cblA Patients Research Articles

Kidney urinary biomarkers in patients with branched-chain amino acid and cobalamin metabolism defects.

There is a clinical need for early detection of chronic kidney disease (CKD) in patients with organic acidurias. We measured kidney markers in a longitudinal study over 5 years in 40 patients with methylmalonic aciduria (Mut0 ), propionic aciduria (PA), cobalamin A (CblA), and cobalamin C (CblC) deficiencies. Neutrophil gelatinase-associated lipocalin (NGAL), calprotectin (CLP), kidney injury molecule-1 (KIM-1), dickkopf-3 (DKK-3), albumin and beta-2-microglobulin (B2MG) in urine, as well as cystatin C (CysC) in serum were quantified. In Mut0 patients, mean concentrations of B2MG, KIM-1, and DKK-3 were elevated compared with healthy controls, all markers indicative of proximal tubule damage. In PA patients, mean B2MG, albumin, and CLP were elevated, indicating signs of proximal tubule and glomerulus damage and inflammation. In CblC patients, mean B2MG, NGAL, and CLP were increased, and considered as markers for proximal and distal tubule damage and inflammation. B2MG, was elevated in all three diseases, and correlated with DKK-3 in Mut0 /CblA and with eGFR(CysC) and KIM-1 in PA patients, respectively. None of the markers were elevated in CblA patients. Significant deterioration of kidney function, as determined by steady increase in CysC concentrations was noted in seven patients within the observation period. None of the investigated biomarker profiles showed a clear increase or added value for early detection. In conclusion, we identified disease-specific biomarker profiles for inflammation, tubular, and proximal damage in the urine of Mut0 , PA, and CblC patients. Whether these biomarkers can be used for early detection of CKD requires further investigation, as significant kidney function deterioration was observed in only a few patients.

Delineating the clinical spectrum of isolated methylmalonic acidurias: cblA and mut.

Long-term outcome is postulated to be different in isolated methylmalonic aciduria caused by mutations in the MMAA gene (cblA type) compared with methylmalonyl-CoA mutase deficiency (mut), but case definition was previously difficult. Cross-sectional analysis of data from the European Registry and Network for Intoxication type Metabolic Diseases (Chafea no. December 1, 2010). Data from 28 cblA and 95 mut patients in most cases confirmed by mutation analysis (including 4 new mutations for cblA and 19 new mutations for mut). Metabolic crisis is the predominant symptom leading to diagnosis in both groups. Biochemical disturbances during the first crisis were similar in both groups, as well as the age at diagnosis. Z scores of body height and body weight were similar in both groups at birth, but were significantly lower in the mut group at the time of last visit. Glomerular filtration rate was significantly higher in cblA; and as a consequence, chronic renal failure and related complications were significantly less frequent and renal function could be preserved even in older patients. Neurological complications were predominantly found in the mut subgroup. Methylmalonic acidemia (MMA) levels in urine and plasma were significantly lower in cblA. 27/28 cblA patients were reported to be responsive to cobalamin, only 86% of cblA patients were treated with i.m. hydroxocobalamin. In total, 73% of cblA and 98% of mut patients followed a calculated diet with amino acid supplements in 27% (cblA) and 69% (mut). During the study interval, six patients from the mut group died, while all cblA patients survived. Although similar at first, cblA patients respond to hydroxocobalamin treatment, subsequently show significantly lower levels of MMA and a milder course than mut patients.

357. A Mouse Model of Cobalamin A (cblA) Class Isolated Methymalonic Academy (MMA) Provides Unique Platform for Testing Gene and Cell Therapies

Isolated Methylmalonic Acidemia (MMA) comprises a relatively common and heterogeneous group of inborn errors of metabolism. Most affected individuals display severe multisystemic disease with episodes of metabolic instability, chronic renal disease, and neurological complications. The most severe clinical presentations are typically associated with complete loss of methylmalonyl-CoA mutase (MUT) enzymatic activity. MUT converts methylmalonyl-CoA to succinyl-CoA within mitochondria but defects in the transport or synthesis of its cobalamin co-factor also variably impair MUT activity. Previous mouse models of Mut MMA generated by our group have been critical to developing gene therapy for MMA but have either displayed neonatal lethality or required concomitant transgenesis for viability. Thus, the need for ameliorated models to further investigate disease-associated pathophysiology and facilitate gene therapy studies exists. We have therefore constructed a mouse model of an attenuated form of isolated MMA, the cblA subtype, by using homologous recombination to create a deletion allele of Mmaa, the enzyme that performs the gated transfer of adenosylcobalamin to Mut and protects Mut from oxidative inactivation. Patients with the cblA subtype of MMA can have variable presentations, spanning the full spectrum of MMA associated symptoms and pathology, yet always harbor an element of clinical and biochemical responsiveness to injectable B12.Mmaa-/- mice were born in mendelian proportions, exhibited decreased survival after weaning (P<0.0001), weighed 50% less than littermates at 6 months (P<0.0001), lacked immunoreactive Mmaa and Mmaa mRNA in multiple tissues, including the liver, and manifested severe metabolic perturbations. Plasma methylmalonic acid concentrations ranged between 63.88-1641.91 µM, representing a 62-fold or more elevation over age matched littermates. Mmaa-/- mice also display diminished 1-C-13 propionate oxidative capacity (P<0.0001) and hepatic complex IV activity (P=0.002). Electron microscopy confirmed the presence of hepatic megamitochondria and abnormal proximal renal tubular mitochondria. The glomerular filtration rate, measured using FITC-sinistrin decay, showed that Mmaa-/- mice have diminished kidney function compared to heterozygous littermates. Following hydroxycobalamin administration, Mmaa-/- mice had improved survival and increased weight (P=0.0003), which was associated with improved 1-13C-propionate oxidative capacity.This new mouse model fully recapitulates the clinical and biochemical features seen in cblA patients, and provides a robust platform for testing gene and cell therapies for MMA because affected mice are relatively severe but survive until weaning. These animals also offer the opportunity to longitudinally examine disease biomarkers and more easily assay vectors across a wide age range.

