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M Ugarte

Publications and source records attributed to M Ugarte.

At least 19 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↗
PubMed2002

Splicing mutations, mainly IVS6-1(G>T), account for 70% of fumarylacetoacetate hydrolase (FAH) gene alterations, including 7 novel mutations, in a survey of 29 tyrosinemia type I patients.

Hereditary tyrosinemia type I (HTI) is an autosomal recessive disease characterized by a deficiency in fumarylacetoacetate hydrolase (FAH) activity. In this work, the FAH genotype was established in a group of 29 HTI patients, most of them from the Mediterranean area. We identified seven novel mutations-IVS8-1(G>A, IVS10-2(A>T), 938delC, E6/I6del26, W78X, Q328X, and G343W-and two previously described mutations-IVS6-1(G>T) and IVS12+5(G>A). Fully 92.8% of the patients were carriers of at least one splice site mutation, with IVS6-1(G>T) accounting for 58.9% of the total number of alleles. The splice mutation group of patients showed heterogeneous phenotypic patterns ranging from acute forms with severe liver malfunction to chronic forms with renal manifestations and slow progressive hepatic alterations. Qualitative FAH cDNA expression was the same in all IVS6-1(G>T) homozygous patients regardless of their clinical picture. One patient with a heterozygous combination of a nonsense (Q328X) and a frameshift (938delC) mutation showed an atypical clinical picture of hypotonia and repeated infections. Despite the high prevalence of IVS12+5(G>A) in the northwestern European population, we found only two patients with this mutation in our group.

Alternative Splicing↗
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↗
PubMed2002

Screening for adenylosuccinate lyase deficiency: clinical, biochemical and molecular findings in four patients.

Adenylosuccinate lyase deficiency is an autosomal recessive defect of purine metabolism. Succinyladenosine (S-Ado) and succinylaminoimidazole carboxamide riboside (SAICAr) are the disease marker metabolites in physiological fluids. The Bratton-Marshall test for detection of SAICAr in urine has been added to the selective screening for inborn errors of metabolism that is carried out in our lab. During the last three years, around 2,000 patients have been screened by this method, resulting in the detection of four new cases with this disease. They all presented with severe psychomotor delay, hypotonia and refractory epilepsy since the neonatal period. The S-Ado/SAICAr ratio in cerebrospinal fluid was below 2, indicating that they correspond to the most severe form of the disease. New missense mutations were found in a heterozygous fashion in three patients. The study of purines in all patients with neurological disease of unknown etiology is highly recommended.

Adenylosuccinate Lyase↗
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

Molecular basis of phenylketonuria in Cuba.

In this study we report the mutation analysis performed in Cuban PKU patients using DGGE and direct sequencing. Sixteen different mutations have been detected, which account for 91% of the total mutant alleles. Haplotype analysis and genealogical data support the European (mainly Spanish) origin of the mutations. Two mutations were found at unexpectedly high frequencies, E280K and R261Q, possibly due to consanguinity and genetic drift, among other factors. Hum Mutat 18:252, 2001.

Cuba↗
PubMed2001

A novel single-base substitution (380C>T) that activates a 5-base downstream cryptic splice-acceptor site within exon 5 in almost all transcripts in the human mitochondrial acetoacetyl-CoA thiolase gene.

Most mutation-related aberrant splicing occurs in the conserved splice-acceptor and -donor sites and some exonic mutations also affect splicing. We identified and characterized a point mutation (380C>T) in a Spanish patient (GK25) with mitochondrial acetoacetyl-CoA thiolase (T2) deficiency. GK25 is a homozygote of 380C>T, which activates a cryptic splice-acceptor site 5 bases downstream from 380C>T within exon 5, causing aberrant splicing in 94% of transcripts. The aberrant splicing results in a 17-amino acids deletion, including the active-site 126Cys. The 380C>T mutation also results in A127V mutation in 6% of transcripts. Transient expression analysis showed that the A127V mutation did not retain T2 activity, indicating that 380C>T was a null mutation. Although this cryptic splice site has a higher Shapiro and Senapathy's score (86) in even a normal sequence than the authentic splice-acceptor site of intron 4 (78), it is not used in normal controls. While the 380C>T mutation increases the score slightly (90), the cryptic splice site is used in almost all transcripts in GK25 fibroblasts. This is an example in which a point mutation activates a cryptic splice-acceptor site motif that is used preferentially over the upstream authentic splice site.

Acetyl-CoA C-Acyltransferase↗
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↗
PubMed2001

Zinc in the retina.

