003 1-399819 1/300 1-000 1$03.00/0 PEDIATRIC RESEARCH Vol. 30, No. 1, 1991 Copyright 0 199 1 International Pediatric Research Foundation, Inc. Printed in U.S.A. Pyruvate Carboxylase Deficiency: A Benign Variant with Normal Development R. N. VAN COSTER, P. M. FERNHOFF, D. C. DE VIVO Division of Pediatric Neurology, Columbia Presbyterian Medical Center, New York, New York 10032 [R.N. V.C., D.C.D. V.] and Division of Medical Genetics, Department of Pediatrics, Emory University School ofMedicine, Atlanta, Georgia 30322 [P.M.F.] ABSTRACT. The devastating nature of a pyruvate carbox- hyperammonemia (12-22). Essentially all these patients died ylase deficiency is underscored by the uniformly fatal within the first 4 mo of life. Eighteen patients had clinical outcome of the neonatal (French) type and the severely symptoms with lactic acidosis during infancy (North American disabling and ultimately fatal outcome of the infantile phenotype) (7-9, 18, 23-31). These patients had severe mental (North American) type. We report a 7-y-old girl with and motor developmental delay and death during infancy or metabolic and biochemical features of the North American early childhood. phenotype. Remarkably, the clinical course has been be- We have encountered a patient, now 6'12 years of age, with nign with preservations of motor and mental abilities. The severe PC deficiency documented in cultured skin fibroblasts residual enzyme activity in cultured skin fibroblast homog- who has had normal mental and motor development. To our enates was 1.8% and cross-reacting material was present knowledge, this child is the only patient with a "benign form" of in normal abundance and electrophoretic mobility. She has PC deficiency. had several episodes of metabolic acidosis with elevated lactate, pyruvate, alanine, 0-hydroxybutyrate, acetoace- CASE REPORT tate, lysine, and proline values, and undetectably low as- partate concentrations. These crises have been managed This 7-y-old girl is the only child of a nonconsanguineous by rehydration and bicarbonate therapy. We are unable to marriage. The full-term pregnancy, labor, and delivery were provide a satisfactory explanation for the uniquely benign uncomplicated. Birth weight was 4500 g. Growth and develop- clinical course that has been experienced by this patient. ment during the first year of life were normal. At 7 mo of age, (Pediatr Res 30: 1-4, 1991) she was hospitalized briefly for vomiting and dehydration, and was hospitalized again at 14 mo for evaluation of a 1-d history Abbreviations of vomiting and rapid respirations. Physical examination at that time revealed an acutely ill infant. Body weight and length were CRM, cross-reacting material above the 95th percentile, and the head circumference was 48 MW, molecular weight cm (90-95th percentile). Although afebrile, her respiratory rate PC, pyruvate carboxylase was 60/min. Auscultatory and radiographic findings were con- SSC, standard saline citrate sistent with bronchiolitis. Serum glucose was 14 mM, sodium TBS, Tris-buffered saline 132 mM, chloride 111 mM, bicarbonate 1.5 mM, and urea nitrogen 4.1 mM. Blood gases were 1.7 kPa (13 mm Hg) PCOZ and 17.3 kPa (130 mm Hg) PO,. The pH was 6.89 and the base excess -28.5 mM. Urinary pH was 5.0; large quantities of ketones were present. Treatment included oxygen, bronchodila- PC is a mitochondria1 matrix enzyme that catalyzes the ATP- tors, sodium bicarbonate, and a single dose of intramuscular dependent fixation of C02to pyruvate yielding oxaloacetate (1- insulin. She responded quickly to treatment and was discharged 3). This reaction is essential to the function of the Krebs cycle, on the 4th day of hospitalization. One wk later she was readmit- gluconeogenesis (4), and lipogenesis (5, 6). The enzyme is ex- ted for a metabolic evaluation. Serum chemistry profile was pressed in brain, liver, kidney, adrenal gland, lactating mammary normal, but the blood lactic acid value was 5.5 mM (normal 0.5 gland, fibroblasts, white blood cells, adipose, and other tissues to 2.2). Blood ammonia was normal. Plasma amino acid profile (5, 7-9). Low activities are found in skeletal muscle. Tissue- by anion exchange chromatography, however, revealed elevated specific isoenzymes are not known. The PC holoenzyme is a alanine [852 pM (normal 99 to 313)], lysine [I60 pM (normal tetramer composed of four identical monomers (014) each with a 45 to 144)], and proline [28 1 pM (normal 5 1 to 185)l. Citrulline MW of 125 kD (10). Partial cDNA clones corresponding to the was normal and aspartate was undetectably low. The 24-h urine 3' terminal of PC mRNA are available (6, 1I), and the gene is lactic acid content was 1.48 mg/mg creatinine (normal <0.1) as located on the long arm of chromosome 11 (6). analyzed by gas chromatography/mass spectrometry. Thirty-four patients with PC deficiency have been reported. The child was discharged but readmitted 7 d later with a 24-h Sixteen were severely ill in the neonatal period (French pheno- history of decreased appetite, restlessness, and unsteady gait. On type) with lactic acidosis, citrullinemia, hyperlysinemia, and admission she was afebrile, tachypneic (60/min), tachycardic (178/min), and ataxic. Serum glucose was 8 mM, bicarbonate Received July 27, 1990; accepted February 2 1, 199 1. Correspondence and reprint requests: Danyl C. De Vivo, M.D., Neurological 4.9 mM, lactic acid 7.3 mM, and pyruvic acid 0.306 mM (normal Institute. 