CAPITULO III MATERIALES Y MÉTODOS
3.4. PERTINENCIA DE LA METODOLOGÍA DE INVESTIGACIÓN
3.4.2. METODOLOGÍA PARA DETERMINAR LA DIFERENCIA SIGNIFICATIVA
The essential role of genes is in encoding structural proteins and enzymes which enable the cell or organism to maintain homeostasis in the face of the environmental challenges experienced (Humphries et al, 1995a). DNA containing such genetic information varies from one species to another. Even within a species, there are inter-individual differences in DNA sequence, giving rise for example to variation in fitness and appearance. Naturally occurring sequence variants, many of which are neutral sequence changes, have been estimated to exist approximately every 200- 300bp throughout the human genome (Cooper et al, 1985). However, there are some sequence variants which affect the control of gene expression, the biosynthesis and transport of the gene product, or the function of the gene product itself (Cooper and Krawczak, 1993b and 1993c). Consequently, individuals within a population will have different abilities to maintain homeostasis, and the failure of some individuals to do so will lead to the development of disease.
Geneticists have long been endeavouring to define the molecular basis of inherited disorders. Many disease genes have now been identified, predominately through linkage analysis approaches which test for cosegregation within families of the disease trait with a marker locus. The majority of these are genes involved in monogenic Mendelian diseases with simple patterns of inheritance. Now, human geneticists are beginning to study the genetics of multifactorial disorders, such as coronary heart disease, hypertension, diabetes, arthritis and multiple sclerosis, which are common in the population and account for the majority of mortality and morbidity (Weeks and Lathrop, 1995). Multifactorial disorders are caused by multiple genes interacting with each other and with environmental factors to create a gradient of genetic susceptibility to disease. Most genetic variants involved in such complex diseases are common in the population, each having a small effect that is not on its own enough to precipitate disease in an individual (Green and Humphries, 1994). However, such genetic variants may increase the predisposition to disease and, being common, would make a large contribution to the incidence of disease in the population by increasing the risk by a small amount in a large number of
individuals, resulting in many individuals going over the disease threshold. This is in contrast with the pathogenesis of single gene defects in which a gene mutation causes a severe inborn error of metabolism. Although mutations causing monogenic diseases can have a large impact on the disease susceptibility of an individual, they have little effect on the population's morbidity since they only occur at a very low frequency (Boerwinkle and Chan, 1991).
Accordingly, when analysing the genetic component of a multifactorial, polygenic disease, such as coronary heart disease, the major aim is to identify those common genetic variants that will alter gene expression or the function of its products in a relatively small but significant way. Once such variants are thoroughly studied and the mechanisms underlying their roles in disease pathogenesis are better understood, it should be possible to create a "risk factor profile" for an increased risk of disease based not merely on plasma levels of lipids, thrombogenic and other factors but also on genotypes for common functional variants (Chamberlain and Galton, 1991). This is likely to be a more reliable risk profile than measurements of plasma levels of risk factors alone, since the levels of lipids and proteins in the blood often fluctuate over time. The ultimate goal of such studies is to develop a system for assessing the likelihood of developing disease, which should in turn, aid the development of molecular diagnostics, preventive medicine, and novel therapeutic strategies (Weeks and Lathrop, 1995).
However, the more immediate aim of identifying common functional genetic variants is to analyze the effect of these on the development of disease, in order to gain better understanding of the underlying biological mechanisms. It is now clear that the prevalence of atherosclerosis-related diseases is increased by environmental factors such as high fat intake, smoking, stress and lack of exercise, but it is largely unknown why with similar life-style, some individuals develop disease and others do not. The genetic contribution to the increased susceptibility of some individuals to these diseases and the relative resistance of others is beginning to be exposed. In the last ten years, a number of genetic variants have been found to be associated with the development of atherosclerosis and its complications, most of which are
located in the genes encoding proteins and enzymes involved in lipid metabolism and coagulation. However, since the formation of atherosclerotic lesions involves numerous biological processes, many more candidate genes remain to be studied (Chamberlain and Galton, 1991).
The major aim of this project was to identify and analyze variation in the gene of stromelysin-1, which has been implicated in atherogenesis (Henney et al, 1991 ; Galis et al, 1994a). Although the matrix metalloproteinases (MMPs) including stromelysin- 1 had been found to be involved in a number of diseases such as arthritis, atherosclerosis, tumour invasion and metastasis (Murphy and Reynolds, 1993), in which genetic factors might play a role, there had been no reports of studies to examine the possible influence of genetic variation on the expression and activity of these enzymes, and the impact this might have on pathogenesis. The project described here is the first study to address this question. It began with the search for sequence variants in the 5'-flanking region of the stromelysin-1 gene using the PCR- SSCP method. Consequently, two variants were detected in this region, with one being common in the population and the other rare. Since atherosclerosis-related diseases are common disorders, resulting from the additive or synergistic effects of common genetic and environmental factors shared by many individuals, this study focused on the common variant, i.e. the 5A/6A polymorphism.
