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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Feb 1, 2010.
Published in final edited form as:
PMCID: PMC2770844
NIHMSID: NIHMS87456

Lack of Association of Caucasian Rheumatoid Arthritis Susceptibility Loci in a Korean Population

Abstract

Objective

Recent studies have identified a number of novel rheumatoid arthritis (RA) loci in Caucasian populations. In this study, we sought to determine whether the genetic variants at 4q27, 6q23, CCL21, TRAF1/C5, and CD40 identified in Caucasians are also associated with RA in a Korean case-control collection. We also comprehensively evaluated the genetic variation within PTPN22, a well established autoimmune disease-associated gene.

Methods

We designed a Sequenom iPlex experiment to thoroughly evaluate the PTPN22 linkage disequilibrium region using tag SNPs and disease-associated SNPs at 5 other previously reported Caucasian RA-associated loci in 1123 RA Korean RA patients and 1008 ethnically matched controls. We also re-sequenced the PTPN22 gene to look for novel coding variants that might be contributing to disease in this population.

Results

None of the Caucasian RA susceptibility loci contributed significantly to disease in Koreans. Tag SNPs covering the PTPN22 linkage disequilibrium block, while polymorphic, did not reveal any disease association and re-sequencing did not identify any new common coding region variants in this population. The 6q23 and 4q27 SNPs assayed were non-polymorphic in this population and the TRAF1/C5, CD40, and CCL21 SNPs did not show any evidence for association.

Conclusions

Caucasian and Korean rheumatoid arthritis have different genetic risk factors. While patients of different ethnic groups share the HLA region as a major genetic risk locus, most other genes shown to be significantly associated with disease in Caucasians appear not to play a role in Korean RA.

Rheumatoid arthritis (RA) is a chronic autoimmune arthritis characterized by progressive joint destruction. Both genetic and environmental factors have been shown to play a role in the development of RA. While the largest genetic risk factor predisposing to RA, a common set of alleles at HLA-DRB1, has been associated with RA in populations of both Caucasian and Asian ancestry (1, 2), many other risk loci have been shown to confer RA susceptibility in only one ethnic group. For example, although PTPN22 has been consistently shown to be associated with RA in Caucasians, the 620W risk allele in this gene is not found and thus not disease-associated in Asian populations (37). In contrast, PADI4, SLC22A4, and FCRL3 have been associated with RA in studies of Asian patients, but have given weak or negative results in populations of European ancestry (6, 814). While these results suggest that the genetic risk alleles that confer susceptibility to RA are heterogeneous across major ethnic groups, recent reports have identified a common STAT4 haplotype that confers a similar degree of risk for RA in both Asian and Caucasian populations (15, 16).

Outside of the HLA region, the R620W variant of the PTPN22 gene is the most consistently and most strongly disease-associated variant identified in rheumatoid arthritis association studies performed in Caucasian populations. Although this variant is not found in Asians, it is possible that other variants within this important candidate gene could contribute to genetic susceptibility to this disease in Asian populations. In addition to PTPN22, several recent genetic studies have identified new RA susceptibility loci in Caucasian populations. The TRAF1/C5 locus has been identified both by a genome-wide association study (GWAS) and as a candidate gene (7, 17). Two independent GWAS follow-up studies have identified a susceptibility locus on 6q23 near TNFAIP3 (18, 19). A large meta-analysis using GWAS and replication collections has identified the CD40 and CCL21 loci (20), and a 4q27 region including IL2/IL21 has been identified through a candidate gene approach after being shown to be associated with other autoimmune diseases (21). To date, there has been no study of SNPs in these new regions associated with RA in an Asian population. Therefore, the aim of this study was to determine whether PTPN22 variants or any of five disease-associated SNPs recently identified in Caucasians also contribute to Korean RA.

PATIENTS AND METHODS

Study Population

We included 1128 Korean RA patients who were enrolled consecutively from the outpatient clinic of The Hospital for Rheumatic Diseases, Hanyang University, Seoul, South Korea and 1022 ethnically matched controls that had no history of RA and other autoimmune diseases in the self reported questionnaire at the enrollment. All RA patients met the American College of Rheumatology 1987 classification criteria for RA (22). The study was approved by the institutional review board of Hanyang University Hospital. Written informed consent was obtained from all participants. All subjects were typed for HLA-DRB1 subtypes using polymerase chain reaction and sequence-specific oligonucleotide probe hybridization.

