Structural insights on pathogenic effects of novel mutations causing pyruvate carboxylase deficiency.

Pyruvate carboxylase (PC), a key enzyme for gluconeogenesis and anaplerotic pathways, consists of four domains, namely, biotin carboxylase (BC), carboxyltransferase (CT), pyruvate carboxylase tetramerization (PT), and biotin carboxyl carrier protein (BCCP). PC deficiency is a rare metabolic disorder inherited in an autosomal recessive way. The most severe form (form B) is characterized by neonatal lethal lactic acidosis, whereas patients with form A suffer chronic lactic acidosis with psychomotor retardation. Diagnosis of PC deficiency relies on enzymatic assay and identification of the PC gene mutations. To date, six mutations of the PC gene have been identified. We report nine novel mutations of the PC gene, in five unrelated patients: three being affected with form B, and the others with form A. Three of them were frameshift mutations predicted to introduce a premature termination codon, the remaining ones being five nucleotide substitutions and one in frame deletion. Impact of these mutations on mRNA was assessed by RT-PCR. Evidence for a deleterious effect of the missense mutations was achieved using protein alignments and three-dimensional structural prediction, thanks to our modeling of the human PC structure. Altogether, our data and those previously reported indicate that form B is consistently associated with at least one truncating mutation, mostly lying in CT (C-terminal part) or BCCP domains, whereas form A always results from association of two missense mutations located in BC or CT (N-terminal part) domains. Finally, although most PC mutations are suggested to interfere with biotin metabolism, none of the PC-deficient patients was biotin-responsive.

The molecular basis of pyruvate carboxylase deficiency: mosaicism correlates with prolonged survival.

Pyruvate carboxylase (PC) deficiency (OMIM, 266150) is a rare autosomal recessive disease. The revised PC gene structure described in this report consists of 20 coding exons and four non-coding exons at the 5'-untranslated region (5'-UTR). The gene codes for three transcripts due to alternative splicing: variant 1 (NM_000920.3), variant 2 (NM_022172.2) and variant 3 (BC011617.2). PC deficiency is manifested by three clinical phenotypes-an infantile form (Type A), a neonatal form (Type B), and a benign form (Type C). We report the molecular basis for eight cases (one Type A, five Type B and two Type C) of PC deficiency. Eight novel complex mutations were identified representing different combinations of missense mutations, deletions, a splice site substitution and a nonsense mutation. The classical phenotypes (A, B and C) correlated poorly with clinical outcomes. Mosaicism was found in five cases (one Type A, three Type B and one Type C) and four of these cases had prolonged survival. Death in the fifth case resulted from unrelated medical complications. The discrepancy between the current findings and the existing classification system should be addressed to accommodate these new observations.

A case of pyruvate carboxylase deficiency with atypical clinical and neuroradiological presentation.

Pyruvate carboxylase (PC) is a key enzyme for gluconeogenesis and anaplerotic pathways in brain. PC deficiency is a rare autosomal recessive neurometabolic disorder with three described characteristic presentations. We report a patient with atypical clinical and neuroradiological aspects. He survived from neonatal lactic acidemia and is alive at 9 years of age with a mild developmental delay. A brain MRI performed by the age of 18 months disclosed an unusual subcortical leucodystrophic process.

Intron retention and frameshift mutations result in severe pyruvate carboxylase deficiency in two male siblings.

This paper describes the molecular characterization of two male siblings displaying the complex (Type B) form of pyruvate carboxylase (PC) deficiency in which severe neonatal lactic acidosis and redox abnormalities results in death within the first few weeks of life. The two male siblings were found to be compound heterozygous for a TAGG deletion at the exon15/intron15 splice site (IVS15+2-5delTAGG) and a dinucleotide deletion in exon 16 (2491-2492delGT) of the PC gene. We also demonstrate through RT-PCR and sequencing of aberrant transcripts that the IVS15+2-5delTAGG results in the retention of intron 15 during pre-mRNA splicing. In addition, both deletions are predicted to result in a frameshift to generate a premature termination codon such that the encoded mRNA could be subject to nonsense mediated decay.

