ABSTRACT
We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.
Subject(s)
Death, Sudden, Cardiac/etiology , Inorganic Pyrophosphatase/deficiency , Inorganic Pyrophosphatase/genetics , Mitochondrial Diseases/genetics , Mitochondrial Proteins/deficiency , Mitochondrial Proteins/genetics , Mutation, Missense/genetics , Acidosis, Lactic/genetics , Adolescent , Adult , Amino Acid Sequence , Animals , Arrhythmias, Cardiac/genetics , Cardiomyopathies/enzymology , Cardiomyopathies/genetics , Cardiomyopathies/pathology , Cardiomyopathies/physiopathology , Child , Child, Preschool , Death, Sudden, Cardiac/pathology , Ethanol/adverse effects , Exome/genetics , Female , Fibroblasts/cytology , Fibroblasts/pathology , Fibrosis/enzymology , Fibrosis/genetics , Fibrosis/pathology , Humans , Infant , Infant, Newborn , Inorganic Pyrophosphatase/chemistry , Inorganic Pyrophosphatase/metabolism , Male , Mitochondria/enzymology , Mitochondria/genetics , Mitochondria/pathology , Mitochondrial Diseases/enzymology , Mitochondrial Diseases/pathology , Mitochondrial Diseases/physiopathology , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Models, Molecular , Pedigree , Phenotype , Seizures , Young AdultABSTRACT
Isolated methylmalonic acidurias comprise a heterogeneous group of inborn errors of metabolism caused by defects of methylmalonyl-CoA mutase (MCM) (mut0, mut-) or deficient synthesis of its cofactor 5'-deoxyadenosylcobalamin (AdoCbl) (cblA, cblB). The aim of this study was to compare the long-term outcome in patients from these four enzymatic subgroups. Eighty-three patients with isolated methylmalonic acidurias (age 7-33 y) born between 1971 and 1997 were enzymatically characterized and prospectively followed to evaluate the long-term outcome (median follow-up period, 18 y). Patients with mut0 (n = 42), mut- (n = 10), cblA (n = 20), and cblB (n = 11) defects were included into the study. Thirty patients (37%) died, and 26 patients survived with a severe or moderate neurologic handicap (31%), whereas 27 patients (32%) remained neurologically uncompromised. Chronic renal failure (CRF) was found most frequently in mut0 (61%) and cblB patients (66%), and was predicted by the urinary excretion of methylmalonic acid (MMA) before CRF. Overall, patients with mut0 and cblB defects had an earlier onset of symptoms, a higher frequency of complications and deaths, and a more pronounced urinary excretion of MMA than those with mut- and cblA defects. In addition, long-term outcome was dependent on the age cohort and cobalamin responsiveness.
Subject(s)
Alkyl and Aryl Transferases/genetics , Amino Acid Metabolism, Inborn Errors/genetics , Cobamides/metabolism , Membrane Transport Proteins/genetics , Methylmalonic Acid/metabolism , Methylmalonyl-CoA Mutase/genetics , Mitochondrial Proteins/genetics , Mutation , Adolescent , Adult , Age of Onset , Alkyl and Aryl Transferases/metabolism , Amino Acid Metabolism, Inborn Errors/complications , Amino Acid Metabolism, Inborn Errors/drug therapy , Amino Acid Metabolism, Inborn Errors/enzymology , Amino Acid Metabolism, Inborn Errors/mortality , Child , Disease Progression , Female , Follow-Up Studies , Gastrointestinal Diseases/etiology , Genetic Predisposition to Disease , Humans , Kaplan-Meier Estimate , Kidney Failure, Chronic/etiology , Male , Membrane Transport Proteins/metabolism , Methylmalonic Acid/urine , Methylmalonyl-CoA Mutase/metabolism , Mitochondrial Membrane Transport Proteins , Mitochondrial Proteins/metabolism , Nervous System Diseases/etiology , Prognosis , Prospective Studies , Time Factors , Vitamin B 12/therapeutic use , Vitamin B Complex/therapeutic useABSTRACT
Isolated methylmalonic acidurias (MMA-urias) comprise a group of rare autosomal recessively inherited disorders characterised by accumulation of MMA in urine and other body fluids, resulting from deficient activity of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM). Isolated MMA-uria results from either MCM apoenzyme defects (mut(0) and mut(-)) or defects in synthesis of its cofactor 5-deoxyadenosylcobalamin, i.e. cblA, cblB and cblD-variant 2. To date various studies have identified 171 disease-causing mutations in the MCM gene (MUT). We report mutation analysis in 32 probands with mut MMA-uria including 13 probands with a mut(-) defect. Sixty two of 64 possible mutant alleles were identified, seven of which were novel missense alleles. We found three novel mutations (c.427C>T/p.H143Y; c.862T>C/p.S288P; c.1361G>A/p.G454E) among 19 probands with a mut(0) defect and four novel mutations (c.299A>G/p.Y100C; c.1031C>T/p.S344F; c.1097A>G/p.N366S; c.2081G>T/p.R694L) among 13 probands with a mut(-) defect. Our study provides evidence that the p.Y100C, p.R108H, p.N366S, p.V633G, p.R694W, p.R694L and p.M700K mutations are associated with a mut(-) phenotype.
