Magdalena Ugarte

Glutaryl-CoA Dehydrogenase Deficiency in Spain: Evidence of Two Groups of Patients, Genetically, and Biochemically Distinct

Glutaryl-CoA dehydrogenase (GCDH) deficiency causes glutaric aciduria type I (GA I), an inborn error of metabolism that is characterized clinically by dystonia and dyskinesia and pathologically by neural degeneration of the caudate and putamen. Studies of metabolite excretion allowed us to categorize 43 GA I Spanish patients into two groups: group 1 (26 patients), those presenting with high excretion of both glutarate and 3-hydroxyglutarate, and group 2 (17 patients) , those who might not be detected by routine urine organic acid analysis because glutarate might be normal and 3-hydroxyglutarate only slightly higher than controls. Single-strand conformation polymorphism (SSCP) screening and sequence analysis of the 11 exons and the corresponding intron boundaries of the GCDH gene allowed us to identify 13 novel and 10 previously described mutations. The most frequent mutations in group 1 were A293T and R402W with an allele frequency of 30% and 28%, respectively. These two mutations were also found in group 2, but always in heterozygosity, in particular in combination with mutations V400M or R227P. Interestingly, mutations V400M and R227P were only found in group 2, and at least one of these mutations was found in 11 of 15 unrelated alleles, accounting together for 53% of the mutant alleles in group 2. Therefore, it seems clear that two genetically and biochemically distinct groups of patients exist. The severity of the clinical phenotype seems to be closely linked to the development of encephalopathic crises rather than to residual enzyme activity or genotype. Comparison of GCDH protein with other acyl-CoA dehydrogenases (whose x-ray crystal structure has been determined) reveals that most of the mutations identified in GCDH protein seem to affect folding and tetramerization, as has been described for a number of mutations affecting mitochondrial β-oxidation acyl-CoA dehydrogenases.

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 inE. 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.

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 α and β 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 α and β subunits by Western blot of many PCCA patients, as well as conventional mRNA‐minus result of Northern blots for either gene or β 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. Hum Mutat 14:275–282, 1999.

Maternal polymorphisms 677C-T and 1298A-C of MTHFR, and 66A-G MTRR genes : Is there any relationship between polymorphisms of the folate pathway, maternal homocysteine levels, and the risk for having a child with down syndrome?

This study was aimed at analyzing the effect of mutations in three non‐synonymous SNP genes (677C > T and 1298A > C of the methylenetetrahydrofolate reductase (MTHFR) gene, and 66A > G in the MTRR gene) on total plasmatic homocysteine (Hcy), in 91 mothers of Down syndrome (DS) infants and 90 control mothers. The comparison of both groups of mothers is a new way to determine if those mutations and their interactions increase the risk for DS. Material came from the case‐control network of the Spanish Collaborative Study of Congenital Malformations (ECEMC).

Genetic and cellular studies of oxidative stress in methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC)

Methylmalonic aciduria (MMA) cobalamin deficiency type C (cblC) with homocystinuria (MMACHC) is the most frequent genetic disorder of vitamin B12 metabolism. The aim of this work was to identify the mutational spectrum in a cohort of cblC‐affected patients and the analysis of the cellular oxidative stress and apoptosis processes, in the presence or absence of vitamin B12. The mutational spectrum includes nine previously described mutations: c.3G>A (p.M1L), c.217C>T (p.R73X), c.271dupA (p.R91KfsX14), c.331C>T (p.R111X), c.394C>T (p.R132X), c.457C>T (p.R153X), c.481C>T (p.R161X), c.565C>A (p.R189S), and c.615C>G (p.Y205X), and two novel changes, c.90G>A (p.W30X) and c.81+2T>G (IVS1+2T>G). The most frequent change was the known c.271dupA mutation, which accounts for 85% of the mutant alleles characterized in this cohort of patients. Owing to its high frequency, a real‐time PCR and subsequent high‐resolution melting (HRM) analysis for this mutation has been established for diagnostic purposes. All cell lines studied presented a significant increase of intracellular reactive oxygen species (ROS) content, and also a high rate of apoptosis, suggesting that elevated ROS levels might induce apoptosis in cblC patients. In addition, ROS levels decreased in hydroxocobalamin‐incubated cells, indicating that cobalamin might either directly or indirectly act as a scavenger of ROS. ROS production might be considered as a phenotypic modifier in cblC patients, and cobalamin supplementation or additional antioxidant drugs might suppress apoptosis and prevent cellular damage in these patients. Hum Mutat 30:1–9, 2009.

