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P Rodríguez-Pombo

Publications and source records attributed to P Rodríguez-Pombo.

17 recordsLinked to original sources
PubMed2004

Prenatal diagnosis of propionic acidemia.

In this report we summarize our experience in prenatal diagnosis of propionic acidemia (PA) since 1987. Overall, we have investigated 25 pregnancies at risk from 19 unrelated families. Until genetic structure of the genes involved in PA was elucidated, prenatal diagnosis has been successfully performed by means of metabolite quantitation and/or enzymatic assays in foetal issue. Today, direct propionyl-CoA carboxylase activity assay in combination with molecular analysis in chorion villi can be regarded as a fast and reliable method of choice for prenatal diagnosis of this organic acidemia.

Amniotic Fluid↗
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↗
PubMed2003

Functional analysis of MCCA and MCCB mutations causing methylcrotonylglycinuria.

Methylcrotonylglycinuria (MCG; MIM 210200) is an autosomal recessive inherited human disorder caused by the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC, E.C.6.4.1.4), involved in leucine catabolism. This mitochondrial enzyme is one of the four biotin-dependent carboxylases known in humans. MCC is composed of two different types of subunits, alpha and beta, encoded by the nuclear genes MCCA and MCCB, respectively, recently cloned and characterized. Several mutations have been identified, in both genes, the majority are missense mutations along with splicing mutations and small insertions/deletions. We have expressed four missense mutations, two MCCA and two MCCB mapping to highly evolutionarily conserved residues, by transient transfection of SV40-transformed deficient fibroblasts in order to confirm their pathogenic effect. All the missense mutations expressed resulted in null or severely diminished MCC activity providing direct evidence that they are disease-causing ones. The MCCA mutations have been analysed in the context of three-dimensional structural information modelling the changes in the crystallized biotin carboxylase subunit of the Escherichia coli acetyl-CoA carboxylase. The apparent severity of all the MCC mutations contrasts with the variety of the clinical phenotypes suggesting that there are other cellular and metabolic unknown factors that affect the resulting phenotype.

Amino Acid Sequence↗
PubMed2002

Transfection screening for defects in the PCCA and PCCB genes encoding propionyl-CoA carboxylase subunits.

Propionic acidemia can result from mutations in the PCCA or PCCB genes encoding the alpha and beta subunits, respectively, of propionyl-CoA carboxylase. We have developed a method based on complementation of the enzyme defect using a lipid-mediated transient transfection of the normal human PCCA or PCCB cDNA into primary fibroblasts. We demonstrate the reliability of this method for identification of the defective PCC gene in order to unequivocally approach the mutational analysis in the corresponding PCCA and PCCB genes.

Carboxy-Lyases↗
PubMed2001

The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine catabolism.

3-Methylcrotonylglycinuria is an inborn error of leucine catabolism and has a recessive pattern of inheritance that results from the deficiency of 3-methylcrotonyl-CoA carboxylase (MCC). The introduction of tandem mass spectrometry in newborn screening has revealed an unexpectedly high incidence of this disorder, which, in certain areas, appears to be the most frequent organic aciduria. MCC, an heteromeric enzyme consisting of alpha (biotin-containing) and beta subunits, is the only one of the four biotin-dependent carboxylases known in humans that has genes that have not yet been characterized, precluding molecular studies of this disease. Here we report the characterization, at the genomic level and at the cDNA level, of both the MCCA gene and the MCCB gene, encoding the MCC alpha and MCC beta subunits, respectively. The 19-exon MCCA gene maps to 3q25-27 and encodes a 725-residue protein with a biotin attachment site; the 17-exon MCCB gene maps to 5q12-q13 and encodes a 563-residue polypeptide. We show that disease-causing mutations can be classified into two complementation groups, denoted "CGA" and "CGB." We detected two MCCA missense mutations in CGA patients, one of which leads to absence of biotinylated MCC alpha. Two MCCB missense mutations and one splicing defect mutation leading to early MCC beta truncation were found in CGB patients. A fourth MCCB mutation also leading to early MCC beta truncation was found in two nonclassified patients. A fungal model carrying an mccA null allele has been constructed and was used to demonstrate, in vivo, the involvement of MCC in leucine catabolism. These results establish that 3-methylcrotonylglycinuria results from loss-of-function mutations in the genes encoding the alpha and beta subunits of MCC and complete the genetic characterization of the four human biotin-dependent carboxylases.

