Genome-wide association analyses identify 13 new susceptibility loci for generalized

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1 Supplementary information for: Genome-wide association analyses identify 13 new susceptibility loci for generalized vitiligo Ying Jin 1,2, Stanca A Birlea 1,3, Pamela R Fain 1,2,4, Tracey M Ferrara 1, Songtao Ben 1, Sheri L Riccardi 1, Joanne B Cole 1, Katherine Gowan 1, Paulene J Holland 1, Dorothy C Bennett 5, Rosalie M Luiten 6, Albert Wolkerstorfer 6, JP Wietze van der Veen 6, Anke Hartmann 7, Saskia Eichner 7, Gerold Schuler 7, Nanja van Geel 8, Jo Lambert 8, E Helen Kemp 9, David J Gawkrodger 10, Anthony P Weetman 9, Alain Taïeb 11, Thomas Jouary 11, Khaled Ezzedine 11, Margaret R Wallace 12, Wayne T McCormack 13, Mauro Picardo 14, Giovanni Leone 14, Andreas Overbeck 15, Nanette B Silverberg 16,17 & Richard A Spritz 1,2 1. Human Medical Genetics Program, University of Colorado School of Medicine, Aurora, Colorado, USA. 2.Department of Pediatrics, University of Colorado School of Medicine, Aurora, Colorado, USA. 3.Department of Dermatology, University of Colorado School of Medicine, Aurora, Colorado, USA. 4. Barbara Davis Center for Childhood Diabetes, University of Colorado School of Medicine, Aurora, Colorado, USA. 5. Division of Biomedical Sciences, St. George s, University of London, London, UK. 6. Department of Dermatology and Netherlands Institute for Pigment Disorders, Academic Medical Centre, University of Amsterdam, The Netherlands. 7. Department of Dermatology, University Hospital Erlangen, Erlangen, Germany. 8. Department of Dermatology, Ghent University Hospital, Ghent, Belgium. 9. Department of Human Metabolism, School of Medicine, University of Sheffield, Sheffield, UK. 10. Department of Dermatology, Royal Hallamshire Hospital, Sheffield, UK. 11. Centre de Référence des maladies rares de la peau, Department of Dermatology, Hôpital St.-André, Bordeaux, France. 12. Department of Molecular Genetics & Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA. 13. Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, Florida, USA. 14. Laboratorio Fisiopatologia Cutanea, Istituto Dermatologico San Gallicano, Rome, Italy. 15. Lumiderm, Madrid, Spain. 16. Department of Dermatology, Columbia University College of Physicians and Surgeons, New York, New York, USA. 17. Pediatric and Adolescent Dermatology, St. Luke's-Roosevelt Hospital Center, New York, New York, USA.

2 Supplementary Figure 1 Genome-wide meta-analysis results. The genome-wide distribution of log 10 P values from the Cochran-Mantel-Haenszel meta-analysis for 495,821 SNPs from GWAS1 and GWAS2 is shown across the chromosomes. The dotted line indicates the threshold for suggestive significance (P < 10-4 ) and the solid line indicates the threshold for genome-wide significance (P < 5 x 10-8 ). The inset shows quantile-quantile plots of the observed versus expected log 10 P values, in red for all 495,821 SNPS, and in blue excluding 1,944 SNPs spanning the extended major histocompatibility complex (chromosome 6, nt 25,895,575-33,427,350 in GRCh37/hg19).

3 Supplementary Figure 2 Working model for the mechanism relating OCA2 protein level to risk of vitiligo versus melanoma. Melanocytes from individuals with rs GG or GA (tan/brown eye color; left) express more OCA2 protein and melanin than melanocytes from individuals with rs AA (blue eye color; right). Accordingly, quantitatively more OCA2 peptide-loaded HLA-A*02:01 molecules are likely presented on the surface of melanocytes from individuals with tan/brown eye color than with blue eye color, potentially modulating greater versus lesser recognition of melanocytes by effector immune cells, and corresponding greater susceptibility to vitiligo versus melanoma, analogous to TYR 1,2.

