title: Functional assays testing pathogenicity of 14 cystathionine-beta synthase mutations.
abstract: 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.
authors.affiliation: Departament de Genètica, Universitat de Barcelona, Barcelona, Spain.
authors.forename: Roser
authors.initials: R
authors.lastname: Urreizti
authors.forename: Carla
authors.initials: C
authors.lastname: Asteggiano
authors.forename: Mónica
authors.initials: M
authors.lastname: Cozar
authors.forename: Nina
authors.initials: N
authors.lastname: Frank
authors.forename: María Antonia
authors.initials: MA
authors.lastname: Vilaseca
authors.forename: Daniel
authors.initials: D
authors.lastname: Grinberg
authors.forename: Susana
authors.initials: S
authors.lastname: Balcells
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D015153:Blotting, Western; D003541:Cystathionine beta-Synthase / Q000235:genetics*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D020125:Mutation, Missense; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017386:Sequence Homology, Amino Acid
chemical_list: D003541:Cystathionine beta-Synthase