Sir Peter MacCallum Department of Oncology - Theses

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Type: PhD thesis × Subject: Genomics × Author: Subramanian, Deepak Naga ×

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    Discovery and validation of novel ovarian carcinoma predisposition genes
    Subramanian, Deepak Naga ( 2021)
    Epithelial ovarian carcinoma (EOC) has a significant hereditary component, over half of which cannot be explained by known hereditary breast and ovarian cancer (HBOC) genes (e.g. BRCA1 and BRCA2). Gene discovery studies to date have generally been limited to a small number of candidate genes in relatively few families and have failed to consistently identify any compelling new genes that may account for this missing heritability. The underlying hypothesis of this thesis is that the remaining unexplained EOC families are due to individually rare deleterious variants in numerous genes, each explaining a small proportion of families. To overcome the limitations of earlier targeted panel sequencing efforts, germline whole exome sequencing (WES) was performed on 516 likely familial high-grade serous ovarian cancer (HGSOC) patients with no pathogenic variants in BRCA1 or BRCA2 to discover novel predisposition genes. Forty-three candidate genes enriched for rare loss-of-function (LoF) variants were identified, along with LoF variants in several proposed EOC predisposition genes (e.g. ATM, PALB2). A high degree of genetic heterogeneity was observed, with no single gene harbouring LoF variants in more than 1% of cases. These candidate genes represent diverse functional pathways, with relatively few involved in DNA repair and only a small enrichment for genes involved in homologous recombination (HR) repair. This suggests that many of the remaining HGSOC families are explained by genes in pathways that have been previously under-explored. Since candidate gene variants were individually very rare, orthogonal approaches of tumour sequencing and segregation analysis were undertaken to validate these genes. WES and/or Sanger sequencing was performed on tumour DNA from 105 germline variant carriers, along with bisulphite sequencing of promoter CpG islands for selected genes, to identify evidence of biallelic inactivation and mutational signatures that might support a causative role for that gene. Two genes previously implicated as HGSOC predisposition genes, PALB2 and ATM, displayed biallelic inactivation in nearly every germline variant carrier tumour, associated with characteristic mutational signatures defined principally by the presence or absence of HR repair deficiency, respectively. Of the candidate genes, 19 out of 38 demonstrated biallelic inactivation in at least one tumour from affected carriers, but only three- LLGL2, SCYL3 and MIPOL1- displayed this result consistently in multiple samples, with the others showing loss of the variant allele or returning inconclusive results. Distinctive mutational signatures were found in the LLGL2 and SCYL3 tumours, similar to those for ATM and PALB2, respectively. In the limited number of segregation studies performed amongst six families, none of the tested germline variants consistently segregated with disease. In conclusion, these studies provided data supporting PALB2 and ATM as likely moderate-risk HGSOC predisposition genes, demonstrating the utility of this approach for validating novel familial cancer genes. Several candidate genes showed evidence to indicate a potential predisposing role, but the extreme genetic heterogeneity of unexplained familial HGSOC will necessitate larger studies to confirm these findings.