James Pitt

Successful treatment of molybdenum cofactor deficiency type A with cPMP

Molybdenum cofactor deficiency (MoCD) is a rare metabolic disorder characterized by severe and rapidly progressive neurologic damage caused by the functional loss of sulfite oxidase, 1 of 4 molybdenum-dependent enzymes. To date, no effective therapy is available for MoCD, and death in early infancy has been the usual outcome. We report here the case of a patient who was diagnosed with MoCD at the age of 6 days. Substitution therapy with purified cyclic pyranopterin monophosphate (cPMP) was started on day 36 by daily intravenous administration of 80 to 160 μg of cPMP/kg of body weight. Within 1 to 2 weeks, all urinary markers of sulfite oxidase (sulfite, S-sulfocysteine, thiosulfate) and xanthine oxidase deficiency (xanthine, uric acid) returned to almost normal readings and stayed constant (>450 days of treatment). Clinically, the infant became more alert, convulsions and twitching disappeared within the first 2 weeks, and an electroencephalogram showed the return of rhythmic elements and markedly reduced epileptiform discharges. Substitution of cPMP represents the first causative therapy available for patients with MoCD. We demonstrate efficient uptake of cPMP and restoration of molybdenum cofactor–dependent enzyme activities. Further neurodegeneration by toxic metabolites was stopped in the reported patient. We also demonstrated the feasibility to detect MoCD in newborn-screening cards to enable early diagnosis.

Expanded newborn screening: Outcome in screened and unscreened patients at age 6 years

OBJECTIVE: Tandem mass spectrometry is widely applied to routine newborn screening but there are no long-term studies of outcome. We studied the clinical outcome at six years of age in Australia. METHODS: In a cohort study, we analyzed the outcome at 6 years for patients detected by screening or by clinical diagnosis among >2 million infants born from 1994 to 1998 (1 017 800, all unscreened) and 1998 to 2002 (461 500 screened, 533 400 unscreened) recording intellectual and physical condition, school placement, other medical problems, growth, treatment, diet, and hospital admissions. Results were analyzed separately for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and other disorders, and grouped patients as those who presented clinically or died in the first 5 days of life; patients presented later or diagnosed by screening, and those with substantially benign disorders. RESULTS: Inborn errors, excluding phenylketonuria, were diagnosed in 116 of 1 551 200 unscreened infants (7.5/100 000 births) and 70 of 461 500 screened infants (15.2/100 000 births). Excluding MCADD, 21 unscreened patients with metabolic disorders diagnosed after 5 days of life died or had a significant intellectual or physical handicap (1.35/100 000 population) compared with 2 of the screened cohort (0.43/100 000; odds ratio: 3.1 [95% CI: 0.73–13.32]). Considering the likely morbidity or mortality among the expected number of never-diagnosed unscreened patients, there would be a significant difference. Growth distribution was normal in all cohorts. CONCLUSION: Screening by tandem mass spectrometry provides a better outcome for patients at 6 years of age, with fewer deaths and fewer clinically significant disabilities.

Metabolic profiling of infant urine using comprehensive two-dimensional gas chromatography: Application to the diagnosis of organic acidurias and biomarker discovery

Comprehensive two-dimensional gas chromatography (GCxGC) time-of-flight mass spectrometry (ToFMS) was applied to the analysis of urinary organic acids from patients with inborn errors of metabolism. Abnormal profiles were obtained from all five patients studied. Methylmalonic academia and deficiencies of 3-methylcrotonyl-CoA carboxylase and medium chain acyl-CoA dehydrogenase gave diagnostic profiles while deficiencies of very long chain acyl-CoA dehydrogenase and mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase gave profiles with significant increases in dicarboxylic acids suggestive of these disorders. The superior resolving power of GCxGC with ToFMS detection was useful in separating isomeric organic acids that were not resolved using one-dimensional GC. A novel urinary metabolite, crotonyl glycine, was also discovered in the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase sample which may be a useful specific diagnostic marker for this disorder. The quantitative aspects of GCxGC were investigated using stable isotope dilution analyses of glutaric, glyceric, orotic, 4-hydroxybutyric acids and 3-methylcrotonylglycine. Correlation coefficients for linear calibrations of the analytes ranged from 0.9805 to 0.9993 (R(2)) and analytical recoveries from 77% to 99%. This study illustrates the potential of GCxGC-ToFMS for the diagnosis of organic acidurias and detailed analysis of the complex profiles that are often associated with these disorders.

