A 31 bp VNTR in the cystathionine β-synthase (CBS) gene is associated with reduced CBS activity and elevated post-load homocysteine levels

Abstract

Molecular defects in genes encoding enzymes involved in homocysteine metabolism may account for mild hyperhomocysteinaemia, an independent and graded risk factor for cardiovascular disease (CVD). Although heterozygosity for cystathionine b-synthase (CBS) deficiency has been excluded as a major genetic cause of mild hyperhomocysteinaemia in vascular disease, mutations in (non-)coding DNA sequences may lead to a mildly decreased CBS expression and, consequently, to elevated plasma homocysteine levels. We assessed the association between a 31 bp VNTR, that spans the exon 13-intron 13 boundary of the CBS gene, and fasting, post-methionine load and increase upon methionine load plasma homocysteine levels in 190 patients with arterial occlusive disease, and in 381 controls. The 31 bp VNTR consists of 16, 17, 18, 19 or 21 repeat units and shows a significant increase in plasma homocysteine concentrations with an increasing number of repeat elements, in particular after methionine loading. In 26 vascular disease patients the relationship between this 31 bp VNTR and CBS enzyme activity in cultured fibroblasts was studied. The CBS enzyme activity decreased with increasing number of repeat units of the 31 bp VNTR. RT ± PCR experiments showed evidence of alternative splicing at the exon 13-intron 13 splice junction site. The 31 bp VNTR in the CBS gene is associated with post-methionine load hyperhomocysteinaemia that may predispose individuals to an increased risk of cardiovascular diseases.

Association of a 31 bp VNTR in the CBS gene with postload homocysteine concentrations in the Framingham Offspring Study

“…In the present study, we observed six different alleles, of which alleles 16 to 21 showed similar allele frequencies compared with those in our previous report of a Dutch population 7 and those of earlier reports. 8,19 Allele 15 was not reported before, probably because its frequency is rather low.…”

“…However, we observed that this most common allele in fact consist of 18 repeat units, resulting in a different numbering in which our allele 18 is the most common. 7 They reported that the genotypes 17 -18 and 18 -19 had lower delta (ie increase upon methionine loading) tHcy concentrations that the most common 18 -18 genotype, and that this observation is probably caused by linkage disequilibrium with upstream transcriptional regulatory elements. They argued that the occurrence of alternative splicing is prevented by a G to A substitution at the exon -intron border of the second repeat unit.…”

“…17,18 Recently, we described the association of a 31 bp VNTR in the CBS gene with postmethionine load tHcy concentrations. 7 In a Dutch population, of 190 vascular patients and 381 population-based controls, we distinguished five different alleles, that is, consisting of 16, 17, 18, 19 or 21 repeat units of 31 bp and observed higher 6-h post-methionine load tHcy concentrations with increasing number of repeat units. This observation was further substantiated by the observation of decreased CBS activities in fibroblasts of individuals with genotypes that include a high number of repeat elements.…”

“…Furthermore, we provided evidence that alternative splicing does occur, since the first part of intron 13 is still present in a significant proportion of the CBS mRNA population. 7 Still, we do not know whether the entire intron 13 is retained in some mRNAs, which would lead to a premature stop codon, or that alternative splice donor sites downstream of this repeat are also used, which will cause frame shifts.…”

“…However, very recently, we and others described the association of a 31 bp variable number of tandem repeats (VNTR) in the CBS gene with tHcy concentrations. 7,8 In our previous study, we assessed the relationship between the CBS 31 bp VNTR genotype and both fasting and post-methionine load tHcy concentrations in 190 CVD patients and 381 population-based controls, and found a positive association between postload tHcy concentration and increasing number of repeat units of the 31 bp VNTR. 7 In the present study, we determined the CBS 31 bp VNTR genotype in more than 2500 samples of the Framingham Offspring Study and studied the association between this genotype and fasting, post-methionine load and delta (ie increase upon methionine loading) tHcy concentrations in 1416 subjects.…”