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1.  Identification of two new arthritis severity loci that regulate levels of autoantibodies, IL-1β and joint damage 
Arthritis and Rheumatism  2012;64(5):1369-1378.
Objective
Cia3 is a locus on rat chromosome 4 that regulates severity and joint damage in collagen and pristane-induced arthritis (CIA and PIA). This study aimed to refine the Cia3 gene-containing interval towards gene identification and obtain insights into its mode of action.
Methods
Five DA.F344(Cia3) subcongenic strains were generated and studied in PIA and CIA. Levels of antibodies against type II collagen (both allo- and autoantibodies) were measured. Joints and synovial tissues were collected 32 days after the induction of PIA (chronic stage) for histology and qPCR for IL-1β and matrix metalloproteases (MMPs).
Results
Three subcongenics sharing the centromeric Cia3d interval were protected, while two subcongenics sharing the telomeric Cia3g interval, which did not overlap with Cia3d, were also protected, developing significantly less severe CIA and PIA. DA.F344(Cia3) and DA.F344(Cia3d) congenics with PIA preserved a normal joint architecture, while DA rats had pronounced synovial hyperplasia, angiogenesis, inflammatory infiltration, bone or cartilage erosions. DA.F344(Cia3d) and DA.F344(Cia3g) strains had significantly lower synovial levels of IL-1β (5-fold), MMP-1 (expressed predominantly in DA), MMP-3 (79-fold) and MMP-14 (21-fold) and reduced levels of pathogenic autoantibodies against type II collagen, compared with DA.
Conclusions
We have identified two new arthritis severity and articular damage loci within Cia3. These loci regulate pathogenic processes in two different models of RA, and the identification of these genes has the potential to generate new targets for therapies aimed at reducing disease severity and articular damage, and for prognostication in RA.
doi:10.1002/art.33468
PMCID: PMC3288617  PMID: 22076633
Autoimmunity; Rheumatoid arthritis; animal model; erosion; IL-1β
2.  Cold Urticaria, Immunodeficiency, and Autoimmunity Related to PLCG2 Deletions 
The New England Journal of Medicine  2012;366(4):330-338.
Background
Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance.
Methods
We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing.
Results
Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures.
Conclusions
Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.)
doi:10.1056/NEJMoa1102140
PMCID: PMC3298368  PMID: 22236196
5.  Effective Sample Size: Quick Estimation of the Effect of Related Samples in Genetic Case-Control Association Analyses 
Summary
Affected relatives are essential for pedigree linkage analysis, however, they cause a violation of the independent sample assumption in case-control association studies. To avoid the correlation between samples, a common practice is to take only one affected sample per pedigree in association analysis. Although several methods exist in handling correlated samples, they are still not widely used in part because these are not easily implemented, or because they are not widely known. We advocate the effective sample size method as a simple and accessible approach for case-control association analysis with correlated samples. This method modifies the chi-square test statistic, p-value, and 95% confidence interval of the odds-ratio by replacing the apparent number of allele or genotype counts with the effective ones in the standard formula, without the need for specialized computer programs. We present a simple formula for calculating effective sample size for many types of relative pairs and relative sets. For allele frequency estimation, the effective sample size method captures the variance inflation exactly. For genotype frequency, simulations showed that effective sample size provides a satisfactory approximation. A gene which is previously identified as a type 1 diabetes susceptibility locus, the interferon-induced helicase gene (IFIH1), is shown to be significantly associated with rheumatoid arthritis when the effective sample size method is applied. This significant association is not established if only one affected sib per pedigree were used in the association analysis. Relationship between the effective sample size method and other methods – the generalized estimation equation, variance of eigenvalues for correlation matrices, and genomic controls – are discussed.
doi:10.1016/j.compbiolchem.2010.12.006
PMCID: PMC3119257  PMID: 21333602
6.  Genetic variants at CD28, PRDM1, and CD2/CD58 are associated with rheumatoid arthritis risk 
Nature genetics  2009;41(12):1313-1318.