710 West 168 Street. New York. NY 10032 <O. 15). She responded rapidly to i.v. fluids and bicarbonate, and ~uppbrtedby a grant frdm the Colleen Giblin Charitable Foundation for was diSchargedeafter2 d of h&pitalization. Pediatric Neurological Research. R.N.V.C. was supported by grants from the ~h~ was hospitalized again at 16, 25, 31, and 47 mo of National Foundation of Scientific Research of Belgium and from the Fulbright Foundation, Washington, DC. He currently is located at the University Hospital, age for acute episodes of lactic acidosis. Each episode was pre- Kinderkliniek "C. Hooft," De Pintelaan 185, 9000 Gent, Belgium. ceded by a brief illness and decreased appetite and responded 2 VAN COSTER ET AL. rapidly to i.v. fluids and bicarbonate therapy. Several milder NaCl (pH 7.0-7.5) for 30 min. Transfer proceeded for 20 h. The episodes were treated at home with oral bicarbonate therapy and filter was heated at 80°C for 2 h in vacuo. Prehybridization was increased fluids. At 47 mo of age, the child weighed 24.5 kg carried out in a solution containing 40% formamide, 0.6 M (>95th percentile) and her neurologic development was normal sodium chloride and 0.06 M sodium citrate (4 x SSC), 5 x except for a mild dysarthria. When last seen at 6'12 y of age, the Denhardt's solution, 0.5 mg/mL denatured salmon sperm DNA, child was attending the first grade and performing all age-appro- 0.2% SDS, and 0.1 M Tris-HC1 (pH 7.4) at 42°C for 6 h. The priate activities. She was doing well in school except for some filter was then hybridized overnight at 42°C in 40% formamide, difficulty with mathematics. She continued to be free of severe 4 x SSC, 1 x Denhardt's, 0.1 M Tris-HC1 (pH 7.4), 2.5 g/100 episodes of lactic acidosis, but was given supplemental oral fluids mL dextran sulfate, and lo6 cpm/lane of 32P-labeledPC probe and bicarbonate whenever she was febrile. She was a large child prepared from denatured double-stranded cDNA using the ran- whose height was 132 cm (>95th percentile) and who weighed dom priming method. The filter was washed in 4 x SSC, 0.1 % 42 kg (>95th percentile). Except for macrosomia, obesity, and SDS, then in 2 X SSC, 0.1 % SDS, and in 0.2 x SSC, 0.1 % SDS mild dysarthria, her physical and neurologic examinations were (at room temperature for 15 min). The filter was blot-dried and normal. hybridization signals were visualized by autoradiography using a Dupont Cronex Lightning Intensifying screen. Protein was de- termined by the Lowrv method (35). A cDNA robe. 1.1 kb~in length, corresponding to the 3" teiminal of PC ~RNAwas a MATERIALS AND METHODS generous gift of Svend Freytag (University of Michigan Medical Tissue culture supplies were purchased from Gibco Laborato- School, Ann Arbor, MI) (6). ries (Grand Island, NY). Laboratory reagents and chemicals were purchased from Sigma Chemical Company (St. Louis, MO), RESULTS SDS-polyacrylamide electrophoresis reagents and the streptavi- din-horseradish peroxidase conjugate from Bio-Rad (Rockville Mitochondria1 enzyme activities measured in cultured skin Center, NY), DNA polymerase and NADH from Boehringer- fibroblast homogenates of our patient and controls are shown in Mannheim Biochemicals (Indianapolis, IN), and 32P-nucleotides Table 1. PC residual activity in the patient's cells was only 1.8% from New England Nuclear (Wilmington, DE). of the control cell activity. The activities of other mitochondrial Fibroblasts were cultured from biopsied skin specimens and enzymes were normal. trypsinized as described previously (32). The cell pellet was The electrophoretic profile of biotin-containing mitochondrial resuspended in nine volumes of homogenizing buffer (0.25 M proteins is shown in Figure 1. Three discernible bands were sucrose, 10 mM Tris-HC1, pH 7.4, and 2 mM EDTA). The visualized in the control cells and PC-deficient cell lines. The suspension was homogenized in a glass tube with a Teflon pestle band with the highest MW represents the biotin-containing for 10 passes at 300 rpm. Triton X-100 was added to a 0.01% subunit of PC (MW = 125 kD); the doublet corresponds to the homogenate, which was allowed to sit on ice for 30 min before a-subunits of P-methyl-crotonyl CoA carboxylase (MW = 75 assay.