Complete deletion of ornithine transcarbamylase gene confirmed by CGH array of X chromosome.

Ornithine transcarbamylase deficiency is an X-linked semidominant trait that is the most frequent inborn error of the urea cycle. Three hundred and fifty different mutations, including mostly point mutations and a small proportion of large rearrangements have been reported. Conventional molecular diagnosis is highly reliable for point mutations but can miss gross rearrangements. We describe a contiguous gene syndrome involving the RPGR, OTC and TM4SF2 genes in a male patient with severe neonatal OTC deficiency identified by the conventional molecular approach. Molecular characterization was ascertained by X chromosome CGH array and confirmed by MLPA. Complete deletion of the OTC gene led to absent OTC enzymatic activity in liver and to a severe clinical phenotype. The maternal phenotype, characterized by less severe hyperammonaemic crises associated with neurological impairment would result from a deficient but not null OTC activity due to random X chromosome inactivation in the liver. Our cases are similar toothers described presenting with OTC deficient phenotype in which OTC and contiguous genes are affected. Clinical expression would be conditioned by complete OTC deficiency in males and by X chromosome inactivation in females, leading to compensation by the normal allele in tissues such as blood or muscle but not sufficiently in liver. The application of high-resolution genetic techniques allows the characterization of causative mutations such as large deletions in order to guide genetic counselling and prenatal diagnosis.

Estimation of the total number of disease-causing mutations in ornithine transcarbamylase (OTC) deficiency. Value of the OTC structure in predicting a mutation pathogenic potential.

Ornithine transcarbamylase deficiency (OTCD), the X-linked, most frequent urea cycle error, results from mutations in the OTC gene, encoding a 354-residue polypeptide. To date 341 OTCD clinical mutations, including 222 missense single nucleotide changes (mSNCs), have been compiled (Hum Mutat 2006;27:626). OTCD mutation detection might be simplified if the entire repertoire of OTCD-causing mutations were known. We estimate the size of this repertoire from 23 new OTCD patients exhibiting 22 different mutations, of which 9, including 4 mSNCs, are novel. The complete repertoire of OTCD-causing mutations is estimated as 560 mutations (95% confidence interval, 422-833 mutations), including 290 mSNCs (95% confidence interval, 230-394 mSNCs). Thus, OTCD diagnosis based on the screening for known mutations might attain 90% sensitivity in <5 years. Since disease-causing mSNCs represent <20% of the 2064 possible OTC mSNCs, simple approaches are essential for discrimination between causative and trivial mSNCs. Observation of the OTC structure appears a simple approach for such discrimination, comparing favourably in our sample with three formalized structure-based and/or sequence-based in silico assessment methods, and supporting the causation of complete deficiency by the mutations p.Pro305Arg and p.Ser96Phe, and of partial deficiency by p.Asp41Gly, p.Glu122Gly, p.Leu179Phe, p.Pro220Thr and p.Glu273del. Five non-mSNC novel mutations (p.Gly71X, a 7-nucleotide and a 10-nucleotide duplication and deletion in exon 5, G>A transitions at bases +1 and +5 of introns 4 and 9, respectively) are obviously pathogenic. The previously reported mSNCs p.Arg26Gln, p.Arg40His, p.Glu52Lys, pLys88Asn, p.Arg129His, p.Asn161Ser, p.Thr178Met, p.His202Tyr, p.Ala208Thr and p.His302Arg, found in our cohort, are also discussed.

Plasma urea-cycle-related amino acids, ammonium levels, and urinary orotic acid excretion in short-bowel patients managed with an oral diet.

BACKGROUND AND AIMS: The small intestine contains several enzymes involved in arginine synthesis and converts glutamine to citrulline, the major compound for endogenous arginine synthesis. This study was conducted to assess the plasma status of urea-cycle intermediates and orotic urinary excretion in short-bowel patients. METHODS: Thirteen stable short-bowel syndrome patients (7 men; 60.2+/-15.2 years) were studied. Patients were divided into moderately resected (Group A; n=6) and severely resected (Group B; n=7) according to their remnant bowel length (Group A: 61-150 cm; Group B: < or =60 cm). All subjects were consuming an oral diet plus dietetic supplements. Plasma urea-cycle amino acids, ammonium and urinary orotic acid were determined. RESULTS: Plasma glutamine levels were significantly higher in both patient groups than in the control group (P<0.001). Regarding citrulline, Group B levels were significantly lower vs. controls (P<0.001). Comparisons between patient groups showed higher arginine in Group A (P<0.05) and non-statistically lower citrulline in Group B. Blood ammonium and orotic urinary excretion were normal. CONCLUSIONS: Although plasma citrulline and glutamine alterations were found, patients showed no hyperammonemia or orotic aciduria, which suggests a certain degree of adaptation in arginine and related amino acid metabolism, when an adequate dietary supply of arginine is provided.

