 131 (25.3)
392 (75.4) 119 (22.9)
1.00 1.14 (0.86–1.52)
0.358
AA
8 (1.5)
9 (1.7)
0.92 (0.35–2.42)
0.871
GA?AA
139 (26.9)
128 (24.6)
1.13 (0.85–1.49)
0.397
GG?GA
509 (98.4)
511 (98.3)
1.00
8 (1.5)
9 (1.7)
147 (14.2)
137 (13.2)
A allele
0.80 (0.56–1.16)
0.236
REL rs702873 G/A
AA A allele
0.89 (0.34–2.34)
0.817
Bold values are statistically significant (p \ 0.05) * Adjusted for age and sex
susceptibility risk loci identified in ACPA -negative RA samples would be interesting. However, robustly confirmed susceptibility loci for sero-negative RA are lacking.
Genetic polymorphisms often vary between ethnic groups. In the present study employing 520 controls, we reported that the allele frequency of HLA-C rs10484554
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C/T (0.115) was similar to that reported in Chinese populations (0.107). However, the mutant homozygote among controls was higher than that reported in controls in the UK (0.07). Considering HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A mutant alleles in the control group, OR, RA samples and control samples, the power of our analyses (a = 0.05) was 0.352, 0.274, 0.352, 0.099 and 0.105 in 520 RA cases and 520 controls with adjusted OR = 0.72 for HLA-C rs10484554 C/T, adjusted OR = 0.73 for HLA-C rs12212594 T/C, adjusted OR = 0.72 for HLA-C rs12191877 C/T, adjusted OR = 0.92 for JAM2 rs2829866 A/T and adjusted OR = 1.13 for REL rs702873 G/A, respectively. Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study and selection bias was therefore unavoidable. Thus, the subjects were not fully representative of the general population. Second, the polymorphisms investigated, which was based on their functional considerations, may not offer a comprehensive view of the genetic variability of HLA-C, JAM2 and REL. Third, a single case–control study is not sufficient to fully interpret the relationship between HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and susceptibility to RA because of the relatively small number of patients evaluated. Studies with more subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and other populations. This is because the risk of RA is likely to be influenced by gene–gene and gene–environment interactions; HLA-C, JAM2 and REL genes may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, the present study provided strong evidence that HLA-C polymorphisms may be associated with a decreased risk of RA. However, further studies are needed to confirm the results of this preliminary study. Acknowledgments This study was supported in part by National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (2012NJMU128). Conflict of interest None of the authors have a potential financial conflict of interest related to the publication of this manuscript.
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Human leukocyte antigen (HLA)-C polymorphisms are associated with a decreased risk of rheumatoid arthritis Yiqun Zhang • Hui Zhang • Yong Huang Rongbin Sun • Ruiping Liu • Jie Wei
•
Received: 10 February 2013 / Accepted: 13 February 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Rheumatoid arthritis (RA) is an autoimmune rheumatological disease thought to have substantial genetic contributions. Several genetic factors involved in the susceptibility to psoriasis and psoriatic arthritis (PsA) have been identified with genome-wide association studies, including human leukocyte antigen (HLA)-C, junction adhesion molecule 2 (JAM2) and REL. Psoriasis and PsA may share many features in common with RA. We hypothesized that this polymorphism may contribute to RA susceptibility in a Chinese population. We studied HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL
