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Objective To report an adolescent with infantile‐onset carnitine palmitoyltransferase 2 (CPT2) deficiency and cerebral malformations and to review the occurrence of brain malformations in CPT2 deficiency. The patient presented clinically at age 5 months with dehydration and hepatomegaly. He also has an unrelated condition, X‐linked nephrogenic diabetes insipidus. He had recurrent rhabdomyolysis but normal psychomotor development. At age 17 years, he developed spontaneous focal seizures. Cerebral magnetic resonance imaging revealed extensive left temporo‐parieto‐occipital polymicrogyria, white matter heterotopias, and schizencephaly. Neuronal migration defects were previously reported in lethal neonatal CPT2 deficiency but not in later‐onset forms. Design and Methods We searched PubMed, Google Scholar, and the bibliographies of the articles found by these searches, for cerebral malformations in CPT2 deficiency. All antenatal, neonatal, infantile, and adult‐onset cases were included. Exclusion criteria included insufficient information about age of clinical onset and lack of confirmation of CPT2 deficiency by enzymatic assay or genetic testing. For each report, we noted the presence of cerebral malformations on brain imaging or pathological examination. Results Of 26 neonatal‐onset CPT2‐deficient patients who met the inclusion criteria, brain malformations were reported in 16 (61.5%). In 19 infantile‐onset cases, brain malformations were not reported, but only 3 of the 19 reports (15.8%) include brain imaging or neuropathology data. In 276 adult‐onset cases, no brain malformations were reported. Conclusion To the best of our knowledge, this is the first report of cerebral malformations in an infantile onset CPT2‐deficient patient. Brain imaging should be considered in patients with CPTII deficiency and neurological manifestations, even in those with later clinical onset.

Owing to the role of pyruvate and the tricarboxylic acid (TCA) cycle in energy metabolism, as well as in gluconeogenesis, lipogenesis and amino acid synthesis, defects in pyruvate metabolism and in the TCA cycle almost invariably affect the central nervous system. The severity and the different clinical phenotypes vary widely among patients and are not always specific, with the range of manifestations extending from overwhelming neonatal lactic acidosis and early death to relatively normal adult life and variable effects on systemic functions. The same clinical manifestations may be caused by other defects of energy metabolism, especially defects of the respiratory chain (Chap. 15). Diagnosis depends primarily on biochemical analyses of metabolites in body fluids, followed by definitive enzymatic assays in cells or tissues, and DNA analysis. The deficiencies of pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) constitute defects in gluconeogenesis, and therefore fasting results in hypoglycemia with worsening lactic acidosis. Deficiency of the pyruvate dehydrogenase complex (PDHC) impedes glucose oxidation and aerobic energy production, and ingestion of carbohydrate aggravates lactic acidosis. Treatment of disorders of pyruvate metabolism comprises avoidance of fasting (PC and PEPCK) or minimizing dietary carbohydrate intake (PDHC) and enhancing anaplerosis. In some cases, vitamin or drug therapy may be helpful. Dihydrolipoamide dehydrogenase (E3) deficiency affects PDHC as well as KDHC and the branched-chain 2-ketoacid dehydrogenase (BCKD) complex (Chap. 19), with biochemical manifestations of all three disorders. The deficiencies of the TCA cycle enzymes, the 2-ketoglutarate dehydrogenase complex (KDHC) and fumarase, interrupt the cycle, resulting in accumulation of the corresponding substrates. Succinate dehydrogenase deficiency represents a unique disorder affecting both the TCA cycle and the respiratory chain. Recently, defects of mitochondrial transport of pyruvate and glutamate (▸ Chap. 29) have been identified. Treatment strategies for the TCA cycle defects are limited.

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

Mitochondrial fatty acid β-oxidation defects (FAODs) are a group of severe inherited metabolic diseases, most of which can be treated with favorable prognosis following diagnosis. A description of the broad range of phenotypes resulting from these defects remains incomplete, and for this study, we sought to investigate the semiology at diagnosis in a country without a newborn screening program for FAODs.Using a retrospective French multicentre study, we analyzed 187 children aged6 years at diagnosis with FAOD confirmed by enzymatic and/or molecular analyses. Clinical and biological parameters at diagnosis were assessed to screen liver, heart, neurological, and muscle symptoms. Information concerning the long-term prognosis was also collected.Predominant hepatic symptoms were observed in 89 % of patients regardless of the underlying defect. The most frequent symptoms observed were hepatomegaly (92 %), increased blood alanine aminotransferase (ALAT) level (82 %), and steatosis (88 %). Other frequent features included Reye syndrome (49 %), increased gamma-glutamyltranspeptidase (GGT) (37 %), and liver failure (27 %). Extrahepatic features were often associated in the foreground. Hypoglycemia (75 %), neurological (64 %), muscle (61 %), or cardiac features (55 %) [as either cardiomyopathy (47 %) or arrhythmias (31 %)] were frequently documented. Hemodynamic events (41 %) were represented by shock (31 %) or sudden death (35 %). Hyperammonemia (73 %) and hyperlactacidemia (57 %) were the two main biochemical features. Total, very-long-chain acyl-CoA dehydrogenase (VLCADD), long-chain 3-hydroxyacylCoA dehydrogenase (LCHADD), and medium-chain acyl-CoA dehydrogenase (MCADD) deficiency mortality rates were 48 %, 60 %, 63 %, and 20 % respectively.This study presents clinical features of a large cohort of patients with FAODs in a country without neonatal screening for FAODs. Our results highlight liver as the main organ involved at diagnosis regardless of age at diagnosis, classical phenotype (i.e., cardiac, hepatic, or muscular), or enzyme deficiency. Although steatosis may be observed in various inherited metabolic defects, it is a reliable indicator of FAOD and should prompt systematic screening when the diagnosis is suspected. The poor long-term prognoses reported are a strong argument for inclusion of FAODs in newborn screening programs.

La galactosa es uno de los componentes del disacarido lactosa, que es el carbohidrato mas abundante en la leche de los mamiferos. Existen tres enfermedades hereditarias del metabolismo de la galactosa. El deficit de galactocinasa provoca un cuadro de catarata aislada. La galactosemia es la enfermedad debida al deficit de galactosa-1-fosfato-uridil-transferasa; se revela a traves de la aparicion de manifestaciones hepaticas, renales u oculares y presenta una evolucion favorable con una dieta exenta de galactosa; su pronostico a largo plazo depende de la afectacion neurologica. El deficit de uridina-difosfato-galactosa-4-epimerasa es excepcional y es responsable de formas benignas o graves de galactosemia. La fructosa es un azucar muy abundante en la alimentacion humana. Existen tres enfermedades hereditarias que afectan a su metabolismo. El deficit de fructocinasa es asintomatico. La intolerabilidad a la fructosa, debida al deficit de fructosa aldolasa, ocasiona sintomas digestivos agudos, con o sin hipoglucemias, o manifestaciones hepaticas o renales que pueden comprometer el pronostico vital si no se instaura rapidamente una dieta exenta de fructosa. El deficit de fructosa-1,6-difosfatasa es una anomalia de la gluconeogenesis que se manifiesta a traves de hipoglucemias con hiperlactacidemia.