4 Supplementary Figure 3 Newly identified associations in generalized vitiligo. (a-m) Regional plots of Cochran-Mantel-Haenszel GWAS1 and GWAS2 meta-analysis results for genotyped (black) and imputed (MAF > 0.01; blue) SNPs on the y axis versus chromosomal nucleotide position (GRCh37/hg19) on the x axis surrounding (a) chr16q24.3 (MC1R); (b) chr11q21 (rs rs ); (c) IFIH1; (d) CD80; (e) CLNK; (f) BACH2; (g) TG/SLA; (h) CASP7; (i) CD44; (j) IKZF4; (k) SH2B3; (l) chr22q13.2 (TOB2); (m) TICAM1. The red circles indicate Cochran-Mantel-Haentzel GWA1, GWA2, and replication study meta-analysis P values for the SNPs at each locus shown in Table 1. Arrows indicate gene positions and transcriptional orientation. Dotted arrows indicate genes extending beyond the figure.

7 Supplementary Figure 4 Bioinformatic functional interaction network analysis of proteins encoded by all confirmed and suggestive generalized vitiligo susceptibility genes. Unsupervised functional interaction network analysis was carried out using STRING 9.0 3, considering each of the corresponding proteins as a node and considering only first-order interactions. BTNL2 was chosen to represent the MCH class II gene region. All known genes across the 16q24.3 and 22q13.2 association regions as well as FAM76B as potentially representing the 11q21 association region were iteratively included as a means of potential gene identification. No firstorder edges were shared with other nodes by FOXD3, RERE, CLNK, NLRP1, FAM76B, and no proteins encoded by genes in the 16q24.3 and 22q13.2 association regions shared edges with any other nodes; however, NLRP1 can be connected via addition of either XIAP or IL2. Green, neighborhood; blue, databases; red, experimental evidence 3.

Supplementary Figure 5 Shared genetic associations of identified generalized vitiligo susceptibility loci and other autoimmune diseases.

8 Supplementary Figure 5 Shared genetic associations of identified generalized vitiligo susceptibility loci and other autoimmune diseases. Yellow, same high-risk allele; orange, different high-risk allele(s); unfilled, genetic association uncertain. Note that CTLA4 is only associated with vitiligo in the subset of patients who have other concomitant autoimmune diseases 4. Susceptibility loci that are thus far specific to vitiligo are not shown.

9 Supplementary Table 1 GWAS1 and GWAS2 results for new generalized vitiligo susceptibility loci GWAS 1 GWAS 2 Chr SNP Gene Region RA EA EAF TREND P OR TREND P OR 2q24.2 rs IFIH1 G A x x rs T C x x q13.33 rs CD80 A C x 10-4 * 1.29* 1.93 x 10-4 * 1.46* rs C A x x p16.1 rs CLNK C T x 10-6 * 1.24* 5.81 x 10-3 * 1.23* rs T C x x q15 rs BACH2 G A x x q24.22 rs SLA A G x x q25.3 rs CASP7 G A x x rs T C x x p13 rs CD44 G A x x rs A G x x q21 rs TYR regulation T C x x rs T C x 10-8 * 1.39* 1.37 x 10-2 * 1.25* 12q13.2 rs IKZF4 A C x x rs A C x 10-8 * 1.30* 6.37 x 10-3 * 1.23* 12q24.12 rs SH2B3 T C x x rs T A x 10-6 * 0.80* 5.93 x 10-8 * 0.66* 15q rs OCA2-HERC2 T C x 10-5 * 1.24* 1.91 x 10-3 * 1.29* rs G A x x q24.3 rs MC1R G A x 10-6 * 0.80* 2.83 x 10-4 * 0.76* rs A G x 10-6 * 0.80* 8.83 x 10-7 * 0.69* 19p13.3 rs TICAM1 C T x x q13.2 rs TOB2 G A x 10-4 * 0.80* 3.72 x 10-5 * 0.66* rs79008 G A x x This table reports GWAS1 5 and GWAS2 data for newly identified vitiligo susceptibility loci that had P < 1 x 10-4 in the GWAS meta-analysis (GWAS1 + GWAS2), P < 0.05 in the replication study, P < in the overall metaanalysis (GWAS1 + GWAS2 + replication study), and consistent effect allele and non-significant Breslow-Day heterogeneity statistics for the ORs across all three studies (P > 1.09 x 10-3 ). RA, reference allele; EA, effect allele; EAF, effect allele frequency (among all cases and controls). *Imputed from 1000 Genomes Project data. The association signal at 19p13.3 does not quite achieve the criterion for genome-wide significance but is included for completeness.

