Partial response to biotin therapy in a patient with holocarboxylase synthetase deficiency: clinical, biochemical, and molecular genetic aspects.

We report the clinical course and biochemical findings of a 10-year-old, mentally retarded girl with late-onset holocarboxylase synthetase (HCS, gene symbol HLCS) deficiency and only partial response to biotin. On treatment, even with an unusually high dose of 200mg/day, activities of the biotin-dependent mitochondrial carboxylases in lymphocytes remained below 50% of the mean control values. Not only urinary 3-hydroxyisovaleric acid excretion has been persistently elevated, but also plasma and, with even higher concentrations, cerebrospinal fluid 3-hydroxyisovaleric acid have not normalized. The unusual and insufficient response of this patient to biotin treatment can be explained by the effect of the combination of the common HLCS allele IVS10 +5 g>a on one chromosome and a truncating mutation on the other. This case illustrates mechanisms involved in the genotype-phenotype correlation that unequivocally exists in HCS deficiency.

The molecular basis of human 3-methylcrotonyl-CoA carboxylase deficiency.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that appears to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing alpha subunits and smaller beta subunits. Here, we report cloning of MCCA and MCCB cDNAs and the organization of their structural genes. We show that a series of 14 MCC-deficient probands defines two complementation groups, CG1 and 2, resulting from mutations in MCCB and MCCA, respectively. We identify five MCCA and nine MCCB mutant alleles and show that missense mutations in each result in loss of function.

Relationship between kinetic properties of mutant enzyme and biochemical and clinical responsiveness to biotin in holocarboxylase synthetase deficiency.

Holocarboxylase synthetase (HCS) deficiency is a metabolic disorder that causes a biotin-responsive multiple carboxylase deficiency. We analyzed the kinetic properties of seven mutant HCS proteins. Two of these enzymes harbored mutations within the putative biotin-binding region of HCS and showed elevated Km values for biotin compared with that of the wild-type form (Km mutant; Gly581Ser: 45 times, delThr610: 3 times). The remaining five mutations (Arg183Pro, Leu216Arg, Leu237Pro, Val333Glu, and Val363Asp) were located outside the biotin-binding region. The enzymes containing these mutations showed normal or low Km values for biotin (non-Km mutant). Symptoms of patients who have the non-Km, mutants, as well as those of patients who have the Km, mutants, responded to biotin therapy. This is probably because the Km value for biotin of normal HCS is higher than the physiologic concentration of biotin in human cells. The Vmax values of all mutant HCS proteins were considerably decreased, but to a variable degree. The responsiveness to biotin supplementation of propionyl-CoA carboxylase activity in cultured cells bearing the mutations correlated well with the degree of reduction in the Vmax of HCS. Patients who have mutant HCS proteins with lower Vmax showed poorer clinical and biochemical responses to biotin therapy. These observations suggest that the reduction of Vmax is an essential factor for pathophysiology and prognosis of HCS deficiency under treatment with large amounts of biotin. The determination of HCS genotype can be valuable for characterizing the clinical phenotype in HCS deficient patients.

Isolated biotin-resistant 3-methylcrotonyl-CoA carboxylase deficiency: long-term outcome in a case with neonatal onset.

UNLABELLED: A patient with early-onset 3-methylcrotonyl coenzyme A carboxylase (MCC) deficiency showing a severe clinical course is described. Abnormal eye and head movements suggestive of seizures were noticed soon after birth. Tonic convulsions at the age of 10 weeks led to admission. Urinary organic acid analysis using gas chromatography-mass spectrometry at 3 months of age revealed elevated concentrations of 3-hydroxyisovaleric acid (3HIVA) and 3-methylcrotonylglycine but normal levels of lactate, 3-hydroxypropionate and methylcitrate suggesting isolated MCC deficiency. This was confirmed by enzyme assays in lymphocytes and cultured skin fibroblasts: MCC activity was virtually undetectable whereas activities of propionyl-CoA and pyruvate carboxylases were within the normal range. A low protein (0.8-1.5 g/kg/day) diet supplemented with a leucine-free amino acid mixture resulted in a marked decrease of 3HIVA excretion. L-Carnitine and biotin administration had no effect on the clinical condition or metabolite excretion. Supplementation with glycine resulted in only a temporary fall of 3HIVA excretion and was therefore discontinued. L-Carnitine therapy was reintroduced later because of secondary carnitine deficiency. Compliance with treatment was poor until the age of 27 months resulting in a severe episode with seizures and coma. The general clinical condition of the patient was always good but his psychomotor development was delayed and seizures were not continuously under good control due to poor therapy compliance. The boy is now 10.5 years old and attending a school for children with learning handicaps. CONCLUSION: Isolated MCC deficiency of early-onset is a rare condition exhibiting a more severe clinical course than the later-onset form described in most other cases. The prognostic value of 3 HIVA measurements in CSF and serum should be evaluated in future cases.