Background Pyruvate dehydrogenase complex (PDHc) deficiencies are an important cause of primary lactic acidosis. Most cases result from mutations in the X-linked gene for the pyruvate dehydrogenase E1α subunit ( PDHA1 ) while a few cases result from mutations in genes for E1β ( PDHB ), E2 ( DLAT ), E3 ( DLD ) and E3BP ( PDHX ) subunits or PDH-phosphatase ( PDP1 ). Aim To report molecular characterization of 82 PDHc-deficient patients and analyze structural effects of novel missense mutations in PDHA1. Methods PDHA1 variations were investigated first, by exon sequencing using a long range PCR product, gene dosage assay and cDNA analysis. Mutation scanning in PDHX , PDHB, DLAT and DLD cDNAs was further performed in unsolved cases. Novel missense mutations in PDHA1 were located on the tridimensional model of human E1 protein to predict their possible functional consequences. Results PDHA1 mutations were found in 30 girls and 35 boys. Three large rearrangements, including two contiguous gene deletion syndrome were identified. Novel missense, frameshift and splicing mutations were also delineated and a nonsense mutation in a mosaic male. Mutations p.Glu75Ala, p.Arg88Ser, p.Arg119Trp, p.Gly144Asp, p.Pro217Arg, p.Arg235Gly, p.Tyr243Cys, p.Tyr243Ser, p.Arg245Gly, p.Pro250Leu, p.Gly278Arg, p.Met282Val, p.Gly298Glu in PDHA1 were predicted to impair active site channel conformation or subunit interactions. Six out of the seven patients with PDHB mutations displayed the recurrent p.Met101Val mutation; 9 patients harbored PDHX mutations and one patient DLD mutations. Conclusion We provide an efficient stepwise strategy for mutation screening in PDHc genes and expand the growing list of PDHA1 mutations analyzed at the structural level.

Background Deficiency of mitochondrial trifunctional protein (MTP) is caused by mutations in the HADHA and HADHB genes, which have been mostly delineated at the genomic DNA level and have not been always elucidated. Aim To identify mutations in a French cohort of 52 MTP deficient patients and the susceptibility of mutations generating premature termination codons (PTCs) to the nonsense mRNA mediated decay (NMD). Methods Mutation screening in fibroblasts was performed at the cDNA level and real-time RT-PCR was used to compare the levels of the different PTC-bearing mRNAs before and after a treatment of fibroblasts by emetine, a translation inhibitor. Results A mutation detection rate of 100% was achieved. A total of 22 novel mutations were identified, including a large-sized genomic deletion in HADHB gene. A high proportion of all identified mutations were non-sense, frameshift and splicing mutations, generating (PTCs), distributed essentially on HADHA coding regions. We could demonstrate that the majority of mutations resulting in PTCs conform to the established rules governing the susceptibility to NMD. Conclusion Our results emphasize the value of cDNA analysis in the characterization of HADHA and HADHB mutations and further strengthen the model of haploinsufficiency as a major pathomechanism in MTP defects.

Aim To describe the phenotype and genotype of pyruvate dehydrogenase complex (PDHc) deficiency. Method Twenty-two participants with enzymologically and genetically confirmed PDHc deficiency were analysed for clinical and imaging features over a 15-year period. Results Four groups were identified: (1) those with neonatal encephalopathy with lactic acidosis (one male, four females; diagnosis at birth); (2) those with non-progressive infantile encephalopathy (three males, three females; age at diagnosis 2–9mo); (3) those with Leigh syndrome (eight males; age at diagnosis 1–13mo); and (4) those with relapsing ataxia (three males; 18–30mo). Seventeen mutations involved PDHA1 (a hotspot was identified in exons 6, 7, and 8 in seven males with Leigh syndrome or recurrent ataxia). Mutations in the PDHX gene (five cases) were correlated with non-progressive encephalopathy and long-term survival in four cases. Interpretation Two types of neurological involvement were identified. Abnormal prenatal brain development resulted in severe non-progressive encephalopathy with callosal agenesis, gyration anomalies, microcephaly with intrauterine growth retardation, or dysmorphia in both males and females (12 cases). Acute energy failure in infant life produced basal ganglia lesions with paroxysmal dystonia, neuropathic ataxia due to axonal transport dysfunction, or epilepsy only in males (11 cases). The ketogenic diet improved only paroxysmal dysfunction, providing an additional argument in favour of paroxysmal energy failure.

Very Long-Chain Acyl-CoA dehydrogenase (VLCAD) deficiency is an inborn error of mitochondrial long-chain fatty acid oxidation (FAO) most often occurring in childhood with cardiac or liver involvement, but rhabdomyolysis attacks have also been reported in adults. We report in this study the clinical, biochemical and molecular studies in 13 adult patients from 10 different families with VLCAD deficiency. The enzyme defect was demonstrated in cultured skin fibroblasts or lymphocytes. All patients exhibited exercise intolerance and recurrent rhabdomyolysis episodes, which were generally triggered by strenuous exercise, fasting, cold or fever (mean age at onset: 10 years). Inaugural life-threatening general manifestations also occurred before the age of 3 years in four patients. Increased levels of long-chain acylcarnitines with tetradecenoylcarnitine (C14:1) as the most prominent species were observed in all patients. Muscle biopsies showed a mild lipidosis in four patients. For all patients but two, molecular analysis showed homozygous (4 patients) or compound heterozygous genotype (7 patients). For the two remaining patients, only one mutation in a heterozygous state was detected. This study confirms that VLCAD deficiency, although being less frequent than CPT II deficiency, should be systematically considered in the differential diagnosis of exercise-induced rhabdomyolysis. Measurement of fasting blood acylcarnitines by tandem mass spectrometry allows accurate biochemical diagnosis and should therefore be performed in all patients presenting with unexplained muscle exercise intolerance or rhabdomyolysis.

Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

Carnitine-acylcarnitine translocase (CACT) deficiency is a rare disorder of fatty acid oxidation associated with high mortality. Two female newborns of different ethnic origin (the first Anglo-Celtic and the second Palestinian Arab) both died after sudden collapse on day 2 of life. Both had elevated bloodspot long-chain acylcarnitines consistent with either CACT or carnitine palmitoyltransferase II (CPT2) deficiency; the latter was excluded by demonstrating normal CPT2 activity in fibroblasts. Direct sequencing of all SLC25A20 (CACT) gene exons and exon-intron boundaries revealed that Patient 1 was compound heterozygous for a novel c.609-3c>g (IVS6-3c>g) mutation on the paternal allele and a previously described c.326delG mutation on the maternal allele. Patient 2 was homozygous for the same, novel c.609-3c>g mutation. Previously reported SLC25A20 mutations have been almost exclusively confined to a single family or ethnic group. Analysis of fibroblast cDNA by RT-PCR, agarose gel electrophoresis and sequencing of extracted bands showed that both mutations produce aberrant splicing. c.609-3C>G results in exon 7 skipping leading to a frameshift with premature termination seven amino acids downstream. c.326delG was confirmed to produce skipping of exons 3 or 3 plus 4. CACT activity in both patients' fibroblasts was near-zero. For both families, prenatal diagnosis of an unaffected fetus was performed by mutation analysis on CVS tissue in a subsequent pregnancy. Due to the urgency of prenatal diagnosis in the second family, molecular diagnosis was performed prior to demonstration of CACT enzyme deficiency, illustrating that mutation analysis is a rapid and reliable approach to first-line diagnosis of CACT deficiency.

Objective To describe the clinical course, neuroradiological presentation, biochemical and molecular studies of a new patient with pyruvate dehydrogenase complex (PDHc) deficiency. To compare this case with the data on other published cases. Methods Brain magnetic resonance imaging (MRI), basal metabolic investigations with lactate measurements in body fluids, PDHc activity assay on cultured skin fibroblasts, immunoblot analysis and molecular studies (polymerase chain reaction [PCR] and sequencing procedures). Results Our patient accused an unspecific encephalopathy for years and presented at 13 years of age an acute deterioration with basal ganglia necrosis and subcortical white matter involvement. PDHc deficiency was secondary to a large deletion (3913 bp) in the PDHX gene, which encodes E3 binding protein (E3BP) subunit. Interpretation These data provide an additional case of E3BP deficiency with a unique and previously unreported deletion in the PDHX gene. Ann Neurol 2006;59:709–714

Resume Le galactose est un des constituants du disaccharide lactose, lequel est le carbohydrate le plus abondant dans le lait des mammiferes. Il existe trois maladies hereditaires du metabolisme du galactose. Le deficit en galactokinase est une cause de cataracte isolee. La galactosemie est la maladie due au deficit en galactose-1-phosphate uridyltransferase. Cette maladie se revele par des manifestations hepatiques, renales ou oculaires, dont lʼevolution est favorable sous regime sans galactose. Le pronostic a long terme est lie aux atteintes neurologiques. Le deficit en uridine diphosphate-galactose-4-epimerase est exceptionnel et responsable, soit de formes benignes, soit de formes severes de galactosemies. Le fructose est un sucre tres repandu dans lʼalimentation humaine. Trois maladies hereditaires affectent son metabolisme. Le deficit en fructokinase est asymptomatique. Lʼintolerance au fructose, liee au deficit en fructose aldolase, entraine, soit des symptomes digestifs aigus, avec ou sans hypoglycemies, soit des manifestations hepatiques ou renales qui peuvent mettre en jeu le pronostic vital si un regime dʼexclusion du fructose nʼest pas rapidement debute. Le deficit en fructose-1,6-diphosphatase est une anomalie de la neoglucogenese se manifestant par des hypoglycemies avec hyperlactacidemie.

Pyruvate dehydrogenase complex (PDC) deficiency is one of the major recognized causes of congenital lactic acidosis. The most common form is due to PDHA 1 gene (Xp22.12) defects. Here, we report the case of a Polynesian girl presenting with delayed neurological development, cortical atrophy, and posterior corpus callosum agenesis. Elevated lactate and pyruvate levels in blood and cerebrospinal fluid suggested PDC deficiency. However, PDC activity was within the normal range in lymphocytes and the direct sequencing of the 11 exons and intron-exon junctions of the PDHA 1 gene did not show any changes. Long-range PCR amplification of the whole gene (16 kb) from blood DNA revealed a heterozygous deletion of approximately 4.2kb. Fine mapping of the deletion breakpoint was achieved using purified long-range PCR products for restriction enzyme analysis and direct sequencing. The deletion removed a 4,227 bp region covering part of intron 5 to part of intron 9 [g.10,145_14,371 del 4,227]. The deletion breakpoint contained a short direct repeat (GTAG), which may be derived either from the upstream or the downstream homologous sequence. The presence of a GAG triplet and inverted repeats in the vicinity of the deletion suggest replication slippage at a polymerase alpha arrest site. This is the first time that a large intragenic deletion of the PDHA 1 gene has been characterized.

Hypoglycemia occasionally results from oxidative phosphorylation deficiency, associated with liver failure. Conversely, in some cases of respiratory chain defect, the impairment in glucose metabolism occurs with normal hepatic function. The mechanism for this hypoglycemia remains poorly understood. We report here three unrelated children with hypoglycemia as the presenting symptom associated with oxidative phosphorylation deficiency but without liver dysfunction. Two patients had, respectively, complex III and complex IV deficiency and presented with long fast hypoglycemia. During a fasting test, the first patient showed evidence for impaired gluconeogenesis (progressive increase of plasma lactate and no decrease of alanine levels), whereas the second patient appeared to have impaired fatty acid oxidation (hypoketotic hypoglycemia with increased levels of non esterified fatty acids). The third patient presented with both long and short fast hypoglycemia related to complex IV deficiency. The mechanism of hypoglycemia for this patient may have been partly related to GH insufficiency, whereas impaired glycogen metabolism possibly accounted for short fast hypoglycemia. We suggest that hypoglycemia can be the presenting symptom for respiratory chain defects, through the possible reduction in cofactors resulting from oxidative phosphorylation deficiency, and that respiratory chain defects should therefore be considered in the differential diagnosis of hypoglycemia.