High resolution melting analysis of the MMAA gene in patients with cblA and in those with undiagnosed methylmalonic aciduria

The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA gene for variants. DNA was scanned by HRMA from 96 unaffected reference individuals, 72 cblA patients confirmed by complementation, and 181 patients with isolated elevated methylmalonic acid, who could not be diagnosed using complementation analysis. Suspected variants were confirmed by Sanger sequencing. In the cblA cohort, HRMA correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (c.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).

Clinical and molecular findings in Thai patients with isolated methylmalonic acidemia

Isolated methylmalonic acidemia (MMA) is a genetically heterogeneous organic acid disorder caused by either deficiency of the enzyme methylmalonyl-CoA mutase (MCM), or a defect in the biosynthesis of its cofactor, adenosyl-cobalamin (AdoCbl). Herein, we report and review the genotypes and phenotypes of 14 Thai patients with isolated MMA. Between 1997 and 2011, we identified 6 mut patients, 2 cblA patients, and 6 cblB patients. The mut and cblB patients had relatively severe phenotypes compared to relatively mild phenotypes of the cblA patients. The MUT and MMAB genotypes were also correlated to the severity of the phenotypes. Three mutations in the MUT gene: c.788G>T (p.G263V), c.809_812dupGGGC (p.D272Gfs*2), and c.1426C>T (p.Q476*); one mutation in the MMAA gene: c.292A>G (p.R98G); and three mutations in the MMAB gene: c.682delG (p.A228Pfs*2), c.435delC (p.F145Lfs*69), and c.585-1G>A, have not been previously reported. RT-PCR analysis of a common intron 6 polymorphism (c.520-159C>T) of the MMAB gene revealed that it correlates to deep intronic exonization leading to premature termination of the open reading frame. This could decrease the ATP:cobalamin adenosyltransferase (ATR) activity resulting in abnormal phenotypes if found in a compound heterozygous state with a null mutation. We confirm the genotype–phenotype correlation of isolated MMA in the study population, and identified a new molecular basis of the cblB disorder.

Long-term outcome in methylmalonic aciduria: A series of 30 French patients

Objective To better delineate the natural history of patients with methylmalonic aciduria (MMA). Study design Thirty patients with vitamin-B 12-unresponsive MMA (25 aged 1.5 to 22.0 years (y) at the end of the study and 5 who died during a metabolic crisis) were managed following standardized guidelines and studied retrospectively. The median follow-up was 8.3 y (range: 1.4–19.5). Patients were investigated with neuropsychological testing, brain MRIs, inulin clearances, biochemical and genetic studies. Results Fifteen patients had a neonatal onset. Thirteen patients (43%) had significant neurological impairment. Chronic renal disease (CRD) occurred in 14 patients (47%) with a median age of onset of 6.5 y (range 1.5–18.6). Renal function further deteriorated in 4 patients within a median period of 5.8 y (range 2–7.4). Of 25 patients investigated at the enzymatic level, 17 were classified mut°, 3 mut- and 5 cblA. Mortality, number of acute decompensations ( p = 0.031), median MMA urinary excretion ( p = 0.006) and neurological impairment ( p < 0.0001) were higher in mut° patients compared to mut-/cblA patients. Concerning the CRD, no difference incidence was found although the onset of CRD occurred earlier in mut° patients and was more severe. Conclusions Our study provides unique data concerning the progression of renal disease in MMA. Patients with mut° phenotype have a more severe phenotype and probably an earlier and more severe CRD than patients with mut-/cblA phenotype.

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

Methylmalonic 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 mut (0) 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 mut (0) 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).

