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Alveolar rhabdomyosarcoma is characterised by a t(2;13)(q35;qI4) chromosome translocation, which leads to the fusion of the P AX3 artd the FKHR genes. The resulting fusion gene encodes a chimeric protein which has aberrant transcriptional activity. The data here describes the molecular definition of the genomic breakpoints on both derivative chromosomes in one case and the derivative chromosome 13 breakpoints in two other cases. The DNA sequences adjacent to the breakpoints on the derivative chromosome 13 are remarkable for their resemblartce to recognition sequences for the protein trartslin. Electrophoretic mobility shift studies (EMSA) confirm that these sequences bind translin. These findings suggest that translin may not only be important in the genesis of chromosomal trartslocations in lymphoid malignancy, but also in translocations found in solid tumours. Mutation analysis of tumour samples and cell lines from patients with embryonal and alveolar rhabdomyosarcoma suggests that there are no subtle disease associated mutations within the P AX3 gene that could contribute towards the neoplastic state.

Mosaic variegated aneuploidy (MVA) syndrome is an autosomal recessive disorder characterised by mosaic aneuploidies, a variety of phenotypic abnormalities and predisposition to cancer. This study aimed to identify the genetic defect underlying MVA and to characterise the associated cellular and clinical phenotypes. Following a candidate gene screening approach, I identified biallelic BUB1B mutations as a cause of MVA. BUB1B encodes BUBR1 which has multiple, crucial roles in the mitotic spindle checkpoint. I subsequently demonstrated that BUBR1 expression was reduced in BUB1B cases. Furthermore, transfer of human chromosome 15, containing the BUB1B locus, complemented the cellular phenotype in BUB1B mutation-positive cells. As hereditary cancer genes are frequently implicated in sporadic cancers, I undertook mutational analyses of sporadic childhood tumours which confirmed that somatic BUB1B mutations are unlikely to be common in cancers of the types typically associated with MVA. To enable stratification of cases into phenotypically characterised subgroups, I evaluated the molecular, cellular and clinical features associated with MVA. Cases can be classified according to the presence (40%) or absence (60%) of BUB1B mutations. Whilst a strong phenotypic overlap of BUB1B and non-BUB1B cases renders the delineation of clinical subgroups difficult, BUB1B cases do appear to be at higher risk of developing cancer. Karyotypic analyses revealed that there was no apparent correlation between mutation status and the extent of aneuploidies. However, assessment of mitotic checkpoint function demonstrated that the checkpoint defect was profound in BUB1B cases; in contrast, most non-BUB1B cases analysed had intermediate or normal checkpoint function. Our results clearly highlight the heterogeneous nature of MVA syndrome. Furthermore, our data were the first to relate germline mutations in a mitotic checkpoint gene with a human disorder and strongly support a causal role for aneuploidy in cancer development. The aim of future work would be to identify the causative gene(s) in non-BUB1B cases.

Tumour suppressor gene (TSG) inactivation plays a major role in the development of cancer. To date there has been no reported systematic approach to the identification of such genes in the development of squamous carcinoma of the head and neck (SCCHN). We have therefore analysed allelic loss of heterozygosity (LOH) in a series of 28 patients with a primary SCCHN. Using the polymerase chain reaction (PCR) to amplify Microsatellite markers we have been able to compare allelic loss between normal and tumour DNA. Additional RNase protection assays were used to screen for APC gene mutations while southern blot analysis was performed to identify cyclin-D gene amplification. Our results demonstrate several regions showing a high frequency of LOH: 3p(44%), 5q(43%), 9q(35%), 11q(45%) and 17p(40%). Further analysis of chromosome 5 has highlighted 5q21 (APC/MCC genes) as its commonest site of LOH. However screening the APC gene failed to detect mutations in the mutation cluster region (MCR). Out of 9 tumours with LOH at 11q13 we were able to exclude, by southern blot analysis, cyclin-D amplification in all but one of these tumours. Our results indicate the locations of several possible TSGs involved in the development of SCCHN.

