LVH is considered a major independent risk factor for CVD (Koren et al., 1991). The assumption was made on the basis of certain clinical characteristics observed in LVH patients, which included the presence of various pathologies such as cardiac fibrosis (Weber et al., 1994), apoptosis (Sharov et al., 1996) and impaired coronary haemodynamics (Marcus et al., 1981). However, the mechanisms that generate LVH in CVD patients are unclear, even though there remains a direct relationship between regression of LVH during treatment and reduction in cardiac events (Liebson and Serry, 2000). According to Korner and Jennings (1998), LVH is due to increased BP, whereby the heart muscle compensates for increased cardiac load. However, there is a poor correlation between BP and cardiac mass (Korner and Jennings, 1998). Additionally, in patients undergoing antihypertensive treatment decreased BP does not always result in a similar reduction in left ventricle mass (LVM), although the prognosis is suggested to be better within these patients (Liebson and Serry, 2000). Several investigators observed unfavourable effects of regressing LVH within animal and human models, by failing to reduce LVM when treated with the antihypertensive drug, hydralazine (Fogari et al., 1995; Norton et al., 1997; Yamazaki et al., 1999; Tsotetsi et al., 2001).
These observations implied the presence of variability in LVM in hypertensive patients (Liebson and Serry, 2000) and indicate that factors independent of haemodynamic effects (BP) should also be examined as potential hypertrophy determinants.
As shown in cultured cells stimulated by ANGII, the RAAS mediates its effects on cardiac growth independently of its actions on BP and induces cardiomyocyte hypertrophy (Sadoshima and Izumo, 1993) through both reactive oxygen species (Takemoto et al., 2001) and calcineurin-dependent pathways (Olson and Molkentin, 1999). In 11 patients (6 males and 5 females) with HCM and 8 healthy asymptomatic individuals, a 4.5-fold increased myocardial aldosterone level was observed in HCM patients versus controls and increased CYP11B2 mRNA levels was also seen in the subjects with HCM, while the cAMP level was normal in both cases and controls (Tsybouleva et al., 2004).
In a study by Schmitt et al., (2003a), the effects of transverse aortic banding on two strains of transgenic mice with and without the cardiac MYH6 Arg403Gln missense mutation (equivalent to the human MYH7 Arg403Gln mutation) were assessed. They found that in transverse aortic banded mice that were not subjected to treatment with the calcineurin-inhibitor cyclosporin A, the hypertrophic response to hypertrophy was uniform. When cyclosporin A (an agent that is known to amplify hypertrophy induced by this sarcomere mutation [Arg403Gln]) (Fatkin et al., 2000) was added to banded mice harbouring either the wild type (WT) (+/+) or the missense (+/-) mutation, it resulted in an augmented hypertrophic response in both groups (Schmitt et al., 2003a).
Consequently, the authors predicted that both hypertension and HCM act independently of one another and not synergistically.
In cultured rat cardiac myocytes and fibroblasts, CYP11B2 provoked expression of hypertrophic and profibrotic effects via activation of protein kinase D (PKD) and upregulation of phosphoinositide 3-kinase (PI3K) (Tsybouleva et al., 2004). Additionally, Tsybouleva et al., (2004) demonstrated that in a cardiac troponin T (cTnT)-Q92 transgenic mouse model of human HCM (in humans, TNNT2 Arg92Trp mutation), blockade with mineralocorticoid receptor (MR) antagonist spironolactone inhibited PKD (a mediator of hypertrophic effect of aldosterone) and PI3K (a mediator of profibrotic effects). This resulted in aldosterone normalised myocardial collagen content and attenuated myocyte disarray phenotypes. The myocytic disarray phenotypes were similar
to those shown by Varnava et al., (2001) and Silvestre et al., (1998) to associate with SCD and heart failure in humans and mouse models of HCM. These studies implicate aldosterone as a molecular link between sarcomeric gene mutations and cardiac phenotypes as demonstrated in human hearts, cultured cells and a genetic animal model of HCM.
Direct role for RAAS in LVH
Thus, there is mounting evidence for the RAAS’s role in LVH, the first being an independent relationship between plasma ACE levels and aldosterone concentrations with either LVH or indices of cardiac growth (Schunkert et al., 1996, 1997). The second line of evidence was the finding by Harrap et al., (1996), which indicated a relationship between plasma ANGII concentrations and LVM in healthy young adults. Numerous data implicate the RAAS as a mediator of LVM by having either direct cellular effect on cardiac growth (Olson and Molkelin, 1999; Takemoto et al., 2001) and/or indirect effects via circulating components of the RAAS on LVM (Harrap et al., 1996; Schunkert et al., 1996, 1997). This evidence has prompted researchers to investigate the role of RAAS gene variants on the development of LVH.
