Biomedical subjects

S Clavero

Publications and source records attributed to S Clavero.

5 recordsLinked to original sources

PubMed2003

Functional analysis of PCCB mutations causing propionic acidemia based on expression studies in deficient human skin fibroblasts.

Propionic acidemia (PA) is a recessive disorder caused by a deficiency of propionyl-CoA carboxylase (PCC), a dodecameric enzyme composed of two different proteins alpha-PCC and beta-PCC, nuclear encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause PA and to date, up to 47 different allelic variations in the PCCB gene have been identified in different populations. In this work, we describe the expression studies of 18 PCCB sequence changes in order to elucidate their functional consequences. We have used a PCCB-deficient transformed fibroblast cell line to target the wild-type and mutant proteins to their physiological situation, analysing the effect of the mutations on PCC activity and protein stability. Of the 18 mutant proteins tested for activity, those carrying the L17M and A497V substitutions showed an activity similar to the wild-type one, which proves that these changes do not have any effect on protein activity. The other 16 mutant proteins exhibited two different functional behaviours, 3 retained substantial activity (K218R, R410W and N536D), and the remaining 13 proteins showed null or very low activity. Western blot analysis demonstrated instability only for the L519P, R512C and G112D mutant proteins. We have proved the pathogenicity of R67S, R165Q and G112D mutation in PCCB gene, expressed for the first time in this work. The information derived from the expression analysis is discussed in the phenotype and genotype context in order to improve the knowledge of this complex disease.

Amino Acid Metabolism, Inborn Errors↗

PubMed2003

Propionic acidemia: identification of twenty-four novel mutations in Europe and North America.

Propionic acidemia is an inherited metabolic disease caused by the deficiency of the mitochondrial protein propionyl-CoA carboxylase (PCC), one of the four biotin-dependent enzymes. PCC is a multimeric protein composed of two different alpha- and beta-PCC subunits, nuclearly encoded by the PCCA and PCCB genes, respectively. Mutations in either gene cause the clinically heterogeneous disease propionic acidemia. In this work we describe the mutational analysis of PCCA and PCCB deficient patients from different European countries (Spain, Italy, Belgium, Croatia, and Austria) and from America (mainly USA). We report 24 novel PA mutations, nine affecting the PCCA gene and 15 affecting the PCCB gene. They include six missense mutations, one nonsense mutation, one point exonic mutation affecting splicing, seven splicing mutations affecting splice sequences, and nine short insertions or deletions, only two in-frame. We have found a highly heterogenous spectrum of PCCA mutations, most of the PCCA deficient patients are homozygous carrying a unique genotype. The PCCA mutational spectrum includes a high proportion of short insertions or deletions affecting one nucleotide. In the PCCA mutant alleles analyzed we have also found one single nucleotide change, a novel nonsynonymous SNP. On the other hand, the PCCB deficient patients carry a more reduced spectrum of mutations, 50% of them are missense. This work represents an extensive update of the mutational study of propionic acidemia providing important information about the worldwide distribution of PA mutations and representing another essential part in the study of the phenotype-genotype correlations for the prediction of the metabolic outcome and for the implementation of treatments tailored to each PA patient.

Alternative Splicing↗

PubMed2001

Effect of PCCB gene mutations on the heteromeric and homomeric assembly of propionyl-CoA carboxylase.

Propionic acidemia is an inherited metabolic disorder caused by deficiency of propionyl-CoA carboxylase, a dodecameric enzyme composed of alpha-PCC and beta-PCC subunits (encoded by genes PCCA and PCCB) that have been associated with a number of mutations responsible for this disease. To clarify the molecular effect associated with gene alterations causing propionic acidemia, 12 different mutations affecting the PCCB gene (R67S, S106R, G131R, R165W, R165Q, E168K, G198D, A497V, R512C, L519P, W531X, and N536D) were analyzed for their involvement in alpha-beta heteromeric and beta-beta homomeric assembly. The experiments were performed using the mammalian two-hybrid system, which was assayed at two different temperatures to distinguish between mutations directly involved in interaction and those probably affecting polypeptide folding, thus indirectly affecting the correct assembly. Mutations R512C, L519P, W531X, and N536D, located at the carboxyl-terminal end of the PCCB gene, were found to inhibit alpha-beta heteromeric and/or the beta-beta homomeric interaction independently of the cultivation temperature, reflecting their primary effect on the assembly. Two mutations A497V and R165Q did not affect either heteromeric or homomeric assembly. The remaining mutations (R67S, S106R, G131D, R165W, E168K, and G198D), located in the amino-terminal region of the beta-polypeptide, resulted in normal interaction levels only when expressed at the lower temperature, suggesting that these changes could be considered as folding defects. From these results and the clinical manifestations associated with patients bearing the mutations described above, several genotype-phenotype correlations may be established. In general, the temperature-sensitive mutations are associated with a less severe, although variable phenotype. This could correlate with the recent hypothesis that the effect of folding mutations can be influenced by the capacity of the cellular protein quality control machinery, which provides clues to our understanding of the variability of the clinical symptoms observed among the patients bearing these mutations.

Amino Acid Metabolism, Inborn Errors↗

PubMed1995

Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef.

The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.

Agar↗

PubMedDate not supplied

Propionic acidemia: mutation update and functional and structural effects of the variant alleles.

Mutations in the PCCA or PCCB genes, encoding both subunits of propionyl-CoA carboxylase, result in propionic acidemia, a life-threatening inborn error of metabolism with autosomal recessive inheritance. To date, 41 mutations in the PCCA gene and 54 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements, and a variety of small insertions and deletions and splicing defects. A greater heterogeneity is observed in the PCCA gene, specially in Caucasians, with no prevalent mutations, while in the Japanese population three mutations account for more than half of the alleles studied. For the PCCB gene a limited number of mutations is responsible for the majority of the alleles characterized in both Caucasian and Oriental populations. These two populations show a different mutational spectrum, only sharing some involving CpG dinucleotides probably as recurrent mutational events. Functional characterization of the mutant missense alleles has been accomplished using different prokaryotic and eukaryotic systems, and the structural consequences have been analyzed in the available crystal models. For the PCCA gene, the main molecular effect of the expressed mutations is related to protein instability, except two mutations in the active site predictably affecting ATP binding. In the PCCB gene the majority of the analyzed mutations are predicted to alter the active site conformation resulting in diminished activity. A few carboxy-terminal PCCB mutations affect the interaction between subunits and the assembly with PCCA to form a functional PCC oligomer. The amount of normal transcripts resulting from some PCCA and PCCB splicing mutations has also been analyzed. Overall, the data generated from the expression analysis reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Alleles↗