Yiqun Zhang and Hui Zhang contributed equally to this study. Y. Zhang Department of Ophthalmology, Affiliated Hospital of Suzhou University, Changzhou No. 4 People’s Hospital, Changzhou 213001, China H. Zhang Y. Huang R. Sun R. Liu (&) Department of Orthopedic Trauma, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou 213003, China e-mail: [email protected] R. Liu Central Laboratory, Affiliated Hospital of Nanjing Medical University, Changzhou Second People’s Hospital, Changzhou 213003, China J. Wei Department of Orthopaedic Trauma, Beijing Jishuitan Hospital, Beijing 100035, China J. Wei (&) Department of Orthopaedic Trauma, Beijing Jishuitan Hospital, Beijing 100035, China e-mail: [email protected]
rs702873 G/A polymorphisms in 520 patients with RA and 520 controls in a Chinese population. HLA-C rs12191877 C/T polymorphism was in complete linkage disequilibrium (LD) (D0 = 1.0, r2 = 1.0) with HLA-C rs10484554 C/T polymorphism. When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the TT/ CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–0.99, p = 0.044). We found that the HLAC rs12191877 C/T polymorphism was also associated with a decreased risk of RA. HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms were not associated with the risk of RA. These results provide evidence that HLA-C polymorphisms are associated with a decreased risk of RA. Keywords HLA-C Polymorphisms Rheumatoid arthritis Molecular epidemiology Abbreviations CI confidence interval HLA human leukocyte antigen LD linkage disequilibrium OR odds ratio SNP single nucleotide polymorphism
Introduction Rheumatoid arthritis (RA) is characterized by synovitis, progressive erosions, and cartilage destruction. RA is an inflammatory disease and thought to have substantial genetic contributions [1]. Psoriasis and psoriatic arthritis (PsA) are also chronic immune-mediated inflammatory
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diseases associated with appreciable morbidity and disability [2, 3]. PsA shares many common features with RA. Some clinical features between RA and PsA overlap, so genetic susceptibility might also (at least in part) overlap. Several epidemiologic studies have examined the association between psoriasis and inflammatory arthritis to determine if PsA is a distinct entity rather than a chance association of psoriasis with an established inflammatory arthritis [4, 5]. PsA (but not RA) is linked with several major histocompatibility complex (MHC) class-I alleles [6]. Genome-wide association studies (GWAS) are the most popular and powerful methods for identifying susceptibility genes for complex diseases. Several genetic factors involved in susceptibility to psoriasis and PsA have been identified with GWAS [7]. The association of human leukocyte antigen (HLA)-C with PsA was found to be linked with early-onset psoriasis [8]. Within the chromosome 6p21 region codes for several genes important in the immune response (including HLA and non-HLA genes), three single nucleotide polymorphisms (SNPs) have shown strong evidence to be associated with psoriasis or PsA approximately 35 kb upstream of HLA-C (rs12212594, rs12191877 and rs10484554) [7, 9, 10]. One study found that HLA-C encoded variants unique to these haplotypes at the level of translated proteins, which increased the brisk of psoriasis, suggesting that HLA-C is a psoriasis susceptibility gene [11]. Furthermore, a meta-analysis of the PsA subsets of three independent GWAS revealed that the junction adhesion molecule 2 (JAM2) rs2829866 A/T polymorphism is associated with PsA [12]. The REL gene, a key modulator of the nuclear factor kappa B (NFjB) pathway, is known to share shared Crohn’s disease (CD) and psoriasis susceptibility loci [13]. However, although HLA-C, JAM2 and REL play important parts in the immune response, few studies have focused on the influences of HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms in the susceptibilities to RA. We therefore undertook a hospital-based case–control study to genotype 520 patients and 520 controls in a Chinese population.
Patients and methods Subjects We obtained approval of the study protocol from the Ethics Committee of Nanjing Medical University (Nanjing, China). All patients provided written informed consent to be included in the study.
