Gene identification for the cblD defect of vitamin B12 metabolism

Background Vitamin B12 (cobalamin) is an essential cofactor in several metabolic pathways. Intracellular conversion of cobalamin to its two coenzymes, adenosylcobalamin in mitochondria and methylcobalamin in the cytoplasm, is necessary for the homeostasis of methylmalonic acid and homocysteine. Nine defects of intracellular cobalamin metabolism have been defined by means of somatic complementation analysis. One of these defects, the cblD defect, can cause isolated methylmalonic aciduria, isolated homocystinuria, or both. Affected persons present with multisystem clinical abnormalities, including developmental, hematologic, neurologic, and metabolic findings. The gene responsible for the cblD defect has not been identified. Methods We studied seven patients with the cblD defect, and skin fibroblasts from each were investigated in cell culture. Microcell-mediated chromosome transfer and refined genetic mapping were used to localize the responsible gene. This gene was transfected into cblD fibroblasts to test for the rescue of adenosylcobalamin and methylcobalamin synthesis. Results The cblD gene was localized to human chromosome 2q23.2, and a candidate gene, designated MMADHC (methylmalonic aciduria, cblD type, and homocystinuria), was identified in this region. Transfection of wild-type MMADHC rescued the cellular phenotype, and the functional importance of mutant alleles was shown by means of transfection with mutant constructs. The predicted MMADHC protein has sequence homology with a bacterial ATP-binding cassette transporter and contains a putative cobalamin binding motif and a putative mitochondrial targeting sequence. Conclusions Mutations in a gene we designated MMADHC are responsible for the cblD defect in vitamin B12 metabolism. Various mutations are associated with each of the three biochemical phenotypes of the disorder

Guidelines for diagnosis and management of the cobalamin-related remethylation disorders cblC, cblD, cblE, cblF, cblG, cblJ and MTHFR deficiency

BACKGROUND: Remethylation defects are rare inherited disorders in which impaired remethylation of homocysteine to methionine leads to accumulation of homocysteine and perturbation of numerous methylation reactions. OBJECTIVE: To summarise clinical and biochemical characteristics of these severe disorders and to provide guidelines on diagnosis and management. DATA SOURCES: Review, evaluation and discussion of the medical literature (Medline, Cochrane databases) by a panel of experts on these rare diseases following the GRADE approach. KEY RECOMMENDATIONS: We strongly recommend measuring plasma total homocysteine in any patient presenting with the combination of neurological and/or visual and/or haematological symptoms, subacute spinal cord degeneration, atypical haemolytic uraemic syndrome or unexplained vascular thrombosis. We strongly recommend to initiate treatment with parenteral hydroxocobalamin without delay in any suspected remethylation disorder; it significantly improves survival and incidence of severe complications. We strongly recommend betaine treatment in individuals with MTHFR deficiency; it improves the outcome and prevents disease when given early

Biotin-responsive disorders

Two inherited defects affecting the coenzyme function of biotin are known: holocarboxylase synthetase (HCS) deficiency and biotinidase deficiency. Both lead to deficiency of all biotin-dependent carboxylases, i.e. to multiple carboxylase deficiency (MCD). In HCS deficiency, the binding of biotin to apocarboxylases is impaired. In biotinidase deficiency, biotin depletion ensues from the inability to recycle endogenous biotin and to utilise protein-bound biotin from the diet. As the carboxylases play an essential role in the catabolism of several amino acids, in gluconeogenesis and in fatty-acid synthesis, their deficiency provokes multiple, life-threatening metabolic derangements, eliciting characteristic organic aciduria and neurological symptoms. The clinical presentation is extremely variable in both disorders. Characteristic symptoms include metabolic acidosis, hypotonia, seizures, ataxia, impaired consciousness and cutaneous symptoms, such as skin rash and alopecia

Biotinidase

Biotinidase (EC 3.5.1.12) is required for the recycling of biotin and for the utilization of protein bound biotin from the diet. Biotinidase deficiency (MIM 253260) is inherited as an autosomal recessively trait. Patients become progressively biotin deficient which results in reduced activity of the 4 biotin-dependent carboxylases existing in man, and severe life-threatening illness. Oral biotin substitution effectively protects against disease or reverses symptoms. Delayed treatment may result in irreversible neurological damage. Time of onset and severity of illness depend on the level of residual enzyme activity necessitating early (preferably neonatal) assessment of biotinidase activity. Patients are classified as having profound (0-10% residual activity) or partial (residual activity >10-30%) deficiency, or a Km defect due to reduced affinity of biotinidase for its substrate biocytin. Heterozygous individuals show activities intermediate between the deficient and normal activity. The natural substrate of biotinidase is biocytin but it can also act on artificial biotinyl-derivatives. Biotinidase activity in plasma is usually assayed using biotinyl-p-aminobenzoic acid (biotinyl-PABA) as substrate. Liberated PABA is converted to a purple azo dye and quantitated spectrophotometrically. This simple, reproducible and easy to perform colorimetric assay for the diagnosis of patients with different forms of biotinidase deficiency, including those with a Km defect, is described

Intermediate hyperhomocysteinaemia and compound heterozygosity for the common variant c.677C<T and a MTHFR gene mutation

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of plasma homocysteine levels. MTHFR deficiency, an autosomal recessive disorder, results in homocystinuria and hypomethioninaemia and presents with highly variable symptoms affecting many organs but predominantly the central nervous system. The common polymorphism of the MTHFR gene, c.677C<T, a known risk factor for elevated plasma homocysteine levels, occurs frequently in the caucasian population. In this study we investigated three subjects with moderate hyperhomocysteinaemia (total plasma homocysteine 72 micromol/L in case 1 and 90 micromol/L in case 3, total non-protein-bound homocysteine 144-186 micromol/L in case 2) but different clinical presentation with no symptoms in case 1, muscle weakness at 17 years of age in case 2, and syncopes and cerebral convulsions at 18 years of age in case 3. Each subject was compound heterozygous for the c.677C<T polymorphism and a novel mutation of the MTHFR gene (case 1: c.883G<A [p.D291N]; case 2: c.1552_c.1553delGA [p.E514fsX536]; case 3: c.616C<T [p.P202S]). Moderately decreased fibroblast MTHFR activity was associated with severely reduced affinity for NADPH and increased sensitivity to inhibition by S-adenosylmethionine (AdoMet) in case 2, and with mild FAD responsiveness in case 3. In case 1, fibroblast MTHFR activity was normal but the sensitivity to inhibition by AdoMet was slightly reduced. This study indicates that the sequence alteration c.677C<T combined with severe MTHFR mutations in compound heterozygous state may lead to moderate biochemical and clinical abnormalities exceeding those attributed to the c.677TT genotype and might require in addition to folate substitution further therapy to normalize homocysteine levels