Subject(s)
Amino Acid Metabolism, Inborn Errors/genetics , Amino Acid Metabolism, Inborn Errors/metabolism , Methylmalonic Acid/urine , Methylmalonyl-CoA Mutase/deficiency , Methylmalonyl-CoA Mutase/genetics , Mutation , Adolescent , Apoenzymes/genetics , Child, Preschool , DNA Mutational Analysis , Genotype , Humans , Infant , Infant, Newborn , PhenotypeABSTRACT
We describe 7 Polynesian babies with a unique severe form of holocarboxylase synthetase deficiency characterized by antenatal growth retardation, subependymal cysts, only partial response to biotin, and a poor outcome.
Subject(s)
Biotin/pharmacology , Drug Resistance , Fetal Growth Retardation , Holocarboxylase Synthetase Deficiency/drug therapy , Holocarboxylase Synthetase Deficiency/ethnology , Australia , Central Nervous System Cysts/congenital , Central Nervous System Cysts/diagnostic imaging , Ependyma , Fatal Outcome , Holocarboxylase Synthetase Deficiency/genetics , Holocarboxylase Synthetase Deficiency/pathology , Humans , Infant, Newborn , New Zealand , Samoa/ethnology , UltrasonographyABSTRACT
CONTEXT: Pyruvate dehydrogenase phosphatase (PDP) deficiency has been previously reported as an enzymopathy, but the genetic basis for such a defect has never been established. OBJECTIVE: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency. PATIENTS: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium. RESULTS: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient's fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type). CONCLUSION: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.
Subject(s)
Mutation , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/deficiency , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/genetics , Amino Acid Sequence , Child , Humans , Male , Molecular Sequence Data , Protein Structure, Secondary , Pyruvate Dehydrogenase (Lipoamide)-Phosphatase/chemistry , Recombinant Proteins/therapeutic use , SiblingsABSTRACT
Deficiency of 3-methylcrotonyl-CoA carboxylase (MCC) results in elevated excretion of 3-methylcrotonylglycine (3-MCG) and 3-hydroxyisovaleric acid (3-HIVA). MCC is a heteromeric mitochondrial enzyme comprising biotin-containing alpha subunits and smaller beta subunits, encoded by MCCA and MCCB, respectively. Mutations in these genes cause isolated MCC deficiency, an autosomal recessive disorder with a variable phenotype that ranges from severe neonatal to asymptomatic adult forms. No reported patients have responded to biotin therapy. Here, we describe two patients with a biochemical and, in one case, clinical phenotype of MCC deficiency, both of whom were responsive to biotin. The first patient presented at 3 months with seizures and progressive psychomotor retardation. Metabolic investigation at 2 years revealed elevated excretion of 3-MCG and 3-HIVA, suggesting MCC deficiency. High-dose biotin therapy was associated with a dramatic reduction in seizures, normalization of the electroencephalogram, and correction of the organic aciduria, within 4 weeks. MCC activity in fibroblasts was 25% of normal levels. The second patient, a newborn detected by tandem-mass-spectrometry newborn screening, displayed the same biochemical phenotype and remained asymptomatic with biotin up to the age of 18 months. In both patients, sequence analysis of the complete open reading frames of MCCA and MCCB revealed heterozygosity for MCCA-R385S and for the known polymorphic variant MCCA-P464H but revealed no other coding alterations. MCCA-R385S is unusual, in that it has a normal amount of MCC alpha protein but confers no MCC activity. We show that MCCA-R385S, but not other MCCA missense alleles, reduces the MCC activity of cotransfected MCCA-wild-type allele. Our results suggest that MCCA-R385S is a dominant negative allele and is biotin responsive in vivo.
