We genotyped individuals with primary biliary cirrhosis and unaffected controls for suggestive risk loci (genome-wide association P \u3c 1 × 10−4) identified in a previous genome-wide association study. Combined analysis of the genome-wide association and replication datasets identified IRF5-TNPO3 (combined P = 8.66 × 10−13), 7q12-21 (combined P = 3.50 × 10−13) and MMEL1 (combined P = 3.15 × 10−8) as new primary biliary cirrhosis susceptibility loci. Fine-mapping studies showed that a single variant accounts for the IRF5-TNPO3 association. As these loci are implicated in other autoimmune conditions, these findings confirm genetic overlap among such diseases

Chen, Wei

Coltescu, Catalina

Gorlov, Ivan P.

Han, Younghun

Hirschfield, Gideon M.

Juran, Brian D.

Liu, Xiangdong

Lu, Yan

Lu, Yue

Xu, Chun

English

Scholarworks@UTRGV Univ. of Texas RioGrande Valley

University of Texas Rio Grande Valley ScholarWorks @ UTRGV Health and Biomedical Sciences Faculty Publications and Presentations College of Health Professions 8-2010 Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated with primary biliary cirrhosis Gideon M. Hirschfield Xiangdong Liu Younghun Han Ivan P. Gorlov Yan Lu See next page for additional authors Follow this and additional works at: https://scholarworks.utrgv.edu/hbs_fac  Part of the Medicine and Health Sciences Commons Recommended Citation Hirschfield, G. M., Liu, X., Han, Y., Gorlov, I. P., Lu, Y., Xu, C., Lu, Y., Chen, W., Juran, B. D., Coltescu, C., Mason, A. L., Milkiewicz, P., Myers, R. P., Odin, J. A., Luketic, V. A., Speiciene, D., Vincent, C., Levy, C., Gregersen, P. K., Zhang, J., … Siminovitch, K. A. (2010). Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated with primary biliary cirrhosis. Nature genetics, 42(8), 655–657. https://doi.org/10.1038/ng.631 This Article is brought to you for free and open access by the College of Health Professions at ScholarWorks @ UTRGV. It has been accepted for inclusion in Health and Biomedical Sciences Faculty Publications and Presentations by an authorized administrator of ScholarWorks @ UTRGV. For more information, please contact justin.white@utrgv.edu, william.flores01@utrgv.edu. Authors Gideon M. Hirschfield, Xiangdong Liu, Younghun Han, Ivan P. Gorlov, Yan Lu, Chun Xu, Yue Lu, Wei Chen, Brian D. Juran, and Catalina Coltescu This article is available at ScholarWorks @ UTRGV: https://scholarworks.utrgv.edu/hbs_fac/92 Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated withprimary biliary cirrhosisGideon M Hirschfield1,2, Xiangdong Liu2,3, Younghun Han4, Ivan P Gorlov4, Yan Lu2,3,Chun Xu2,3, Yue Lu4, Wei Chen4, Brian D Juran5, Catalina Coltescu1, Andrew L Mason6,Piotr Milkiewicz7, Robert P Myers8, Joseph A Odin9, Velimir A Luketic10, DanuteSpeiciene11, Catherine Vincent12, Cynthia Levy13, Peter K Gregersen14, Jinyi Zhang2,3, EJenny Heathcote1,2, Konstantinos N Lazaridis5, Christopher I Amos4, and Katherine ASiminovitch2,3,151Liver Centre, Toronto Western Hospital, Toronto, Ontario, Canada. 2Department of Medicine,University of Toronto, Toronto, Ontario, Canada. 3Mount Sinai Hospital, Samuel LunenfeldResearch Institute and Toronto General Research Institute, Toronto, Ontario, Canada. 4Departmentof Epidemiology, University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA. 5Centerfor Basic Research in Digestive Diseases, Division of Gastroenterology and Hepatology, MayoClinic College of Medicine, Rochester, Minnesota, USA. 6Department of Medicine, University ofAlberta, Edmonton, Alberta, Canada. 7Liver Unit, Pomeranian Medical School, Szczecin, Poland.8Liver Unit, University of Calgary, Calgary, Alberta, Canada. 9Division of Liver Diseases, Mount SinaiSchool of Medicine, New York, New York, USA. 10Department of Gastroenterology, VirginiaCommonwealth University, Richmond, Virginia, USA. 11Centre of Gastroenterology and Dietetics,Vilnius University, Lithuania. 12Université de Montréal Hospital Centre, Saint-Luc Hospital, Montréal,Quebec, Canada. 