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Functional assays testing pathogenicity of 14 cystathionine‐beta synthase mutations
Author(s) -
Urreizti Roser,
Asteggiano Carla,
Cozar Mónica,
Frank Nina,
Vilaseca María Antonia,
Grinberg Daniel,
Balcells Susana
Publication year - 2006
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.9395
Subject(s) - biology , mutant , microbiology and biotechnology , western blot , wild type , mutation , gene , allele , genetics
In this study, 14 CBS alleles from homocystinuric patients were expressed heterologously in E. coli and their enzyme activities were assayed in vitro. Additionally, mutant CBS proteins were visualized by Western blot from denaturing and non‐denaturing polyacrylamide gels. The 14 mutations characterized were: p.R125W (c.373C>T), p.G148R (c.442G>A), p.M173V (c.517A>G), p.T191M (c.572C>T), p.A226T (c.676G>A), p.C275Y (c.824G>A), p.R336C (c.1006C>T), p.R336H (c.1007G>A), p.L338P (c.1013T>C), p.S349N (c.1046G>A), p.R379Q (c.1136G>A), p.L456P (c.1367T>C), p.G522fsX540 (c.1566delG), and p.R548Q (c.1643G>A). Eleven of the mutant alleles exhibited an activity lower than 4% of the wild‐type protein. In contrast, mutations p.A226T and p.M173V presented 20% and 40% of the wild‐type activity, respectively, whereas the activity of p.R548Q was up to 60% of the wild‐type. This suggests that it is a new rare variant rather than a pathogenic mutation. Most of the mutated proteins exhibited a decreased signal in Western blot analyses. The non‐denaturing PAGE revealed that the wild‐type protein retained the capacity to form a multimeric quaternary structure, whereas in the mutations p.M173V, p.A226T, and p.G548Q, this structure grade was dramatically reduced and was completely absent in the rest of the mutations. © 2006 Wiley‐Liss, Inc.