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.

Functional analysis of three splicing mutations identified in the PMM2 gene: Toward a new therapy for congenital disorder of glycosylation type Ia

The congenital disorders of glycosylation (CDG) are a group of diseases caused by genetic defects affecting N‐glycosylation. The most prevalent form of CDG—type Ia—is caused by defects in the PMM2 gene. This work reports the study of two new nucleotide changes (c.256–1G>C and c.640–9T>G) identified in the PMM2 gene in CDG1a patients, and of a previously described deep intronic nucleotide change in intron 7 (c.640–15479C>T). Cell‐based splicing assays strongly suggest that all these are disease‐causing splicing mutations. The c.256–1G>C mutation was found to cause the skipping of exons 3 and 4 in fibroblast cell lines and in a minigene expression system. The c.640–9T>G mutation was found responsible for the activation of a cryptic intronic splice‐site in fibroblast cell lines and in a hybrid minigene when cotransfected with certain serine/arginine‐rich (SR) proteins. Finally, the deep intronic change c.640–15479C>T was found to be responsible for the activation of a pseudoexon sequence in intron 7. The use of morpholino oligonucleotides allowed the production of correctly spliced mRNA that was efficiently translated into functional and immunoreactive PMM protein. The present results suggest a novel mutation‐specific approach for the treatment of this genetic disease (for which no effective treatment is yet available), and open up therapeutic possibilities for several genetic disorders in which deep intronic changes are seen. Hum Mutat 0, 1–9, 2009.

2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency : An X-linked inborn error of isoleucine metabolism that may mimic a mitochondrial disease

We describe three patients, from two Spanish families, with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency, a recently described X-linked neurodegenerative inborn error of isoleucine metabolism. Two of them are males with severe lactic acidosis suggestive of a mitochondrial encephalopathy, and the third is a female who was less severely affected, suggesting skewed X-inactivation. Molecular studies revealed a new missense mutation, 740A→G, in one family and a previously described mutation, 388C→T, in the other, causing the amino acid substitutions N247S and R130C, respectively. Both male patients died, one of them despite treatment with an isoleucine-restricted diet, but the disease has remained stable in the female patient after 1 y of treatment.

Inhibition by valproic acid of pyruvate uptake by brain mitochondria.

The anticonvulsive drug, valproic acid, inhibits competitively the pyruvate carrier in rat brain and liver mitochondria. Due to this inhibition the oxygen consumption supported by pyruvate oxidation is also affected. In our experimental conditions, pyruvate oxidation is partially inhibited by VPA concentration as low as 0.05 mM. Valproic acid, however, is unable, even at 10 mM, to fully inhibit pyruvate oxidation. Concentrations of VPA higher than 1 mM have an uncoupling effect on mitochondrial respiration. The oxidation of other mitochondrial substrates such as isocitrate, 2-ketoglutarate, DL-3-hydroxybutyrate and succinate is uncoupled but not inhibited by VPA. The effects of VPA on mitochondrial metabolism may be related to the therapeutic and/or toxicologic properties of this drug.

Methylmalonic acidaemia: Examination of genotype and biochemical data in 32 patients belonging to mut, cblA or cblB complementation group

SummaryMethylmalonic acidaemia (MMA) is a genetic disorder caused by defects in methylmalonyl-CoA mutase or in any of the different proteins involved in the synthesis of adenosylcobalamin. The aim of this work was to examine the biochemical and clinical phenotype of 32 MMA patients according to their genotype, and to study the mutant mRNA stability by real-time PCR analysis. Using cellular and biochemical methods, we classified our patient cohort as having the MMA forms mut (n = 19), cblA (n = 9) and cblB (n = 4). All the mut0 and some of the cblB patients had the most severe clinical and biochemical manifestations, displaying non-inducible propionate incorporation in the presence of hydroxocobalamin (OHCbl) in vitro and high plasma odd-numbered long-chain fatty acid (OLCFA) concentrations under dietary therapy. In contrast, mut− and cblA patients exhibited a milder phenotype with propionate incorporation enhanced by OHCbl and normal OLCFA levels under dietary therapy. No missense mutations identified in the MUT gene, including mut0 and mut− changes, affected mRNA stability. A new sequence variation (c.562G>C) in the MMAA gene was identified. Most of the cblA patients carried premature termination codons (PTC) in both alleles. Interestingly, the transcripts containing the PTC mutations were insensitive to nonsense-mediated decay (NMD).