To discover novel RA risk loci, we systematically examined 370 SNPs from 179 independent loci with p<0.001 in a published meta-analysis of RA GWAS of 3,393 cases and 12,462 controls1. We used GRAIL2, a computational method that applies statistical text mining to PubMed abstracts, to score these 179 loci for functional relationships to genes in 16 established RA disease loci1,3-11. We identified 22 loci with a significant degree of functional connectivity. We genotyped 22 representative SNPs in an independent set of 7,957 cases and 11,958 matched controls. Three validate convincingly: CD2/CD58 (rs11586238, p=1×10−6 replication, p=1×10−9 overall), and CD28 (rs1980422, p=5×10−6 replication, p=1×10−9 overall), PRDM1 (rs548234, p=1×10−5 replication, p=2×10−8 overall). An additional four replicate (p<0.0023): TAGAP (rs394581, p=0.0002 replication, p=4×10−7 overall), PTPRC (rs10919563, p=0.0003 replication, p=7×10−7 overall), TRAF6/RAG1 (rs540386, p=0.0008 replication, p=4×10−6 overall), and FCGR2A (rs12746613, p=0.0022 replication, p=2×10−5 overall). Many of these loci are also associated to other immunologic diseases.
doi:10.1038/ng.479
PMCID: PMC3142887  PMID: 19898481
8.  Functional phosphodiesterase 11A mutations may modify the risk of familial and bilateral testicular germ cell tumors 
Cancer research  2009;69(13):5301-5306.
Inactivating germline mutations in phosphodiesterase 11A (PDE11A) have been implicated in adrenal tumor susceptibility. PDE11A is highly-expressed in endocrine steroidogenic tissues, especially the testis, and mice with inactivated Pde11a exhibit male infertility, a known testicular germ cell tumor (TGCT) risk factor. We sequenced the PDE11A gene-coding region in 95 patients with TGCT from 64 unrelated kindreds. We identified 8 non-synonymous substitutions in 20 patients from 15 families: four (R52T; F258Y; G291R; V820M) were newly-recognized, three (R804H; R867G; M878V) were functional variants previously implicated in adrenal tumor predisposition, and one (Y727C) was a known polymorphism. We compared the frequency of these variants in our patients to unrelated controls that had been screened and found negative for any endocrine diseases: only the two previously-reported variants, R804H and R867G, known to be frequent in general population, were detected in these controls. The frequency of all PDE11A-gene variants (combined) was significantly higher among patients with TGCT (P=0.0002), present in 19% of the families of our cohort. Most variants were detected in the general population, but functional studies showed that all these mutations reduced PDE activity, and that PDE11A protein expression was decreased (or absent) in TGCT samples from carriers. This is the first demonstration of a PDE gene’s involvement in TGCT, although the cAMP signaling pathway has been investigated extensively in other reproductive organs and their diseases. In conclusion, we report that PDE11A-inactivating sequence variants may modify the risk of familial and bilateral TGCT.
doi:10.1158/0008-5472.CAN-09-0884
PMCID: PMC2734464  PMID: 19549888
Phosphodiesterase (PDE); testicular cancer; TGCT; mutations; cAMP
9.  Familial Mediterranean fever with a single MEFV mutation: Where is the second hit? 
Arthritis and rheumatism  2009;60(6):1851-1861.
Objective
FMF has traditionally been considered an autosomal recessive disease; however, it has been observed that a substantial number of patients with clinical FMF possess only one demonstrable MEFV mutation. Here, an extensive search for a second MEFV mutation was performed in 46 patients clinically diagnosed with FMF and carrying only one high-penetrance FMF mutation.
Methods
MEFV and other candidate genes were sequenced by standard capillary electrophoresis. The entire 15 kb MEFV genomic region was re-sequenced in 10 patients using a hybridization-based chip technology. MEFV gene expression levels were determined by qRT-PCR and pyrin protein levels were examined by Western blotting.
Results
A second MEFV mutation was not identified in any of the screened patients. Haplotype analysis did not identify a common haplotype that might be associated with the transmission of a second FMF allele. Western blots did not demonstrate a significant difference in pyrin levels between single and double variant patients; however, FMF patients of both types showed higher protein expression compared to controls and non-FMF patients with active inflammation. Screening of genes encoding pyrin-interacting proteins identified rare variants in a small number of patients, suggesting the possibility of digenic inheritance.