El disacarido lactosa es el carbohidrato mas abundante en la leche de los mamiferos. Uno de sus componentes es la galactosa. Existen tres enfermedades hereditarias del metabolismo de la galactosa. El deficit de galactocinasa provoca un cuadro de catarata aislada. La galactosemia, enfermedad debida al deficit de galactosa-1-fosfato uridiltransferasa, se revela por la aparicion de manifestaciones hepaticas, renales u oculares, que evolucionan de manera favorable con un regimen sin galactosa. El pronostico a largo plazo depende de las lesiones neurologicas. El deficit de uridina difosfato-galactosa-4-epimerasa se revela excepcional, y produce galactosemias benignas o graves. La fructosa es un azucar muy abundante en la alimentacion humana. Existen tres enfermedades hereditarias que afectan a su metabolismo. El deficit de fructocinasa es asintomatico. La intolerancia a la fructosa, debida al deficit de fructosa aldolasa, ocasiona sintomas digestivos agudos con o sin hipoglucemias, o bien manifestaciones hepaticas o renales que pueden causar la muerte si no se instaura de inmediato un regimen sin fructosa. El deficit de fructosa-1,6-difosfatasa es una anomalia de la gluconeogenesis, que se manifiesta mediante hipoglucemias con hiperlactacidemia.

Mitochondrial respiratory chain complex III (ubiquinol-cytochrome c reductase) consists of 11 subunits, only one (cytochrome b) being encoded by the mitochondrial DNA. Disorders of complex III are comparatively rare but are nevertheless present as a clinically heterogeneous group of diseases. To date, no mutation in any of the nuclear-encoded subunits has been described. We report here a deletion in the nuclear gene UQCRB encoding the human ubiquinone-binding protein of complex III (QP-C subunit or subunit VII) in a consanguineous family with an isolated complex III defect. In the proband, a homozygous 4-bp deletion was identified at nucleotides 338-341 of the cDNA predicting both a change in the last seven amino acids and an addition of a stretch of 14 amino acids at the C-terminal end of the protein. Both parents were found to be heterozygous for the deletion, which was absent from 55 controls. Low temperature (-196 degrees C) spectral studies performed on isolated mitochondria from cultured skin fibroblast of the proband showed a decreased cytochrome b content suggestive of a role for the QP-C subunit in the assembly or maintenance of complex III structure.

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69–70insTGTGC, and 609−1g > a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.

Contains fulltext : 138930.pdf (Publisher’s version ) (Open Access) OBJECTIVE: Deficiency of pyruvate dehydrogenase complex (PDHC) is the most common genetic disorder leading to lactic acidosis. PDHC deficiency is genetically heterogenous and most patients have defects in the X-linked E1-alpha gene but defects in the other components of the complex encoded by PDHB, PDHX, DLAT, DLD genes or in the regulatory enzyme encoded by PDP1 have also been found. Phenylbutyrate enhances PDHC enzymatic activity in vitro and in vivo by increasing the proportion of unphosphorylated enzyme through inhibition of pyruvate dehydrogenase kinases and thus, has potential for therapy of patients with PDHC deficiency. In the present study, we investigated response to phenylbutyrate of multiple cell lines harboring all known gene defects resulting in PDHC deficiency. METHODS: Fibroblasts of patients with PDHC deficiency were studied for their enzyme activity at baseline and following phenylbutyrate incubation. Drug responses were correlated with genotypes and protein levels by Western blotting. RESULTS: Large deletions affecting PDHA1 that result in lack of detectable protein were unresponsive to phenylbutyrate, whereas increased PDHC activity was detected in most fibroblasts harboring PDHA1 missense mutations. Mutations affecting the R349-alpha residue were directed to proteasome degradation and were consistently unresponsive to short-time drug incubation but longer incubation resulted in increased levels of enzyme activity and protein that may be due to an additional effect of phenylbutyrate as a molecular chaperone. INTERPRETATION: PDHC enzyme activity was enhanced by phenylbutyrate in cells harboring missense mutations in PDHB, PDHX, DLAT, DLD, and PDP1 genes. In the prospect of a clinical trial, the results of this study may allow prediction of in vivo response in patients with PDHC deficiency harboring a wide spectrum of molecular defects.

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Inherited metabolic disorders are the cause of a small but significant number of sudden infant deaths in infants. We report on a boy who suddenly died at 10 months of age during an acute illness. Parents declined autopsy; nevertheless, they accepted a whole body MRI, which revealed hepatomegaly with steatosis. Acylcarnitine profile of a blood sample from neonatal Guthrie screening led to the diagnosis of type 2 carnitine palmitoyltransferase deficiency. To conclude, whole body MRI is useful in the investigation of some inherited metabolic causes of sudden infant death, which might prevent future deaths in the family. It is a good alternative when autopsy is refused.

Carnitine-acylcarnitine translocase (CATR) deficiency is a severe defect in fatty acid oxidation which presents early in life most frequently with hypoglycemia, hyperammonemia, and severe cardiac abnormalities. CATR exchanges acylcarnitines of various chain lengths for free carnitine across the mitochondrial membrane. In vitro studies in intact fibroblasts from patients with documented deficiency of CATR were probed with stable-isotope-labeled precursors and the resulting acylcarnitines were analyzed by tandem mass spectrometry. After a 72-h incubation with l-[(2)H(3)]carnitine the translocase-deficient cells produced acylcarnitines in which the deuterium was incorporated into short-chain acylcarnitines, C2-C5. Experiments with simultaneous incubation of l-[(2)H(3)]carnitine and l-[(13)C(6)]isoleucine produced [(13)C(5)]2-methylbutyryl-[(2)H(3)]carnitine and [(13)C(3)]propionyl-[(2)H(3)]carnitine indicating exchange of labeled acylcarnitine from inside the mitochondrial matrix with labeled free carnitine. These studies support the possible existence of a "branched-chain" carnitine-acylcarnitine translocator in mitochondria.

The oxidation of long-chain fatty acids in mitochondria plays an important role in energy production, especially in skeletal muscle, heart and liver. Long-chain fatty acids, activated to their CoA esters in the cytosol, are shuttled across the barrier of the inner mitochondrial membrane by the carnitine cycle. This pathway includes four steps, mediated by a plasma membrane carnitine transporter, two carnitine palmitoyltransferases (CPT I and CPT II) and a carnitine-acylcarnitine translocase. Defects in activation and uptake of fatty acids affect these four steps: CPT II deficiency leads to either exercise-induced rhabdomyolysis in adults or hepatocardiomuscular symptoms in neonates and children. The three other disorders of the carnitine cycle have an early onset. Hepatic CPT I deficiency is characterized by recurrent episodes of Reye-like syndrome, whereas severe muscular and cardiac signs are associated with episodes of fasting hypoglycaemia in defects of carnitine transport and translocase. Convenient metabolic investigations for reaching the diagnosis of carnitine cycle disorders are determination of plasma free and total carnitine concentrations, determination of plasma acylcarnitine profile by tandem mass spectrometry and in vitro fatty acid oxidation studies, particularly in fresh lymphocytes. Application of the tools of molecular biology has greatly aided the understanding of the carnitine palmitoyltransferase enzyme system and confirmed the existence of different related genetic diseases. Mutation analysis of CPT II defects has given some clues for correlation of genotype and phenotype. The first molecular analyses of hepatic CPT I and translocase deficiencies were recently reported.