A knock-out mouse model for methylmalonic aciduria resulting in neonatal lethality.

Methylmalonic aciduria is a human autosomal recessive disorder of organic acid metabolism resulting from a functional defect in the activity of the enzyme methylmalonyl-CoA mutase. Based upon the homology of the human mutase locus with the mouse locus, we have chosen to disrupt the mouse mutase locus within the critical CoA binding domain using gene-targeting techniques to create a mouse model of methylmalonic aciduria. The phenotype of homozygous knock-out mice (mut-/-) is one of early neonatal lethality. Mice appear phenotypically normal at birth and are indistinguishable from littermates. By 15 h of age, they develop reduced movement and suckle less. This is followed by the development of abnormal breathing, and all of the mice with a null phenotype die by 24 h of age. Urinary levels of methylmalonic and methylcitric acids are grossly increased. Measurement of acylcarnitines in blood shows elevation of propionylcarnitine with no change in the levels of acetylcarnitine and free carnitine. Incorporation of [14C]propionate in primary fibroblast cultures from mut-/- mice is reduced to approximately 6% of normal level, whereas there is no detectable synthesis of mut mRNA in the liver. This is the first mouse model that recapitulates the key phenotypic features of mut0 methylmalonic aciduria.

Characterization of mutations in 22 females with X-linked dominant chondrodysplasia punctata (Happle syndrome)

Purpose: Human X-linked dominant chondrodysplasia punctata (CDPX2) or Happle syndrome is associated with mutations in the human emopamil binding protein (EBP), a Δ8-Δ7-sterol isomerase involved in cholesterol biosynthesis. The purpose of the current study was to determine the spectrum of EBP mutations in females with CDPX2 and the utility of biochemical screening for the disorder by analysis of plasma sterols.Methods: Genomic sequencing of the coding exons of the human Δ8-Δ7-sterol isomerase gene was performed on DNA from 26 females with suspected X-linked dominant chondrodysplasia punctata. Clinical data and sterol analyses were obtained for 24 and 23 of the patients, respectively.Results: Mutations in the human EBP Δ8-Δ7-sterol isomerase gene were found in 22 (85%) of 26 females studied, including 20 (91%) of 22 patients who demonstrated an abnormal sterol profile. Thirteen of the mutations have not been reported previously. All of the females in whom mutations were found demonstrated typical skin manifestations of CDPX2, and all but one had a skeletal dysplasia.Conclusions: Plasma sterol analysis was a highly specific and sensitive indicator of the presence of an EBP mutation in females with suspected CDPX2, including a clinically unaffected mother of a sporadic case. No clear genotype/phenotype correlations were ascertained, probably because phenotypic expression is influenced substantially by the pattern of X-inactivation in an affected female.

MATRIX-ASSISTED LASER DESORPTION/IONIZATION TIME-OF-FLIGHT MASS SPECTROMETRY OF SIALYLATED GLYCOPEPTIDES AND PROTEINS USING 2,6-DIHYDROXYACETOPHENONE AS A MATRIX

A comparison has been performed between 2,6-dihydroxyacetophenone (DHAP) containing di-ammonium hydrogen citrate (DAHC), dihydroxybenzoic acid and α-cyano-4-hydroxycinnamic acid as matrices for matrix-assisted laser desorption/ionization (MALDI) of sialylated glycopeptides. DHAP/DAHC was found to yield distinct protonated molecules of large sialylated fetuin tryptic peptides by MALDI-linear time-of-flight analyses. By comparison, the other two matrices produced substantially broadened molecular ion envelopes and indications of considerable in-source and post-source fragmentation. Reflector mode analysis revealed protonated molecules for these peptides in addition to discrete successive post-source fragmentation of sialic acid residues using DHAP/DAHC. The other two matrices produced a much greater degree of fragmentation of sialic acids and failed to reveal the molecular ion in reflector mode. DHAP was also found to be of equivalent efficacy to sinapinic acid for linear mode analysis of bovine serum albumin.