Conclusion
Our data underscore the existence of a significant subset of FMF patients who are carriers of only one MEFV mutation and demonstrate that complete MEFV sequencing is not likely to yield a second mutation. Screening for the set of most common mutations appears sufficient in the presence of clinical symptoms to diagnose FMF and initiate a trial of colchicine.
doi:10.1002/art.24569
PMCID: PMC2753538  PMID: 19479870
10.  Lack of Association of Caucasian Rheumatoid Arthritis Susceptibility Loci in a Korean Population 
Arthritis and rheumatism  2009;60(2):364-371.
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.
doi:10.1002/art.24245
PMCID: PMC2770844  PMID: 19180477
11.  Chromosome 7q Region Associated with Female Rheumatoid Arthritis in a British Population Fails to Replicate in a North American Case-Control Series 
Arthritis and rheumatism  2009;60(1):47-52.
Objective
The single nucleotide polymorphism (SNP) rs11761231 on chromosome 7q has been reported as a sexually dimorphic marker for rheumatoid arthritis susceptibility in a British population. We sought to replicate this finding and better characterize susceptibility alleles in the region in a North American population.
Methods
DNA from two North American collections of RA patients and controls (1605 cases and 2640 controls) was genotyped for rs11761231 and 16 additional chromosome 7q tag SNPs using Sequenom iPlex assays. Association tests were performed for each collection and also separately contrasting male cases versus male controls and female cases versus female controls. Principal components analysis (EIGENSTRAT) was used to determine association with RA before and after adjusting for population stratification in the subset of the samples (772 cases and 1213 controls) with whole genome SNP data.
Results
We failed to replicate association of the 7q region with rheumatoid arthritis. Initially, rs11761231 showed evidence for association with RA in the NARAC collection (p=0.0076) and rs11765576 showed association with RA in both the NARAC (p = 0.019) and RA replication (p = 0.0013) collections. These markers also exhibited sexual differentiation. However, in the whole genome subset, neither SNP showed significant association with RA after correction for population stratification.
Conclusion
While two SNPs on chromosome 7q appeared to be associated with RA in a North American cohort, the significance of this finding did not withstand correction for population substructure. Our results emphasize the need to carefully account for population structure to avoid false positive disease associations.
doi:10.1002/art.24180
PMCID: PMC2741408  PMID: 19116934
13.  Data for Genetic Analysis Workshop 16 Problem 1, association analysis of rheumatoid arthritis data 
BMC Proceedings  2009;3(Suppl 7):S2.
For Genetic Analysis Workshop 16 Problem 1, we provided data for genome-wide association analysis of rheumatoid arthritis. Single-nucleotide polymorphism (SNP) genotype data were provided for 868 cases and 1194 controls that had been assayed using an Illumina 550 k platform. In addition, phenotypic data were provided from genotyping DRB1 alleles, which were classified according to the rheumatoid arthritis shared epitope, levels of anti-cyclic citrullinated peptide, and levels of rheumatoid factor IgM. Several questions could be addressed using the data, including analysis of genetic associations using single SNPs or haplotypes, as well as gene-gene and genetic analysis of SNPs for qualitative and quantitative factors.
PMCID: PMC2795916  PMID: 20018009
14.  Common variants at CD40 and other loci confer risk of rheumatoid arthritis 
Nature genetics  2008;40(10):1216-1223.
To identify rheumatoid arthritis risk loci in European populations, we conducted a meta-analysis of two published genome-wide association (GWA) studies totaling 3,393 cases and 12,462 controls1,2. We genotyped 31 top-ranked SNPs not previously associated with rheumatoid arthritis in an independent replication of 3,929 autoantibody-positive rheumatoid arthritis cases and 5,807 matched controls from eight separate collections. We identified a common variant at the CD40 gene locus (rs4810485, P = 0.0032 replication, P = 8.2 × 10−9 overall, OR = 0.87). Along with other associations near TRAF1 (refs. 2,3) and TNFAIP3 (refs. 4,5), this implies a central role for the CD40 signaling pathway in rheumatoid arthritis pathogenesis. We also identified association at the CCL21 gene locus (rs2812378, P = 0.00097 replication, P = 2.8 × 10−7 overall), a gene involved in lymphocyte trafficking. Finally, we identified evidence of association at four additional gene loci: MMEL1-TNFRSF14 (rs3890745, P = 0.0035 replication, P = 1.1 × 10−7 overall), CDK6 (rs42041, P = 0.010 replication, P = 4.0 × 10−6 overall), PRKCQ (rs4750316, P = 0.0078 replication, P = 4.4 × 10−6 overall), and KIF5A-PIP4K2C (rs1678542, P = 0.0026 replication, P = 8.8 × 10−8 overall).
doi:10.1038/ng.233
PMCID: PMC2757650  PMID: 18794853
15.  TRAF1-C5 as a Risk Locus for Rheumatoid Arthritis — A Genomewide Study 
The New England journal of medicine  2007;357(12):1199-1209.