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Five hundred and twenty RA patients who fulfilled the criteria for RA set by the American College of Rheumatology classification in 1987 [14] were consecutively recruited from the Changzhou Second Hospital-Affiliated Hospital of Nanjing Medical University (Changzhou, China), the Changzhou First Hospital (Changzhou, China), and the Changzhou Traditional Chinese Medical Hospital (Changzhou, China), between September 2010 and May 2012. The controls were patients without RA, matched RA for age (±5 years) and sex, and recruited from the same institutions during the same time period; most of the controls were admitted to the hospitals for the treatment of trauma. Each patient was interviewed by trained personnel using a pre-tested questionnaire to obtain information on demographic data and related risk factors for RA. After the interview, 2 mL of peripheral blood was collected from each subject. Isolation of DNA and genotyping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) Blood samples were collected using vacutainers and transferred to test tubes containing ethylenediamine tetraacetic acid (EDTA). Genomic DNA was isolated from whole blood using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). Genotyping was done by MALDI-TOF MS using the MassARRAY system (Sequenom, San Diego, CA, USA) as previously described [15]. Cases and controls at a proportion of &1:1 were assayed. Completed genotyping reactions were spotted onto a 384-well spectroCHIP (Sequenom) using a MassARRAY Nanodispenser (Sequenom), and analyzed by MALDI-TOF MS. Genotype calling was done in real time with MassARRAY RT software (version 3.1; Sequenom), and analyzed using MassARRAY Typer software (version 4.0; Sequenom). For quality control, repeated analyses were undertaken on 10 % of randomly selected samples. Statistical analysis Differences in demographics, variables, and genotypes of the HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphism variants were evaluated using a v2 test. The associations between HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A genotypes and risk of RA was estimated by computing odds ratios (ORs) and 95 % confidence intervals (CIs) using logistic regression analyses, and by using crude ORs. The Hardy–
Mol Biol Rep Table 1 Patient demographics and risk factors in rheumatoid arthritis and controls Variable
RA cases (n = 520)
Controls (n = 520)
p
Age (years)
54.72 (± 15.27)
54.17 (± 10.50)
0.496
Female/male
390/130
385/135
0.722
Age at onset, years, mean ± SD
46.43 (± 13.28)
–
–
Disease duration, years, mean ± SD
8.34 (± 9.40)
–
–
Treatment duration, years, mean ± SD
7.03 (± 7.93)
–
–
RF-positive, no. (%)
414 (79.6 %)
–
–
ACPA-positive, no. (%)
269 (51.7 %)
–
–
CRP-positive, no. (%)
220 (42.3 %)
–
–
ESR, mm/h
36.93 (± 29.41)
–
–
DAS28
4.55 (± 1.53)
–
–
I
68 (13.1 %)
–
–
II III
236 (45.4 %) 183 (35.2 %)
– –
– –
IV
33 (6.3 %)
–
–
Functional class, no. (%)
RF Rheumatoid factor, ACPA Anti-cyclic citrullinated peptide antibody, CRP C-reactive protein, ESR Erythrocyte sedimentation rate, DAS28 RA disease activity score
Weinberg equilibrium (HWE) was tested by a goodness-offit v2 test to compare the observed genotype frequencies to the expected frequencies among controls. All statistical analyses were done with SAS software (version 9.1.3; SAS Institute, Cary, NC, USA).
Results Characteristics of the study population The demographic and clinical characteristics of all subjects are summarized in Table 1. Subjects were adequately matched for age and sex (p = 0.496 and 0.722, respectively) for RA patients and controls. Among 1,040 DNA samples (520 RA patients and 520 controls), we successfully genotyped 1,018 (97.9 %) samples in the first step, then in the second step we genotyping the rest 22 samples and 104 (10 % of all samples) randomly selected samples. At last, the HLA-C rs10484554 C/T polymorphism was successful in 518 (99.6 %) RA patients and 520 (100.0 %) controls. Genotyping for HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A was successful ranging from 99.4 to 99.7 % (Table 2). The concordance rates of repeated analyses were