Since the contribution of a common genetic variant to disease susceptibility is likely to be small, its effect can only be revealed as a trend in the population as a whole (Green and Humphries, 1994). Two types of population association study are commonly used to study genetic variation in relation to multifactorial disorders, one testing association between a genetic variant and inter-individual differences in a quantitative trait such as blood pressure, and the other testing for differences in the frequencies of an allele or genotype in patients and controls which are matched carefully for ethnicity and other factors (Weeks and Lathrop, 1995). An association can arise if the genetic variant is causally implicated in the disease, or if it is in linkage disequilibrium with a susceptibility locus. Both approaches were used in this project to determine whether the stromelysin-1 5 A/6 A polymorphism was associated
with the development of atherosclerosis.
An association study was first carried out in 72 patients recruited in the STARS trial which was originally designed to study the effects of lipid-lowering treatment on the evolution of atherosclerosis assessed by serial quantitative angiography (Watts et al, 1992). An association between the 6A allele and more rapid progression of atherosclerosis was detected in this sample as a whole, and most significantly in those who received "usual care" but not lipid-lowering treatment. The association was then tested in an independent sample, i.e. a group of 58 Swedish MI patients who had undergone repeated angiography, and a trend towards greater progression of disease in those patients with the 6A6A genotype was also observed although it was not statistically significant. These results suggest that genetic variation at the stromelysin-1 locus may have an allele-specific effect on the growth of atherosclerotic plaques, presumably through differences in the expression of stromelysin-1.
Based on the observation that the stromelysin-1 5 A/6A polymorphism was associated with progression of atherosclerosis, it was reasonable to hypothesize that this genetic variant might also be associated with the development of MI, a common complication of atherosclerosis. To address this question, 594 MI patients and 710 controls who participated in the ECTIM study were genotyped, and the distributions of genotype in the two groups were compared. The results showed that in smokers or in individuals with high plasma fibrinogen levels, the 6A6A genotype was more frequent in patients than in controls. It has been firmly established that both smoking and elevated levels of fibrinogen are risk factors for MI (Wilhelmsen et al, 1984; Stone et al, 1985; Meade et al, 1986; Kannel et al, 1987; Yamell et al, 1991). It appears from this study that the 6A6A genotype further increases disease susceptibility.
As mentioned above, an association between a genetic variant and a disease trait does not necessarily indicate that the former is directly the cause of the latter, because association may occur if the genetic variant under study is in linkage
disequilibrium with a causative variant located elsewhere but in the vicinity of the marker. Most sequence changes which have a functional effect lie within gene coding regions and result in dysfunction of the gene products by introducing amino acid substitutions, altering the translational reading frame, or producing truncated proteins (Cooper and Krawczak, 1993c). Some functional genetic variants are, however, located in gene regulatory sequences, most commonly in the 5'-flanking region (Cooper and Krawczak, 1993b). These so-called "regulatory polymorphisms (or mutations)" affect the rate of transcription, thus increasing or decreasing the level of gene product synthesized, rather than altering its nature. Regulatory polymorphisms have been found in the promoters of a number of genes whose products have been implicated in the development of atherosclerosis-related diseases, such as those encoding apo A l, PAI-1 and fibrinogen (Angotti et al, 1994; Dawson et al, 1993; Green et al, 1995). It has been demonstrated that these promoter regulatory polymorphisms influence gene expression in an allele-specific manner, resulting in inter-individual differences in the levels of gene products (Jeenah et al, 1990; Thomas et al, 1991; Dawson et al, 1993). These findings provide new insights into the intricacies of gene regulation as well as the mechanisms involved in the pathogenesis of these common multifactorial, polygenic disorders.
Following the observation of the association between the stromelysin-1 5 A/6 A polymorphism and progression of atherosclerosis and risk of MI, the final part of this study was designed to test the possible regulatory role of this promoter variant in gene expression. The results of transient expression experiments and electrophoretic mobility shift assays indicated that the 6A allelic promoter was less efficient in directing gene expression as compared with the 5A allelic promoter, probably through the preferential binding of what appears to be a transcriptional repressor protein to the 6A allele. These data were supported by experiments in which disruption of the 5A/6A polymorphic site abolished nucleoprotein binding to this region of the promoter and increased promoter activity. Thus it appears that the stromelysin-1 5 A/6 A polymorphism per se is a regulatory variant, capable of influencing gene expression in an allele-specific manner.
