Genetic Variants and Melanoma RiskSimon N. Stacey

SNPs and Association Studies:

AAGGTTA

to

ATGGTTA

Freq SNP-A in Cases / Freq SNP-A in Controls

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Risk of Disease with SNP-A / Risk of Disease without SNP-A

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Relative Risk (RR) or Odds Ratio (OR)

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:

• Related to the different roles of senescence and genome stability in melanocyte and keratinocyte transformation?

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  

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 Candidate Gene(s) Pigmentation Effect Cancer Phenotype1q21 ARNT / SETDB1 Eye Color, Hair Color, (Nevus Count?) Melanoma1q42 PARP1 Melanoma2q33 CASP8 Melanoma5p15 TERT Nevus Count Melanoma Protective / BCC Risk5p13 SLC45A2 Hair Color, Eye Color Melanoma, BCC6p25 IRF4 Freckling, Hair Color Uncertain9p23 TYRP1 Eye Color Melanoma9p21 MTAP-CDKN2A Nevus Count Melanoma (BCC, another SNP)11q13 CCND1 Melanoma11q14 TYR Eye Color, Freckling Melanoma, BCC11q22 ATM Melanoma16q24 MC1R Hair Color, Freckling Melanoma, BCC15q13 OCA2 Eye Color Uncertain20q11 ASIP Hair Color, Freckling Melanoma, BCC21q22 MX2 Melanoma22q13 PLA2G6 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 E318KGROUP P value OR Number Aff Freq Aff Number Ctr Freq CtrICELAND 3.61E-04 2.28 0.0057SWEDEN 7.92E-03 5.47 1057 0.0038 2162 0.0007VALENCIA 1.44E-03 3.90 947 0.0079 1958 0.0020ZARAGOZA 0.05 3.61 288 0.0069 1812 0.0019VIENNA 1.00 1.03 1211 0.0029 1065 0.0028MILAN 1.00 inf 552 0.0009 368 0.0000HOLLAND 0.05 2.68 1339 0.0045 1787 0.0017

P value OR 95%CICOMBINED 2.99E-08 2.52 1.82-3.49

Collaborators:

deCODE Genetics•Patrick Sulem• Gisli Masson •Daniel F. Gudbjartsson•Thorunn Rafnar •Sigurjón Axel Guðjónsson•Guðmar Þorleifsson•Augustine Kong •Unnur Thorsteinsdottir •Kari Stefansson

National University Hospital Iceland •Jon H. Olafsson •Bardur Sigurgeirsson •Kristrun R. Benediktsdottir •Kristin Thorisdottir •Rafn Ragnarsson

Karolinska Institutet •Johan Hansson •Annika Lindblom

Comprehensive Cancer Centre and Radboud University Medical Centre Nijmegen•Lambertus A. Kiemeney•Katja Aben

DKFZ Heidelberg Group•Rajiv Kumar

University Hospital Zaragoza•José I. Mayordomo

Instituto Valenciano de Oncologica, Valencia•Eduardo Nagore

Fondazione IRCCS Istituto Nazionale Tumori, Milan• Monica Roldolfo• Licia Rivoltini

Medical University of Vienna• Ichiro Okamoto• Hubert Pehamberger• Judith Wendt