BACKGROUND
Rheumatoid arthritis has a complex mode of inheritance. Although HLA-DRB1 and PTPN22 are well-established susceptibility loci, other genes that confer a modest level of risk have been identified recently. We carried out a genomewide association analysis to identify additional genetic loci associated with an increased risk of rheumatoid arthritis.
METHODS
We genotyped 317,503 single-nucleotide polymorphisms (SNPs) in a combined case-control study of 1522 case subjects with rheumatoid arthritis and 1850 matched control subjects. The patients were seropositive for autoantibodies against cyclic citrullinated peptide (CCP). We obtained samples from two data sets, the North American Rheumatoid Arthritis Consortium (NARAC) and the Swedish Epidemiological Investigation of Rheumatoid Arthritis (EIRA). Results from NARAC and EIRA for 297,086 SNPs that passed quality-control filters were combined with the use of Cochran-Mantel-Haenszel stratified analysis. SNPs showing a significant association with disease (P<1×10-8) were genotyped in an independent set of case subjects with anti-CCP-positive rheumatoid arthritis (485 from NARAC and 512 from EIRA) and in control subjects (1282 from NARAC and 495 from EIRA).
RESULTS
We observed associations between disease and variants in the major-histocompatibility-complex locus, in PTPN22, and in a SNP (rs3761847) on chromosome 9 for all samples tested, the latter with an odds ratio of 1.32 (95% confidence interval, 1.23 to 1.42; P = 4×10-14). The SNP is in linkage disequilibrium with two genes relevant to chronic inflammation: TRAF1 (encoding tumor necrosis factor receptor-associated factor 1) and C5 (encoding complement component 5).
CONCLUSIONS
A common genetic variant at the TRAF1-C5 locus on chromosome 9 is associated with an increased risk of anti-CCP-positive rheumatoid arthritis.
doi:10.1056/NEJMoa073491
PMCID: PMC2636867  PMID: 17804836
16.  STAT4 and the Risk of Rheumatoid Arthritis and Systemic Lupus Erythematosus 
The New England journal of medicine  2007;357(10):977-986.
BACKGROUND
Rheumatoid arthritis is a chronic inflammatory disease with a substantial genetic component. Susceptibility to disease has been linked with a region on chromosome 2q.
METHODS
We tested single-nucleotide polymorphisms (SNPs) in and around 13 candidate genes within the previously linked chromosome 2q region for association with rheumatoid arthritis. We then performed fine mapping of the STAT1-STAT4 region in a total of 1620 case patients with established rheumatoid arthritis and 2635 controls, all from North America. Implicated SNPs were further tested in an independent case-control series of 1529 patients with early rheumatoid arthritis and 881 controls, all from Sweden, and in a total of 1039 case patients and 1248 controls from three series of patients with systemic lupus erythematosus.
RESULTS
A SNP haplotype in the third intron of STAT4 was associated with susceptibility to both rheumatoid arthritis and systemic lupus erythematosus. The minor alleles of the haplotype-defining SNPs were present in 27% of chromosomes of patients with established rheumatoid arthritis, as compared with 22% of those of controls (for the SNP rs7574865, P = 2.81×10-7; odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.32). The association was replicated in Swedish patients with recent-onset rheumatoid arthritis (P = 0.02) and matched controls. The haplotype marked by rs7574865 was strongly associated with lupus, being present on 31% of chromosomes of case patients and 22% of those of controls (P = 1.87×10-9; odds ratio for having the risk allele in chromosomes of patients vs. those of controls, 1.55). Homozygosity of the risk allele, as compared with absence of the allele, was associated with a more than doubled risk for lupus and a 60% increased risk for rheumatoid arthritis.