Mechanism of RAAS inducing LVH
The mechanism of RAAS’s effects on LVH occurs via an interaction between direct cellular/molecular pathways and indirect haemodynamic changes (Sadoshima and Izumo, 1993; Harrap et al., 1996). Both these effects are considered interdependent and synergistically induce marked LVM changes. Thus, one change is not sufficient to modify LVM as shown in previous preclinical studies (Sen et al., 1974; Sen and Tarazi, 1983; Frohlich and Sasaki, 1990). It is assumed that genetic polymorphisms within the RAAS system could potentially modify RAAS activity in the presence of hypertension and thus produce marked effects on LVM (Tiago et al., 2002). If the RAAS is not the primary cause then it might be a contributor to LVH, a characteristic often associated with HCM. The potential effects of RAAS activation in cardiac hypertrophy include myocardial fibrosis, diastolic dysfunction (Weber et al., 1994), myocyte necrosis (Tan et al., 1991; Ollivier and Bouchet, 1992), myocyte slippage, cardiac dilatation (Mann and
Spinale, 1998) and vasoconstriction (Hall et al., 1986; Hall et al., 1990; Hall and Gayton, 1996).
Some of the risk factors involved in CVD development are known and the RAAS is considered a mediator of these factors leading up to LVH (Tiago et al., 2002). Other studies have sought to determine the genetic factors involved in the development of hypertension and LVH, using genetic approaches.
The following sections will introduce the concept of genetic association studies, linkage disequilibrium (LD), population stratification and using family members as controls in genetic studies, because of their relevance to the present case-control association study in which the role of RAAS as a modifier of the hypertrophic phenotype in HCM was investigated.
Genetic association analyses/ Case-control association studies
Association studies aim to demonstrate a statistical difference in the distribution of allelic variants of selected genes within affected (cases) and unaffected (control) individuals.
The studied individuals are included irrespective of their family members’ disease status in these studies. Genetic association analyses are usually conducted in a population-based setting using larger sample sizes to gain adequate power (Silverman and Palmer, 2000).
For association studies one of two approaches can be used. The first approach is based on an “a priori” candidate gene hypothesis whereby variants within a specific gene are investigated based on known physiological, biochemical or pharmacological evidence.
These studies offer increased power to detect genes of moderate effect, while taking into account the current information known about the tissues, proteins and potential genes involved in the pathogenesis of a condition. The second approach, known as a genome-wide association analysis, involves screening the entire genome in search of the causal genetic variant(s), without prior consideration of pathophysiological mechanisms. This systematic approach is regarded as being unbiased because it also involves no prior
assumptions regarding the localisation of possible susceptibility variants (Hirschhorn and Daly, 2005).
The success of both methods relies on the association found between the selected marker and the susceptibility allele due to a phenomenon known as linkage disequilibrium (LD).
Linkage disequilibrium (LD)
LD is the non-random statistical association of alleles at single linked loci, which co-segregate with a high frequency across a population. LD can be used to investigate human evolution and genetic aetiology of complex disorders (Jorde et al., 1995; Kidd et al., 1998) and can aid in the identification of susceptibility loci, for example, in a group of affected individuals that are descended from a single founder individual. It is considered that, over a restricted period of time, in a group descended from a common ancestor, or single founder individual, a low fraction of meiotic recombination events will have occurred, thereby defining a commonly inherited DNA-region that possesses the susceptibility alleles. In case-control association studies, LD patterns give information on the genetic distance over which signals of causation may be generated. Risch and Merikangas, (1996) regarded LD as a tool to identify the neighborhood surrounding the susceptibility allele. However, there are several caveats that should be considered when performing an association study, one being population stratification (see below).
Population stratification
A study is said to have population stratification, if the allele frequencies between cases and controls differ due to diversity in background of the population (Cardon and Palmer, 2003). Population stratification normally occurs when there are differences in disease prevalence between cases and controls and variation in allele frequencies between population groups (Cardon and Palmer, 2003). In case-control association studies, false positives may occur due to even the slightest differences in genetic ancestry between cases and controls. This is because the source population under investigation might comprise of different clusters of subpopulations resulting in spurious associations. For example any allele which by chance has a higher frequency in the subpopulation that
possesses a larger disease risk than in the control group might appear to have an association with the disease (Wacholder et al., 2002). Alternatively, population stratification may result in a false negative finding: if the frequency of the associated allele is by chance, low in a subgroup of people in which the disease is more prevalent, than in the control group the association with the disease may be masked (Deng et al., 2001). However, population stratification can be avoided by using family members as controls (Thomas and White, 2002); this rationale was implemented in the present study.
Using family members as controls
Transmission disequilibrium test analysis (TDT) makes use of parents and siblings as family-based controls. TDT allows the transmission pattern of selected marker alleles from heterozygous parents to affected offspring to be followed. If transmission frequency of the marker allele exceeds that which is expected by chance alone, the allele is assumed to be associated with the disorder in some way.
The approaches mentioned above have been used in several genetic association studies to determine the influence of genetic factors of the RAAS on the phenotypic expression of hypertension and LVH; as will be discussed in further detail in the following section.