13Division of Gastroenterology, Hepatology and Nutrition, Department ofMedicine, University of Florida, Gainesville, Florida, USA. 14Feinstein Institute for Medical Research,North Shore Long Island Jewish Health System, Manhasset, New York, USA. 15Departments ofImmunology and Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.AbstractWe genotyped individuals with primary biliary cirrhosis and unaffected controls for suggestive riskloci (genome-wide association P < 1 × 10−4) identified in a previous genome-wide association study.Combined analysis of the genome-wide association and replication datasets identified IRF5-TNPO3 (combined P = 8.66 × 10−13), 7q12-21 (combined P = 3.50 × 10−13) and MMEL1 (combinedP = 3.15 × 10−8) as new primary biliary cirrhosis susceptibility loci. Fine-mapping studies showedthat a single variant accounts for the IRF5-TNPO3 association. As these loci are implicated in otherautoimmune conditions, these findings confirm genetic overlap among such diseases.© 2010 Nature America, Inc. All rights reserved.Correspondence should be addressed to K.A.S. (ksimin@mshri.on.ca)..AUTHOR CONTRIBUTIONSG.M.H., X.L., C.I.A. and K.A.S. designed the study. X.L., Y.H., I.P.G., Y.L., C.X., Y.L., W.C., J.Z. and C.I.A. performed the genotypingand data analysis. G.M.H., B.D.J., C.C., A.L.M., P.M., R.P.M., J.A.O., V.A.L., D.S., C.V., C.L., P.K.G., E.J.H., K.N.L. and K.A.S.developed the clinical network and sample collection and processing framework required for case and control accrual. G.M.H., C.I.A.and K.A.S. wrote the manuscript. G.M.H., X.L., C.I.A. and K.A.S. vouch for the data and its analysis. G.M.H., C.I.A. and K.A.S. (thesenior and corresponding author) decided in agreement with all other authors to publish this paper.Note: Supplementary information is available on the Nature Genetics website.COMPETING FINANCIAL INTERESTSThe authors declare competing financial interests: details accompany the full-text HTML version of the paper athttp://www.nature.com/naturegenetics/.NIH Public AccessAuthor ManuscriptNat Genet. Author manuscript; available in PMC 2010 August 27.Published in final edited form as:Nat Genet. 2010 August ; 42(8): 655–657. doi:10.1038/ng.631.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptPrimary biliary cirrhosis (PBC) is the most common autoimmune liver disease, characterizedby chronic nonsuppurative destructive cholangitis, circulating anti-mitochondrial antibodiesand the frequent presence of other autoimmune disorders in affected individuals and theirfamily members1. We previously reported a genome-wide association study (GWAS) for PBCthat identified three susceptibility loci, HLA, IL12A and IL12RB2, as being strongly associatedwith PBC, as well as a number of other loci showing suggestive association with thisdisease2. To replicate these findings and evaluate relevance of these latter loci to PBCsusceptibility, we have now tested an additional independent cohort of 857 individuals withPBC (cases) of European descent and 3,198 controls of European descent (including 1,743historic control subjects from the New York Cancer Project3) for PBC associations with 36SNPs across 24 loci (Supplementary Fig. 1); for each locus, association signals at P < 1 ×10−4 were detected for more than one SNP found in the initial GWAS, with the exception ofIRF5-TNPO3, a well-recognized autoimmune disease risk locus. The combination of thesereplication results and our prior genome-wide association data yielded a genetic dataset derivedfrom 1,351 PBC cases and 4,700 controls (Supplementary Methods).Fifteen SNPs replicated (P < 1.39 × 10−3) after Bonferroni adjustment (Table 1 andSupplementary Table 1). In addition to HLA, IL12A and IL12RB2, genes at the IRF5-TNPO3locus, at chromosome 17q12-21 (containing IKZF3, ZPBP2, GSDMB and ORMDL3) and atMMEL1 loci showed significant association with PBC in both the replication analysis and inan analysis combining the replication and initial GWAS datasets (Fig. 1 and SupplementaryFig. 