CONCLUSIONS
A haplotype of STAT4 is associated with increased risk for both rheumatoid arthritis and systemic lupus erythematosus, suggesting a shared pathway for these illnesses.
doi:10.1056/NEJMoa073003
PMCID: PMC2630215  PMID: 17804842
17.  Common Genetic Determinants of Uveitis Shared with Other Autoimmune Disorders1 
Uveitis is a complex multifactorial autoimmune disease of the eye characterized by inflammation of the uvea and retina, degeneration of the retina, and blindness in genetically predisposed patients. Using the rat model of experimental autoimmune uveitis (EAU), we previously identified three quantitative trait loci (QTL) associated with EAU on rat chromosomes 4, 12, and 10 (Eau1, Eau2, and Eau3). The primary goal of the current study is to delineate additional non-MHC chromosomal regions that control susceptibility to EAU, and to identify any QTLs that overlap with the QTLs of other autoimmune diseases. Using a set of informative microsatellite markers and F2 generations of resistant and susceptible MHC class II-matched rat strains (F344 and LEW), we have identified several new significant or suggestive QTLs on rat chromosomes 2, 3, 7, 10, and 19 that control susceptibility to EAU. A protective allele was identified in the susceptible LEW strain in the Eau5 locus at D7Wox18, and epistatic interactions between QTLs were found to influence the severity of disease. The newly identified regions (Eau4 through Eau9) colocalize with the genetic determinants of other autoimmune disease models, and to disease-regulating syntenic regions identified in autoimmune patients on human chromosomes 4q21-31, 5q31-33, 16q22-24, 17p11-q12, 20q11-13, and 22q12-13. Our results suggest that uveitis shares some of the pathogenic mechanisms associated with other autoimmune diseases, and lends support to the “common gene, common pathway” hypothesis for autoimmune disorders.
PMCID: PMC2493541  PMID: 18453595
18.  Specificity of the STAT4 Genetic Association for Severe Disease Manifestations of Systemic Lupus Erythematosus 
PLoS Genetics  2008;4(5):e1000084.
Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n = 1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r2 = 0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF) = 31.1% in SLE cases compared with 22.5% in controls (OR = 1.56, p = 10−16). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF = 35.1%, OR = 1.86, p<10−19), nephritis (MAF = 34.3%, OR = 1.80, p<10−11), and age at diagnosis<30 years (MAF = 33.8%, OR = 1.77, p<10−13). An association with severe nephritis was even more striking (MAF = 39.2%, OR = 2.35, p<10−4 in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.
Author Summary
Systemic lupus erythematosus is a chronic disabling autoimmune disease, most commonly striking women in their thirties or forties. It can cause a wide variety of clinical manifestations, including kidney disease, arthritis, and skin disorders. Prognosis varies greatly depending on these clinical features, with kidney disease and related characteristics leading to greater morbidity and mortality. It is also complex genetically; while lupus runs in families, genes increase one’s risk for lupus but do not fully determine the outcome. It is thought that the interactions of multiple genes and/or interactions between genes and environmental factors may cause lupus, but the causes and disease pathways of this very heterogeneous disease are not well understood. By examining relationships between subtypes of lupus and specific genes, we hope to better understand how lupus is triggered and by what biological pathways it progresses. We show in this work that the STAT4 gene, very recently identified as a lupus risk gene, predisposes specifically to severe manifestations of lupus, including kidney disease.
doi:10.1371/journal.pgen.1000084
PMCID: PMC2377340  PMID: 18516230
19.  Several Regions in the Major Histocompatibility Complex Confer Risk for Anti-CCP-Antibody Positive Rheumatoid Arthritis, Independent of the DRB1 Locus 
Molecular Medicine  2008;14(5-6):293-300.
