Dissecting the genetic architecture of familial melanoma

Abstract

Cutaneous malignant melanoma (CMM) is a disease that has had a significant impact on the lives of Australians, affecting 1 in 17 people. Around 10% of those diagnosed have a family history of melanoma and in the majority of these cases there is underlying predisposition that makes them more susceptible to melanoma development. Known high-risk loci (CDKN2A, CDK4) account for approximately half of these families. There remains, therefore, a large gap in knowledge regarding the causes of susceptibility to CMM.Uveal malignant melanoma (UMM) is a rare type of melanoma that forms in the uveal tract of the eye. Unlike cutaneous melanoma, ultraviolet radiation does not contribute to the formation of UMM. To date, only one gene has been identified that contributes to risk of uveal melanoma: BAP1.The main objective of this thesis was to discover the underlying germline mutations that contribute to increased risk in high-density CMM and UMM families lacking mutations in any of the known melanoma risk loci. The key techniques that were used to achieve this were whole-genome and exome sequencing of multiple cases from these families. By targeting novel variants that segregated with disease in families, truncating mutations leading to CMM development were found in POT1, ACD and TERF2IP (Chapters 4 and 5). This technique was also used to discover truncating germline BAP1 mutations in a UMM family from Denmark (Chapter 7). A second method of gene discovery that was used was the candidate gene approach. This first involved a literature search to discover likely candidates for melanoma susceptibility. The protein-coding regions of the chosen genes were then Sanger sequenced to identify novel variants in probands from melanoma families. This method was used to interrogate the role of PALB2, SOX10 and MITF in CMM predisposition (Chapter 3).The contribution of known melanoma predisposition genes CDKN2A, CDK4 and BAP1 to a population-based CMM sample from Queensland was also assessed. This was achieved through a combination of targeted pull-down of the selected genes using the Ion Torrent Personal Genome Machine, and also single nucleotide polymorphism analysis using Sequenom mass spectrometry. Chapters 2 and 6 of this thesis detail the contribution of these genes to CMM and UMM predisposition in population-based samples from Australia.Overall this project has made a major contribution to the field of familial melanoma genetics. We described germline mutations in members of the shelterin complex (POT1, ACD and TERF2IP) in CMM, which accounts for 9% of families lacking mutations in previously known risk loci CDKN2A, and CDK4. In combination with a previous finding of mutations in TERT, the work that is presented in this thesis has contributed to the discovery of a new pathway related to melanoma predisposition and thus represents a significant advance in the field.Additionally, we have estimated the contribution of known risk genes CDKN2A and BAP1 to susceptibility in a population-based sample of Queensland melanoma cases (N=1,109). We showed that CDKN2A accounts for around 1.31% of Queensland melanoma families and BAP1 accounts for around 0.63%. Additionally, we showed CDK4 mutations are extremely rare, not being found in 1,550 melanoma families from Queensland.In summary, next-generation sequencing techniques have been an invaluable tool in melanoma predisposition gene discovery. We have identified several new genes that contribute to melanoma susceptibility when mutated in the familial setting. By discovering and characterising these genes, better surveillance and treatment strategies may be available in the future for individuals with familial melanoma.