Adult↗
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↗
PubMed2000

Potential relationship between genotype and clinical outcome in propionic acidaemia patients.

Propionic acidaemia (PA) is an autosomal recessive disorder caused by mutations in either of the PCCA or PCCB genes which encode the alpha and beta subunits, respectively, of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). In this work we have examined the biochemical findings and clinical outcome of 37 Spanish PA patients in relation to the mutations found in both PCCA and PCCB genes. We have detected 27 early-onset and 101 late-onset cases, showing remarkably similar biochemical features without relation to either the age of onset of the disease or the defective gene they have. Twenty-one of the patients have so far survived and three of them, now adolescents, present normal development. Different biochemical procedures allowed us to identify the defective gene in 9 PCCA deficient and 28 PCCB deficient patients. Nine putative disease-causing mutations accounting for 77.7% of mutant alleles were identified among PCCA deficient patients, each one carrying a unique genotypic combination. Of PCCB mutant alleles 98% were characterised. Four common mutations (ins/del, E168K, 1170insT and A497V) were found in 38/52 mutant chromosomes investigated, whereas the remainder of the alleles harbour 12 other different mutations. By examining the mutations identified both in PCCA and PCCB genes and the clinical evolution of patients, we have found a good correlation between certain mutations which can be considered as null with a severe phenotype, while certain missense mutations tend to be related to the late and mild forms of the disease. Expression studies, particularly of the missense mutations identified are necessary but other genetic and environmental factors probably contribute to the phenotypic variability observed in PA.

Adult↗
PubMed1999

Spectrum of CFTR mutations in the Middle North of Spain and identification of a novel mutation (1341G-->A). Mutation in brief no. 252. Online.

We have analyzed 39 unrelated cystic fibrosis (CF) families by denaturing gradient gel electrophoresis (DGGE) and direct sequencing in order to determine the spectrum of CF mutations in our population. This approach has allowed us to detect 72 out of the 78 CF chromosomes (92.3%). The DF508 mutation was found to be present in 51/78 (65.4%) CF chromosomes, in accordance with the predicted Northwest-Southeast gradient within the European population. Another 14 known mutations, and the novel 1341G-->A mutation were identified. Nine out of fifteen non DF508 mutations were present in a single chromosome. The 1341G-->A mutation, found in 2 unrelated patients, is a new mutation associated to severe phenotype, causing pancreatic insufficiency and chronic lung infections. Our data suggest a different distribution of non-DF508 mutations in our population when compared with previous studies carried out in Spanish CF families. Six out of the 14 non-F508 in our study were not present in a recent study carried out in 640 Spanish families with CE These six mutations account for 29.6% non DF508 chromosomes in our sample.

Cystic Fibrosis↗
PubMed1999

Overview of mutations in the PCCA and PCCB genes causing propionic acidemia.

Propionic acidemia is an inborn error of metabolism caused by a deficiency of propionyl-CoA carboxylase, a heteropolymeric mitochondrial enzyme involved in the catabolism of branched chain amino acids, odd-numbered chain length fatty acids, cholesterol, and other metabolites. The enzyme is composed of alpha and beta subunits which are encoded by the PCCA and PCCB genes, respectively. Mutations in both genes can cause propionic acidemia. The identification of the responsible gene, previous to mutation analysis, can be performed by complementation assay or, in some instances, can be deduced from peculiarities relevant to either gene, including obtaining normal enzyme activity in the parents of many patients with PCCB mutations, observing combined absence of alpha and beta subunits by Western blot of many PCCA patients, as well as conventional mRNA-minus result of Northern blots for either gene or beta subunit deficiency in PCCB patients. Mutations in both the PCCA and PCCB genes have been identified by sequencing either RT-PCR products or amplified exonic fragments, the latter specifically for the PCCB gene for which the genomic structure is available. To date, 24 mutations in the PCCA gene and 29 in the PCCB gene have been reported, most of them single base substitutions causing amino acid replacements and a variety of splicing defects. A greater heterogeneity is observed in the PCCA gene-no mutation is predominant in the populations studied-while 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 spectrum of mutations, only sharing some involving CpG dinucleotides, probably as recurrent mutational events. Future analysis of the mutations identified, of their functional effect and their clinical relevance, will reveal potential genotype-phenotype correlations for this clinically heterogeneous disorder.