Since the second most significant risk allele for RA was DRB1*0901 in the Korean population, we regarded DRB1*0901 as a member of the SE group in addition to the following alleles: DRB1*0101, *0102, *0401, *0404, *0405, *0408, *0413, *1001, and *1402. Among 1128 RA patients, 807 patients had CCP data that were quantitatively measured in duplicate by enzyme-linked immunosorbent assay using the DIASTAT Anti-CCP kit FCCP 200 (Axis-Shield Plc, Scotland, UK) according to the manufacturer’s instructions. The staging system proposed by Steinbrocker et al (23) was used as a measure of the radiographic severity of RA.

PTPN22 Re-sequencing

Genomic DNAs from 48 Korean sero-positive (RF+) RA patients were sequenced over the regions representing the promoter region (up to −2000 bp), all 21 exons including exon-intron boundaries and 3’UTR region of PTPN22 using Sanger dideoxy DNA sequencing technique (Polymorphic DNA Technologies, Alameda, California).

SNP selection and assay design

We used Tagger (24) to select SNPs polymorphic in the HapMap CHB/JPT population across the 400 kilobase linkage disequilibrium block that includes PTPN22. In addition, all variants identified from the PTPN22 exon re-sequencing were selected. A total of 34 SNPs/variants were selected from the PTPN22 linkage disequilibrium block. Lastly, we identified SNPs that have been reported as associated with RA in either a Caucasian (TRAF1/C5, 6q23, 4q27, CD40, and CCL21 loci) or Asian (PADI4) population and selected one disease-associated SNP per region (total of 6 SNPs selected). The 40 selected SNPs were used to design multiplex genotyping assays using Sequenom RealSNP (http://www.realsnp.com) and Assay Design software.

Genotyping

Multiplex PCR was used to amplify DNA products containing up to 25 SNPs in one reaction from 5 ng genomic DNA. Synthetic oligonucleotides that bind adjacent to the SNP site were then hybridized and extended with nucleotides complementary to the template SNP site using modified nucleotides that terminate the extension reaction at the interrogated SNP and generate alternate products of sufficiently different mass to be separated by mass spectrometry. The extended products were separated by MALDI-TOF mass spectrometry and the genotypes determined by SpectroTyper software (Sequenom, Inc, San Diego, California). Calls were evaluated and edited by cluster analysis performed with the SpectroTyper software version 4.0.

Data analysis

Association tests for SNPs and haplotypes in cases and controls and regional linkage disequilibrium structure for analyzed SNPs were determined using Haploview version 4.0 (25). We excluded SNPs with significant deviation from Hardy Weinberg equilibrium (P value less than 0.005) and SNPs with minor allele frequency (MAF) less than 0.01. We reanalyzed the association of all SNPs with the following specific subgroups of RA (CCP positive RA, severe erosive RA, or SE positive RA) using multivariate logistic regression with adjustments for age and sex. These subgroup analyses were performed using SAS software, version 9.1 (SAS Institute, Cary, NC).

RESULTS

Patient and control characteristics

Among the patients with RA, the mean ± SD age was 52.4 ± 11.9 years, the mean ± SD age at disease onset was 39.9 ± 11.5 years, 89.7% were female, 81.4% were positive for the SE, 17.0% showed no erosive changes (Steinbrocker stage I), and 83.0% demonstrated erosions (Steinbrocker stages II, III, and IV). Among RA patients with CCP data (N=807), 681 (84.4%) patients were CCP positive. The mean ± SD age of control subjects was 36.8 ± 12.5 years, and 86.3% were female.

Re-sequencing

Nineteen sequence variants (i.e., variants identified in one or more resequenced DNAs) were identified in the exonic (exons and intronic splice junction regions) and the promoter regions of PTPN22 in the resequenced Korean DNA samples. Of these, 13 had previously been identified and 6 were novel. The novel variants were submitted to dbSNP (rs numbers to be added when available). Although the novel variants were all rare (found in only a single DNA of the 48 resequenced), they were included in the case-control genotyping. One of the novel variants was found in the promoter region, four were intronic, and one was a splice junction variant.