10 Supplementary Table 2 Meta-analysis of previous generalized vitiligo susceptibility loci from GWAS1 Chr SNP nt EA RA EAF Gene Region CMH P 1 rs G A 0.40 RERE 3.54 x rs A G 0.10 PTPN x rs G T 0.18 FOXP x rs T G 0.46 LPP 8.83 x rs G A 0.31 HLA-A 2.55 x rs T G 0.39 BTNL x rs T C 0.48 HLA-DRB1-DQA x rs C T 0.46 CCR x rs A G 0.55 IL2RA 4.98 x rs G A 0.75 TYR 9.32 x rs G A 0.25 GZMB 5.60 x rs A G 0.39 UBASH3A 5.81 x rs T G 0.44 C1QTNF x Cochran-Mantel-Haenszel P values were calculated using data from GWAS1 5,6 and GWAS2, as well as data from the original Replication 1 and Replication 2 5,6, carried out following GWAS1 (except for rs , for which Replication 2 data were not available). Chr, chromosome; nt, nucleotide position; EA, effect allele; RA, reference allele; EAF, effect allele frequency; OR, odds ratio.

11 Supplementary Table 3 GWAS meta-analysis and replication data for all SNPs tested Effect Allele Frequency GWAS Meta- Analysis P GWAS Meta- Analysis OR Chr nt SNP Reference Allele Effect Allele Replication Trend P Replication OR Breslow-Day P 1q rs G T E E E-01 2q rs G A E E E rs T C E E E-02 3p rs C T E E E rs T G E E E-02 3q rs A C E-07* 1.33* 3.02E E rs C A E E E-01 4p rs C T E-08* 1.24* 3.03E E rs T C E E E-01 6q rs G A E E E-01 7p rs A G E E E rs T C E E E rs A G E E E rs C T E E E-03 8q rs A G E E E-01 9p rs G T E E E rs C T E-05* 0.82* 1.86E E-04 9q33.3-q rs G A E E E rs T C E E E-03 10q rs G T E E E rs T G E E E-03 10q rs C A E E E rs T C E E E-01 10q rs G A E E E rs T C E E E-01 11p rs G A E E E rs A G E E E-01 11q rs T C E E E rs T C E-10* 1.35* 5.23E E-01 12q rs A C E E E rs A C E-09* 1.28* 4.97E E-01

12 12q rs T C E E E rs T A E-12* 0.76* 4.07E E-01 14q rs T C E E E rs A C E E E-03 15q12-q rs T C E E E rs T C E-07* E E rs G A E E E-01 16q rs G A E-08* 0.80* 1.44E E rs A G E-11* E E-01 18q rs A G E E E-02 19p rs C T E E E-01 19q rs A G E E E rs G A E-07* 0.72* 6.30E E-07 22q rs G A E-07* E E rs79008 G A E E E-01 Chr, chromosome; GWAS meta-analysis, meta-analysis of GWAS1 and GWAS2; asterisk, imputed from 1000 Genomes Project data

13 Supplementary Table 4 In silico functional prediction of all genotyped and imputed missense and splice junction variants in all confirmed non-mhc vitiligo susceptibility loci in EUR Gene Chr nt Position Missense SNP Variant Allele Ancestral Allele VAF CCDS Accession FunctionGVS Amino Acids Amino Acid Position PolyPhen-2 phastcons GERP Most Significant SNP in Region r 2 PTPN rs A G 0.09 CCDS863.1 missense TRP,ARG 620 benign rs IFIH rs C T 0.40 CCDS missense ALA,THR 946 benign rs IFIH rs T C 0.01 CCDS splice-5' rs IFIH rs T C 0.28 CCDS missense HIS,ARG 843 benign rs IFIH rs A G 0.01 CCDS missense THR,ILE 702 benign rs IFIH rs C T 0.01 CCDS missense HIS,ARG 460 benign rs SLA,TG rs T G 0.01 CCDS missense PRO,THR SLA 15 benign rs CASP rs G T 0.10 CCDS missense ASP,GLU 4 benign rs CASP rs G C 0.26 CCDS missense ASP,GLU 255 benign rs CD rs T C 0.01 CCDS missense THR,MET 393 benign rs CD rs A G 0.32 CCDS missense LYS,ARG 417 benign rs CD rs G A 0.00 CCDS missense ILE,VAL 457 benign rs CD rs T C 0.19 CCDS missense ILE,THR 479 benign rs CD rs T C 0.00 CCDS missense PRO,LEU 641 damaging rs TYR rs A C 0.36 CCDS missense SER,TYR 192 damaging rs TYR rs A G 0.21 CCDS missense ARG,GLN 402 damaging rs SH2B rs C T 0.52 CCDS missense TRP,ARG 262 benign rs GZMB rs G A 0.23 CCDS missense TYR,HIS 247 benign rs GZMB rs C G 0.23 CCDS missense PRO,ALA 94 benign rs GZMB rs C T 0.23 CCDS missense ARG,GLN 55 benign rs OCA rs T C 0.00 CCDS missense ALA,THR 481 damaging rs OCA rs T C 0.09 CCDS missense ARG,GLN 419 damaging rs OCA rs A G 0.05 CCDS missense ARG,TRP 305 damaging rs MC1R rs T G 0.06 CCDS missense VAL,LEU 60 damaging rs MC1R rs A C 0.00 CCDS missense ASP,GLU 84 possibly rs

