MOLECULAR AND BIOCHEMICAL APPROACHES IN THE IDENTIFICATION OF HETEROZYGOTES FOR HOMOCYSTINURIA

We compared biochemical and molecular methods for the identification o f heterozygous carriers of mutations in the cystathionine beta-synthas e (CBS) gene. Eleven relatives of seven unrelated patients with homocy stinuria due to homozygous CBS deficiency and controls were studied wi th respect to total homocysteine concentrations before and after methi onine loading. In addition, we determined CBS activity in cultured ski n fibroblasts and tested for the presence of five known mutations by a PCR-based method in these seven patients, their relatives and control s. The results demonstrate that measurement of homocysteine after meth ionine loading and assay of CBS enzyme activity in cultured fibroblast s identify most but not all heterozygotes. There was significant corre lation between homocysteine concentrations and CBS activities only aft er methionine loading (r = 0.12, 0.48, 0.48 and 0.50 at 0, 4, 6 and 8 h, respectively). Among the homozygous patients, molecular approaches identified five T833C and two G(919)A mutations out of 14 independent alleles, confirming the studies of others that these represent the two most prevalent mutations. In addition, we found that three of six het erozygotes with the T833C allele had post-methionine loading homocyste ine levels which overlapped with controls and of the other three, one (as well as an obligate heterozygote who did not carry any of the five mutant alleles tested) had CBS activity comparable to that of control s. These findings demonstrate that genotyping is useful as an adjuncti ve method for the diagnosis of the heterozygous carrier state of CBS d eficiency.