Homozygous nonsense mutation in the MCEE gene and siRNA suppression of methylmalonyl-CoA epimerase expression: A novel cause of mild methylmalonic aciduria

Methylmalonyl-CoA epimerase (MCE) catalyzes the interconversion of d- and l-methylmalonyl-CoA in the pathway responsible for the degradation of branched chain amino acids, odd chain-length fatty acids, and other metabolites. Despite the occurrence of metabolic disorders in the enzymatic step occurring immediately upstream of MCE (propionyl-CoA carboxylase) and downstream of MCE (adenosylcobalamin-dependent methylmalonyl-CoA mutase), no disease-causing mutations have been described affecting MCE itself. A patient, formerly identified as belonging to the cblA complementation group of vitamin B 12 disorders but lacking mutations in the affected gene, MMAA, was tested for mutations in the MCEE gene. The patient’s fibroblasts had normal levels of adenosylcobalamin compared to controls, whereas other cblA cell lines typically had reduced levels of the cofactor. As well, this patient had a milder form of methylmalonic aciduria than usually observed in cblA patients. The patient was found to be homozygous for a c.139C > T (p.R47X) mutation in MCEE by sequence analysis that was confirmed by restriction digestion of PCR products. One sibling, also with mild methylmalonic aciduria, was homozygous for the mutation. Both parents and one other sibling were heterozygous. A nearby insertion polymorphism, c.41–160_161insT, heterozygous in both parents, showed the wild-type configuration on the mutant alleles. To assess the impact of isolated MCE deficiency in cultured cells, HeLa cells were transfected with a selectable vector containing MCEE-specific small interfering RNA (siRNA) to suppress gene expression. The reduced level of MCEE mRNA resulted in the reduction of [ 14C]-propionate incorporation into cellular macromolecules. However, siRNA only led to a small reduction in pathway activity, suggesting that previously postulated non-enzymatic conversion of d- to l-methylmalonyl-CoA may contribute to some flux through the pathway. We conclude that the patient’s MCEE defect was responsible for the mild methylmalonic aciduria, confirming a partial requirement for the enzymatic activity in humans.

Mutations in theMMAAgene in patients with thecblAdisorder of vitamin B12metabolism

Mutations in the MMAA gene on human chromosome 4q31.21 result in vitamin B12-responsive methylmalonic aciduria (cblA complementation group) due to deficiency in the synthesis of adenosylcobalamin. Genomic DNA from 37 cblA patients, diagnosed on the basis of cellular adenosylcobalamin synthesis, methylmalonyl-coenzyme A (CoA) mutase function, and complementation analysis, was analyzed for deleterious mutations in the MMAA gene by DNA sequencing of exons and flanking sequences. A total of 18 novel mutations were identified, bringing the total number of mutations identified in 37 cblA patients to 22. A total of 13 mutations result in premature stop codons; three are splice site defects; and six are missense mutations that occur at highly conserved residues. Eight of these mutations were common to two or more individuals. One mutation, c.433C>T (R145X), represents 43% of pathogenic alleles and a common haplotype was identified. Restriction endonuclease or heteroduplex diagnostic tests were designed to confirm mutations. None of the sequence changes identified in cblA patients were found in 100 alleles from unrelated control individuals.

Vitamin B 12-responsive methylmalonic aciduria due to a new inborn error of adenosylcobalamin synthesis, cblH

Vitamin B12-responsive methylmalonic aciduria is characterized by deficient synthesis of adenosylcobalamin (AdoCbl) and decreased activity of the AdoCbl-dependent mitochondrial enzyme, methylmalonylCoA mutase. Two distinct disorders of AdoCbl synthesized have been characterized (cblA and cblB) and they can be distinguished on the basis of somatic cell complementation analysis. Such studies have clinical usefulness because in general cblA patients have a better prognosis than do cblB patients. We reported a patient whose fibroblasts had the clinical and biochemical features of the cblA class but complemented cells from all known inborn errors of cobalamin metabolism that cause methylmalonic aciduria, including cblA. The disorder in this patient was designated as cblA variant. Cells from this patient were tested against a panel of 29 cell lines from cblA patients and complemented all members of this panel. These studies strongly suggest that this patient represents a novel complementation class which we have called cblH. Detailed complementation analysis of a panel of 10 cblA lines provided evidence of interallelic complementation. The presence of this interallelic complementation indicates that care must be taken in the use of complementation analysis to identify cblA patients. These studies suggest that additional biochemical steps are required for the reduction and adenosylation of cobalamin. The full clinical spectrum of the new cblH class and its implications remains to be defined.

Methylmalonic aciduria due to a new defect in adenosylcobalamin accumulation by cells

A child with methylmalonic aciduria due to failure to accumulate adocbl in mitochondria has a phenotype similar to cblA disease. Deficient utilization of labeled propionate by his fibroblasts is corrected by their fusion with those from cblA patients, indicating that he belongs to a different complementation class and probably is deficient in a different gene product. The defect appears not to be due to reduced affinity of enzymes for adocbl, or for ATP, and the minimal thiol required for adocbl synthesis is not different from that of extracts of normal cells.