Recent evidence suggests that additional risk loci for RA are present in the major histocompatibility complex (MHC), independent of the class II HLA-DRB1 locus. We have now tested a total of 1,769 SNPs across 7.5Mb of the MHC located from 6p22.2 (26.03 Mb) to 6p21.32 (33.59 Mb) derived from the Illumina 550K Beadchip (Illumina, San Diego, CA, USA). For an initial analysis in the whole dataset (869 RA CCP + cases, 1,193 controls), the strongest association signal was observed in markers near the HLA-DRB1 locus, with additional evidence for association extending out into the Class I HLA region. To avoid confounding that may arise due to linkage disequilibrium with DRB1 alleles, we analyzed a subset of the data by matching cases and controls by DRB1 genotype (both alleles matched 1:1), yielding a set of 372 cases with 372 controls. This analysis revealed the presence of at least two regions of association with RA in the Class I region, independent of DRB1 genotype. SNP alleles found on the conserved A1-B8-DR3 (8.1) haplotype show the strongest evidence of positive association (P ~ 0.00005) clustered in the region around the HLA-C locus. In addition, we identified risk alleles that are not present on the 8.1 haplotype, with maximal association signals (P ~ 0.001–0.0027) located near the ZNF311 locus. This latter association is enriched in DRB1*0404 individuals. Finally, several additional association signals were found in the extreme centromeric portion of the MHC, in regions containing the DOB1, TAP2, DPB1, and COL11A2 genes. These data emphasize that further analysis of the MHC is likely to reveal genetic risk factors for rheumatoid arthritis that are independent of the DRB1 shared epitope alleles.
doi:10.2119/2007-00123.Lee
PMCID: PMC2255558  PMID: 18309376
20.  Association of STAT4 with Rheumatoid Arthritis in the Korean Population 
Molecular Medicine  2007;13(9-10):455-460.
A recent study in the North American White population has documented the association of a common STAT4 haplotype (tagged by rs7574865) with risk for rheumatoid arthritis (RA) and systemic lupus erythematosus. To replicate this finding in the Korean population, we performed a case-control association study. We genotyped 67 single nucleotide polymorphisms (SNPs) within the STAT1 and STAT4 regions in 1123 Korean patients with RA and 1008 ethnicity-matched controls. The most significant four risk SNPs (rs11889341, rs7574865, rs8179673, and rs10181656 located within the third intron of STAT4) among 67 SNPs are identical with those in the North American study. All four SNPs have modest risk for RA susceptibility (odds ratio 1.21–1.27). A common haplotype defined by these markers (TTCG) carries significant risk for RA in Koreans [34 percent versus 28 percent, P = 0.0027, OR (95 percent CI) = 1.33 (1.10–1.60)]. By logistic regression analysis, this haplotype is an independent risk factor in addition to the classical shared epitope alleles at the HLA-DRB1 locus. There were no significant associations with age of disease onset, radiographic progression, or serologic status using either allelic or haplotypic analysis. Unlike several other risk genes for RA such as PTPN22, PADI4, and FCRL3, a haplotype of the STAT4 gene shows consistent association with RA susceptibility across Whites and Asians, suggesting that this risk haplotype predates the divergence of the major racial groups.
doi:10.2119/2007-00072.Lee
PMCID: PMC2014726  PMID: 17932559
21.  Genome-wide association analysis identifies new susceptibility loci for Behçet's disease and epistasis between HLA-B*51 and ERAP1 
Nature genetics  2013;45(2):10.1038/ng.2520.
Patients with Behçet's disease (BD) suffer from episodic inflammation often affecting the orogenital mucosa, skin, and eyes. To discover new BD-susceptibility loci, we performed a genome-wide association study (GWAS) of 779,465 SNPs with imputed genotypes in 1,209 Turkish BD patients and 1,278 controls. We identified novel associations at CCR1, STAT4, and KLRC4. Additionally, two SNPs in ERAP1, encoding ERAP1 p.Asp575Asn and p.Arg725Gln, recessively conferred disease risk. These findings replicated in 1,468 independent Turkish and/or 1,352 Japanese samples (combined meta-analysis p < 2 × 10−9). We also found evidence for interaction between HLA-B*51 and ERAP1 (p = 9 × 10−4). The CCR1 and STAT4 variants were associated with gene expression differences. Three risk loci shared with ankylosing spondylitis and psoriasis (MHC-I, ERAP1, and IL23R, and the MHC-I-ERAP1 interaction), as well as two loci shared with inflammatory bowel disease (IL23R and IL10) implicate shared pathogenic pathways in the spondyloarthritides and BD.
doi:10.1038/ng.2520
PMCID: PMC3810947  PMID: 23291587
22.  A Novel Unstable Duplication Upstream of HAS2 Predisposes to a Breed-Defining Skin Phenotype and a Periodic Fever Syndrome in Chinese Shar-Pei Dogs 
PLoS Genetics  2011;7(3):e1001332.
