CHARACTERIZATION OF 5 MISSENSE MUTATIONS IN THE CYSTATHIONINE BETA-SYNTHASE GENE FROM 3 PATIENTS WITH B-6-NONRESPONSIVE HOMOCYSTINURIA

Homocystinuria, due to a deficiency of the enzyme cystathionine beta-s ynthase (CBS), is an inborn error of sulphur-amino acid metabolism, Th is is an autosomal recessive disease which results in hyperhomocystein aemia and a wide range of clinical features, including optic lens disl ocation, mental retardation, skeletal abnormalities and premature thro mbotic events, We report the identification of 5 missense mutations in the protein-coding region of the CBS gene from 3 patients with pyrido xine-nonresponsive homocystinuria. Reverse-transcription PCR was used to amplify CBS cDNA from each patient and the coding region was analys ed by direct sequencing, The mutations detected included 3 novel (1058 C --> T, 992C --> A and 1316G --> A) and 2 previously identified (430G --> A and 833C --> T) base alterations in the CBS cDNA, Each of these mutations predicts a single amino acid substitution in the CBS polype ptide, Appropriate cassettes of patient CBS cDNA, containing each of t he above defined mutations, were used to replace the corresponding cas settes of normal CBS cDNA sequence within the bacterial expression vec tor pT7-7. These recombinant mutant and normal CBS constructs were exp ressed in Escherichia coli cells and the catalytic activities of the m utant proteins were compared with normal. All of the mutant proteins e xhibited decreased catalytic activity in vitro, which confirmed the as sociation between the individual mutation and CBS dysfunction in each patient.