2).Among the PBC loci confirmed by this analysis, the locus at IRF5-TNPO3 (encoding interferonregulatory factor 5 and transportin 3) is of interest because of the integral immunoregulatoryroles for IRF5, the prior association of this locus with systemic lupus erythematosus (SLE)4,5, systemic sclerosis6 and Sjögrens syndrome7, and the strong effect of the disease-associatedSNP at this locus on disease risk (rs10488631) in the replication (P = 1.13 × 10−8, odds ratio(OR) = 1.58) and combined datasets (P = 8.66 × 10−13, OR = 1.57). Association of this locuswas thus explored, initially by resequencing the IRF5 locus intronic and exonic regions andthe 5′ and 3′ flanking regions of IRF5 in genomic pools of 100 subjects so as to delineate thegenetic variation across the locus. This analysis confirmed prior reports of 41 polymorphismsacross this region, but it failed to identify any new variants. Among these 41 SNPs, two(rs3807135 and rs3834330) failed repeatedly in genotyping assays and four (rs10275092,rs12537192, rs1727172 and rs754280) showed negligible polymorphism. Thus, for fine-mapping studies, 1,330 cases and 1,833 controls were geno-typed for 35 SNPs across thisregion. Among the associations identified (Supplementary Table 2), the strongest signals werefrom the rs12539741 and rs2070197 alleles. These variants map to just 3′ of the IRF5 codingregion and are in tight linkage disequilibrium with one another (r2 > 0.95), and theirassociations with PBC reach fine-mapping P values of 1.65 × 10−10 (OR = 1.63) and 3.74 ×10−10 (OR = 1.62), respectively. Conditional logistic regression analysis did not demonstrateany evidence for multiple genetic effects at the IRF5-TNPO3 locus, with the association at thislocus with PBC appearing to be accounted for by a single variant, either rs12539741 orrs2070197 (Supplementary Table 3). Although unidentified variants outside the sequencedregion may also be relevant to disease association or may even be disease causal, the latter twoSNPs are among the SNPs at this locus that are highly associated with SLE, and both arecorrelated with changes in IRF5 expression in transformed B cells4. By contrast, an insertion-deletion polymorphism 64 bp upstream of IRF5 exon 1a, representing a putative disease-causalvariant for SLE, showed modest association with PBC (fine-mapping P = 1.00 × 10−3). Noassociations were detected between PBC and another SLE-associated SNP, rs2004640, or a 3′UTR variant, rs10954213, that has previously been identified as a predictor of IRF5 expressionlevel4.Hirschfield et al. Page 2Nat Genet. Author manuscript; available in PMC 2010 August 27.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptA second region of interest is the 17q12-21 locus, as all eight of the tested SNPs across thisregion achieved significance in the replication analysis (P values between 1.78 × 10−9 and1.88 × 10−5). This chromosomal region has also been associated with asthma8, Crohn'sdisease9 and type 1 diabetes10 and contains four genes, ZPBP2 (encoding zona pellucida–binding protein 2), IKZF3 (encoding IKAROS family zinc finger 3 protein, involved inleukocyte development and IgE production), GSDMB (encoding gasdermin-B, involved inepithelial barrier function) and ORMDL3 (encoding ORM1-like protein 3). All eight of theSNPs tested here were in linkage disequilibrium (pairwise r2 values ranged from 0.66 to 0.96),but the strongest association signal came from a ZPBP2 SNP, rs11557467 (replication P = 1.78× 10−9, OR = 0.71; combined P = 3.50 × 10−13, OR = 0.72). Although further studies areneeded to pinpoint the relevant disease-causative allele(s), results of additive conditionallogistic regression analysis suggest that this SNP fully accounts for the association signal acrossthis region (Supplementary Table 4).The replication and combined association data also identified a strong association of PBC withtwo SNPs (rs3890745 and rs3748816) at the MMEL1 (encoding membrane metallo-endopeptidase–like 