Alternative Splicing↗
PubMed1998

An unusual late-onset case of propionic acidaemia: biochemical investigations, neuroradiological findings and mutation analysis.

UNLABELLED: We report a 5-year-old boy with propionic acidaemia who developed a rapidly fatal necrosis of the basal ganglia after an episode of clinical deterioration. Neither metabolic acidosis nor hyperammonaemia were present. Organic acid analysis in both urine and CSF showed increased levels of methylcitric and 3-hydroxypropionic acids. Propionic acidaemia was confirmed by demonstrating a propionyl-CoA carboxylase deficiency (11% of control value) in skin fibroblasts. DNA analysis revealed that the patient was a compound heterozygote for two mutations in the PCCB gene. CONCLUSION: Propionic acidaemia can present as a sudden and fatal neurological disease and not only as an organic aciduria with severe biochemical dis-turbances and progressive neurological deterioration.

Amino Acid Metabolism, Inborn Errors↗
PubMed1998

Human propionyl-CoA carboxylase beta subunit gene: exon-intron definition and mutation spectrum in Spanish and Latin American propionic acidemia patients.

Propionyl-CoA carboxylase (PCC) is a mitochondrial biotin-dependent enzyme composed of an equal number of alpha and beta subunits. Mutations in the PCCA (alpha subunit) or PCCB (beta subunit) gene can cause the inherited metabolic disease propionic acidemia (PA), which can be life threatening in the neonatal period. Lack of data on the genomic structure of PCCB has been a significant impediment to full characterization of PCCB mutant chromosomes. In this study, we describe the genomic organization of the coding sequence of the human PCCB gene and the characterization of mutations causing PA in a total of 29 unrelated patients-21 from Spain and 8 from Latin America. The implementation of long-distance PCR has allowed us to amplify the regions encompassing the exon/intron boundaries and all the exons. The gene consists of 15 exons of 57-183 bp in size. All splice sites are consistent with the gt/ag rule. The availability of the intron sequences flanking each exon has provided the basis for implementation of screening for mutations in the PCCB gene. A total of 56/58 mutant chromosomes studied have been defined, with a total of 16 different mutations detected. The mutation spectrum includes one insertion/deletion, two insertions, 10 missense mutations, one nonsense mutation, and two splicing defects. Thirteen of these mutations correspond to those not described yet in other populations. The mutation profile found in the chromosomes from the Latin American patients basically resembles that of the Spanish patients.

Amino Acid Substitution↗
PubMedDate not supplied

Primary cultures of astrocytes from rat as a model for biotin deficiency in nervous tissue.

The activities and biotin-dependence of the three mitochondrial biotin-dependent carboxylases: pyruvate carboxylase, propionyl CoA carboxylase, and beta-methylcrotonyl CoA carboxylase of primary culture of astrocytes have been examined. An increase of the three mitochondrial carboxylase activities was observed during cell growth, as was the case for developing rat brain. Mitochondrial carboxylase activities from 3-wk-old primary cultures of astrocytes were higher than those in the neonatal rat brain. When astrocytes were grown in a 10% serum-enriched medium supplemented with avidin to bind biotin, the mitochondrial carboxylase activities were reduced to 15% of control value. Consistent with these results, after 3 wk in culture, the 3-hydroxyisovaleric acid concentration in the growth medium was tenfold higher than the controls. In this culture condition, cellular growth and the nonbiotin-dependent enzyme, glutamine synthetase, were not modified with respect to control. Primary cultures from newborn rat brain hemispheres are suggested as an experimental approach to the study of biotin deficiency in nervous tissue.

Animals↗
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↗