FUNCIONAL CHARACTERIZATION OF 14 CBS MUTATIONS FOUND IN HOMOCYSTINURIC PATIENTS.

URREIZTI, R; ASTEGGIANO,C; COZAR, M; FRANK,N; VILASECA, M.A; GRINBERG, D; BALCELLS, S.

Basel, Switzerland

Conferencia; Fourth International Conference on homocysteine Metabolism; 2005

   Classical homocystinuria (OMIM *236200) is an autosomal recessive disease due to cystathionine beta-synthase (CBS; EC4.2.1.22) deficiency. It is characterized by high levels of homocysteine and methionine in plasma and urine. The worldwide incidence for this disease is estimated at 1/335.000 live births, although there is a wide range of variation depending on the population. The CBS gene was mapped to 21q22.3. More than 130 different mutations have been described until now and the activities of 33 of these were analyzed in an E.coli expression system (CBS Mutation Database: http://www.uchsc). The aim of this study was to analyze the enzymatic activity of 14 mutations found in our patients’ cohort. The cDNA of the CBS gene, cloned in the pkk3.88 plasmid was directly mutated generating these 14 alleles. The mutations were expressed in the E.coli strain XL-Blue and their enzymatic activity was measured in an in-vitro reaction. Besides, these expressions were tested in SDS-PAGE and in non-denaturing PAGE followed by Western blot. The 14 mutations characterized were: R125W (c.373C>T), G148R (c.442G>A), M173V (c.517A>G), T191M (c. 572C>T), A226T (c. 676G>A), C275Y (c.824G>A), R336C (c. 1006C>T), R336H(C. 1007G>A), L338P (c.1013T>C), S349N (c.1046G>A), R379Q (c. 1136G>A), L456P (c.1367T>C), G522fsX540 (c.1566delG) and R548Q (c.1643G>A). Eleven of them showed an activity lower than 5% of the wt protein. In contrast, mutations A226T and M173V presented near 20% of the wt activity, whereas the activity of R548Q was higher than 68% of wt. This activity, together with the fact that this change was found in a control chromosome, suggest that it is a new polymorphism rather than a pathogenic mutation. In the majority of cases, the mutated protein showed a decreased signal in Western blot analysis compared with the wt. The non-denaturing PAGE showed that the wt protein kept the capacity to form a multimeric quaternary structure, whereas in two mutations this structure grade was dramatically reduced and it was completely absent in the rest of them.