In a personal series of 107 patients, we describe clinical presentations, methods of recognition and therapeutic management of inherited fatty acid oxidation (FAO) defects. As a whole, FAO disorders appear very severe: among the 107 patients, only 57 are still living. Including 47 siblings who died early in infancy, in total 97 patients died, of whom 30% died within the first week of life and 69% before 1 year. Twenty-eight patients presented in the neonatal period with sudden death, heart beat disorders, or neurological distress with various metabolic disturbances. Hepatic presentations were observed in 73% of patients (steatosis, hypoketotic hypoglycaemia, hepatomegaly, Reye syndrome). True hepatic failure was rare (10%); cholestasis was observed in one patient with LCHAD deficiency. Cardiac presentations were observed in 51% of patients: 67% patients presented with cardiomyopathy, mostly hypertrophic, and 47% of patients had heart beat disorders with various conduction abnormalities and arrhythmias responsible for collapse, near-miss and sudden unexpected death. All enzymatic blocks affecting FAO except CPT I and MCAD were found associated with cardiac signs. Muscular signs were observed in 51% of patients (of whom 64% had myalgias or paroxysmal myoglobinuria, and 29% had progressive proximal myopathy). Chronic neurologic presentation was rare, except in LCHAD deficiency (retinitis pigmentosa and peripheral neuropathy). Renal presentation (tubulopathy) and transient renal failure were observed in 27% of patients. The diagnosis of FAO disorders is generally based on the plasma acylcarnitine profile determined by FAB-MS/MS from simple blood spots collected on a Guthrie card. Urinary organic acid profile and total and free plasma carnitine can also be very helpful, mostly in acute attacks. If there is no significant disturbance between attacks, the diagnosis is based upon a long-chain fatty acid loading test, fasting test, and in vitro studies of fatty acid oxidation on fresh lymphocytes or cultured fibroblasts. Treatment includes avoiding fasting or catabolism, suppressing lipolysis, and carnitine supplementation. The long-term dietary therapy aims to prevent periods of fasting and restrict long-chain fatty acid intake with supplementation of medium-chain triglycerides. Despite these therapeutic measures, the long-term prognosis remains uncertain.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is an enzyme catalyzing the dehydrogenation of long-chain fatty acids in the first step of mitochondrial fatty acid oxidation. Using an ETF (electron transfer flavoprotein, the physiological electron acceptor of VLCAD) reduction assay, we identified VLCAD deficiency in cultured skin fibroblasts or liver tissue from 30 patients in 27 families. They clinically presented two phenotypes: a 'severe' presentation characterized by an early onset of symptoms, with hypertrophic cardiomyopathy and a high incidence of death, and a 'mild' form with hypoketotic hypoglycaemia, resembling MCAD (medium-chain acyl-CoA dehydrogenase) deficiency. Cells isolated from patients who develop cardiomyopathy characteristically accumulate longer-chain length acylcarnitines (hexadecanoylcarnitine and tetradecanoylcarnitine) when incubated with palmitate. However, cells from patients with the hypoglycaemic presentation produced relatively shorter-chain-length intermediates (mainly dodecanoylcarnitine). Inhibition of carnitine palmitoyl transferase I, in vitro, eliminated these intermediates with cells from both phenotypes indicating their intramitochondrial origin. Although the explanation for these distinct biochemical findings is not obvious, the correlation with the two phenotypes provides an opportunity for accurate prognosis and early implementation of appropriate treatment. Prenatal diagnosis of this life-threatening disorder was successfully performed in seven pregnancies in six of those families by assay of trophoblasts or amniocytes. In an at risk family, diagnosis of an affected fetus by measurement of VLCAD activity in noncultured chorionic villi allowed termination of the pregnancy before 13 weeks of gestation.

The mitochondrial trifunctional protein (MTP) catalyzes the last three steps in the long-chain fatty acid beta-oxidation pathway. We report herein a patient with an inherited MTP deficiency and hypoparathyroidism that were both revealed at 4 months of age. Although parathyroid function appeared to be normalized following nutritional management of the fatty acid beta-oxidation defect, persistent gland dysfunction was suggested by frequent mild episodes of hypocalcaemia without increase in plasma intact parathyroid hormone (iPTH) levels during recurrent fasting-induced episodes of rhabdomyolysis and by our finding of a bilateral cataract at 5 years of age. An acute provocation test conducted to stimulate iPTH release with sodium bicarbonate infusion resulted in a subnormal rise in iPTH release, which further supported a partial hypoparathyroidism. This case is the third report of inherited MTP deficiency associated with hypoparathyroidism, thus raising the possibility of a link between these two rare disorders.

Four steps are required for the transport of long-chain fatty acids (LCFA) into the mitochondrial matrix : a plasma membrane carnitine transporter maintains the intracellular supply of carnitine ; an outer mitochondrial membrane carnitine palmitoyltransferase I (CPT I) converts acyl-CoA compounds to their acylcarnitine analogues ; a carnitine-acylcarnitine translocase (CATR) mediates the transmembrane transfer of acylcarnitines ; an inner mitochondrial membrane carnitine palmitoyltransferase II (CPT II) allows the re-esterification of acylcarnitines to form acyl-CoA esters. Inherited metabolic diseases affecting CATR and CPT II are recognized as a contributing cause of life-threatening events in infancy. In these two disorders, long-chain acylcarnitines are not efficiently converted to their corresponding acyl-CoA esters, and prominent long-chain acylcarnitine species are seen in plasma. It has been speculated that increased concentrations of long-chain acylcarnitines may promote cardiac arrhythmia (Corr et al 1989). Tandem mass spectrometry has enabled quantitative analysis of acylcarnitines in small volumes of blood, including filter paper blood spots used for neonatal screening (Millington et al 1991). Determination of the acylcarnitine profile in the Guthrie card is particularly useful in case of sudden infant death when no other biological specimen had been preserved. The finding of abnormal accumulation of C 18 and C 16 species argues in favour of CATR or profound CPT II deficiency. However, the definitive diagnosis of these two disorders requires documentation of the specific enzymatic defect in cellular material. Both defects are transmitted as autosomal recessive traits. The enzymatic activities of obligate heterozygotes are approximately 50% of normal activity (Stanley et al 1992; Elpeleg et al 1993). Thus, enzymatic determinations in parents' cells can be used to establish the diagnosis.

