Celia Pérez-Cerdá

Proposed guidelines for the diagnosis and management of methylmalonic and propionic acidemia

Methylmalonic and propionic acidemia (MMA/PA) are inborn errors of metabolism characterized by accumulation of propionic acid and/or methylmalonic acid due to deficiency of methylmalonyl-CoA mutase (MUT) or propionyl-CoA carboxylase (PCC). MMA has an estimated incidence of ~ 1: 50,000 and PA of ~ 1:100-000 -150,000. Patients present either shortly after birth with acute deterioration, metabolic acidosis and hyperammonemia or later at any age with a more heterogeneous clinical picture, leading to early death or to severe neurological handicap in many survivors. Mental outcome tends to be worse in PA and late complications include chronic kidney disease almost exclusively in MMA and cardiomyopathy mainly in PA. Except for vitamin B12 responsive forms of MMA the outcome remains poor despite the existence of apparently effective therapy with a low protein diet and carnitine. This may be related to under recognition and delayed diagnosis due to nonspecific clinical presentation and insufficient awareness of health care professionals because of disease rarity.These guidelines aim to provide a trans-European consensus to guide practitioners, set standards of care and to help to raise awareness. To achieve these goals, the guidelines were developed using the SIGN methodology by having professionals on MMA/PA across twelve European countries and the U.S. gather all the existing evidence, score it according to the SIGN evidence level system and make a series of conclusive statements supported by an associated level of evidence. Although the degree of evidence rarely exceeds level C (evidence from non-analytical studies like case reports and series), the guideline should provide a firm and critical basis to guide practice on both acute and chronic presentations, and to address diagnosis, management, monitoring, outcomes, and psychosocial and ethical issues. Furthermore, these guidelines highlight gaps in knowledge that must be filled by future research. We consider that these guidelines will help to harmonize practice, set common standards and spread good practices, with a positive impact on the outcomes of MMA/PA patients.

The enigmatic role of tafazzin in cardiolipin metabolism

The mitochondrial phospholipid cardiolipin plays an important role in cellular metabolism as exemplified by its involvement in mitochondrial energy production and apoptosis. Following its biosynthesis, cardiolipin is actively remodeled to achieve its final acyl composition. An important cardiolipin remodeling enzyme is tafazzin, of which several mRNA splice variants exist. Mutations in the tafazzin gene cause the X-linked recessive disorder Barth syndrome. In addition to providing an overview of the current knowledge in literature about tafazzin, we present novel experimental data and use this to discuss the functional role of the different tafazzin variants in cardiolipin metabolism in relation to Barth syndrome. We developed and performed specific quantitative PCR analyses of different tafazzin mRNA splice variants in 16 human tissues and correlated this with the tissue cardiolipin profile. In BTHS fibroblasts we showed that mutations in the tafazzin gene affected both the level and distribution of tafazzin mRNA variants. Transient expression of selected human tafazzin variants in BTHS fibroblasts showed for the first time in a human cell system that tafazzin lacking exon5 indeed functions in cardiolipin remodeling.

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.

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.

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).

Human propionyl-CoA carboxylase β 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.

A novel congenital disorder of glycosylation type without central nervous system involvement caused by mutations in the phosphoglucomutase 1 gene

Recent years have seen great advances in our knowledge of congenital disorders of glycosylation (CDG), a clinically and biochemically heterogeneous group of genetic diseases caused by defects in the synthesis (CDG-I) or processing (CDG-II) of glycans that form glycoconjugates. This paper reports a new subtype of non-neurological CDG involving the impaired cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. A patient presented with muscle fatigue, elevated creatine kinase, growth hormone deficiency, and first branchial arch syndrome. These findings, together with the abnormal type II plasma transferrin isoform profile detected, was compatible with a CDG. Functional testing and clinical analyses suggested a deficiency in the interconversion of glucose-1-phosphate and glucose-6-phosphate catalyzed by phosphoglucomutase (PGM1), a defect previously described as glycogenosis type XIV (GSDXIV, MIM 612934). PGM1 activity in patient-derived fibroblasts was significantly reduced, as was the quantity of immunoreactive PGM1 protein (Western blot assays). Mutation analysis of PGM1 and subsequent functional analysis investigating transient expression of PGM1 in immortalized patient fibroblasts, followed by ex vivo splicing assays using minigenes, allowed the characterization of two novel pathogenic mutations: c.871G>A (p.Gly291Arg) and c.1144 + 3A>T. The latter represents a severe splicing mutation leading to the out-of-frame skipping of exon 7 and the formation of a truncated protein (p.Arg343fs). MALDI mass spectra of permethylated protein N-glycans from the patient’s serum suggested a marked hypoglycosylation defect. The present findings confirm that, in addition to a rare muscular glycolytic defect, PGM1 deficiency causes a non-neurological disorder of glycosylation.