Characterization of a cystathionine beta-synthase allele with three mutations in cis in a patient with B6 nonresponsive homocystinuria.

We used SSCP to survey reverse transcribed-PCR amplified cystathionine synthase cDNAs from patients with homocystinuria. In a single CBS allele, we identified one synonymous and two missense mutations in a portion of the cDNA encoded by a single 135 bp exon which also encodes K119, the putative site of cofactor, pyridoxal 5'-phosphate, binding. The patient, a B6-nonresponsive homocystinuric of Irish descent, is homozygous for a G-->A transition at cDNA position 374, a G-->A transversion at position 393, and a G-->A transition at position 453 resulting 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-acetylserine(thiol)-lyase, from a variety of plant and bacterial species. Expression studies showed that both R125Q and E131D, either individually or together, inactivate CBS. The apparently simultaneous appearance of more than one mutation in a single exon suggests they may have arisen by a gene conversion event or by nonhomologous recombination.