Five patients with a biotin-responsive defect in holocarboxylase formation: evaluation of responsiveness to biotin therapy in vivo and comparative biochemical studies in vitro.

Biochemical studies in five patients with a defect in biotin-responsive holocarboxylase synthesis are reported. The age of onset (2 d to 6 y) as well as the severity of illness varied considerably. In all patients diagnosis was established by the finding of organic aciduria typical for multiple carboxylase deficiency in a catabolic state. In four patients the response to biotin therapy was evaluated by measurement of mitochondrial carboxylase activities in lymphocytes and by monitoring urinary organic acid excretion. In three patients clinical symptoms disappeared with 10-20 mg biotin/d, whereas normalization of the biochemical parameters required higher doses (20-40 mg/d). The fourth patient required a dose of 100 mg biotin/d before her skin rash disappeared. She remains mentally retarded and shows slightly elevated urinary organic acid excretion. Carboxylase activities were clearly deficient in fibroblasts grown in the commonly used medium which contains 10 nmol/L biotin (contributed by FCS in medium) in two patients. Fibroblasts of the other three patients became deficient only in a low biotin medium (0.1 nmol/L). Reactivation of deficient carboxylase activities in relation to time and biotin concentration correlated well with the severity and age of onset of illness in four patients. In one patient, however, carboxylase reactivation followed a more complex pattern requiring the longest incubation time but only a moderately increased biotin concentration of 19 nmol/L compared with 3-5 nmol/L in normal cells and 34-4000 nmol/L in the other four patients. The results in the five patients are in accordance with a primary defect of holocarboxylase synthetase due to a decreased affinity for biotin, in one patient combined with a decreased Vmax.

Prolonged survival in pyruvate carboxylase deficiency: lack of correlation with enzyme activity in cultured fibroblasts.

OBJECTIVE: To report the clinical history and laboratory evaluation of a patient presenting with lactic acidosis secondary to pyruvate carboxylase deficiency. METHODS AND RESULTS: Enzyme analysis of cultured skin fibroblasts revealed 2-5% of normal pyruvate carboxylase activity. Although most patients with this condition die in early infancy, this child has survived to age 8-1/2 years, with only occasional episodes of metabolic acidosis, usually responding rapidly to intravenous hydration and bicarbonate. Despite having a seizure disorder and moderate mental retardation, he continues to thrive and make progress in his acquisition of motor and language skills. Of the 35 patients described in the literature with pyruvate carboxylase deficiency, only two other patients have lived beyond 5 years of age. CONCLUSION: There does not seem to be a correlation of prolonged survival with residual pyruvate carboxylase activity on assay of cultured fibroblasts. Possible explanations for this patient's prolonged survival include tissue heterogeneity, increased residual enzyme activity in vivo, or partial stabilization of the enzyme by supplemental biotin.

Leigh encephalopathy: histologic and biochemical analyses of muscle biopsies.

To elucidate the pathogenesis of Leigh encephalopathy, histologic, biochemical, and mitochondrial DNA analyses were performed on biopsied muscles from 33 patients with the clinical characteristics of this disorder. On muscle histochemistry, cytochrome c oxidase activity was decreased or absent in 7 patients (21%), although none had ragged-red fibers. In 2 patients with cytochrome c oxidase deficiency, staining for this enzyme was poor in the muscle fibers and fibroblasts but was normal in the arterial wall, indicating tissue-specific involvement. Ten patients (30%) had biochemical defects, including 2 with pyruvate dehydrogenase complex, 4 with cytochrome c oxidase, 1 with NADH-cytochrome c reductase (complex I), and 3 with multiple complex deficiencies. None of the 28 patients in whom muscle mitochondrial (mt)DNA was analyzed had DNA deletions or point mutation at nucleotide positions 3,243 or 8,344. These results indicate that the underlying defect in Leigh encephalopathy is heterogeneous because only 30% of patients had enzyme defects demonstrable in muscle biopsy material.