Genotyping

Of the 40 SNPs included in the assay design, assays for two SNPs failed (Table 1). The genotype call rate was greater than 95% for the remaining markers with the exception of rs6537798 which had a 77.7% call rate. No cases or controls were polymorphic at rs2476601 (R620W) while between 1 and 9 minor alleles were present out of 4178 alleles in the 6 novel PTPN22 variants, the 6q23 SNP rs6920220, and the 4q27 SNP rs6822844 (Table 1 and Table 2). Additionally, the PTPN22 tag SNP rs1018592 was not analyzed because it exhibited significant deviation from Hardy-Weinberg equilibrium.

Table 1
Association tests of SNPs in the PTPN22 region in 1123 Korean rheumatoid arthritis cases and 1008 controls.
Table 2
Disease variants from recent studies in Caucasian populations are not associated with disease in Korean rheumatoid arthrtitis cases and controls.

RA association tests

No statistically significant association (p<0.05) with RA was detected between any of the 24 polymorphic PTPN22 tag SNPs (Table 1) from the linkage disequilibrium block covering the AP4B1/C1orf178/PTPN22/RSBN1/PTHF1/MAG13 genes. In addition, Caucasian RA-associated SNPs at TRAF1/C5, CD40, and CCL21 were found to be polymorphic in the Korean population but not to be associated with disease (Table 2). As noted above, the 4q27 and 6q23 variants were too rare in this population to be evaluated for association (Table 2). The PADI4 SNP rs2240320 was noted to be strongly disease-associated with a p-value of 1.15*10−8 and with an odds ratio of 1.43 (95% confidence limits: 1.27 to 1.62) for the presence of the disease-associated T allele on case chromosomes compared with control chromosomes (Table 2). In the subgroup analyses, there were no significant genotypic or allelic associations of any of these SNPs in the radiographic severity subgroups (Steinbrocker stage 1 and stages 2–4), the SE status subgroups (SE positive and negative group), and the CCP subgroups (CCP positive and negative group) compared with controls, except for PADI4 SNP rs2240310, which was significantly associated with RA irrespective of SE status (p-value <0.01) as well as CCP positivity (p-value < 0.0001).

PTPN22 linkage disequilibrium

The linkage disequilibrium structure of the broad genomic region containing the PTPN22 gene was similar in HapMap CEU and CHB+JPT individuals with a 400 kb region of strong linkage disequilibrium containing 6 genes (MAGI3, PHTF1, RSBN1, PTPN22, C1orf178, AP4B1) identified in both populations (see Supplemental Figure 1). The 24 PTPN22 tag SNPs, which passed quality control, captured with r2 greater than 0.8, 90% of the variation (166 of 185 alleles with HapMap CHB+JPT minor allele frequency greater than 5%) in the 400 kb region. Although the linkage disequilibrium structure of the PTPN22 gene region was similar in HapMap CEU and CHB+JPT individuals, the frequencies of the common haplotypes in these two populations were very different. Eleven of the 13 tag SNPs used for the haplotype association analysis of the 85kb PTPN22 gene region (see below) were genotyped in the HapMap CHB+JPT and CEU samples. These 11 SNPs defined only four common haplotypes (those with greater than 5% frequency) in both populations. All four common haplotypes found in the Asian samples were also common haplotypes in the CEU samples, but the haplotype frequencies were strikingly different between the two populations (Table 3). Interestingly, the 620W autoimmune disease-associated variant is found only on a portion (approximately half) of the copies of Haplotype 1 in the CEU samples, but is not found on any of the Haplotype 1 copies in the CHB+JPT samples (Table 3).

Table 3
PTPN22 gene region haplotypes and frequencies in HapMap populations.

Haplotype analysis

PTPN22 haplotypes were constructed using the 13 SNPs from Table 1 numbers 6 through 25 with minor allele frequency greater than 0.05 using Haploview software. These 13 SNPs covered the entire PTPN22 gene region. The four common haplotypes (with frequency >5%) together accounted for 96.6% of the chromosomes of the individuals genotyped. Association tests were performed for each of these four haplotypes and none were found to be associated with disease (Table 4).

Table 4
PTPN22 haplotype association analysis in Korean RA cases and controls.

DISCUSSION

Recent whole genome association studies in European ancestry case-control collections have shown that after HLA variants, SNPs within the PTPN22 genetic region are consistently the second most significantly disease-associated variants in the genome (3, 7). The proposed causative variant, a SNP encoding a structural variant R620W of the PTPN22 protein, however, is not present in the Asian population. We therefore sought to determine whether other PTPN22 alleles are associated with Asian RA. We also evaluated variants in five other loci that have strong evidence for association in Caucasian RA in a large Korean RA case-control series.