Letting Pyruvate In Transport of pyruvate is an important event in metabolism whereby the pyruvate formed in glycolysis is transported into mitochondria to feed into the tricarboxylic acid cycle (see the Perspective by Murphy and Divakaruni ). Two groups have now identified proteins that are components of the mitochondrial pyruvate transporter. Bricker et al. (p. 96 , published online 24 May) found that the proteins mitochondrial pyruvate carrier 1 and 2 (MPC1 and MPC2) are required for full pyruvate transport in yeast and Drosophila cells and that humans with mutations in MPC1 have metabolic defects consistent with loss of the transporter. Herzig et al. (p. 93 , published online 24 May) identified the same proteins as components of the carrier in yeast. Furthermore, expression of the mouse proteins in bacteria conferred increased transport of pyruvate into bacterial cells.

The present work presents a “from gene defect to clinics” pathogenesis study of a patient with a hitherto unreported mutation in the CPT1A gene. In early childhood, the patient developed a life-threatening episode (hypoketotic hypoglycemia, liver cytolysis, and hepatomegaly) evocative of a mitochondrial fatty acid oxidation disorder, and presented deficient fibroblast carnitine palmitoyltransferase 1 (CPT1) activity and homozygosity for the c.1783 C > T nucleotide substitution on exon 15 of CPT1A (p.R595W mutant). While confirming CPT1A deficiency, whole blood de novo acylcarnitine synthesis and the levels of carnitine and its esters formally linked intracellular free-carnitine depletion to intracellular carnitine esterification. Sequence alignment and modeling of wild-type and p.*R595W CPT1A proteins indicated that the Arg595 targeted by the mutated codon is phylogenetically well conversed. It contributes to a hydrogen bond network with neighboring residues Cys304 and Met593 but does not participate in the catalysis and carnitine pocket. Its replacement by tryptophan induces steric hindrance with the side chain of Ile480 located in α-helix 12, affecting protein architecture and function. This hindrance with Ile480 is also originally described with tryptophan 304 in the known mutant p.C304W CPT1A, suggesting that the mechanisms that invalidate CPT1A activity and underlie pathogenesis could be common in both the new (p.R595W) and previously described (p.C304W) mutants.

Pyruvate dehydrogenase deficiency is one of the genetic defects of mitochondrial energy metabolism. Clinical features are heterogeneous, ranging from fatal lactic acidosis in the newborn period to chronic neurodegenerative abnormalities. Most cases have mutations in the gene for the E1alpha subunit of the pyruvate dehydrogenase complex. Primary defects of the E3 binding protein component of the pyruvate dehydrogenase complex are rarier. We describe two unrelated Moroccan patients with the same new mutation c.1182 + 2T > C in the E3 binding protein gene PDHX and different clinical forms.

Deficiency of the enzymes of mitochondrial fatty acid oxidation and related carnitine dependent steps have been shown to be one of the causes of the fasting-induced hypoketotic hypoglycemia. We describe here carnitine-acylcarnitine translocase deficiency in a neonate who died eight days after birth. The proband showed severe fasting-induced hypoketotic hypoglycemia, high plasma creatine kinase, heartbeat disorder, hypothermia, and hyperammonemia. The plasma-free carnitine on day three was only 3 microM, and 92% of the total carnitine (37 microM) was present as acylcarnitine. Treatments with intravenous glucose, carnitine, and medium-chain triglycerides had been tried without improvements. Measurements in fibroblasts confirmed deficient oxidation of palmitate and showed normal activities of the carnitine palmitoyltransferases I and II and of the three acyl-CoA dehydrogenases. A total deficiency of the carnitine-acyl-carnitine translocase was found in fibroblasts using the carnitine acetylation assay (1986. Biochem. J. 236:143-148). This assay has been further simplified by seeking conditions permitting application to permeabilized fibroblasts and lymphocytes.

A female patient, with normal familial history, developed at the age of 30 months an episode of diarrhoea, vomiting and lethargy which resolved spontaneously. At the age of 3 years, the patient re-iterated vomiting, was sub-febrile and hypoglycemic, fell into coma, developed seizures and sequels involving right hemi-body. Urinary excretion of hexanoylglycine and suberylglycine was low during this metabolic decompensation. A study of pre- and post-prandial blood glucose and ketones over a period of 24 hours showed a normal glycaemic cycle but a failure to form ketones after 12 hours fasting, suggesting a mitochondrial β-oxidation defect. Total blood carnitine was lowered with unesterified carnitine being half of the lowest control value. A diagnosis of mild MCAD deficiency (MCADD) was based on rates of 1-14C-octanoate and 9, 10-3H-myristate oxidation and of octanoyl-CoA dehydrogenase being reduced to 25% of control values. Other mitochondrial fatty acid oxidation proteins were functionally normal. De novo acylcarnitine synthesis in whole blood samples incubated with deuterated palmitate was also typical of MCADD. Genetic studies showed that the patient was compound heterozygous with a sequence variation in both of the two ACADM alleles; one had the common c.985A>G mutation and the other had a novel c.145C>G mutation. This is the first report for the ACADM gene c.145C>G mutation: it is located in exon 3 and causes a replacement of glutamine to glutamate at position 24 of the mature protein (Q24E). Associated with heterozygosity for c.985A>G mutation, this mutation is responsible for a mild MCADD phenotype along with a clinical story corroborating the emerging literature view that patients with genotypes representing mild MCADD (high residual enzyme activity and low urinary levels of glycine conjugates), similar to some of the mild MCADDs detected by MS/MS newborn screening, may be at risk for disease presentation.