Germline mutations in the tumor suppressor gene PTEN (phosphatase and tensin homology deleted on chromosome 10) cause Cowden and Bannayan-Riley- Ruvalcaba (BRR) syndromes, two dominantly inherited disorders characterized by mental retardation, multiple hamartomas, and variable cancer risk. Here, we modeled three sentinel mutant alleles of PTEN identified in patients with Cowden syndrome and show that the nonsense PtenΔ4-5 and missense PtenC124R and PtenG129E alleles lacking lipid phosphatase activity cause similar developmental abnormalities but distinct tumor spectrawith varying severity and age of onset. Allele-specific differences may be accounted for by loss of function for PtenΔ4-5, hypomorphic function for PtenC124R, and gain of function for Pten G129E. These data demonstrate that the variable tumor phenotypes observed in patients with Cowden and BRR syndromes can be attributed to specificmutations in PTEN that alter protein function through distinct mechanisms.

Colorectal cancer (CRC) is common, with >1 million annual incidence worldwide and is associated with significant morbidity and mortality. Prevention is a particularly appealing strategy to combat CRC, but there is a paucity of well-founded mechanistic research in the area. CRC is a complex disorder, with both genetic and environmental factors influencing incidence. The heritable component of CRC variance is estimated to be ~35%, with ~5% due to highly penetrant mutations. Common genetic variance likely makes up the majority of the heritable component, yet a large proportion of heritability remains unexplained. One possible explanation is gene-environment interactions (GxE) where-by both genetic and environmental factors interact to influence risk. Observational data implicate vitamin D as an environmental risk factor in CRC aetiology and in-house data indicates that genotype at the GWAS identified CRC risk locus rs9929218 influences this association, i.e. a GxE. The rs9929218 locus is intronic within CDH1, a tumour suppressor gene, and present evidence suggests a gene-environment interaction model of vitamin D-induced CDH1 transcription dependent on genotype at the rs9929218 locus and mediated by VDR and FOXO transcription factors and SIRT1, a FOXO regulator. To test this model, two broad approaches were employed - an observational approach to assess the association between human plasma vitamin D status, rs9929218 genotype and normal colorectal mucosa CDH1 expression and an interventional approach treating cell lines, organoids and human subjects with vitamin D to assess genotype-specific effects. Observational analysis of vitamin D level (25OHD) in CRC cases identified a significant influence of age, gender, BMI and selected vitamin D pathway genetic variation, while analysis of 424 normal colorectal mucosa samples from CRC cases and cancer-free subjects demonstrated strong sampling, gender, age and site differences in gene expression. 25OHD was not significantly associated with mucosa gene expression at individual gene level. However, association with a number of pathways relevant to tumourigenesis, including 'cell adhesion', 'migration' and 'cell death' was seen, providing possible mechanism to the published observational data. Circulating 25OHD level was not associated with mucosa CDH1 expression, yet crucially, analysis demonstrated a strong additive gene-environment interaction effect between plasma 25OHD, the rs9929218 genotype and NM expression of VDR, FOXO and SIRT1 explaining ~70% of the variance of mucosal CDH1 expression. The interventional approach first investigated vitamin D effects on CRC cell lines and human colorectal mucosa organoids. Calcitriol, the active form of vitamin D, induced CDH1 expression in 4 CRC cell lines, with interaction effects explaining 66% of the variance of CDH1 expression. CDH1 induction was replicated in human colorectal epithelial organoids, a non-aberrant tissue model, while gene enrichment analysis from both cells and organoids implicate vitamin D in a number of processes relevant to CRC tumourigenesis including regulation of cell proliferation, differentiation, migration and apoptosis, consistent with the pleiotropic effects of vitamin D reported in the published literature. Finally, a novel human intervention study was undertaken to investigate the impact of vitamin D supplementation in human blood and rectal mucosa. Short-term high-dose supplementation of 50 participants significantly induced CDH1 expression in rectal mucosa, with significant gene interaction effects between 25OHD, rs992818 genotype and CDH1, VDR and FOXO expression, thus independently replicating the same gene interaction effects seen in the human observational study. Meanwhile, transcriptome profiling identified numerous pathways relevant to tumourigenesis significantly enriched after supplementation, validating several pathways also associated with vitamin D status in the observational study. In summary, the research presented in this thesis demonstrates that vitamin D treatment of cells, epithelial organoids and human subjects induces CDH1 expression, and that strong gene interaction effects involving the colorectal cancer risk locus rs9929218 modulate this effect. FOXO transcription factors influence the gene interaction effect, consistent with the proposed model of ligand dependent regulation of FOXO by VDR and transcription activation of the CDH1 gene by the FOXO complex dependent on rs9929218 genotype. These data provide support for rectal CDH1 expression as an intermediate biomarker for vitamin D chemopreventive studies and suggest that gene environment interactions underlie some of the missing heritability of CRC.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Cutaneous melanoma is a cancer of melanocytes, the pigment-producing cells in our skin. It is one of the most aggressive human malignancies, constituting only about 2% of all dermatological cancers but being responsible for over 75% of all deaths from skin cancer. It has recently become a major public health problem, as it is now the fifth most common cancer in the United Kingdom after its incidence more than quadrupled in the last three decades. For these reasons, understanding the biological processes that are involved in its development is of great importance for devising novel treatments and for the management of patients in the clinic. The study of the genetic factors that influence melanoma risk can uncover mechanisms that are relevant in the transition from a benign melanocyte to a malignant melanoma. Approximately 10% of all melanoma cases are familial, and about half of these familial cases can be explained by pathogenetic variants in genes such as cyclin-dependent kinase inhibitor 2A (CDKN2A), cyclin-dependent kinase 4 (CDK4), breast cancer 2 (BRCA2), BRCA1-associated protein-1 (BAP1) and in the promoter of the telomerase reverse transcriptase (TERT). However, about 50% of all familial melanoma cannot be explained by mutations in known genes. In this dissertation, I detail the methodology I followed in an effort to uncover additional high-penetrance melanoma susceptibility genes. I analysed exome and genome sequence data from a total of 184 individuals that belong to 105 melanoma-prone families from the United Kingdom, The Netherlands and Australia that did not have any pathogenetic variants in known susceptibility genes. I applied different gene prioritisation strategies and developed novel software tools in order to devise a list of plausible melanoma susceptibility candidate genes; these analyses suggested that genes regulating telomere function could be influencing melanoma risk. After performing functional experimental analyses, our research team was able to determine that carriers of rare variants in the protection of telomeres (POT1) gene, a member of the shelterin complex that safeguards telomere integrity, are at high risk for developing melanoma. We successfully described the mechanism by which this happens, showing that the variants identified either disrupt POT1 mRNA splicing or abolish the ability of POT1 to bind to telomeres, and lead to increased telomere length in carriers when compared to melanoma cases with wild-type POT1. The main finding of the work described in this dissertation is the identification of telomere dysfunction as an important contributor to the risk of developing melanoma, and possibly other cancers. Our analyses suggest that POT1 is the second most commonly mutated high-penetrance melanoma susceptibility gene reported thus far, and moreover, that rare variants in this gene constitute the first hereditary mechanism for telomere lengthening in humans.

