Postgraduate Profiles

Luke Diepeveen

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Thesis: Analysis of functional SNPs in the VNN1 gene, and the development of a novel functional SNP screen

Functional SNPs have been identified in the VNN1 gene by Göring et al. (2007) and Kerkel et al. (2008). Functional SNPs within the VNN1 gene have been associated with hypertension (Zhu and Cooper, 2007) and cardiovascular disease (Göring et al., 2007).

Cell lines from the San Antonio Family Heart Study and the American Type Culture Collection, which had genotyped at the -137 and -587 SNPs in the VNN1 gene promoter, were assessed for functionality using CHART-PCR. These promoter SNPs had moderate-high chromatin accessibility when compared to SPA-2 and B-Actin controls, which is indicative that the VNN1 gene promoter is transcriptionally active. Further CHART-PCR analysis of -137 and -587 homozygotes using two chromatin accessibility agents (DNase I and MNase) identified allele-specific chromatin accessibility at both loci indicating SNP functionality.

To screen for functional VNN1 SNPs, a novel deep sequencing-based methodology (“Cha-seq”) was developed to measure chromatin accessibility. This method captured 42 heterozygous SNPs of which 21 represented candidate functional SNPs. One of the 21 candidate functional SNPs, namely rs2294757 matched the results of Kerkel et al. (2008). A targeted extension of this novel SNP screening method was used to validate the findings of Göring et al. (2007) who identified the -137 VNN1 SNP as functional. A candidate functional SNP (rs2267951) captured in the Cha-seq screen was verified using EMSA, which identified an additional DNA-protein complex in the presence of the T allele.

Bisulfite sequencing of the VNN1 gene promoter revealed allele-specific DNA methylation at the -720 CpG site. The methylation status at the -720 CpG site was dependent on the allele (G/A) present at the -587 SNP. The size of the CpG island predicted by BISMA was also dependent on the -587 allele present, with the CpG island length larger (263bp) in the presence of the -587G allele.

Why my research is important

The findings from this research will add to the number of functional SNPs known in the VNN1 gene, provide further insight to other candidate SNPs in the genome and to add novel methodologies for identifying candidate functional SNPs.

The long term aim of functional genetics is to provide targets for the development of future therapeutic interventions.

Mar 2009

Dec 2012