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Screening for mutations by expressing patient cDNA segments in E. coli : Homocystinuria due to cystathionine β‐synthase deficiency
Author(s) -
Kožich Viktor,
Kraus Jan P.
Publication year - 1992
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/humu.1380010206
Subject(s) - biology , cystathionine beta synthase , transversion , complementary dna , homocystinuria , microbiology and biotechnology , genetics , mutation , frameshift mutation , transition (genetics) , exon , mutant , gene , amino acid , methionine
Deficiency of cystathionine β‐synthase (CBS) causes the most common form of inherited homocystinuria. We developed a simple CBS expression system in E. coli to screen for pathogenic mutations in affected individuals. Portions of patient cDNAs were amplified by PCR and used to replace the corresponding segments of normal human CBS cDNA in the bacterial expression plasmid pHCS3. Hybrid CBS was expressed in E. coli and the segments of patient's cDNA which extinguished CBS activity were sequenced to identify the mutation. The first study of a pyridoxine‐responsive patient using this screen revealed that of the clones which contained either the middle or the 3′‐portion of his cDNA, about half were devoid of catalytic activity. Subsequent sequencing of the affected segments confirmed a compound heterozygosity for a maternal T 833 →C transition (I278T) and for a paternal A→C transversion in the intron 11 splice acceptor. The latter mutation leads to an in‐frame deletion of exon 12 (nt 1224‐1358, amino acids W408 to G453). This bacterial expression system proved to be a rapid screening method for localizing pathogenic mutations in CBS, allowing us to sequence the affected portions of mutant cDNA within 7–10 days of harvesting cultured fibroblasts. © 1992 Wiley‐Liss, Inc.