1) locus on chromosome 1p36 (replication P = 3.31 × 10−6, OR = 1.31,combined P = 2.28 × 10−9, OR = 1.32 and replication P = 8.14 × 10−5, OR = 1.31, combinedP = 3.15 × 10−8, OR = 1.33, respectively) (Table 1 and Supplementary Table 1). These SNPsare in linkage disequilibrium with one another (r2 > 0.88) and one of them (rs3748816) is anonsynonymous SNP in exon 16 that encodes a potentially functional methionine-to-threoninesubstitution, whereas the other (rs3890745) maps within intron 2 of MMEL1 and has beenassociated with risk for rheumatoid arthritis and for celiac disease11,12.A suggestive association signal (combined P = 9.12 × 10−7, OR = 1.27) was observed in thecombined case-control cohort for rs3745516, an intronic SNP in SPIB, which is a geneencoding the Spi-B transcription factor. This SNP did not achieve significance in the replicationcohort after Bonferroni correction (P = 6.21 × 10−3, OR = 1.18), but the strength of associationin the combined analysis as well as the role of Spi-B in dendritic cell development13 and B-cell receptor signaling are in keeping with potential relevance of this locus to PBCpathogenesis.Anti-mitochondrial antibodies (AMA) are found in most individuals with PBC, but nocorrelation of specific PBC genotypes with AMA status was observed in our prior2 or currentassociation studies (data not shown). PBC is also associated with specific nuclear antibodies,with some 20% of individuals with PBC manifesting glycoprotein-210 antibodies (anti-gp210)directed against the human nuclear pore complex and/or sp100 antibodies that recognize a 53-kDa nuclear antigen14. Evaluation of genotype status in the subset of 462 cases typed fornuclear antibodies revealed a strong association of the HLA locus (rs9277535 at HLA-DPB1)with disease (P = 4.25 × 10−8, OR = 2.25) in anti-sp100–positive individuals (SupplementaryTable 5a) in contrast to anti-sp100–negative individuals (P = 3.52 × 10−4, OR = 1.36). A furtheranalysis incorporating GWAS data available for 412 of the individuals with known anti-sp100status also revealed that among 13 MHC-region SNPs tested, three SNPs at the HLA-DPB1locus were the most strongly associated with disease in anti-sp100–positive individuals but notin anti-sp100–negative individuals (Supplementary Table 5b). In particular, for these SNPs,the OR associations with PBC risk were much higher in anti-sp100– positive cases comparedto anti-sp100–negative cases. By contrast, no genetic distinctions were apparent for the anti-gp210–positive subgroup (Supplementary Table 6). These findings need to be interpretedcautiously in view of the relatively small sample size used here, but they suggest a relevanceof the HLA locus to anti-sp100 status, which parallels previously reported correlations of anti-CCP and HLA-DRB1 status in rheumatoid arthritis15.Hirschfield et al. Page 3Nat Genet. Author manuscript; available in PMC 2010 August 27.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptIn conclusion, we identify IRF5-TNPO3, 17q12-21 and MMEL1 as three new risk loci for PBC.Our data also suggest some genetic sub-structure may exist in PBC in relation to anti-sp100status. Notably, all the PBC risk loci replicated or identified here have been implicated in otherautoimmune diseases. Our data provide further evidence for the existence of sharedautoimmunity susceptibility loci that contribute to the frequent appearance of additionalautoimmune diseases in individuals with PBC and their families.Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.AcknowledgmentsThis research was supported by grants from the Canadian Institutes for Health Research (MOP 74621), the OntarioResearch Fund (RE01-061), the Canadian Primary Biliary Cirrhosis Society, the Canadian Foundation for Innovation,the Ben and Hilda Katz Charitable Foundation, the US National Institutes of Health (K23 DK68290, RO3 DK78527and RO1 DK80670), the American Gastroenterological Association and the A.J. and Sigismunda Palumbo CharitableTrust. K.A.S. is a recipient of a Canada Research Chair award. We thank all the study subjects and supportingphysicians as well as the technical staff of the University Health Network Analytical Genetics Technology Centre fortheir assistance in this research.References1. Lindor KD, et al. Hepatology 2009;50:291–308. [PubMed: 19554543]2. Hirschfield GM, et al. N. Engl. J. Med 2009;360:2544–2555. [PubMed: 19458352]3. Mitchell MK, Gregersen PK, Johnson S, Parsons R, Vlahov D. J. Urban Health 2004;81:301–310.[PubMed: 15136663]4. Graham RR, et al. Proc. Natl. Acad. Sci. USA 2007;104:6758–6763. [PubMed: 17412832]5. Sigurdsson S, et al. Hum. Mol. Genet 2008;17:872–881. [PubMed: 18063667]6. Dieudé P, et al. Arthritis Rheum 2009;60:225–233. [PubMed: 19116937]7. Miceli-Richard C, et al. Arthritis Rheum 2009;60:1991–1997. [PubMed: 19565491]8. Bouzigon E, et al. N. Engl. J. Med 2008;359:1985–1994. [PubMed: 18923164]9. Barrett JC, et al. Nat. Genet 2008;40:955–962. [PubMed: 18587394]10. Barrett JC, et al. Nat. Genet 2009;41:703–707.11. Raychaudhuri S, et al. Nat. Genet 2008;40:1216–1223. [PubMed: 18794853]12. Coenen MJ, et al. Hum. Mol. Genet 2009;18:4195–4203. [PubMed: 19648290]13. Schotte R, Nagasawa M, Weijer K, Spits H, Blom B. J. Exp. Med 2004;200:1503–1509. [PubMed:15583020]14. Muratori L, Granito A, Muratori P, Pappas G, Bianchi FB. Clin. Liver Dis. vii 2008;12:261–276.15. Snir O, et al. Ann. Rheum. Dis 2009;68:736–743. [PubMed: 18635594]Hirschfield et al. Page 4Nat Genet. Author manuscript; available in PMC 2010 August 27.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptfigure 1.Association plots for the IRF5-TNPO3, 17q12-21 and MMEL1 loci. (a–c) Strength of theassociations and recombination rates estimated from HapMap data for genotyped SNPs areshown for the (a) IRF5-TNPO3, (b) 17q12-21 and (c) MMEL1 loci. Both genome-wideassociation (circles) and combined fine-mapping (diamonds) data are shown where available.The extent of linkage disequilibrium with the most significant polymorphisms are indicatedby the size of each data point; larger data points indicate stronger linkage disequilibrium. Genepositions for each gene region are indicated by the arrows, with the arrow direction representingthe orientation of translation. Linkage disequilibrium was calculated using observed data inPLINK.Hirschfield et al. Page 5Nat Genet. Author manuscript; available in PMC 2010 August 27.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptHirschfield et al. Page 6Table 1PBC GWAS, replication and combined association analysesGWASReplicationCombined stageEuropeanNorth AmericanTotalSNPGene lociRisk alleleRAF case/controlP GWARAF case/controlPRAF case/controlPRAF case/controlPaORb  (95% CI)PcRAF case/controlPdOR (95% CI)Pcrs10488631IRF5-TNPO3C0.168/0.1184.23 × 10–50.193/0.1131.43 × 10–20.158/0.1082.20 × 10–70.164/0.1081.13 × 10–81.58 (1.35–1.85)0.480.165/0.1118.66 × 10–131.57 (1.38–1.77)0.85rs11557467ZPBP2G0.434/0.5113.00 × 10–50.478/0.5519.39 × 10–20.425/0.5107.25 × 10–90.433/0.5121.78 × 10–90.71 (0.64–0.80)0.790.434/0.5113.50 × 10–130.72 (0.66–0.79)0.9rs3748816MMEL1C0.410/0.3398.50 × 10–50.355/0.2831.30 × 10–10.395/0.3342.63 × 10–40.389/0.3328.14 × 10–51.31 (1.15–1.50)0.760.399/0.3343.15 × 10–81.33 (1.20–1.47)0.93RAF, risk allele frequency; OR, odds ratio. The GWAS included 494 cases and 1,502 controls, the replication included 857 cases and 3,198 controls, and the combined collection included 1,351 cases and 4,700 controls.a Replication P.b Odds ratios are given for the risk allele.c Breslow-Day P.d Combined P. This refers to analyses incorporating the combined GWAS and replication datasets.Nat Genet. Author manuscript; available in PMC 2010 August 27.

Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated with primary biliary cirrhosis

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Variants at IRF5-TNPO3, 17q12-21 and MMEL1 are associated with primary biliary cirrhosis

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