Frequent Inborn Errors Of Metabolism Research Articles

Structure-Based Analysis of Five Novel Disease-Causing Mutations in 21-Hydroxylase-Deficient Patients

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most frequent inborn error of metabolism, and accounts for 90–95% of CAH cases. The affected enzyme, P450C21, is encoded by the CYP21A2 gene, located together with a 98% nucleotide sequence identity CYP21A1P pseudogene, on chromosome 6p21.3. Even though most patients carry CYP21A1P-derived mutations, an increasing number of novel and rare mutations in disease causing alleles were found in the last years. In the present work, we describe five CYP21A2 novel mutations, p.R132C, p.149C, p.M283V, p.E431K and a frameshift g.2511_2512delGG, in four non-classical and one salt wasting patients from Argentina. All novel point mutations are located in CYP21 protein residues that are conserved throughout mammalian species, and none of them were found in control individuals. The putative pathogenic mechanisms of the novel variants were analyzed in silico. A three-dimensional CYP21 structure was generated by homology modeling and the protein design algorithm FoldX was used to calculate changes in stability of CYP21A2 protein. Our analysis revealed changes in protein stability or in the surface charge of the mutant enzymes, which could be related to the clinical manifestation found in patients.

Evidence for Physical Association of Mitochondrial Fatty Acid Oxidation and Oxidative Phosphorylation Complexes

Fatty acid β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in cellular energetics. Reducing equivalents from FAO enter OXPHOS at the level of complexes I and III. Genetic disorders of FAO and OXPHOS are among the most frequent inborn errors of metabolism. Patients with deficiencies of either FAO or OXPHOS often show clinical and/or biochemical findings indicative of a disorder of the other pathway. In this study, the physical and functional interactions between these pathways were examined. Extracts of isolated rat liver mitochondria were subjected to blue native polyacrylamide gel electrophoresis (BNGE) to separate OXPHOS complexes and supercomplexes followed by Western blotting using antisera to various FAO enzymes. Extracts were also subjected to sucrose density centrifugation and fractions analyzed by BNGE or enzymatic assays. Several FAO enzymes co-migrated with OXPHOS supercomplexes in different patterns in the gels. When palmitoyl-CoA was added to the sucrose gradient fractions containing OXPHOS supercomplexes in the presence of potassium cyanide, cytochrome c was reduced. Cytochrome c reduction was completely blocked by myxothiazol (a complex III inhibitor) and 3-mercaptopropionate (an inhibitor of the first step of FAO), but was only partially inhibited by rotenone (a complex I inhibitor). Although palmitoyl-CoA and octanoyl-CoA provided reducing equivalents to OXPHOS-containing supercomplex fractions, no accumulation of their intermediates was detected. In contrast, short branched acyl-CoA substrates were not metabolized by OXPHOS-containing supercomplex fractions. These data provide evidence of a multifunctional FAO complex within mitochondria that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling.

Prenatal diagnosis of propionic acidemia by measuring methylcitric acid in dried amniotic fluid on filter paper using GC/MS

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.

Inhibition of energy metabolism in cerebral cortex of young rats by the medium-chain fatty acids accumulating in MCAD deficiency

Patients affected by medium-chain acyl CoA dehydrogenase (MCAD) deficiency, a frequent inborn error of metabolism, suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. In the present study, we investigated the in vitro effect of the medium-chain fatty acids (MCFA), at concentrations varying from 0.01 to 3 mM, accumulating in MCAD deficiency on some parameters of energy metabolism in cerebral cortex of young rats. 14CO 2 production from [U 14] glucose, [1- 14C] acetate and [1,5- 14C] citrate was evaluated by incubating cerebral cortex homogenates from 30-day-old rats in the absence (controls) or presence of octanoic acid, decanoic acid or cis-4-decenoic acid. OA and DA significantly reduced 14CO 2 production from acetate by around 30–40%, and from glucose by around 70%. DA significantly reduced 14CO 2 production from citrate by around 40%, while OA did not affect this parameter. cDA inhibited 14CO 2 production from all tested substrates by around 30–40%. The activities of the respiratory chain complexes and of creatine kinase were also tested in the presence of DA and cDA. Both metabolites significantly inhibited cytochrome c oxidase activity (by 30%) and complex II–III activity (DA, 25%; cDA, 80%). Furthermore, only cDA inhibited complex II activity (by 30%), while complex I–III and citrate synthase were not affected by these MCFA. On the other hand, only cDA reduced the activity of creatine kinase in total homogenates, as well as in mitochondrial and cytosolic fractions from cerebral cortex (by 50%). The data suggest that the major metabolites which accumulate in MCAD deficiency, with particular emphasis to cDA, compromise brain energy metabolism. We presume that these findings may contribute to the understanding of the pathophysiology of the neurological dysfunction of MCAD deficient patients.