Splicing mutations, mainly IVS6-1(G>T), account for 70% of fumarylacetoacetate hydrolase (FAH) gene alterations, including 7 novel mutations, in a survey of 29 tyrosinemia type I patients.

Hereditary tyrosinemia type I (HTI) is an autosomal recessive disease characterized by a deficiency in fumarylacetoacetate hydrolase (FAH) activity. In this work, the FAH genotype was established in a group of 29 HTI patients, most of them from the Mediterranean area. We identified seven novel mutations-IVS8-1(G>A, IVS10-2(A>T), 938delC, E6/I6del26, W78X, Q328X, and G343W-and two previously described mutations-IVS6-1(G>T) and IVS12+5(G>A). Fully 92.8% of the patients were carriers of at least one splice site mutation, with IVS6-1(G>T) accounting for 58.9% of the total number of alleles. The splice mutation group of patients showed heterogeneous phenotypic patterns ranging from acute forms with severe liver malfunction to chronic forms with renal manifestations and slow progressive hepatic alterations. Qualitative FAH cDNA expression was the same in all IVS6-1(G>T) homozygous patients regardless of their clinical picture. One patient with a heterozygous combination of a nonsense (Q328X) and a frameshift (938delC) mutation showed an atypical clinical picture of hypotonia and repeated infections. Despite the high prevalence of IVS12+5(G>A) in the northwestern European population, we found only two patients with this mutation in our group.

Influence of dose and age on the response of the allopurinol test for ornithine carbamoyltransferase deficiency in control infants.

Measurement of urinary orotidine and orotic acid after an oral allopurinol challenge is an important diagnostic test for ornithine carbamoyltransferase deficiency that is sometimes used in infants (< 1 year of age), although there is little information on normal test results in this age group. We found higher orotidine excretion in normal infants than in older children given the test, whereas orotate excretion was similar in both groups. The increased orotidine excretion appears to be due to the use in the infants of higher allopurinol doses per kilogram of body weight than in the children. The normalized-dose dependency of the orotidine response extends even to adult age. Thus, dose-normalized responses should be used in the test and there is no need for careful age-matching of the controls.

Optimization of allopurinol challenge: sample purification, protein intake control, and the use of orotidine response as a discriminative variable improve performance of the test for diagnosing ornithine carbamoyltransferase deficiency.

BACKGROUND: The diagnosis of heterozygosity for X-linked ornithine carbamoyltransferase (OCT) deficiency has usually been based on measurement of the increase of orotate and orotidine excretion after an allopurinol load. We examined the choices of analyte, cutoff, and test conditions to obtain maximal test accuracy. METHODS: Urine orotate/orotidine responses to allopurinol load in 37 children (13 OCT-deficient and 24 non-OCT-deficient) and 24 women (7 at risk for carrier status and 17 not related to OCT-deficient children) were analyzed by liquid chromatography after sample purification by anion-exchange chromatography. Diagnostic accuracy was evaluated by nonparametric ROC curves. RESULTS: Sample purification was necessary to prevent interferences. Orotate and orotidine excretion increased with increased protein intake during the test. At a cutoff of 8 mmol orotidine/mol creatinine, sensitivity was 1.0 and specificity was 0. 92 in mild forms of OCT deficiency. Results in monoplex carrier women may differ greatly from those expected because of the genetics of this deficiency. CONCLUSIONS: Standardization of protein intake is required in the allopurinol loading test. A negative response in the face of clinical suspicion should be followed with a repeat test during a protein intake not <2.5 g x kg-1 x day-1. Measurements of orotidine provide better clinical sensitivity than measurements of orotate.

Identification of four new mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene in two patients: one of the variant alleles, 511C-->T, is present at an unexpectedly high frequency in the general population, as was the case for 625G-->A, together conferring susceptibility to ethylmalonic aciduria.