14 damaging MC1R rs A G 0.11 CCDS missense VAL,MET 92 benign rs MC1R rs T C 0.07 CCDS missense ARG,CYS 151 damaging rs MC1R rs T C 0.04 CCDS missense ARG,TRP 160 benign rs MC1R rs A G 0.05 CCDS missense ARG,GLN 163 benign rs UBASH3A rs A G 0.35 CCDS missense SER,GLY 18 benign rs UBASH3A rs T C 0.06 CCDS missense LEU,PHE 28 damaging rs UBASH3A rs A G 0.08 CCDS missense ARG,GLN 324 possibly damaging rs UBASH3A rs G C 0.04 CCDS missense ASP,GLU 466 benign rs C1QTNF rs T C 0.01 CCDS missense ARG,HIS 226 damaging rs C1QTNF rs A G 0.07 CCDS missense PRO,LEU 138 benign rs C1QTNF rs T C 0.18 CCDS missense GLY,ASP 55 benign rs C1QTNF rs C G 0.22 CCDS missense PRO,ARG 42 damaging rs C1QTNF rs A C 0.42 CCDS missense GLY,VAL 21 possibly damaging rs Chr, chromosome; nt,nucleotide; VAF, variant allele frequency; CCDS, Consensus Coding Sequence; Function GVS, Functional Genome Server; phastcons, PHylogenetic Analysis with Space/Time models Conservation; GERP, Genomic Evolutionary Rate Profiling; r 2, Linkage disequilibrium between functional SNP and the most significant SNP in the region. RERE, FOXP1, IFIH1, LPP, CD80, CLNK, BACH2, CCR6, IL2RA, IKZF4, GZMB, and TOB2 contain no genotyped or imputed missense or splice junction variants in the EUR population. MHC loci were not included in this analysis because of the very large amount of variation at each locus.

15 1. Jin, Y., et al. Next-generation DNA re-sequencing identifies common variants of TYR and HLA-A that modulate the risk of generalized vitiligo via antigen presentation. J. Investig. Dermatol. (2012). advance online publication 8 March 2012; doi: /jid Spritz, R.A. The genetics of generalized vitiligo: autoimmune pathways and an inverse relationship with malignant melanoma. Genome Med. 19, 2(10):78 (2010). 3. Szklarczyk, D. et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucl. Acids Res. 39 (Database Issue), D561-D568 (2011). 4. Birlea, S.A., Laberge, G.S., Procopciuc, L.M., Fain, P.R., & Spritz, R.A. CTLA4 and generalized vitiligo: two genetic association studies and a meta-analysis of published data. Pigment Cell Melanoma Res. 22, (2009). 5. Jin, Y. et al. Variant of TYR and autoimmunity susceptibility loci in generalized vitiligo. New Engl. J. Med. 362, (2010). 6. Jin, Y. et al. Common variants in FOXP1 are associated with generalized vitiligo. Nat. Genet. 42, (2010).

This is a repository copy of Genome-wide association analyses identify 13 new susceptibility loci for generalized vitiligo.

This is a repository copy of Genome-wide association analyses identify 13 new susceptibility loci for generalized vitiligo. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/85888/