100 %. The genotype distributions of HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A in all subjects are illustrated in Table 3. The observed genotype frequencies for the polymorphism in controls were in HWE for HLA-C rs12212594 T/C (p = 0.895), HLAC rs12191877 C/T (p = 0.431), JAM2 rs2829866 A/T (p = 0.413) and REL rs702873 G/A (p = 0.993). However, the observed genotype frequencies for the polymorphism in controls were not in HWE for HLA-C rs10484554 C/T (p = 0.009). Associations between HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and the risk of RA The genotype frequencies of the HLA-C rs10484554 C/T polymorphism were 84.6 % (CC), 14.5 % (CT) and 1.0 % (TT) in RA patients, and 79.4 % (CC), 18.1 % (CT) and 2.5 % (TT) in controls (p = 0.040) (Table 3). When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the TT/CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–0.99, p = 0.044). When the HLA-C rs10484554 CC homozygote genotype was used as the reference group, the CT or TT genotype was not associated with the risk for RA (CT vs. CC, adjusted OR = 0.77, 95 % CI = 0.55–1.07, p = 0.116; TT vs. CC, adjusted OR = 0.38, 95 % CI = 0.13–1.08, p = 0.068). In the recessive model, when the HLA-C rs10484554 CC/CT genotypes were used as the reference group, the TT homozygote genotype was not associated with susceptibility to RA (adjusted OR = 0.40, 95 % CI = 0.14–1.12, p = 0.082). HLA-C rs12191877 C/T polymorphism was in complete linkage disequilibrium (LD) (D0 = 1.0, r2 = 1.0) with HLA-C rs10484554 C/T polymorphism. The genotype frequencies of the HLA-C rs12191877 C/T polymorphism were 84.5 % (CC), 14.5 % (CT) and 1.0 % (TT) in RA patients, and 79.4 % (CC), 18.1 % (CT) and 2.5 % (TT) in controls (p = 0.043) (Table 3). When the HLAC rs12191877 CC homozygote genotype was used as the reference group, the TT/CT genotypes were associated with a significantly decreased risk for RA (adjusted OR = 0.72, 95 % CI = 0.52–1.00, p = 0.047). When the HLA-C rs12191877 CC homozygote genotype was used as the reference group, the CT or TT genotype was not associated with the risk for RA (CT vs. CC, adjusted OR = 0.77, 95 % CI = 0.55–1.07, p = 0.123; TT vs. CC, adjusted OR = 0.38, 95 % CI = 0.13–1.08, p = 0.069). In the recessive model, when the HLA-C rs12191877 CC/CT genotypes were used as the reference group, the TT
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Mol Biol Rep Table 2 Primary information for HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms Genotyped SNPs
Chr Pos
Regulome DB scorea
Location
MAF for Chinese in database
MAF in our controls (n = 520)
p value for HWE test
% genotyping value
HLA-C rs10484554 C/T
6p21.3
No data
Intergenic region
0.102
0.115
0.655
99.8
HLA-C rs12212594 T/C
6p21.3
No data
Intergenic region
0.065
0.090
1.000
99.7
HLA-C rs12191877 C/T JAM2 rs2829866 A/T
6p21.3 21q21.2
6 6
Intergenic region Intron
0.094 0.375
0.115 0.369
0.752 0.403
99.6 99.4
REL rs702873 G/A
2p16.1
No Data
Intron
0.131
0.132
0.752
99.7
a
http://www.regulomedb.org/, HLA-C rs10484554 C/T polymorphism was in complete linkage disequilibrium (LD) (D = 1.0, r2 = 1.0) with HLA-C rs12191877 C/T polymorphism 0
MAF minor allele frequency HWE Hardy–Weinberg equilibrium
homozygote genotype was not associated with susceptibility to RA (adjusted OR = 0.40, 95 % CI = 0.14–1.132, p = 0.083). None of the HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms achieved a significant difference in the genotype distributions between cases and controls. Logistic regression analyses revealed that HLA-C rs12212594 T/C, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms were not associated with the risk of RA (Table 3).