We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele, and a single point mutation, 511C-->T, in the other. The 1147C-->T mutation was not present in 98 normal alleles, but was detected in three alleles of 133 patients with elevated EMA excretion, consistently as a 625A-1147T allele. On the other hand, the 511C-->T mutation was present in 13 of 130 and 15 of 67 625G alleles, respectively, of normal controls and patients with elevated EMA excretion, and was never associated with the 625A variant allele. This over-representation of the haplotype 511T-625G among the common 625G alleles in patients compared with controls was significant ( P < 0.02), suggesting that the allele 511T-625G-like 511C-625A-confers susceptibility to ethylmalonic aciduria. Expression of the variant R147W SCAD protein, encoded by the 511T-625G allele, in COS-7 cells showed 45% activity at 37 degrees C in comparison with the wild-type protein, comparable levels of activity at 26 degrees C, and 13% activity when incubated at 41 degrees C. This temperature profile is different from that observed for the variant G185S SCAD protein, encoded by the 511C-625A allele, where higher than normal activity was found at 26 and 37 degrees C, and 58% activity was present at 41 degrees C. These results corroborate the notion that the 511C-625A variant allele is one of the possible underlying causes of ethylmalonic aciduria, and suggest that the 511C-->T mutation represents a second susceptibility variation in the SCAD gene. We conclude that ethylmalonic aciduria, a commonly detected biochemical phenotype, is a complex multifactorial/polygenic condition where, in addition to the emerging role of SCAD susceptibility alleles, other genetic and environmental factors are involved.

Mild or absent clinical signs in twin sisters with short-chain acyl-CoA dehydrogenase deficiency.

UNLABELLED: Two HLA-identical twin sisters are reported, of whom one has remained essentially asymptomatic, and an episode of hypotonia and decreased level of conciousness being the only relevant clinical finding in the other. Organic acid-analysis revealed that ethylmalonate was constantly, although sometimes only slightly, increased. No abnormal acylglycines or acylcarnitines could be detected. Enzyme assay in cultured skin fibroblasts confirmed short-chain acyl-CoA dehydrogenase deficiency. CONCLUSION: The lack of appropriate biochemical markers for this deficiency makes the diagnosis difficult and consequently, the low number of patients described may be the result of underdiagnosis.

Plasma free fatty acids in mitochondrial fatty acid oxidation defects.

Plasma free fatty acid profiles from patients suffering from various mitochondrial beta-oxidation deficiencies were analyzed by gas chromatography-mass spectrometry. cis-4-Decenoic acid (10:1n-6) in medium-chain acyl-CoA dehydrogenase deficiency and cis-5-tetradecenoic acid (14:1n-9) in very-long-chain and 3-hydroxy-long chain acyl-CoA dehydrogenase deficiencies are characteristic of these diseases. In addition, patients with 3-hydroxy-long chain acyl-CoA dehydrogenase deficiency showed a specific increase of 3-hydroxy-long chain fatty acids. The study of plasma free fatty acids is an easy and useful methodology for the diagnostic approach of some mitochondrial beta-oxidation deficiencies, allowing us to establish a quick differentiation between medium- and long-chain defects.

The clinical spectrum of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

Four patients with long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency are presented. Clinical onset in the form of acute encephalopathy occurred between the ages of 9 months and 3 years. The clinical course included recurrent metabolic crises in 4 patients, cardiac involvement and retinopathy in 3, and myopathy in 2. None had signs of peripheral neuropathy. Three patients died and one is currently well. Hypoketotic hypoglycemia with C6-C14 3-hydroxy-dicarboxylic aciduria during metabolic crises associated with decreased plasma carnitine levels was the main biochemical finding. Enzymologic studies disclosed long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency in all patients. Homozygosity for a G to C mutation at position 1528 in the encoding region of the enzyme was found in 2 patients. Histologic and electron microscopic studies of liver biopsy specimens revealed steatosis in 3 patients and mitochondrial abnormalities in 2. Skeletal muscle biopsies disclosed nonspecific degenerative changes in 2 patients and were normal in the remaining 2. Ultrastructural abnormalities in mitochondria were found in 3 patients. A review of the literature combined with the data from our series (total 22 patients) disclosed acute clinical onset in 77% of cases and subacute in 23%. In the combined series, the average age at onset was 11 months, family history was positive in 32% of patients and overall mortality was 50%. We describe the clinical spectrum of this disease and emphasize that, among patients with suspected beta-oxidation defects the finding of pigmentary retinopathy should lead to the suspicion of long-chain 3-hydroxyacyl-coenzyme A-dehydrogenase deficiency.

Bilateral striatal lesions in childhood.

From 1983 to 1991, 13 patients were identified with a clinical radiologic association characterized by acute or persistent neurologic dysfunction and bilateral lesions in the basal ganglia region demonstrated by ultrasound, computed tomography, or magnetic resonance imaging. Initial clinical manifestations of this group of patients were characterized by extrapyramidal signs (i.e., dystonia 9, hypotonia 2, athetosis 1, rigidity 1), altered state of consciousness in 5, and seizures in 3. The outcomes of most of these patients were poor: 10 had motor sequelae, 9 cognitive impairment, and 4 died. The outcomes of 2 patients, however, were much better than what was expected from the initial presentation. Based on current and previous reports, the diagnostic approach and classification of patients with neurologic dysfunction and bilateral striatal lesions are presented.