Subject(s)
Alleles , Biotin/therapeutic use , Carbon-Carbon Ligases/deficiency , Carbon-Carbon Ligases/genetics , Glycine/analogs & derivatives , Mitochondrial Diseases/drug therapy , Base Sequence , Biotin/metabolism , DNA Mutational Analysis , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Gene Expression , Genetic Vectors/genetics , Germany , Glycine/urine , Greece , Humans , Infant, Newborn , Male , Molecular Sequence Data , Mutation/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Transfection , Valerates/urineABSTRACT
Profound biotinidase deficiency (PBD) is an autosomal recessively inherited disorder of biotin metabolism, which can be detected by newborn screening and treated with biotin supplementation. Children were investigated in whom PBD was detected by newborn screening and who were treated presymptomatically, or who were not screened but were diagnosed and treated after experiencing initial clinical symptoms (symptomatic children). In a follow-up of our study group, differences in development, social and behavioural adaptation, and signs of residual impairment were examined. Parents and physicians of children with PBD completed questionnaires which included the Child Behavior Checklist and Vineland Adaptive Behavior Scales. Information was obtained for 37 children (24 males, 13 females; median age at recruitment 6 years 8 months, range to 6 months-20 years; median length of follow-up 6 years 6 months, range 5 months to 18 years 3 months). All 11 symptomatic children had residual enzyme activity of <1%, or variants of the Michaelis-Menten constant which were not detected by newborn screening. Some symptomatic children showed residual impairments: hearing impairment (n=2), optic atrophy (n=2), both hearing impairment and optic atrophy (n=2). In addition, symptomatic children had a higher risk of delayed motor and speech development. No child with PBD detected by newborn screening (n=25) had auditory or visual loss; and milestones of speech development and motor skills were reached at an appropriate age. There was no significant difference in social adaptation or behavioural problems between symptomatic and asymptomatic children. Symptomatic children often have developmental delay and are at risk of irreversible damage to auditory, visual, or central nervous functions; whereas children with PBD (established presymptomatically following newborn screening) treated with biotin supplementation, do not experience these effects.
Subject(s)
Biotinidase Deficiency/drug therapy , Biotinidase Deficiency/epidemiology , Neonatal Screening/methods , Adaptation, Psychological , Adolescent , Adult , Biotin/metabolism , Biotin/therapeutic use , Biotinidase Deficiency/genetics , Child , Child, Preschool , Female , Follow-Up Studies , Hearing Disorders/diagnosis , Hearing Disorders/epidemiology , Humans , Infant , Infant, Newborn , Male , Motor Skills Disorders/diagnosis , Motor Skills Disorders/epidemiology , Optic Atrophy/diagnosis , Optic Atrophy/epidemiology , Outcome Assessment, Health Care , Severity of Illness Index , Social Adjustment , Speech Disorders/diagnosis , Speech Disorders/epidemiologyABSTRACT
The validity of various transformed and untransformed CNS and skin-derived cell cultures as a model for studying effects of biotin deficiency was tested. In biotin-sufficient conditions (0.1-10 mumol/L) all cell types showed considerable activities of the four biotin-dependent carboxylases. Notably, pyruvate carboxylase activity was also present in the different neuronal cells. One passage in low-biotin medium (6-130 pmol/L) lowered mitochondrial carboxylase activities in all cell types, but to varying degrees. Sensitivity to biotin depletion was greatest in three neuronal cell types, Roc-1 oligodendroglia, and three keratinocyte cell types (carboxylase activities decreased to 2-11% of maximal); intermediate in primary astrocytes and C6 glioma (decreased to 12-28%), and least in SAOS2 sarcoma and skin fibroblasts (decreased to 32-85%). Transformed and untransformed cell lines of the same cell type showed similar sensitivity. We conclude that cultures of different transformed CNS and keratinocyte cell types allow the study of effects of biotin deprivation. Carboxylase activities of neurons, oligodendroglia, and keratinocytes were much more sensitive to biotin depletion than fibroblasts. This may be an important factor in the pathogenesis of neurological and cutaneous abnormalities in congenital biotinidase deficiency where recycling of biotin is deficient.
Subject(s)
Biotin/deficiency , Biotin/metabolism , Carboxy-Lyases/metabolism , Fibroblasts/metabolism , Keratinocytes/metabolism , Oligodendroglia/metabolism , Animals , Brain/cytology , Brain/metabolism , Cell Line , Cells, Cultured , Culture Media , Fibroblasts/enzymology , Humans , Kinetics , Mice , Mitochondria/enzymology , Mitochondria/metabolism , Neurons/enzymology , Neurons/metabolism , Oligodendroglia/enzymology , Rats , Skin/cytology , Skin/metabolismABSTRACT
Two siblings, a boy age 12 and his sister age 4 years, presented with proteinuria and hematuria, hypertension, and chronic hemolytic anemia. At age 13 years, the boy developed an episode of severe hypertensive encephalopathy and transient renal failure. Both children are attending normal school, have no neurologic symptoms, and only minimal pigmentary retinal abnormalities. Renal biopsy showed a chronic thrombotic microangiopathic nephropathy. Both patients had hyperhomocysteinemia and mild methylmalonic aciduria. Fibroblasts showed decreased cobalamin uptake, reduced methyl- and adenosyl-cobalamin formation, and deficient incorporation of formate and propionate, compatible with the Cbl-C complementation group, but milder than that found in cells from most patients. Both patients and their father carry a balanced reciprocal translocation. Parenteral hydroxycobalamin treatment reduced the homocysteine levels, and methylmalonic acid disappeared. Increasing the dosage of hydroxycobalamin from 1 to 2.5, then 5 mg daily together with betaine, further reduced homocysteine levels (boy from 118 to 23 microM and girl from 59 to 14 microM). With this treatment, hemolysis has stopped, hematuria has disappeared, proteinuria has almost normalized, and creatinine clearance has been stable. Investigations for chronic thrombotic microangiopathy should include testing for this unusual but treatable disorder, regardless of age of presentation.
