Four novel mutations in the cystathionine beta-synthase gene: Effect of a second linked mutation on the severity of the homocystinuric phenotype

Homocystinuria due to cystathionine beta-synthase (CBS) deficiency is frequ ently caused by missense mutations. In this article, we report four novel m issense mutations in the CBS gene: 172C-->T (R58W) linked in cis with A114V ; 376A-->G (M126V); 904G-->A (E302K); and 1006C-->T (R336C). The CBS activi ty of the corresponding mutant enzymes expressed in Escherichia coli was gr eatly diminished, confirming the pathogenicity of these mutations. Western analysis showed that the R58W+A114V and M126V mutant enzymes were un stable in E. coli, while the E302K subunits were partially degraded to shorter pr oducts. Using site-directed mutagenesis we found that CBS containing either the R58W or A114V as the only mutations demonstrated 18% and 46% of normal activity, respectively. Both mutant forms of CBS were stable in E, coli. W hen these two mutations were expressed in cis, the resultant mutant protein exhibited activity 1.3% that of a control. All these in vitro results were in good agreement with the clinical manifestation in these patients. The I talian patient 2241, an A114V + R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian pati ent 2242, with an A114V/E302K genotype, responded to pyridoxine treatment a nd had a much milder phenotype. The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsiv e. This report of a ninth homocystinuric allele carrying two mutations in c is raises the possibility that double mutant alleles may be underestimated in homocystinuric patients. In this context, a search for additional mutati ons in cis may sometimes be necessary to establish a good genotype phenotyp e relationship. Hum Mutat 13:453-457, 1999. (C) 1999 Wiley-Liss, Inc.