Hereditary periodic fever syndromes are characterized by recurrent episodes of fever and inflammation with no known pathogenic or autoimmune cause. In humans, several genes have been implicated in this group of diseases, but the majority of cases remain unexplained. A similar periodic fever syndrome is relatively frequent in the Chinese Shar-Pei breed of dogs. In the western world, Shar-Pei have been strongly selected for a distinctive thick and heavily folded skin. In this study, a mutation affecting both these traits was identified. Using genome-wide SNP analysis of Shar-Pei and other breeds, the strongest signal of a breed-specific selective sweep was located on chromosome 13. The same region also harbored the strongest genome-wide association (GWA) signal for susceptibility to the periodic fever syndrome (praw = 2.3×10−6, pgenome = 0.01). Dense targeted resequencing revealed two partially overlapping duplications, 14.3 Kb and 16.1 Kb in size, unique to Shar-Pei and upstream of the Hyaluronic Acid Synthase 2 (HAS2) gene. HAS2 encodes the rate-limiting enzyme synthesizing hyaluronan (HA), a major component of the skin. HA is up-regulated and accumulates in the thickened skin of Shar-Pei. A high copy number of the 16.1 Kb duplication was associated with an increased expression of HAS2 as well as the periodic fever syndrome (p<0.0001). When fragmented, HA can act as a trigger of the innate immune system and stimulate sterile fever and inflammation. The strong selection for the skin phenotype therefore appears to enrich for a pleiotropic mutation predisposing these dogs to a periodic fever syndrome. The identification of HA as a major risk factor for this canine disease raises the potential of this glycosaminoglycan as a risk factor for human periodic fevers and as an important driver of chronic inflammation.
Author Summary
Shar-Pei dogs have two unique features: a breed defining “wrinkled” skin phenotype and a genetic disorder called Familial Shar-Pei Fever (FSF). The wrinkled phenotype is strongly selected for and is the result of excessive hyaluronan (HA) deposited in the skin. HA is a molecule that may behave in a pro-inflammatory manner and create a “danger signal” by being analogous to molecules on the surface of pathogens. FSF is characterized by unprovoked episodes of fever and/or inflammation and resembles several human autoinflammatory syndromes. Here we show that the two features are connected and have the same genetic origin, a regulatory mutation located close to a HA synthesizing gene (HAS2). The mutation is a 16.1 Kb duplication, the copy number of which correlates with HAS2 expression and disease. We suggest that the large amount of HA responsible for the skin condition predisposes to sterile fever and inflammation. HAS2 was previously not known to associate with autoinflammatory disease, and this finding is of wide interest since approximately 60% of human patients with periodic fever syndrome remain genetically unexplained. This investigation also demonstrates how strong artificial selection may affect not only desired and selected phenotypes, but also the health of domestic animals.
doi:10.1371/journal.pgen.1001332
PMCID: PMC3060080  PMID: 21437276
23.  Meta-Analysis of Genome-Wide Association Studies in Celiac Disease and Rheumatoid Arthritis Identifies Fourteen Non-HLA Shared Loci 
PLoS Genetics  2011;7(2):e1002004.
Epidemiology and candidate gene studies indicate a shared genetic basis for celiac disease (CD) and rheumatoid arthritis (RA), but the extent of this sharing has not been systematically explored. Previous studies demonstrate that 6 of the established non-HLA CD and RA risk loci (out of 26 loci for each disease) are shared between both diseases. We hypothesized that there are additional shared risk alleles and that combining genome-wide association study (GWAS) data from each disease would increase power to identify these shared risk alleles. We performed a meta-analysis of two published GWAS on CD (4,533 cases and 10,750 controls) and RA (5,539 cases and 17,231 controls). After genotyping the top associated SNPs in 2,169 CD cases and 2,255 controls, and 2,845 RA cases and 4,944 controls, 8 additional SNPs demonstrated P<5×10−8 in a combined analysis of all 50,266 samples, including four SNPs that have not been previously confirmed in either disease: rs10892279 near the DDX6 gene (Pcombined = 1.2×10−12), rs864537 near CD247 (Pcombined = 2.2×10−11), rs2298428 near UBE2L3 (Pcombined = 2.5×10−10), and rs11203203 near UBASH3A (Pcombined = 1.1×10−8). We also confirmed that 4 gene loci previously established in either CD or RA are associated with the other autoimmune disease at combined P<5×10−8 (SH2B3, 8q24, STAT4, and TRAF1-C5). From the 14 shared gene loci, 7 SNPs showed a genome-wide significant effect on expression of one or more transcripts in the linkage disequilibrium (LD) block around the SNP. These associations implicate antigen presentation and T-cell activation as a shared mechanism of disease pathogenesis and underscore the utility of cross-disease meta-analysis for identification of genetic risk factors with pleiotropic effects between two clinically distinct diseases.
