Ye, Shu; (1996) Variation in the promoter of the human stromelysin-1 gene influences expression and is associated with progression of coronary atherosclerosis. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis describes studies on the promoter of the gene encoding human stromelysin-1, a potent proteinase which plays an important role in extracelluar matrix turnover which has been implicated in the connective tissue remodelling during the pathogenesis of atherosclerosis. The 5'-flanking region of the stromelysin gene was examined in an attempt to identify variants which may influence the expression of the proteinase. Two novel sequence variants were detected, one being common in the population and the other rare. The former arises from an insertion/deletion of an adenosine, giving rise to one allele having a run of 6As and the other 5As, whereas the latter is a deletion of 7 base pairs(TTATAAC). Since atherosclerosis is a common disorder and our interests focus on the effects of common genetic variation, further studies focused on the 5 A/6 A polymorphism. The frequencies of the 5A and 6A alleles were 0.52 (95% Cl from 0.48 to 0.56) and 0.48 (95% Cl from 0.44 to 0.52) respectively in 354 healthy men. These were similar in a group of 74 male patients with coronary heart disease, recruited in the St Thomas' Atherosclerosis Regression Study (STARS). However, an association was detected in this group between the 5A/6A polymorphism and progression of global and focal atherosclerosis as defined by serial quantitative angiography, with patients homozygous for the 6A allele showing greater decrease in luminal width in the 3 years study period. A similar trend was also observed in 58 Swedish patients who had myocardial infarction and who underwent serial angiography. The genotypes of the stromelysin 5A/6A polymorphism were also determined in 594 patients with myocardial infarction and 710 control subjects participating in the ECTIM (Etude Cas-Têmoins sur I'Infarctus du Myocarde) study. No difference in allele and genotype frequencies was detected in the entire patient and control groups. However, in smokers, the frequency of the 6A6A genotype was higher in patients than in controls. This difference was also observed in those with a plasma fibrinogen level above the 80th percentile of the fibrinogen distribution. Functional analyses were carried out to determine whether the 5A/6A polymorphism has a direct effect on gene expression, or whether it is merely acting as a genetic marker for functional variants elsewhere. In transiently transfected human foetal foreskin fibroblasts and rat vascular smooth muscle cells, expression of the chloramphenicol acetyl transferase reporter gene driven by the 6A allelic stromelysin-1 promoter was lower compared with the expression directed by the 5A allelic promoter. Electrophoretic mobility shift assays and DNase I footprinting revealed the interaction of one or more nuclear protein(s) with the DNA sequence at the 5A/6A polymorphic site. The binding affinity of one of the nucleoprotein factors appeared to be higher to an oligonucleotide probe corresponding to the 6A allele as compared with a probe corresponding to the 5A allele. Replacing the core binding sequence with a random DNA sequence abolished the interaction between the nuclear protein(s) and the probe and also increased reporter gene expression in transiently transfected cells, suggesting that the nuclear protein with different binding affinities to the 5A and 6A alleles was a transcriptional repressor. These data provide the first evidence linking genetic variation at the stromelysin-1 locus with progression of coronary atherosclerosis, and support the hypothesis that connective tissue remodelling mediated by metalloproteinases contributes to the pathogenesis of atherosclerosis.

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