Holocarboxylase synthetase deficiency: early diagnosis and management of a new case.

We present a new case of holocarboxylase synthetase (HCS) deficiency, a rare autosomal recessive metabolic disorder, causing the "early-onset" form of multiple carboxylase deficiency. The patient was born at term of healthy consanguineous parents after an uncomplicated pregnancy. On the 2nd day of life she refused oral feeding, became tachydyspnoeic and showed excessive weight loss. Laboratory studies showed metabolic acidosis, marked lactic acidaemia, hyperammonaemia and increased urinary excretion of 3-hydroxyisovaleric acid, 3-methylcrotonylglycine, 3-hydroxpropionic acid and methylcitric acid. Peritoneal dialysis combined with oral supplementation of biotin (10 mg/day) started on the 3rd day of life resulted in rapid clinical recovery and normalisation of biochemical parameters. HCS deficiency was established in lymphocytes and skin fibroblasts. The activities of all biotin-dependent carboxylases were severely decreased in fibroblasts grown in medium with moderate biotin concentration (10(-8) mol/l) but normal in a high biotin medium (10(-5) mol/l). Mitochondrial carboxylase activities in lymphocytes were 23%-29% of mean normal during therapy with 20 mg of biotin/day, with the higher dose of 40 mg/day they were within (3-methylcrotoryl-CoA carboxylase, pyruvate carboxylase) or slightly below (propionyl-CoA carboxylase) the normal range. At the age of 3 years the patient's physical and psychomotor development are normal. Early biotin supplementation should be considered in newborns with lactic acidosis and organoaciduria until a final diagnosis has been established. Furthermore, the required individual dose of biotin has to be carefully evaluated biochemically for the individual patient.

Intestinal absorption and renal excretion of biotin in patients with biotinidase deficiency.

We have investigated four patients from three unrelated families with typical clinical and biochemical features of "late-onset" multiple carboxylase deficiency. All patients suffered from biotinidase deficiency (plasma biotinidase activities 1.4%-3% of normal). Intestinal absorption of biotin, measured in three of the patients using a single load of 1.5 micrograms/kg, was found to be normal. Deficient activities of the mitochondrial biotin-dependent carboxylases in lymphocytes of one of these patients increased from 25% of mean basal control values to 33%-36% within 45 min and to 46%-47% within 2 h of the 1.5 micrograms/kg biotin load. After a high biotin load of 100 micrograms/kg, the values normalised within 45 min in all three patients studied. These results indicate normal cellular transport of biotin and normal holocarboxylase synthesis. After cessation of biotin supplementation, the plasma and urinary biotin in patients decreased to subnormal levels. In one patient, available for more detailed studies, both plasma and urinary biotin declined about twice as fast as in controls (apparent half-life 12-14 h in the patient and 26 h in controls). These results point to increased excretion of free biotin in our patient. Renal loss of biotin is one of the factors contributing to the high biotin requirement observed in patients with biotinidase deficiency.

Biotinidase deficiency: factors responsible for the increased biotin requirement.

Inability to recycle biotin from endogenous biocytin in congenital biotinidase deficiency is associated with increased requirement of exogenous free biotin. We have observed that severe biotin depletion with clinical and biochemical consequences occurs within 12 days after birth in a newborn patient and within 15-20 days after withdrawal of biotin supplementation in four other patients. Our studies have shown that: Urinary loss of biotin and biocytin are major causes for this rapid biotin depletion. Intestinal absorption of biotin seems to be normal at least at the loading dose of 1.5 micrograms/kg. At normal or subnormal plasma biotin concentrations biocytin is found in low concentrations (below 1 nmol l-1) in plasma of patients but at much higher concentrations in urine (100-600 nmol l-1). An oral load of biocytin results in patients in unchanged biotin levels but in a marked rise of biocytin in plasma followed by rapid renal excretion of biocytin whereas in controls biotin levels in plasma increase rapidly and biocytin remains below detection levels.

Biotinidase deficiency associated with renal loss of biocytin and biotin.

Clinical and biochemical investigations in six patients with congenital biotinidase deficiency are presented. The time course of biotin depletion in relation to carboxylase activities and clinical onset of symptoms was studied after withdrawal of biotin supplementation. Renal biotin clearance studies were performed in patients and controls. Renal loss of biocytin and biotin itself are shown to be a major cause for the increased biotin requirement in patients with congenital biotinidase deficiency.