MAOB: a modifier gene in phenylketonuria?

Phenylketonuria (PKU), the most frequent inborn error of metabolism (1/15,000 live births), is an autosomal recessive condition caused by phenylalanine hydroxylase deficiency. Despite early and strict dietary control, some PKU children still exhibit behavioral and cognitive difficulties suggestive of a partly prenatal brain injury. The reported variability between the cognitive and clinical phenotypes within the same family raises the question of modifying genes in PKU. We suggest here that monoamine oxidase type B, MAOB, an enzyme degrading phenylethylamine, a very toxic metabolite of phenylalanine, could act as a modifying gene since a variant enzymatic activity of MAOB in PKU patients with similar phenylalanine levels would result in different phenylethylamine levels and different clinical outcomes. Finally the report of low MAOB, and consequently expectedly high phenylethylamine levels in neonates is consistent with a phenylethylamine-mediated brain injury possibly causing irreversible damages in PKU newborns prior to onset of the low protein diet.

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography–mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography–mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC–MS measurement and provides final results within 1.5 h.

Neurodevelopmental outcome of long-term therapy of urea cycle disorders in Japan.

In Japan, urea cycle disorders (UCDs) are one of the most frequent inborn errors of metabolism, estimated to have a prevalence of 1 per 50,000 live births. In an attempt to develop more effective treatment and enhance the quality of life, we investigated the clinical manifestations and prognosis of 216 patients with UCDs diagnosed and treated between 1978 and 1995. These included 92 cases of neonatal-onset UCD and 116 of late-onset UCD. Two cases of ornithine transcarbamylase (OTC) deficiency in males and 2 cases of argininosuccinase (AL) deficiency were diagnosed prospectively. By far the most common disorder was OTC deficiency, accounting for 2/3 of all cases. At the end of 1995, the 5-year survival rate was 22% for the neonatal-onset type and 41% for the late-onset type. Among the 20 long-term survivors with neonatal-onset UCD, 18 (90%) had moderate to severe neurodevelopmental deficits; this contrasts with 13 of 47 (28%) survivors with the late-onset type. In analysing 108 UCD cases, peak blood ammonia level during the first hyperammonaemic attack was correlated with neurodevelopmental outcome. When the concentration of blood ammonia was less than 180 mumol/L (5 times normal), there was no severe neurological damage. When the concentration of blood ammonia exceeded 350 mumol/L (10 times normal) at the first hyperammonaemic attack, the patients died or had severe neurological deficits. Our data point to the importance of early diagnosis and aggressive treatment.

Metabolic pigmentary retinopathies: Diagnosis and therapeutic attempts

Retinal degeneration in children occurs in errors of lipid, peroxisomal and mitochondrial (including respiratory chain) metabolism. In this review the most frequent inborn errors of metabolism with retinal degeneration are discussed including abetalipoproteinaemia, classical Refsum disease, neuronal ceroid lipofuscinosis, hydroxydicarboxylic aciduria, Sjögren-Larsson syndrome, infantile Refsum disease, Kearns-Sayre syndrome and gyrate atrophy. These metabolic disorders must be differentiated from those with retinal degeneration but without known metabolic basis. In patients with such a disorder metabolic investigations should be considered whenever atypical manifestations are encountered.