Chromosome Instability (CIN) is defined as an increase in the rate at which whole chromosomes or large parts are gained or lost. CIN is not only associated with virtually all tumor types, but it is associated with aggressive tumors, tumor recurrence, acquisition of multidrug resistance and poor patient prognosis. However, the genes and molecular defects that contribute to CIN are poorly understood. I hypothesize that KIF11 is an essential gene for chromosomes integrity during mitosis and therefore any defect in KIF11 expression or function will induce CIN and contribute to tumorigenesis. Accordingly, KIF11 was either silenced using siRNA or inhibited using monastrol within two distinct human cell lines and was investigated for CIN associated phenotypes. Here, I have identified and validated KIF11 as a novel CIN gene. This study represents the first steps necessary to identify and develop novel treatments design to target origins of CIN in CIN associated cancers. / February 2017

This paper discusses the lessons learned by our collaborative, transdisciplinary team while developing a pilot/demonstration educational health campaign geared toward underserved communities in the Columbus, Ohio metropolitan area. The objective of the current study was to determine the feasibility of a campaign to raise awareness of the association between family history and cancer risk and to inform individuals of the availability of Jameslink, an online familial cancer risk assessment tool. The research team included members of The Ohio State University Primary Care Research Institute, which includes a unique combination of expertise in Genetics, Behavioral Science, Social and Health Psychology, Communication, Medicine, and Methodology. The experience of the team in developing university and community partnerships, identifying stakeholders and formulating campaign messages is described. Groups who aided in this process as well as the perspectives they brought to the project are discussed. The lessons learned may be helpful to those developing similar community health projects.