Ethylmalonic encephalopathy (EE) is a rare metabolic disorder caused by dysfunction of ETHE1, a mitochondrial dioxygenase involved in hydrogen sulfide (H2S) detoxification. Patients present in infancy with psychomotor retardation, chronic diarrhea, orthostatic acrocyanosis and relapsing petechiae. High levels of lactic acid, ethymalonic acid (EMA) and methylsuccinic acid (MSA) are detected in body fluids. Several pathways may contribute to the pathophysiology, including isoleucine, methionine and fatty acid metabolism. We report on a 15-month-old male presenting with typical EE associated with a homozygous ETHE1 mutation. We investigated oral isoleucine (150 mg/kg), methionine (100 mg/kg), fatty acid loading tests and isoleucine-restricted diet (200 mg/day) for any effects on several metabolic parameters. Before loading tests or specific dietary interventions, EMA, C4-C5 acylcarnitines and most acylglycines were elevated, indicating functional deficiency of short chain acyl-CoA (SCAD) as well as all branched acyl-CoA dehydrogenases. Excretion of EMA and n-butyrylglycine increased following each of the loads, and isoleucine led to increased levels of derivative metabolites. An isoleucine-restricted diet for 8 days corrected some of the abnormalities but led to no obvious clinical improvement and only partial effects on EMA. A principal component analysis supports the inference that these dietary conditions have consistent effects on the global metabolic profile. Our results suggest that multiple pathways modulate EMA levels in EE. They might all interact with H2S toxicity. Prolonged dietary interventions involving the restriction for branched aminoacids, fatty acids and methionine could be discussed as auxiliary therapeutical strategies in EE.

International audience; Autosomal recessive LPIN1 mutations have been recently described as a novel cause of rhabdomyolysis in a few families. The purpose of the study was to evaluate the prevalence of LPIN1 mutations in patients exhibiting severe episodes of rhabdomyolysis in infancy. After exclusion of primary fatty acid oxidation disorders, LPIN1 coding sequence was determined in genomic DNA and cDNA. Among the 29 patients studied, 17 (59%) carried recessive nonsense or frameshift mutations, or a large scale intragenic deletion. In these 17 patients, episodes of rhabdomyolysis occurred at a mean age of 21 months. Secondary defect of mitochondrial fatty oxidation or respiratory chain was found in skeletal muscle of two patients. The intragenic deletion, c.2295-866_2410-30del, was identified in 8/17 patients (47%), all Caucasians, and occurred on the background of a common haplotype, suggesting a founder effect. This deleted human LPIN1 form was unable to complement Delta pah1 yeast for growth on glycerol, in contrast to normal LPIN1. Since more than 50% of our series harboured LPIN1 mutations, LPIN1 should be regarded as a major cause of severe myoglobinuria in early childhood. The high frequency of the intragenic LPIN1 deletion should provide a valuable criterion for fast diagnosis, prior to muscle biopsy.

The chemiluminescence (CL) response of normal peripheral blood polymorphonuclear cells (PMN) in ascitic fluids (cirrhotic = 32; malignant = 17) was studied independently of the ascitic fluid complement activity. CL response and fibronectin levels were higher in malignant ascitic fluid than in cirrhotic ascitic fluid (p less than 0.001). Addition of pure fibronectin or malignant ascitic fluids to cirrhotic ascitic fluids increased the CL response of normal PMN. These findings suggest that the susceptibility of cirrhotic patients to spontaneous bacterial peritonitis (SBP) is a multifactorial defect involving factors distinct from low C3 levels. Fibronectin is an important factor in the promotion of the respiratory burst of normal PMN stimulated by opsonized zymosan or PMA in ascitic fluid. Our results suggest that low levels of ascitic fluid fibronectin could partly explain the high susceptibility of cirrhotic patients to spontaneous bacterial peritonitis.

To describe the phenotype and genotype of pyruvate dehydrogenase complex (PDHc) deficiency.Twenty-two participants with enzymologically and genetically confirmed PDHc deficiency were analysed for clinical and imaging features over a 15-year period.Four groups were identified: (1) those with neonatal encephalopathy with lactic acidosis (one male, four females; diagnosis at birth); (2) those with non-progressive infantile encephalopathy (three males, three females; age at diagnosis 2-9mo); (3) those with Leigh syndrome (eight males; age at diagnosis 1-13mo); and (4) those with relapsing ataxia (three males; 18-30mo). Seventeen mutations involved PDHA1 (a hotspot was identified in exons 6, 7, and 8 in seven males with Leigh syndrome or recurrent ataxia). Mutations in the PDHX gene (five cases) were correlated with non-progressive encephalopathy and long-term survival in four cases.Two types of neurological involvement were identified. Abnormal prenatal brain development resulted in severe non-progressive encephalopathy with callosal agenesis, gyration anomalies, microcephaly with intrauterine growth retardation, or dysmorphia in both males and females (12 cases). Acute energy failure in infant life produced basal ganglia lesions with paroxysmal dystonia, neuropathic ataxia due to axonal transport dysfunction, or epilepsy only in males (11 cases). The ketogenic diet improved only paroxysmal dysfunction, providing an additional argument in favour of paroxysmal energy failure.

Classical galactosemia is an autosomal recessive disorder caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase. Undoubtedly, some of the short term complications are linked to the toxic effects of the accumulated abnormal metabolites (galactose-1-phosphate and galactitol). However, the physiopathology of neonatal liver failure remains unclear. We report the case of a 7-week-old girl who was first diagnosed with liver failure, hypoprotidaemia, ascites and generalized edemas. High citrulline (293 micromol/L), on initial plasma amino acid, suggested the diagnosis of citrin deficiency. As the citric acid cycle intermediates were non-detectable (oxoglutarate, succinate and citrate), a cataplerotic state was suspected. As a result, citrate (as an anaplerotic treatment) induced a clear improvement in her liver function. Four weeks later, this patient was switched to a galactose-free formula (as recommended in citrin deficiency with galactosemia) and her pathological status returned to normal. Citrin deficiency was later ruled out by molecular biology studies; then we reintroduced a galactose-containing formula which re-evoked rapidly vomiting, galactose aversion and hepatic cytolysis and the diagnosis of classical galactosemia was established. Our case clearly shows that cataplerosis could play a role in the pathophysiology of the neonatal liver disease observed in classical galactosemia.

Background Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. Methods and results One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5′ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5′ splice site by site-directed mutagenesis. Conclusion These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.

The long interspersed element-1 (LINE-1 or L1) retrotransposition has altered the human genome in many ways. In particular, recent in vitro studies have demonstrated that the retrotranspositional insertion of L1 elements has resulted in significant genomic deletions. Here we provide evidence for its operation in the human genome by identifying a approximately 46-kb pathological genomic deletion in the PDHX gene directly linked to the insertion of a full-length L1 element, in a patient with pyruvate dehydrogenase complex (PDHc) deficiency. Both the deduced bottom and top strand cleavage sites in the PDHX gene coincide with the consensus L1 endonuclease (EN) target sequence 5'-TTTT/A-3', while the full-length L1 element is followed by a 67-bp poly(A) tail. Interestingly, two hairpin structures, potentially formed by the inverted repeats present immediately 5' to the top strand nick site and 3' to the bottom strand nick site, may have facilitated the accessibility of L1 EN to the target sequences and also brought the two otherwise distantly located sequences into close proximity. Since the L1 element inserted in the PDHX gene is full-length, we favor the model of the template jumping as opposed to that of the microhomology-mediated end-joining for linking the 5' end of the nascent L1 copy to its genomic target. Our finding not only serves as an important complement to the in vitro approaches to studying L1 retrotransposition, but also reveals a novel mechanism causing human genetic disease.