SNP variants within the PTPN22 linkage disequilibrium block were evaluated using a tag SNP approach to interrogate the known common variants in the region. None of the 24 polymorphic SNPs covering the genomic region including PTPN22 were associated with susceptibility to rheumatoid arthritis in this Korean population. Of note, we have confirmed that the R620W variant is non-polymorphic in this population and have shown that a promoter SNP, rs2488457, which has been reported as associated with RA in Caucasians and type 1 diabetes in Japanese (26), had no evidence for association in Korean RA. The genotyped SNPs cover 90% of the known polymorphic SNPs in the 400 kilobase PTPN22 linkage disequilibrium block (with r2 > 0.8), and none were associated with disease. Furthermore, none of the 4 common PTPN22 haplotypes were associated with disease. Coupled with the fact that no common PTPN22 structural variants were identified by re-sequencing, these data provide strong evidence that common PTPN22 region genetic variants are not associated with RA in this population. This is in stark contrast to the very strong association signals seen in multiple Caucasian RA case-control studies. Although none of the common PTPN22 variants were associated with disease, the novel rare variants are more difficult to evaluate. Interestingly, three of the novel variants identified (one promoter and two intronic) were found in a single RA case but in no controls. Further work will be required to determine whether any of the rare variants of the PTPN22 gene might be private mutations that contribute to disease susceptibility.

In addition, this study has shown that SNPs in five additional regions (TRAF1/C5, 6q23, 4q27, CD40, and CCL21) that are strongly associated with RA in Caucasians are either non-polymorphic (6q23 and 4q27) or not associated with Korean RA (TRAF1/C5, CD40, and CCL21). For these SNPs, we can only say with certainty that the SNP associated with disease in Caucasians is not associated with disease in Koreans. Further investigation and fine mapping similar to what we have done with PTPN22 would be needed to determine whether other polymorphic SNPs in these regions are associated with RA in Koreans. By comparison, we genotyped the PADI4 SNP rs2240340 for which we have previously reported an association with a subset (545 cases and 392 controls) of this case-control series and found a striking association with a p-value for association of 1.15*10−8 in this larger collection (9).

These data make a convincing argument that the genetic risk factors for RA differ substantially between Asian and Caucasian populations. While the HLA region has been clearly demonstrated to be the most substantial genetic risk factor in populations of both ancestries, the other genetic contributions seem to be very different. PTPN22 is consistently the second strongest genetic effect in Caucasian whole genome association studies and yet our data indicate that not only is the R620W variant not present, but no other common polymorphisms in the region are significant risk alleles in Koreans. The other Caucasian loci for which we assayed SNPs (TRAF1/C5, 6q23, 4q27, CD40, CCL21) were derived from rigorously interpreted and well-replicated studies and probably represent many of the next tier of susceptibility loci in European ancestry populations. The fact that none of these SNPs demonstrated association in this large Korean study reveals a large degree of genetic heterogeneity of RA across continental ancestry differences, especially when juxtaposed with the PADI4 locus, which has been strongly associated with RA in this and other Asian populations, but which has shown little evidence for association in Caucasians (14).

In conclusion, we found no association between Korean RA and tag SNPs covering the entire PTPN22 region or with individual SNPs shown to be strongly associated with disease in Caucasians at the TRAF1/C5, 6q23, 4q27, CD40, and CCL21 loci. When contrasted with other loci such as PADI4, FCRL3, and SLC22A4, which have been shown to be associated with RA in Asian but not Caucasian populations, these data indicate that most of the disease-associated variants have arisen independently in different continental populations and that different points in disease-associated pathways are influenced by genetic risk factors in the different populations. Therefore, one would expect that whole genome association studies in Asian populations would reveal more and different susceptibility loci than those that have been described in Caucasian disease; identification of such loci should expand our understanding of the processes that lead to the development of rheumatoid arthritis.

Supplementary Material

Supp Fig1

ACKNOWLEDGEMENTS

This work was supported in part by a grant of the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (01-PJ3-PG6-01GN11-0002) and by the Intramural Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases at the National Institutes of Health. Bethesda, Maryland, USA. B.D.K. was supported by the NIH Clinical Research Training Program, a public-private partnership between the Foundation for the National Institutes of Health and Pfizer, Inc. This work was also supported by grants from the National Institutes of Health: R01 AR44422 and N01 AI95386 (P.K.G.)

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