Experimental evidence exists to suggest that zinc can have positive and negative effects on the physiology of cells depending on the "local" concentration, localisation (extracellular vs. intracellular) and/or state (bound vs. free). The retina contains particularly high amounts of zinc suggesting a pivotal role in the tissue. There is also suggestive evidence that zinc deficiency in humans may result in abnormal dark adaptation and/or age-related macular degeneration. The purpose of this article is to provide an overview of various proposed functions for zinc, particularly in the retina. Endogenous chelatable zinc in the retina is localised mainly to the photoreceptors and retinal pigment epithelial cells. Moreover, the zinc localisation in the photoreceptors varies in dark and light, suggesting a role for zinc in a light-regulated process. Some zinc is also located to other areas of the retina but clearly defined zinc-enriched neurones could not be identified as has been shown to occur in certain areas of the brain. Neurones post-synaptic to zinc-enriched neurones in the brain have been suggested to be particularly vulnerable in ischaemia. The role of zinc in retinal ischaemia has been investigated to determine how it is involved in the process. It would appear that when zinc is administered in low concentrations it generally has a positive effect on an insulted retina as in ischaemia. However, higher concentrations of zinc exacerbates the influence of the insult and also acts as a toxin. Use of zinc supplements in diet must, therefore, be taken with caution.

Animals↗
PubMed2001

Outcome of tyrosinaemia type III.

Tyrosinaemia type III is a rare disorder caused by a deficiency of 4-hydroxyphenylpyruvate dioxygenase, the second enzyme in the catabolic pathway of tyrosine. The majority of the nine previously reported patients have presented with neurological symptoms after the neonatal period, while others detected by neonatal screening have been asymptomatic. All have had normal liver and renal function and none has skin or eye abnormalities. A further four patients with tyrosinaemia type III are described. It is not clear whether a strict low tyrosine diet alters the natural history of tyrosinaemia type III, although there remains a suspicion that treatment may be important, at least in infancy.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed2000

Expression analysis of phenylketonuria mutations. Effect on folding and stability of the phenylalanine hydroxylase protein.

Phenylketonuria is an autosomal recessive human genetic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. In the present work we have used different expression systems to reveal folding defects of the PAH protein caused by phenylketonuria mutations L348V, S349L, and V388M. The amount of mutant proteins and/or the residual activity can be rescued by chaperonin co-overexpression in Escherichia coli or growth at low temperature in COS cells. Thermal stability profiles and degradation time courses of PAH expressed in E. coli show that the mutant proteins are less stable than the wild-type enzyme, also confirmed by pulse-chase experiments using a coupled in vitro transcription-translation system. Size exclusion chromatography shows altered oligomerization, partially corrected with chaperonins coexpression, except for the S349L mutant protein, which is recovered as inactive aggregates. PAH subunit interaction is affected in the S349L protein, as demonstrated in a mammalian two-hybrid assay. In conclusion, serine 349, located in the three-dimensional structure lining the active site and involved in the structural maintenance of the iron binding site, is essential for the structural stability and assembly and also for the catalytic properties of the PAH enzyme, whereas the L348V and V388M mutations affect the folding properties and stability of the protein. The experimental modulation of mutant residual activity provides a potential explanation for the existing inconsistencies in the genotype-phenotype correlations.

Escherichia coli↗
PubMed2000

Mutations in the 4-hydroxyphenylpyruvate dioxygenase gene (HPD) in patients with tyrosinemia type III.

Tyrosinemia type III (OMIM 276710) is an autosomal recessive disorder caused by the deficiency of 4-hydroxyphenylpyruvate dioxygenase (HPD), the second enzyme in the tyrosine catabolic pathway. The enzyme deficiency results in an accumulation and increased excretion of tyrosine and phenolic metabolites. Only a few cases with the disorder have been described, and the clinical spectrum of the disorder is unknown. Reported patients have presented with mental retardation or neurological symptoms or have been picked up by neonatal screening. We have identified four presumed pathogenic mutations (two missense and two nonsense mutations) in the HPD gene in three unrelated families encompassing four homozygous individuals and one compound heterozygous individual with tyrosinemia type III. Furthermore, a number of polymorphic mutations have been identified in the HPD gene. No correlation of the severity of the mutation and enzyme deficiency and mental function has been found; neither do the recorded tyrosine levels correlate with the clinical phenotype.

4-Hydroxyphenylpyruvate Dioxygenase↗