As mentioned above, the major aim of this study was to investigate whether there was an genetic effect on stromelysin-1 expression and hence on the pathogenesis of atherosclerosis-related diseases. The results presented here suggest the existence of such genetic influence. In the population studies the 6A allele was associated with more rapid progression of atherosclerosis and increased risk of MI, whereas in the functional analysis gene expression driven by the 6A allelic promoter was less efficient. A extrapolation from these data is that reduced stromelysin-1 expression favours progression of atherosclerosis and development of MI. But what are the mechanisms?
A typical atheroma contains a lipid-rich core and on its luminal aspect, a fibrous cap which is mainly composed of vascular smooth muscle cells and extracellular matrix macromolecules including interstitial collagens, elastin and proteoglycans (Libby, 1995b). Thus, an atherosclerotic lesion consists of two components: atherosis and sclerosis. The sclerotic component is hard, collagenous, and much bulkier, whereas the atheromatous component is soft, lipid-rich, fragile, but occupies less space (Falk, 1992). Although almost all plaques contain these two components, there is a large variation in their relative proportions between plaques, giving rise to different morphological expression (Richardson et al, 1989; Falk, 1992; Libby, 1995b). At one end of the spectrum are the plaques with a thin fibrous cap and a relatively well- preserved lumen. The thin cap in this type of plaque makes it less resistant to mechanical stress and therefore more prone to rupture in systole. At the other end of the spectrum are the atherosclerotic plaques with a thick fibrous cap containing a substantial amount of extracellular matrix. The bulk of the plaque protrudes into the lumen, and restricts coronary flow (Figure 6.1).
N o n -o c c lu siv e le sio n
M o re lip id rich
P o te n tia lly u n sta b le ( i f fib ro u s cap th in ) W ith a w e ll-p re s e rv e d lu m en L ittle o r n o a n g io g ra p h ic a b n o rm a lity ’’Im m a tu re ” T h ro m b u s fo rm a tio n re su lts fro m p la q u e ru p tu re F ibrous c a p Lipid c o r e Lumei T unica m edia T hrom bus S te n o tic le sio n M o re fib ro tic O fte n s ta b le ( i f fib ro u s c ap th ic k ) C o ro n a ry flo w re s tric te d A b n o rm a l a n g io g ra m ’’M a t u r e ”
I
T h ro m b u s fo rm a tio n r e su lts fro m d e c re a s e in c o ro n a ry c irc u la tio n (s ta s is )Atherosclerosis develops over many years, involving numerous pathophysiological processes (reviewed in Ross, 1993). One of the fundamental processes which takes place during atherogenesis is connective tissue remodelling, involving the deposition and removal of extracellular matrix proteins (Reviewed in Dollery et al, 1995). Although matrix degradation may exceed synthesis at certain focal areas such as the "shoulder regions" where plaque rupture occurs most frequently, the global connective tissue turnover during atherogenesis favours deposition rather than degradation, since there is a net increase in the content of extracellular matrix in all plaques (Galis et al, 1994a).
Several MMPs, including stromelysin-1, interstitial collagenase, and gelatinases A and B, have been localized to atherosclerotic plaques, suggesting that these enzymes are involved in connective tissue remodelling during atherogenesis (Henney et al, 1991; Galis et al, 1994a & 1995a; Brown et al, 1995, Nikkari et al, 1995, Tyagi et al, 1995). These enzymes have been found to be highly expressed in rupture-prone areas (such as the shoulders of plaque) where there is loss of connective tissue. Therefore, there have been suggestions that over-expression of MMPs in certain lesional regions may contribute to plaque rupture (Henney et al, 1991; Galis et al, 1994a & 1995a; Brown et al, 1995, Nikkari et al, 1995). In the majority of the plaque, however, expression of MMPs is coupled with a large increase in extracellular matrix deposition. Tyagi et al (1995) recently studied the MMP levels in relation to the extracellular matrix contents in normal and atherosclerotic arteries, and found that weight-for-weight atherosclerotic vessels contained more collagen and proteoglycans but lower collagenolytic activity than normal vascular tissues. These findings suggest that during atherogenesis, the increase in matrix proteins outstrips the increase in matrix-degrading enzymes, which may in turn create an imbalance in connective tissue turnover, favouring matrix deposition and contributing to plaque growth. In this context, it may be envisaged that in most lesional areas, MMPs are under-expressed rather than over-expressed.
Based on these observations and the results of the present study, the putative model illustrated in Figure 6.2 is proposed to explain how the stromelysin-1 polymorphism