CMA rs1800875 and rs1885108 polymorphisms

The results of the within-family association tests with single markers suggested that none of the polymorphisms within CMA was associated with any of the 17 LVH parameters investigated.

The present study’s results are in agreement with studies of Pfeufer et al., (1996) and Ortlepp et al., (2002), where no independent association was found between CMA rs1800875 and LVH in HCM subjects. Similarly, no evidence was found to support a possible association between the CMA rs1800875 polymorphism and regression of LVH when 157 unrelated Chinese hypertensive patients were treated with the same antihypertensive drug benazepril (He et al., 2005). The usual arguments are discussed in the He et al., (2005) paper to explain why their observed results are different from the studies of Pfeufer et al., (1996) and Gumprecht et al., (2002). Reasons such as the different population groups used, genetic heterogeneity, different diagnosis criteria and the level of hypertension and LVH were posited. Finally, the dosage of the ACE inhibitor (ACEI) and other medicines used could also have contributed to spurious results observed between studies where treatment of LVH was investigated. These findings indicate that LVH formation/development is a complex process with the possibility of various genes interacting to produce a clinical phenotype, for example CMA and other genes encoding RAAS products.

4.8.5. AGTR1 rs2640539, rs3772627and rs5182 polymorphisms

The AGTR1 rs2640539 and rs3772627 polymorphisms have not been previously studied in LVH or in hypertension, as far as is known. In the present study, the results of the within-family association tests with single markers suggested that the AGTR1 rs2640539 polymorphism in intron 1 is associated with AWmit (table 3.11), and the AGTR1 rs3772627 polymorphism in intron 2 is associated with aIVSpap (table 3.16).

Additionally, the association tests with single markers suggested that the AGTR1 rs5182 polymorphism in exon 3 is associated with IWpap and AWapx (tables 3.19 and 3.23).

Further statistical analysis of rs2640539 polymorphism revealed that subjects homozygous for the G-allele were at a lower risk of developing increased AWmit compared to homozygotes for the A-allele, indicating a protective role for the G-allele in LVH susceptibility. Moreover, statistical analysis of rs3772627 polymorphism revealed that subjects homozygous for the A-allele were at a higher risk of developing increased aIVSpap compared to homozygotes for the G-allele, making the A-allele the risk allele.

Statistical analysis of rs5182 polymorphism revealed that subjects homozygous for the T-allele were at a lower risk of developing increased IWpap and AWapx compared to homozygotes for the C-allele, indicating a protective role for the T-allele of the rs5182 polymorphism.

The T-allele of the AGTR1 rs5182 (573 C/T) polymorphism was shown to be a protective factor for urinary albumin excretion in EHT patients (Chaves et al., 2001). Additionally, Chaves et al., (2001) demonstrated evidence of linkage disequilibrium between rs5186 (1166A/C) and 573C/T polymorphisms. However, Redon and colleagues (2005) found no association between rs5182 and rs5186 polymorphisms and BP reduction following telmisartan treatment. The present results are in contrast with those of Kupari et al., (1994), Lindpaintner et al., (1996) and Hamon et al., (1997) that showed no significant influence of AGTR1 genotypes on LVH.

It should be noted that genotyping the rs5182 polymorphism using restriction enzyme MnlI generated incongruous results. For technical reasons the fragment sizes differed on the gel from the expected digested product sizes generated using the software package

DNAMAN. A possible reason for the banding pattern is that the MnlI enzyme has the ability to remain associated with the cleaved DNA, thus resulting in DNA band shifting during electrophoresis (http://www.fermentas.com/catalog/re/mnli.htm). Sequencing the AGTR1 fragment 3 containing the SNP revealed that the correct region of the gene containing the SNP of interest was amplified. These discrepancies were also confirmed by another laboratory (Dr P. Fernandez, Department of Urology, US), which also found that the 325bp and 422bp fragments did not migrate as expected by size. Additionally, no disparities between the AGTR1 nucleotide sequence of fragment 3 and the NCBI database sequence were observed. After confirming that the different sized fragment on the gels was not due to technical errors, and that mobilities were constant, the genotyping of rs5182 polymorphism was continued.

The functional significance of the association of the AGTR1 rs5182 polymorphism with cardiac hypertrophy remains unknown. This polymorphism is a synonymous substitution and does not alter the sense codon 191 (CTC →CTT) that codes for leucine but it can be speculated that it is in LD with an unknown polymorphism in AGTR1 or adjacent genes.

The AGTR1 rs2640539 and rs3772627 polymorphisms are intronic and so far the functional significance of these polymorphic alleles is unclear. However, introns may contain regulatory elements that affect gene transcription and translation (Ying and Lin et al., 2006). Moreover, microRNAs derived from introns (Ying and Lin et al., 2006) have the ability to bind mRNA, interfering with transcription, degrading mRNA and thereby reducing transcript levels (Sevignani et al., 2006), indicating that intronic polymorphisms can also influence gene splicing by affecting a splice donor or acceptor sites. Of course, these polymorphisms could also be in LD with other functional variants that affect mRNA and protein expression.

Animal models

In a rat model, it was demonstrated that overexpression of AGTR1 under physiological conditions resulted in no change of the cardiac structure, whereas pressure and volume overload produced hypertrophic growth in the model (Hoffmann et al., 2001). In a study by Harada et al., (1999), AGTR1 subtype a (AGTR1a) specific receptor knockout mice

displayed less LV remodelling and improved survival rates at four weeks after myocardial infarction (MI) compared to wild type (WT) MI mice. However, Yoshiyama et al., (2005) demonstrated that ACE inhibitors prevent LV remodelling in a non-AGTR1 mediated mechanism after MI in AGTR1 knockout mice. In experimental animal model studies, ACE inhibitors and AGTR1 antagonists induce regression and prevent cardiac hypertrophy development in hypertensive animal models (Pfeffer et al., 1982 and 1983;

Dunn et al., 1984; Nakashima et al., 1984). Harada et al., (1998) demonstrated that AGTR1 is not essential for the development of pressure overload-induced cardiac hypertrophy in transgenic animal models. Similarly, Katada and colleagues (2005) observed elevated cardiac CYP11B2 levels in AGTR1a receptor knockout mice after MI inducing cardiac remodelling via ANGII- independent mechanisms.

Tsuchida and colleagues (1998) generated AGTR1 knockout mice by gene target disruption creating a null mutation gene. Similarly to homozygous (AGT -/-) knockout mice, (AGTR1 subtype a and b specific receptors, are products of different genes) double null-zygote mice developed a ventricular septum defect in combination with abnormal phenotypes, namely, severe hypotension (low BP), growth retardation, renal arterial wall thickening and hypolastic papilla (Tsuchida et al 1998). Paradis et al., (2000) generated transgenic mice expressing human AGTR1 under the control of the mouse (α-MHC) promoter and observed ventricular hypertrophy and fibrosis in cardiac overexpressed AGTR1 (by more than 200 fold) in absence of external stimuli.