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Abstract
Neuroblastoma is the most common solid tumour in infancy, yet the cause of the disease remains largely unknown. This thesis examines whether genetic factors involved in folate metabolism including DNA methylation, folate gene polymorphisms and gene expression influence the risk and clinical outcome of childhood neuroblastoma. Furthermore, the regulation of a major folate transporter, solute carrier family 19A1 (SLC19A1) was investigated in several in vitro systems in neuroblastoma.
DNA or RNA samples were isolated from primary neuroblastoma tissues for 174 children diagnosed in Australia and New Zealand between 1985 and 2000. Methylation levels of 48 candidate tumour-associated genes were measured using a customised Illumina GoldenGate Assay, while polymorphisms of genes in the folate pathway were examined using real-time PCR.
Promoter DNA hypermethylation of FOLH1, MTRR, MYOD1 and THBS1 were found to be independent predictors of poorer clinical outcome, after adjusting for known prognostic factors such as MYCN status, age and stage at diagnosis (p≤0.05). Moreover, over 40% of patients displayed hypermethylation in two or more genes and were three times more likely to relapse or die (HR=3.04; 95% C1=1.69-5.46; p<0.001), independent of known prognostic factors.
Analysis of folate gene polymorphisms showed that children with the MTHFR haplotype, which encodes for low MTHFR enzyme activity, were 1.5 times more likely to develop neuroblastoma (p=0.025), while children with one or more copies of the SLC19A1 80A variant allele had a reduced risk of developing neuroblastoma compared to those without the SLC19A1 80A allele (p=0.027). Patients with SLC19A1 80AA genotype were also less likely to relapse or die compared to those with SLC19A1 80GG genotype, after adjusting for MYCN status, stage and age at diagnosis (p=0.048). Although these observations could not be replicated in an independent patient cohort, the discovery of a functional link between SLC19A1 and MYCN in neuroblastoma cell lines and the ability to impact on folate and antifolate uptake suggested the potential importance of the SLC19A1 gene in neuroblastoma. Furthermore, elevated expression of SLC19A1 was prognostic of poor survival in patients with neuroblastoma.
Taken together, the genetic modification of folate uptake and metabolism has an important role in the tumourigenesis of neuroblastoma.