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Genetic Variants and Melanoma Risk Simon N. Stacey SNPs and Association Studies: Freq SNP-A in Cases / Freq SNP-A in Controls AAGGTTA to ~ Risk of Disease with SNP-A / Risk of Disease without SNP-A ~ Relative Risk (RR) or Odds Ratio (OR) ATGGTTA Marker of Predisposition Location, Location, Location Themes of large scale SNP association studies in melanoma : Searching for melanoma variants among: • Genes affecting variation in pigmentation in Europeans • Genes affecting nevus counts • Genes affecting related cancers: Basal Cell Carcinoma (BCC) • Genes directly associated with melanoma risk • Rare variants detected by large scale sequencing Multiple Testing in Genome Wide Association Studies • Bonferroni adjusted threshold for genome-wide significance: • 300,000 SNPs is 1.7 x 10-7 • 1M SNPs is 5 x 10-8 • 5M SNPs is 1 x 10-8 Genetic variants that affect pigmentation traits in Europeans Using pigmentation to detect cancer loci: chr20 ASIP locus: Melanoma risk variants in MC1R pathway: ASIP Pigmentation-associated variants in MC1R, ASIP and Tyrosinase loci confer risk of both Melanoma and BCC Variants affecting nevus count confer risk of melanoma: Loci near CDKN2A and PLA2G6 (22p13) implicated Do variants that confer risk of BCC also confer risk of melanoma? BCC variant in TERT-CLPTM1L associated with lung, bladder, prostate cancer Variants in CDKN2A/B are also associated with melanoma, breast cancer, NPC and glioma BCC and multi-cancer TERT variant is protective against melanoma: Association of TERT-CLPTM1L rs401681 with BCC and CM Number Frequency Sample Group Risk Allele Cases Controls Cases Controls OR 95% CI P Iceland BCCa C 1,850 34,998b 0.60 0.55 1.23 (1.14, 1.33) 6.0 x 10-8 Eastern Europe BCCa C 525 525 0.62 0.57 1.19 (1.00, 1.42) 4.5 x 10-2 U.S. BCC C 908 826 0.59 0.56 1.12 (0.98, 1.28) 0.10 Spain BCC C 185 1,758 0.55 0.54 1.08 (0.86, 1.35) 0.50 All Non-Icelandic BCC C 1,618 3,109 NA NA 1.13 (1.03, 1.25) 1.1 x 10-2 All BCC Combined C 3,468 38,107 NA NA 1.20 (1.13, 1.27) 4.8 x 10-9 Iceland CMa C 591 34,998b 0.52 0.55 0.90 (0.80, 1.01) 7.9 x 10—2 Sweden CMa C 1,056 2,631 0.49 0.54 0.85 (0.77, 0.94) 1.2 x 10—3 Spain CMa C 748 1,758 0.51 0.54 0.90 (0.80, 1.02) 9.4 x 10—2 Holland CM C 736 1,832 0.53 0.57 0.83 (0.73,0.94) 3.9 x 10—3 Austria CM C 152 376 0.53 0.53 0.98 (0.75,1.27) 0.88 Italy CM C 560 368 0.49 0.56 0.74 (0.62, 0.89) 1.2 x 10—3 All CM Combined C 3,843 41,963 NA NA 0.86 (0.81, 0.91) 5.0 x 10—8 • Related to the different roles of senescence and genome stability in melanocyte and keratinocyte transformation? GWAS directly for melanoma associated variants: • • • • Bishop et al. 2009 Nature Genetics 41, 920-925 Amos et al. 2011 Human Molecular Genetics 20, 5012-5023 MacGregor 2011 Nature Genetics 43, 1114-1118 Barrett et al 2011 Nature Genetics 43, 1108-1113 Chromosomal region 1q21 1q42 2q33 5p15 5p13 6p25 9p23 9p21 11q13 11q14 11q22 16q24 15q13 20q11 21q22 22q13 • • • • • Candidate Gene(s) ARNT / SETDB1 PARP1 CASP8 TERT SLC45A2 IRF4 TYRP1 MTAP-CDKN2A CCND1 TYR ATM MC1R OCA2 ASIP MX2 PLA2G6 Pigmentation Effect Cancer Phenotype Eye Color, Hair Color, (Nevus Count?) Melanoma Melanoma Melanoma