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The stroke-prone spontaneously hypertensive rat (SHRSP) is an inbred model of hypertension. Renal microarrays and functional genomic strategies investigated chromosome 2 candidate hypertension genes, focussing on the oxidative-stress defence gene, glutathione s-transferse mu type 1 (Gstm1). Ingenuity pathway analysis of renal microarrays in 5 and 16-week SHRSP, normotensive Wistar Kyoto (WKY) and chromosome 2 congenic rats identified differential expression of several glutathione cycling genes. The Gstm1 promoter was investigated by luciferase and Transfac bioinformatic analysis, implicating two polymorphism clusters and several transcription factors in reduced SHRSP Gstm1 expression. Recombinant adenoviruses expressing Gstm1 and short-hairpin RNA-interference sequences to reduce Gstm family expression were produced. In-vivo overexpression of Gstm1 did not improve endothelial nitric-oxide bioavailability in SHRSP carotid arteries. Bacterial artificial chromosome and linear expression constructs were purified for production of Gstm1 transgenic rats, putative transgenic rats were screened by PCR. The strategies developed in this project are an example of thorough functional genomic analysis in experimental hypertension research.

Background Essential hypertension remains a major risk factor for coronary heart disease (CHO) and stroke, and its prevalence is greater, more severe, occurs earlier, and is less well controlled among black individuals than among white individuals, at all ages after young adult hood (Comoni-Huntley et al, 1989). In Caucasians, studies have shown that β2-adrenoceptor polymorphism accounts for the variability in the vascular responsiveness to the agonist isoprenaline (Cockcroft et al, 1994 and Lang et al, 1995). Individuals homozygous for Gln 27 β2-adrenoceptor showed reduced responses due to chronic down regulation of β2-adrenoceptor in the vasculature. Therefore, variability in response to isoprenaline was determined by β2-adrenoceptor gene polymorphism. Aim and Objectives This study investigated whether there is a relationship between the ArglGly16 and Gln/Glu 27 β2-adrenoceptor polymorphisms by examining whether the incidence of occurrence is prevalent in African Trinidadians. In addition, comparison data of vascular responses with arterial compliance using pulse wave analysis (PWA) was correlated. The study aimed to give evidence if these polymorphisms contributed fully or in part, to determine the disease severity, or response to therapy in hypertensive individuals. It also aimed to prove that PWA is a reliable and therapeutic tool, in diagnosing and treating blood pressure, as reliance on brachial artery recording of blood pressure, alone, is becoming a poor indicator and predictor of risk. Methods The study genotyped 408 African Trinidadian subjects for the β2-adrenoceptor polymorphism and used the technique of applanation tonometry to analyse the central pulse wave, generating information on arterial compliance, left ventricular function and coronary perfusion. Blood pressure was measured in triplicate using a semiautomatic blood pressure meter after 15 minutes of supine rest and bloods lipids assessed using a validated portable lipid cartridge. This was achieved by subjects attending a nurse-led cardiovascular risk clinic. Results There is no significant association between the Arg-Glyl6 polymorphism and the Gln-Glu27 polymorphism and hypertension in African Trinidadians. Interestingly, the appearance of the Glu27 polymorphism was very uncommon in African Trinidadians and this is constant with findings by Candy et al, 2000. Conclusion There is no difference in the frequency of β2-polymorphisms between normotensive and hypertensive African Trinidadians, and are unlikely to be a contributing factor for essential hypertension. Therefore, hypertension would indicate that it is polygenic with complex gene to gene and gene environmental interactions, through multiple, indirect and intermediate phenotypes and interactions.

The studies reported in this thesis aimed to identify the pattern of variation across the CYP11B1/CYP11B2 locus in order to determine the genetic variation responsible for the observed impaired 11ß-hydroxylation and its link with aldosterone levels and hypertension. In Chapter 3, an attempt was made to identify polymorphisms in CYP11B1 that associate with hypertension and a raised aldesterone-to-renin ratio (ARR) and therefore might contribute to the genetic component of these phenotypes. The study in Chapter 4 aimed to analyze the pattern of variation across the CYP11B2/B1 locus in detail. In Chapter 5, the frequency and phenotypic associations of the newly identified CYP11B1 5’UTR polymorphisms -1889 G/T and -1859 A/G were investigated in a hypertensive population. The frequencies were found to be -1889 G-0.53, T- 0.47; -1859 A-0.5, G-0.5. Interestingly, the -1889 G/T and -1859 A/G polymorphisms were found to be associated with impaired 11ß-hydroxylase efficiency in a hypertensive population. The THS/total F ratio (index of 11ß-hydroxylase activity) was significantly higher in -1889 TT homozygotes than in GG homozygotes (p=0.025). A similar pattern was seen with the -1859 SNP, with GG homozygotes tending to have a higher ratio than AA homozygotes (p=0.056). These steroid data were supported by the results from Chapter 6 that examined the effects of these polymorphisms in vitro using luciferase reporter gene constructs transfected into Y1 mouse adrenal cells. In summary, these studies have confirmed that the CYP11B2 and CYP11B1 genes contain a high frequency of genetic variation. Many of the variants identified in CYP11B1 have been shown to alter activity significantly. There is strong evidence to suggest that the impaired 11ß-hydroxylase efficiency previously associated with the -344 C/T and IC variants in CYP11B2 is due to linkage with the -1889 G/T and -1859 A/G polymorphisms upstream of CYP11B1.