Author Summary
Celiac disease (CD) and rheumatoid arthritis (RA) are two autoimmune diseases characterized by distinct clinical features but increased co-occurrence in families and individuals. Genome-wide association studies (GWAS) performed in CD and RA have identified the HLA region and 26 non-HLA genetic risk loci in each disease. Of the 26 CD and 26 RA risk loci, previous studies have shown that six are shared between the two diseases. In this study we aimed to identify additional shared risk alleles and, in doing so, gain more insight into shared disease pathogenesis. We first empirically investigated the distribution of putative risk alleles from GWAS across both diseases (after removing known risk loci for both diseases). We found that CD risk alleles are non-randomly distributed in the RA GWAS (and vice versa), indicating that CD risk alleles have an increased prior probability of being associated with RA (and vice versa). Next, we performed a GWAS meta-analysis to search for shared risk alleles by combing the RA and CD GWAS, performing both directional and opposite allelic effect analyses, followed by replication testing in independent case-control datasets in both diseases. In addition to the already established six non-HLA shared risk loci, we observed statistically robust associations at eight SNPs, thereby increasing the number of shared non-HLA risk loci to fourteen. Finally, we used gene expression studies and pathway analysis tools to identify the plausible candidate genes in the fourteen associated loci. We observed remarkable overrepresentation of T-cell signaling molecules among the shared genes.
doi:10.1371/journal.pgen.1002004
PMCID: PMC3044685  PMID: 21383967
24.  STAT4: Genetics, Mechanisms, and Implications for Autoimmunity Review for Current Allergy and Asthma Reports 
Recent advances in genetics and technology have led to breakthroughs in understanding the genes that predispose individuals to autoimmune diseases. A common haplotype of the signal transducer and activator of transcription 4 (STAT4) gene has been shown to be associated with susceptibility to rheumatoid arthritis, systemic lupus erythematosus, and primary Sjögren’s syndrome. STAT4 is a transcription factor that transduces interleukin-12, interleukin-23, and type I interferon cytokine signals in T cells and monocytes, leading to T-helper type 1 and T-helper type 17 differentiation, monocyte activation, and production of interferon-γ. Although the evidence for this association is very strong and well replicated, the exact mechanism by which polymorphisms in this gene lead to disease remains unknown. In concert with the identification of other disease-associated loci, elucidating how the variant form of STAT4 modulates immune function should lead to an improved understanding of the pathophysiology of autoimmunity.
PMCID: PMC2562257  PMID: 18682104
25.  Two independent alleles at 6q23 associated with risk of rheumatoid arthritis 
Nature genetics  2007;39(12):1477-1482.
To identify susceptibility alleles associated with rheumatoid arthritis, we genotyped 397 individuals with rheumatoid arthritis for 116,204 SNPs and carried out an association analysis in comparison to publicly available genotype data for 1,211 related individuals from the Framingham Heart Study1. After evaluating and adjusting for technical and population biases, we identified a SNP at 6q23 (rs10499194, ∼150 kb from TNFAIP3 and OLIG3) that was reproducibly associated with rheumatoid arthritis both in the genome-wide association (GWA) scan and in 5,541 additional case-control samples (P = 10−3, GWA scan; P < 10−6, replication; P = 10−9, combined). In a concurrent study, the Wellcome Trust Case Control Consortium (WTCCC) has reported strong association of rheumatoid arthritis susceptibility to a different SNP located 3.8 kb from rs10499194 (rs6920220; P = 5 × 10−6 in WTCCC)2. We show that these two SNP associations are statistically independent, are each reproducible in the comparison of our data and WTCCC data, and define risk and protective haplotypes for rheumatoid arthritis at 6q23.
doi:10.1038/ng.2007.27
PMCID: PMC2652744  PMID: 17982456

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