Hyperhomocysteinemia in premature arterial disease: examination of cystathionine β-synthase alleles at the molecular level

Hyperhomocysteinemia occurs in approximately 30% of the patients with premature occlusive arterial disease (POAD). Some of these exhibit significantly reduced fibroblast cystathionine β-synthase (CBS) activities, suggesting that they may be heterozygous for CBS deficiency. To test this possibility, we studied cDNA derived from four well characterized patients with POAD, exhibiting hyperhomocysteinemia and reduced CBS activities, from four normal controls, and from four obligatory heterozygotes for CBS deficiency. Lysates of individual colonies of E.coli, containing full-length PCR-amplification products in the expression vector, pKK388.1, were tested for CBS activity. cDNA from at least seven of the eight possible independent POAD alleles encoded catalytically active, stable CBS which exhibited normal response to both PLP and AdoMet. The sequences of all 3'-untranslated regions of all seven isolated POAD alleles were identical to the normal, wild-type CBS sequences. The results of the expression studies were confirmed for one POAD patient by determining the full-length cDNA sequences for both alleles; these were entirely normal over the complete length of the cDNA. In contrast, the screening method correctly distinguished mutant from normal alleles in all four obligatory heterozygotes studied. We conclude that CBS mRNAs from POAD individuals are free from inactivating mutations, including all 33 previously identified in heterozygous carriers and homocystinuric patients.