Chromatin accessibility is considered an excellent measure of the functionality of genetic variants (Mikkelson et al., 2007; Birney et al., 2010; Pang et al., 2013). It is common to use reporter gene and gel shift assays for determining the functionality of genetic variants, however, both approaches are limited by scalability and are lacking in chromatin context. The present study uses a novel next-gen sequencing-based approach ('CHA-seq') which translates Chromatin Accessibility by Real-Time PCR (CHARTPCR) into a robust method in order to assess allele-specific differences in chromatin arrangement across an extended genomic region. CHA-seq is a means of simultaneously sequencing genetic variants, mapping nucleosomes and nuclease hypersensitivity sites, and assessing SNP functionality. Genetic variants within the VNN1 gene have been associated with cardiovascular disease (Jacobo-Albavera et al., 2012; Fava et al., 2010; Zhu and Cooper, 2007), and as such, VNN1 represents an ideal gene model for functional assessment. Göring et al. (2007) identified two candidate functional SNPs, namely -137 (rs4897612) and -587 (rs2050153), in the VNN1 gene promoter that have been experimentally validated using chromatin arrangement assays as part of this current research. Genotyped cell lines derived from the San Antonio Family Heart Study (SAFHS) (Göring et al., 2007), carrying different -137 and -587 alleles, were characterised for allele-dependent differences in chromatin arrangement as an indicator of function. The CHART-PCR and CHA-seq results showed allele-specific chromatin accessibility at the -137 and the -587 variants. Further CHA-seq analysis across an extended genomic interval revealed several candidate functional genetic variants, including the rs2267951 SNP in intron 2 of VNN1. Experimental validation using EMSA showed allele-specific DNA-complexes at the rs2267951 locus, therefore supporting the CHA-seq results. Of the genetic variants selected for functional assessment, both CHA-seq and other experimental validation assays have given an equivalent measure of function in VNN1. Bioinformatic analysis of the VNN1 promoter showed the presence of a CpG island, of which one CpG site was disrupted by the -587A allele. Bisulfite sequencing showed allele-specific DNA methylation patterns at the -720 CpG site which were dependent on the allele at the -587 locus. Therefore, this current study provides evidence that allelespecific chromatin differences can be seen at sites remote from the actual genetic variation. The development of enhanced genome-wide in vivo approaches such as CHA-seq allows for a more cost and time-effective assessment of whether a variant does indeed regulate chromatin arrangement and gene expression, yielding insights into the long range allele-specific mechanisms of action.
|Qualification||Doctor of Philosophy|
|Publication status||Unpublished - 2013|