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 frequently caused by missense mutations. In this article, we report four novel missense 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 activity of the corresponding mutant enzymes expressed in Escherichia coli was greatly diminished, confirming the pathogenicity of these mutations. Western analysis showed that the R58W+A114V and M126V mutant enzymes were unstable in E. coli, while the E302K subunits were partially degraded to shorter products. 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. When 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 Italian patient 2241, an A114V+R58W/M126V compound heterozygote, exhibited severe pyridoxine nonresponsive homocystinuria, while another Italian patient 2242, with an A114V/E302K genotype, responded to pyridoxine treatment and had a much milder phenotype. The third patient 3064, an English compound heterozygote for two severe mutations R336C and G307S, was B6 nonresponsive. This report of a ninth homocystinuric allele carrying two mutations in cis raises the possibility that double mutant alleles may be underestimated in homocystinuric patients. In this context, a search for additional mutations in cis may sometimes be necessary to establish a good genotype-phenotype relationship.
de Franchis
R
R
Department of Pediatrics, University of Colorado School of Medicine, Denver, USA.
Kraus
E
E
Kozich
V
V
Sebastio
G
G
Kraus
J P
JP
D000483:Alleles; D002648:Child; D002675:Child, Preschool; D003541:Cystathionine beta-Synthase / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts; D005838:Genotype; D006712:Homocystinuria / Q000235:genetics*; D006801:Humans; D008297:Male; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype
D003541:Cystathionine beta-Synthase