Characterization of the promoter region of the HAMP gene implicated in iron metabolism and its possible association with Oesophageal cancer in the black South African population

[摘要] ENGLISH ABSTRACT: Oesophageal cancer (OC) is the sixth leading cause of cancer related deaths in the world with approximately 300 000 new cases reported each year. OC may be characterized into two forms with 90% of cases presenting as squamous-cell carcinoma (SCC) and the remaining 10% as adenocarcinoma (ADC). Several factors have been attributed to the development of OC, including oesphageal injury and/or irritation, chronic inflammation and excess iron associated with enhanced tumour growth.The HAMP gene codes for a 25 amino-acid protein found to be primarily expressed in the liver and crucial to regulation of bodily iron status. Defects occurring in the HAMP gene could therefore lead to the dysregulation of the gene, resulting in an iron overload status. Iron overload is a previously described risk factor in the development of various cancers, including OC, and therefore the aim of this study was to investigate whether dysregulation of the HAMP gene may be involved in the cancer phenotype exhibition.The study cohort comprised of 48 unrelated patients presenting with SCC and a control group of 51 healthy, unrelated population-matched individuals. Mutation detection techniques included polymerase chain reaction (PCR) amplification, heteroduplex single-stranded conformation polymorphism (HEX-SSCP) analysis and bi-directional semi-automated DNA sequencing analysis. Screening of the 5' regulatory region (5'UTR) of the HAMP gene revealed one known (-582A/G) and two novel (-188C/T and -429G/T) variants with the -429G/T variant showing statistically significant reduction in expression in patients relative to controls. Iron parameters were correlated between patient and control cohorts, as well as for variant presence and absence within individuals. Luciferase reporter constructs were used to investigate the functional implications of the presence of a variant on HAMP gene expression, and how these results correlated to the iron parameter statistics obtained.Luciferase reporter assay results indicated the -188C/T and -429G/T variants to result in under-, and the -582A/G variant to result in over-expression at the basal level, relative to the respective wild-type sequence constructs. Correlation of the luciferase data with the iron parameter statistics, indicate the -429G/T variant to be coupled to significantly higher levels of ferritin and C-reactive protein (CRP) and significantly lower levels of serum-iron and transferrin when compared to individuals without the variant. Considering only the patient group, the presence of the -188C/T and -429G/T variants were coupled to significantly lower levels of transferrin in patients with either variant, compared to patients without. The variants found within the HAMP promoter region are therefore able to alter gene regulation to an extent where iron parameters deviate between healthy and OC afflicted individuals, and also between patients with and without a variant. This dysregulation in iron homeostasis may play a role in the development and/ or progression of OC. Characterisation of the 5' UTR of the HAMP gene may contribute to linking iron regulation to the establishment of an effective screening program, facilitating the early detection of OC.

[发布机构] Stellenbosch University