Discussion We determined the association between the HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and the risk of RA in a Chinese population. We found that the HLA-C rs10484554 C/T and rs12191877 C/T polymorphisms may be associated with a decreased risk of RA. We have identified DNA features and regulatory elements that contain the coordinate of the SNP (http://www. regulomedb.org/), we find that HLA-C rs12191877 C/T and JAM2 rs2829866 A/T SNPs are functional (Table 2). While HLA-C rs10484554 C/T polymorphism was in complete LD with HLA-C rs12191877 C/T polymorphism. HLA-C plays a key part in the presentation of antigens to CD8 ? T cells, which predominate in the epidermis of psoriatic lesions. Several other studies have also identified oligoclonal T cell receptor rearrangements in psoriasis [16]. HLA-C also has a role in binding to killer immunoglobulin-like receptors (KIRs) upon natural killer (NK) cells, and KIRs genes have been associated with PsA [17, 18]. However, it could also be argued that inflammatory arthritis may be an outcome of different immune responses
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in joints caused by different triggers with different underlying genetic susceptibilities. Indeed, enthesitis is thought by many researchers to be the primary abnormality in PsA, with synovitis being a secondary phenomenon. This is in contrast to RA, in which synovitis takes primacy. The results of the present genetic study support the notion of the opposite allele between RA and psoriasis/PsA. In a recently conducted psoriasis GWAS, evidence for interaction between the HLA-C and endoplasmic reticulum aminopeptidase 1 (ERAP1) loci was reported [9]. ERAP1 variants influenced psoriasis susceptibility in individuals carrying only the HLA-C risk allele [19]. Association of different autoimmune diseases with opposite alleles of the same susceptibility variant has been reported. For example, the minor T allele of the protein tyrosine phosphatase (PTPN22) rs2476601 SNP confers susceptibility to RA, type-1 diabetes and autoimmune thyroid disease, whereas the major C allele confers susceptibility to Crohn’s disease [20]. Similarly, association at the REL locus SNP, rs13017599, has been reported in psoriasis but the opposite allele has also been associated with RA [9, 21]. These findings may suggest that RA clusters with the classical autoantibody-associated autoimmune diseases, whereas PsA shares greater genetic similarity to psoriasis and sero-negative diseases such as Crohn’s disease. These SNPs could be markers for susceptibility to psoriasis and may not be specific to PsA. Testing these markers in psoriasis patients screened to exclude cases with evidence of inflammatory arthritis would be very interesting. The REL locus encoding the NFjB inflammatory pathway member c-REL has been reported in a large GWAS to be associated with type-I psoriasis but with a different SNP, i.e., rs702873 (OR 1.12). Most RA risk loci are identified using patients who have anti-cyclic citrullinated peptide antibodies (ACPA). Given the importance of seronegativity in the classification of PsA, evaluating the
Mol Biol Rep Table 3 Logistic regression analysis of associations between HLA-C rs10484554 C/T, HLA-C rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and risk of rheumatoid arthritis Genotype
RA (n = 520)
Controls (n = 520)
No. (%)
No. (%)
Adjusted OR* (95 % CI)
CC
438 (84.6)
413 (79.4)
1.00
CT TT
75 (14.5) 5 (1.0)
94 (18.1) 13 (2.5)
0.77 (0.55–1.07) 0.38 (0.13–1.08)
0.116 0.068
CT?TT
80 (15.4)
107 (20.6)
0.72 (0.52–0.99)
0.044
CC?CT
513 (99.0)
507 (97.5)
1.00
5 (1.0)
13 (2.5)
85 (8.2)
120 (11.5)
TT
450 (87.0)
430 (82.7)
TC
66 (12.8)
86 (16.5)
0.75 (0.53–1.06)
0.098
p
HLA-C rs10484554 C/T
TT T allele
0.40 (0.14–1.12)
0.082
HLA-C rs12212594 T/C
CC
1.00
1 (0.2)
4 (0.8)
0.26 (0.03–2.31)
0.225
TC?CC
67 (13.0)
90 (17.3)
0.73 (0.51–1.02)
0.067
