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

Hyperhomocysteinemia occurs in approximately 30% of the patientswith premature occlusive arterial disease (POAD). Some of theseexhibit significantly reduced fibroblast cystathionine ß-synthase(CBS) activities, suggesting that they may be heterozygous forCBS deficiency. To test this possibility, we studied cDNA derivedfrom four well characterized patients with POAD, exhibitinghyperhomocysteinemia and reduced CBS activities, from four normalcontrols, and from four obligatory heterozygotes for CBS deficiency.Lysates of individual colonies of E.coli, containing full-lengthPCR-amplification products in the expression vector, pKK388.1,were tested for CBS activity. cDNA from at least seven of theeight possible independent POAD alleles encoded catalyticallyactive, stable CBS which exhibited normal response to both PLPand AdoMet. The sequences of all 3'-untranslated regions ofall seven isolated POAD alleles were identical to the normal,wild-type CBS sequences. The results of the expression studieswere confirmed for one POAD patient by determining the full-lengthcDNA sequences for both alleles; these were entirely normalover the complete length of the cDNA. In contrast, the screeningmethod correctly distinguished mutant from normal alleles inall four obligatory heterozygotes studied. We conclude thatCBS mRNAs from POAD individuals are free from inactivating mutations,including all 33 previously identified in heterozygous carriersand homocystinuric patients.