Subject(s)
Hemolytic-Uremic Syndrome/etiology , Thrombosis/etiology , Vitamin B 12 Deficiency/complications , Age of Onset , Child , Child, Preschool , Female , Hematinics/therapeutic use , Hematuria/prevention & control , Hemolysis/drug effects , Hemolytic-Uremic Syndrome/diagnosis , Hemolytic-Uremic Syndrome/metabolism , Homocysteine/metabolism , Humans , Hydroxocobalamin/therapeutic use , Kidney/pathology , Male , Methylmalonic Acid/metabolism , Microcirculation/pathology , Proteinuria/prevention & control , Thrombosis/diagnosis , Thrombosis/metabolism , Vitamin B 12 Deficiency/diagnosis , Vitamin B 12 Deficiency/metabolismABSTRACT
We report the first case of isolated biotin resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency in Argentina. The diagnosis was established at 14 months of age by urinary organic-acid analysis and confirmed by enzyme assay in fibroblasts. The patient suffered from severe psychomotor retardation, hypotonia, areflexia, and failure to thrive, and died unexpectedly at 3 years 4 months of life. Brain MRI at 14 months showed signals of the white matter on cerebral T2-weighted, which were indicative of confluent and multiple foci of leukodystrophy, a pattern not previously described in this entity. In addition, high levels of oxypurines were detected in cerebrospinal fluid. This might be related to energetic consequences of the enzyme deficiency in the brain. This case extends the phenotype of isolated MCC deficiency in infancy and suggests this entity should be considered to be one of the possible causes of "metabolic leukodystrophies."
Subject(s)
Brain/pathology , Carbon-Carbon Ligases/deficiency , Nervous System Diseases/enzymology , Nervous System Diseases/pathology , Purines/cerebrospinal fluid , Fatal Outcome , Fibroblasts/enzymology , Genetic Complementation Test , Humans , Infant , Magnetic Resonance Imaging , Male , Mitochondria/enzymologyABSTRACT
Uptake of biocytin and biotin was investigated in cultured transformed variants of neuronal (NB2a neuroblastoma) and glial (C6 astrocytoma) CNS cells. NB2a cells took up both compounds but biocytin was transported more efficiently than biotin in the nanomolar concentration range. In NB2a cells a single transport mechanism was found for biocytin with different kinetic parameters in the presence of high extracellular Na+ (Km 0.4 microM, Vmax 20 pmol/min/mg), K+ (Km 1.7 microM, Vmax 32 pmol/min/mg), or choline+ (Km 0.1 microM, Vmax 5 pmol/min/mg). Two transport systems (Km1 17 microM, Vmax1 53 pmol/min/mg; Km2 314 microM, Vmax2 360 pmol/min/mg) were identified for biotin with only system 1 being Na+-dependent. Biocytin uptake was competitively inhibited by excess biotin but not vice versa. Inhibition studies with structural analogs indicated different specificities for biotin and biocytin uptake. Biocytin uptake into C6 cells was hardly detectable whereas biotin was taken up by diffusion (kD 0.6 microl/min/mg) and a single saturable mechanism (Km 70 microM, Vmax 119 pmol/min/mg) at high extracellular Na+. High extracellular K+ enhanced biotin diffusion into C6 cells. Inhibition studies with structural analogs revealed a less specific biotin uptake mechanism in C6 than in NB2a cells. Biocytin normalized deficient biotin-dependent propionyl-CoA carboxylase activity within 4 h in biotin-deficient NB2a cells whereas in C6 cells reactivation was <20% thereby confirming that biocytin is only poorly transported into C6 cells. Specific biocytin uptake into NB2a cells is to our knowledge the first demonstration of a carrier-mediated transport mechanism for this compound. Neuronal biocytin uptake might contribute to the pathogenesis of biotinidase deficiency where biocytin is present in elevated levels.