Mykyta Artomov1,2, Alexander J Stratigos3, Ivana Kim4, Raj Kumar5, Martin Lauss4,6, Bobby Y Reddy5, Benchun Miao5, Carla Daniela Robles-Espinoza7,8, Aravind Sankar7, Ching-Ni Njauw5, Kristen Shannon9, Evangelos S Gragoudas4, Anne Marie Lane4, Vivek Iyer7, Julia A Newton-Bishop10, D Timothy Bishop5,10, Elizabeth A Holland11, Graham J Mann11,12, Tarjinder Singh13, Mark J Daly1, Hensin Tsao9. 1. MGH Analytic and Translational Genetics Unit, MGH and Broad Institute, Boston, MA. 2. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA. 3. 1st Department of Dermatology, University of Athens School of Medicine, Andreas Sygros Hospital, Athens, Greece. 4. Retina Service, Massachusetts Eye and Ear Infirmary, Boston, MA. 5. Department of Dermatology, Wellman Center for Photomedicine, MGH, Boston, MA. 6. Department of Oncology, Clinical Sciences, Lund University, Lund, Sweden. 7. Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Cambridge, UK. 8. Laboratorio Internacional de Investigación sobre el Genoma Humano, Universidad Nacional Autónoma de México, Santiago de Querétaro, Mexico. 9. Melanoma Genetics Program, MGH Cancer Center, MGH, Boston, MA. 10. Section of Epidemiology and Biostatistics, Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UK. 11. Centre for Cancer Research, Westmead Institute for Medical Research, University of Sydney, Westmead, Australia. 12. Melanoma Institute Australia, University of Sydney, North Sydney, NSW, Australia. 13. Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK.
Abstract
Background: Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM). Methods: Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three human melanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of human melanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided. Results: Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10-6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10-4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10-4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10-5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels. Conclusions: The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene.
Background: Extraordinary progress has been made in our understanding of common variants in many diseases, including melanoma. Because the contribution of rare coding variants is not as well characterized, we performed an exome-wide, gene-based association study of familial cutaneous melanoma (CM) and ocular melanoma (OM). Methods: Using 11 990 jointly processed individual DNA samples, whole-exome sequencing was performed, followed by large-scale joint variant calling using GATK (Genome Analysis ToolKit). PLINK/SEQ was used for statistical analysis of genetic variation. Four models were used to estimate the association among different types of variants. In vitro functional validation was performed using three humanmelanoma cell lines in 2D and 3D proliferation assays. In vivo tumor growth was assessed using xenografts of humanmelanoma A375 melanoma cells in nude mice (eight mice per group). All statistical tests were two-sided. Results: Strong signals were detected for CDKN2A (Pmin = 6.16 × 10-8) in the CM cohort (n = 273) and BAP1 (Pmin = 3.83 × 10-6) in the OM (n = 99) cohort. Eleven genes that exhibited borderline association (P < 10-4) were independently validated using The Cancer Genome Atlas melanoma cohort (379 CM, 47 OM) and a matched set of 3563 European controls with CDKN2A (P = .009), BAP1 (P = .03), and EBF3 (P = 4.75 × 10-4), a candidate risk locus, all showing evidence of replication. EBF3 was then evaluated using germline data from a set of 132 familial melanoma cases and 4769 controls of UK origin (joint P = 1.37 × 10-5). Somatically, loss of EBF3 expression correlated with progression, poorer outcome, and high MITF tumors. Functionally, induction of EBF3 in melanoma cells reduced cell growth in vitro, retarded tumor formation in vivo, and reduced MITF levels. Conclusions: The results of this large rare variant germline association study further define the mutational landscape of hereditary melanoma and implicate EBF3 as a possible CM predisposition gene.
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