CHARACTERIZATION OF A CYSTATHIONINE BETA-SYNTHASE ALLELE WITH 3 MUTATIONS IN CIS IN A PATIENT WITH B-6 NONRESPONSIVE HOMOCYSTINURIA

We used SSCP to survey reverse transcribed-PCR amplified cystathionine synthase cDNAs from patients with homocystinuria. In a single CBS all ele, we identified one synonymous and two missense mutations in a port ion of the cDNA encoded by a single 135 bp exon which also encodes K11 9, the putative site of cofactor, pyridoxal 5'-phosphate, binding. The patient, a B-6-nonresponsive homocystinuric of Irish descent, is homo zygous for a G --> A transition at cDNA position 374, a G --> C transv ersion at position 393, and a G --> A transition at position 453 resul ting in R125Q, E131D and P145P, respectively. Family studies confirmed that all three mutations are present in cis and none were present in 54 Irish and 58 North American controls. R125 is conserved in rat CBS while E131D is conserved in rat CBS, and a related enzyme, O-acetylser ine(thiol)-lyase, from a variety of plant and bacterial species. Expre ssion studies showed that both R125Q and E131D, either individually or together, inactivate CBS. The apparently simultaneous appearance of m ore than one mutation in a single exon suggests they may have arisen b y a gene conversion event or by nonhomologous recombination.