Trimethylaminuria, fish odour syndrome: a new method of detection and response to treatment with metronidazole

SummaryTrimethylaminuria is an autosomal recessive disorder involving deficientN-oxidation of the dietary-derived amine trimethylamine (TMA). TMA, a volatile tertiary amine, accumulates and is excreted in urine of patients with deficient TMA oxidase activity. Treatment strategies for this condition are limited. We report a new stable-isotope dilution method for rapid sequential analysis of TMA concentrations and the clinical and biochemical response to treatment with metronidazole.

ECHS1 mutations in Leigh disease: a new inborn error of metabolism affecting valine metabolism

Two siblings with fatal Leigh disease had increased excretion of S-(2-carboxypropyl)cysteine and several other metabolites that are features of 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency, a rare defect in the valine catabolic pathway associated with Leigh-like disease. However, this diagnosis was excluded by HIBCH sequencing and normal enzyme activity. In contrast to HIBCH deficiency, the excretion of 3-hydroxyisobutyryl-carnitine was normal in the children, suggesting deficiency of short-chain enoyl-CoA hydratase (ECHS1 gene). This mitochondrial enzyme is active in several metabolic pathways involving fatty acids and amino acids, including valine, and is immediately upstream of HIBCH in the valine pathway. Both children were compound heterozygous for a c.473C > A (p.A158D) missense mutation and a c.414+3G>C splicing mutation in ECHS1. ECHS1 activity was markedly decreased in cultured fibroblasts from both siblings, ECHS1 protein was undetectable by immunoblot analysis and transfection of patient cells with wild-type ECHS1 rescued ECHS1 activity. The highly reactive metabolites methacrylyl-CoA and acryloyl-CoA accumulate in deficiencies of both ECHS1 and HIBCH and are probably responsible for the brain pathology in both disorders. Deficiency of ECHS1 or HIBCH should be considered in children with Leigh disease. Urine metabolite testing can detect and distinguish between these two disorders.

Mitochondrial Carbonic Anhydrase VA Deficiency Resulting from CA5A Alterations Presents with Hyperammonemia in Early Childhood

Four children in three unrelated families (one consanguineous) presented with lethargy, hyperlactatemia, and hyperammonemia of unexplained origin during the neonatal period and early childhood. We identified and validated three different CA5A alterations, including a homozygous missense mutation (c.697T>C) in two siblings, a homozygous splice site mutation (c.555G>A) leading to skipping of exon 4, and a homozygous 4 kb deletion of exon 6. The deleterious nature of the homozygous mutation c.697T>C (p.Ser233Pro) was demonstrated by reduced enzymatic activity and increased temperature sensitivity. Carbonic anhydrase VA (CA-VA) was absent in liver in the child with the homozygous exon 6 deletion. The metabolite profiles in the affected individuals fit CA-VA deficiency, showing evidence of impaired provision of bicarbonate to the four enzymes that participate in key pathways in intermediary metabolism: carbamoylphosphate synthetase 1 (urea cycle), pyruvate carboxylase (anaplerosis, gluconeogenesis), propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase (branched chain amino acids catabolism). In the three children who were administered carglumic acid, hyperammonemia resolved. CA-VA deficiency should therefore be added to urea cycle defects, organic acidurias, and pyruvate carboxylase deficiency as a treatable condition in the differential diagnosis of hyperammonemia in the neonate and young child.

Atypical pyroglutamic aciduria: possible role of paracetamol

Batshaw, M. L. and Brusilow, S. W. Valproate-induced hyperammonemia. Ann. Neurol. 11 (1982) 319-321 Brusilow, S. W. Inborn errors of ureagenesis. In Scriver, C. and Lloyd J. (eds.), Genetic and Metabolic Disease in Pediatrics, Butterworth, Stoneham, Mass., 1985, pp. 140-165 Coude, F. X., Sweetman, L. and Nyhan, W. L. Inhibition of propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. J. Clin. Invest. 64 (1979) 1544-1551