Nevus Count Melanoma Protective / BCC Risk Hair Color, Eye Color Melanoma, BCC Freckling, Hair Color Uncertain Eye Color Melanoma Nevus Count Melanoma (BCC, another SNP) Melanoma Eye Color, Freckling Melanoma, BCC Melanoma Hair Color, Freckling Melanoma, BCC Eye Color Uncertain Hair Color, Freckling Melanoma, BCC Melanoma Nevus Count Melanoma Majority of variants are associated with known risk factors Effect on risk factors often does not fully account for effect of variant on melanoma Some pigmentation variants do not appear to be associated with melanoma risk Increasing number of variants have no discernable effect on pigmentation Variants are common and with modest effects Search for rare variants through genome sequencing: TA Manolio et al. Nature 461, 747-753 (2009) doi:10.1038/nature08494 High power is required to detect association of rare variants: • Shows OR required to detect variants to P= 2x10-9 at 80% power • Bonferroni GWS for 30M variants • Rare variants need large effects and/or large sample sizes to detect by association • Sequence data from large numbers of people are required • Integrated approach using both statistical and biological data is required Icelandic Sequencing Project: • Whole genome sequencing of 1,800 Icelanders • Average coverage 18x • Identified 23 million SNPs and 8 million short indels and added functional annotation • Used as a training set to impute genotypes of 72,000 SNP phased, chip-typed Icelanders • Phasing and imputation allows us to carry out case:control associations studies based on full-resolution genomic sequence data (i.e. 30 million variants) Icelandic Sequencing Project discovers BCC variant in TP53: Icelandic Sequencing Project results for MITF E318K: • Bertolotto et al 2011 Nature and Yokoyama et al 2011 Nature reported variant E318K in MITF in familial and sporadic melanoma • Integrated association and biological data • Investigated in Icelandic sequencing project data and replication cohorts: MITF E318K GROUP ICELAND SWEDEN VALENCIA ZARAGOZA VIENNA MILAN HOLLAND P value 3.61E-04 7.92E-03 1.44E-03 0.05 1.00 1.00 0.05 OR 2.28 5.47 3.90 3.61 1.03 inf 2.68 Number Aff COMBINED P value 2.99E-08 OR 2.52 95%CI 1.82-3.49 1057 947 288 1211 552 1339 Freq Aff Number Ctr 0.0038 0.0079 0.0069 0.0029 0.0009 0.0045 2162 1958 1812 1065 368 1787 Freq Ctr 0.0057 0.0007 0.0020 0.0019 0.0028 0.0000 0.0017 Collaborators: deCODE Genetics •Patrick Sulem • Gisli Masson •Daniel F. Gudbjartsson •Thorunn Rafnar •Sigurjón Axel Guðjónsson •Guðmar Þorleifsson •Augustine Kong •Unnur Thorsteinsdottir •Kari Stefansson Comprehensive Cancer Centre and Radboud University Medical Centre Nijmegen •Lambertus A. Kiemeney •Katja Aben National University Hospital Iceland •Jon H. Olafsson •Bardur Sigurgeirsson •Kristrun R. Benediktsdottir •Kristin Thorisdottir •Rafn Ragnarsson Instituto Valenciano de Oncologica, Valencia •Eduardo Nagore Karolinska Institutet •Johan Hansson •Annika Lindblom Medical University of Vienna • Ichiro Okamoto • Hubert Pehamberger • Judith Wendt DKFZ Heidelberg Group •Rajiv Kumar University Hospital Zaragoza •José I. Mayordomo Fondazione IRCCS Istituto Nazionale Tumori, Milan • Monica Roldolfo • Licia Rivoltini