TT ? TC
516 (99.8)
516 (99.2)
CC
1.00
1 (0.2)
4 (0.8)
68 (6.6)
94 (9.0)
CC CT
436 (84.5) 75 (14.5)
413 (79.4) 94 (18.1)
1.00 0.77 (0.55–1.07)
0.123
TT
5 (1.0)
13 (2.5)
0.38 (0.13–1.08)
0.069
CT?TT
80 (15.5)
107 (20.6)
0.72 (0.52–1.00)
0.047
CC?CT
511 (99.0)
507 (97.5)
1.00
5 (1.0)
13 (2.5)
85 (8.2)
120 (11.5)
TT
220 (42.7)
211 (40.7)
1.00
TA
234 (45.4)
233 (44.9)
0.96 (0.74–1.25)
0.754
C allele
0.27 (0.03–2.42)
0.241
HLA-C rs12191877 C/T
TT T allele
0.40 (0.14–1.13)
0.083
JAM2 rs2829866 A/T
AA
61 (11.8)
75 (14.5)
0.79 (0.53–1.16)
0.223
TA?AA
295 (57.3)
308 (59.3)
0.92 (0.72–1.18)
0.493
TT?TA
454 (88.2)
444 (85.5)
1.00
AA
61 (11.8)
75 (14.5)
366 (35.5)
383 (36.9)
GG GA
378 (73.1) 131 (25.3)
392 (75.4) 119 (22.9)
1.00 1.14 (0.86–1.52)
0.358
AA
8 (1.5)
9 (1.7)
0.92 (0.35–2.42)
0.871
GA?AA
139 (26.9)
128 (24.6)
1.13 (0.85–1.49)
0.397
GG?GA
509 (98.4)
511 (98.3)
1.00
8 (1.5)
9 (1.7)
147 (14.2)
137 (13.2)
A allele
0.80 (0.56–1.16)
0.236
REL rs702873 G/A
AA A allele
0.89 (0.34–2.34)
0.817
Bold values are statistically significant (p \ 0.05) * Adjusted for age and sex
susceptibility risk loci identified in ACPA -negative RA samples would be interesting. However, robustly confirmed susceptibility loci for sero-negative RA are lacking.
Genetic polymorphisms often vary between ethnic groups. In the present study employing 520 controls, we reported that the allele frequency of HLA-C rs10484554
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C/T (0.115) was similar to that reported in Chinese populations (0.107). However, the mutant homozygote among controls was higher than that reported in controls in the UK (0.07). Considering HLA-C rs10484554 C/T, HLAC rs12212594 T/C, HLA-C rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A mutant alleles in the control group, OR, RA samples and control samples, the power of our analyses (a = 0.05) was 0.352, 0.274, 0.352, 0.099 and 0.105 in 520 RA cases and 520 controls with adjusted OR = 0.72 for HLA-C rs10484554 C/T, adjusted OR = 0.73 for HLA-C rs12212594 T/C, adjusted OR = 0.72 for HLA-C rs12191877 C/T, adjusted OR = 0.92 for JAM2 rs2829866 A/T and adjusted OR = 1.13 for REL rs702873 G/A, respectively. Several limitations of the present study need to be addressed. First, this was a hospital-based case–control study and selection bias was therefore unavoidable. Thus, the subjects were not fully representative of the general population. Second, the polymorphisms investigated, which was based on their functional considerations, may not offer a comprehensive view of the genetic variability of HLA-C, JAM2 and REL. Third, a single case–control study is not sufficient to fully interpret the relationship between HLAC rs10484554 C/T, HLA-C rs12212594 T/C, HLAC rs12191877 C/T, JAM2 rs2829866 A/T and REL rs702873 G/A polymorphisms and susceptibility to RA because of the relatively small number of patients evaluated. Studies with more subjects are necessary to confirm our findings. Finally, environmental factors differ between Chinese populations and other populations. This is because the risk of RA is likely to be influenced by gene–gene and gene–environment interactions; HLA-C, JAM2 and REL genes may be associated with different degrees of genetic risk in different ethnic groups and under different environmental exposures. In conclusion, the present study provided strong evidence that HLA-C polymorphisms may be associated with a decreased risk of RA. However, further studies are needed to confirm the results of this preliminary study. Acknowledgments This study was supported in part by National Natural Science Foundation of China (81371927) and Nanjing Medical University Foundation for Development of Science and Technology (2012NJMU128). Conflict of interest None of the authors have a potential financial conflict of interest related to the publication of this manuscript.
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