Dantas, Maria Fernanda. Molecular basis and functional characterization of human 3-methylcrotonyl-CoA carboxylase deficiency. 2009, PhD Thesis, University of Basel, Faculty of Science.
Official URL: http://edoc.unibas.ch/diss/DissB_8949
MCC is one of the four biotin-dependent carboxylases present in humans. MCC is a mitochondrial enzyme composed of biotin containing subunits and smaller subunits, encoded by MCCA and MCCB, respectively. In order to understand the molecular biology of human MCC, including the molecular defects causing MCC deficiency and their functional consequences, MCC-deficient patients were studied and attempts were made to correlate molecular defects with the phenotypic variability in our patient population.
In this thesis mutation analysis is described in 30 MCC-deficient probands, of whom 10 came to attention because of clinical symptoms, 18 were asymptomatic newborns detected by TMS based newborn screening, and 2 were affected but asymptomatic mothers diagnosed by abnormal metabolites in the neonatal screening samples of their healthy babies. Among these 30 probands, 11 have mutations in MCCA, and 19 in MCCB. We identified 10 novel MCCA and 14 novel MCCB mutant alleles including missense, nonsense, frameshift and splice site mutations.
In order to confirm the functional consequences of MCCA and MCCB missense mutations, we used transient transfection of SV40T-transformed MCCA and MCCB deficient skin fibroblasts to express 1 MCCA and 3 MCCB missense alleles. The MCCB missense alleles expressed showed no or very low residual activity, whereas the MCCA missense allele had 26% residual activity of wild type, thus confirming that 3 out of 4 missense alleles expressed have a deleterious effect on enzyme activity. The apparent severity of MCC mutations contrasts with the variety of clinical phenotypes found in MCC-deficient patients. Our data demonstrate no clear correlation between genotype and phenotype suggesting that factors other than the genotype at the MCC loci have a major influence on the phenotype of MCC deficiency.
To analyse the MCC polypeptides under steady-state condition in fibroblasts of MCCA deficient patients, we carried out Western blot analysis. Our results demonstrate that the MCC protein was absent in 5 patients homozygous or compound heterozygous for nonsense or frameshift mutations that are expected to result in a truncated protein. The MCC protein was also absent in 2 further compound heterozygous patients in whom only one missense allele could be identified in the RT-PCR products. The second allele could not be detected because presumably this mutant allele is unstable and degraded by the mechanisms of nonsense-mediated mRNA decay. In contrast, Western blot analysis of the MCC protein was normal in amount and size in 4 patients carrying MCCA-p.R385S. This is in agreement with previous studies, which show that the protein product of this allele is stable.
Finally, we provide evidence that the missense mutation MCCA-p.R385S has a dominant negative effect on the activity of wild type. Biotin can partially reverse this negative effect and result in biotin responsiveness in vivo. This is the first example of biotin responsiveness for an isolated partial deficiency of any of the biotin-dependent carboxylases, suggesting that therapeutic trials with biotin in patients carrying this mutant allele are warranted.
|Advisors:||Meyer, Urs Albert|
|Committee Members:||Baumgartner, Matthias and Wermuth, Bendicht|
|Faculties and Departments:||03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Hospital Basel|
|Bibsysno:||Link to catalogue|
|Number of Pages:||94 Bl.|
|Last Modified:||30 Jun 2016 10:41|
|Deposited On:||30 Apr 2010 10:37|
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