Human essential hypertension is a highly heritable disorder with complex aetiology and is a major risk factor for cardiovascular events such as ischaemic heart disease and stroke. A combination of multiple environmental and lifestyle factors contribute to blood pressure variation alongside a strong genetic component. Only a small proportion of the genetic factors that regulate blood pressure in the population are currently known, although there is strong evidence that the adrenal cortex and the steroid hormones it produces contribute. Several research strategies have been utilised to dissect the genetic aetiology of hypertension, including candidate gene studies and association studies. Two recent genome-wide association studies aimed to identify variations associated with altered blood pressure and hypertension. A total of ten variants were identified with genome-wide significance after a combined analysis between the two consortia, including a polymorphism located within intron 3 of the CYP17A1 gene. This variant was reported to be associated with a systolic blood pressure increase of 1.16 mmHg. The CYP17A1 gene codes for a dual-function enzyme (17α-hydroxylase/17,20 lyase) expressed primarily in the adrenal cortex and gonads that plays a key role in the steroidogenic pathway. Mutations in its coding region and splice sites are known to cause a rare form of congenital adrenal hyperplasia, suggesting that more common genetic variations at this locus might result in more subtle effects on blood pressure. A detailed examination of the variation across the CYP17A1 locus was required to establish patterns of linkage disequilibrium and is presented in Chapter 3. Some information on the polymorphic variation in this region was already available in public databases but precise details on linkage disequilibrium and the corresponding haplotype blocks were lacking. The entire CYP17A1 gene was directly sequenced, including approximately 2.5kb upstream from the transcriptional start site, in 62 subjects drawn from a normotensive population. Polymorphic variations were identified mainly in the promoter and introns. Two seemingly unrelated blocks of SNPs were identified as being worthy of follow-up investigations, particularly those located in the promoter region, as these could be responsible for alterations in the transcriptional activity of the gene. A total of seven promoter polymorphisms were then genotyped in a larger hypertensive population where the relationship between SNPs was less clear. In Chapter 4 the effect of CYP17A1 genotype on intermediate corticosteroid phenotype is explored in a hypertensive population. Corticosterone, cortisol and androgen production were not significantly altered in the population when stratified by genotype for each polymorphism. However when further split by gender, increased cortisol excretion rates were found to associate with the minor allele at position -362 in males and at positions -1204 and -2205 in females. Ratios of various corticosteroid intermediary metabolites were also compared as indices of CYP17A1 enzymatic activity. Ratios of THDOC:THS were significantly reduced in the presence of the minor allele at positions -34, -1204 and -2205, suggesting increased 17α-hydroxylase efficiency. In addition, aldosterone excretion was significantly elevated in individuals with CC genotype at position -1877; an indirect genotype-dependent effect has been speculated. A bioinformatic search was conducted to identify putative transcription factor binding sites at the polymorphic locations. This is presented in Chapter 5. This confirmed the hypothesis that single base changes at each of the seven polymorphic sites could lead to altered transcriptional activity. Using reporter gene assays in vitro, the G allele at position -362 (rs248658) associated with greater transcriptional activity than the A allele. The T allele at position -1877 (rs138009835) was transcriptionally less active than its alternative C allele. Similarly, the C allele at position -2205 (rs2150927) showed lower activity than the T allele. These data provide strong evidence that common variation at this locus may be of functional significance. The studies in Chapter 6 investigate a potential regulatory role of microRNA (miRNA) at the CYP17A1 locus. MiRNAs are a class of small non-coding RNA molecules that have recently emerged as novel post-transcriptional regulators of gene expression. They function by targeting the 3’ untranslated region (3’UTR) of specific mRNAs and cause repression either through mRNA destabilisation followed by degradation, or by mRNA translational repression. Previous research utilised a siRNA approach to knock down Dicer, a protein required for miRNA maturation, and noted significantly increased CYP17A1 mRNA levels in the H295R human adrenocortical cell line. The investigation presented here cross-referenced bioinformatic analysis with microarray expression data in order to predict which adrenal miRNAs are most likely to regulate CYP17A1 expression. Predicted miRNAs also shown to be differentially expressed between normal and diseased adrenal tissue were then selected for further analysis. In vitro investigation involved artificial manipulation of the specific miRNA levels in H295R cells followed by measurement of CYP17A1 mRNA levels. Increased amounts of hsa-miR-320a significantly raised CYP17A1 mRNA levels, although subsequent reporter construct assays showed that this was not due to direct miRNA binding of the CYP17A1 3’UTR. The studies in this chapter are the first to demonstrate miRNA-mediated regulation of CYP17A1 expression. In summary, this work aimed to investigate polymorphic variation in the human CYP17A1 gene and its association with hypertension. Patterns of linkage disequilibrium across the CYP17A1 gene were identified and the association of several polymorphisms with intermediate corticosteroid phenotype examined. The functional effects of candidate polymorphisms have also been assessed in vitro. Further studies will be required to determine whether observed changes in transcriptional activity are the direct result of altered transcription factor binding at polymorphic sites. Finally, the role of miRNA in the post-transcriptional regulation of CYP17A1 has been confirmed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:601573 |
| Date | January 2014 |
| Creators | Diver, Louise A. |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/5016/ |
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