We report here the molecular analysis of a pyruvate dehydrogenase E3-binding protein (PDH-E3BP) deficiency in a new patient, born to first cousin parents. She has initially presented with a non-progressive and unspecific encephalopathy, followed by an acute neurological deterioration at 14 years of age. E3BP subunit was undetectable on Western blot. The sequence of exons 1-9 and exon 11 of the PDHX gene were normal, but exon 10 was impossible to amplify with standard PCR. Long-range PCR including exons 9-11 (11.5 kb) was performed. The patient's sample displayed a unique PCR product of 7.5 kb, whereas the parents' samples displayed two bands (11.5 and 7.5 kb). The deletion breakpoints were determined by restriction analysis followed by direct sequencing. The homozygous deletion covered the end of intron 9, exon 10 and the beginning of intron 10 and was found to be 3913 bp long. The cDNA sequencing confirmed the deletion of exon 10. The most probable mechanism for this gross deletion appears to be a slipped mispairing mediated by an exact direct repeat CCACTG. It is the first time that a non-homologous recombination is reported in the PDHX gene causing pyruvate dehydrogenase complex (PDHc) deficiency.

The activity of deoxyguanosine kinase (DGUOK), a mitochondrial enzyme involved in the anabolism of mitochondrial (mt) deoxyribonucleotides, governs the maintenance of the mtDNA. Deleterious mutations of the DGUOK gene are thus associated with mtDNA depletion and result in combined deficiencies of mtDNA-encoded respiratory chain enzymes. With the aim to estimate the prevalence of DGUOK mutations in a cohort of 30 patients with hepatocerebral disease and combined respiratory chain deficiencies, we studied the DGUOK gene and identified previously unreported mutations in five families. Two patients and their affected sibs, born to non-consanguineous parents, were homozygous for a missense mutation (M1T, and L250S, respectively). One patient presented a homozygous 4 pb insertion (796 insTGAT) and two other patients, and their affected sibs, were compound heterozygous (E165V/L266R and E211G/L266R, respectively). These findings allowed us to propose prenatal diagnosis in two families. In conclusion, we observed a high prevalence of DGUOK mutations (17%) in patients with hepatic involvement and combined respiratory chain deficiencies with hepatic involvement.

Received 6 December 2004/Returned for modification 4 January 2005/Accepted 14 January 2005 Pyruvate dehydrogenase (PDH) complex deficiency is a major cause of lactic acidosis and Leigh’s encephalomyelopathies in infancy and childhood, resulting in early death in the majority of patients. Most of the molecular defects have been localized in the coding regions of the E1 PDH gene. Recently, we identified a novel mutation of the E1 PDH gene in a patient with an encephalopathy and lactic acidosis. This mutation, located downstream of exon 7, activates a cryptic splice donor and leads to the retention of intronic sequences. Here, we demonstrate that the mutation results in an increased binding of the SR protein SC35. Consistently, ectopic overexpression of this splicing factor enhanced the use of the cryptic splice site, whereas small interfering RNA-mediated reduction of the SC35 protein levels in primary fibroblasts from the patient resulted in the almost complete disappearance of the aberrantly spliced E1 PDH mRNA. Our findings open the exciting prospect for a novel therapy of an inherited disease by altering the level of a specific splicing factor.

We report on a patient with severe growth retardation and IgF1 deficiency, in which a mitochondrial abnormality was suspected. An isolated mitochondrial respiratory chain complex III deficiency was found in blood lymphocytes and skin fibroblasts. Sequence analysis of the cytochrome b, which is the only mitochondrial DNA-encoded subunit of complex III, revealed a homoplasmic G15498A mutation, resulting in the substitution of a highly conserved amino acid (glycine 251 into an aspartic acid). The mutation was found to be homoplasmic in all tissues examined from the mother and her brother (lymphocytes, fibroblasts, hair roots and buccal cells). Complex III deficiency was also demonstrated in these cells. Nevertheless, the mother and the brother were asymptomatic. This mutation had been considered as a cardiomyopathy-generating mutation in a previously reported case, and its pathogenicity has been demonstrated recently in yeast. However, it seems not to fulfil the classical criteria for pathogenicity of a mitochondrial DNA mutation, especially the heteroplasmic status, and to be clinically silent, albeit present, in nonaffected relatives. We suggest that other factors are contributing to the clinical variability expression of the G15498A mtDNA mutation.

Carnitine palmitoyltransferase 2 (CPT2) deficiency, the most common inherited disease of the mitochondrial long-chain fatty acid (LCFA) oxidation, may result in distinct clinical phenotypes, namely a mild adult muscular form and a severe hepatocardiomuscular disease with an onset in the neonatal period or in infancy. In order to understand the mechanisms underlying the difference in severity between these phenotypes, we analyzed a cohort of 20 CPT2-deficient patients being affected either with the infantile (seven patients) or the adult onset form of the disease (13 patients). Using a combination of direct sequencing and denaturing gradient gel electrophoresis, 13 CPT2 mutations were identified, including five novel ones, namely: 371G>A (R124Q), 437A>C (N146T), 481C>T (R161W), 983A>G (D328G), and 1823G>C (D608H). After updating the spectrum of CPT2 mutations (n=39) and genotypes (n=38) as well as their consequences on CPT2 activity and LCFA oxidation, it appears that both the type and location of CPT2 mutations and one or several additional genetic factors to be identified would modulate the LCFA flux and therefore the severity of the disease.

We report a case of neonatal congenital lactic acidosis associated with pyruvate dehydrogenase E3-binding protein deficiency in a newborn girl. She had a severe encephalopathy, and magnetic resonance imaging of the brain showed large subependymal cysts and no basal ganglia lesions. She died 35 days after birth. We detected a novel homozygous deletion (620delC) in the PDX1 gene, which encodes for the E3BP subunit of the pyruvate dehydrogenase complex.

An intronic point mutation was identified in the E1alpha PDH gene from a boy with delayed development and lactic acidosis, an X-linked disorder associated with a partial defect in pyruvate dehydrogenase (PDH) activity. Protein analysis demonstrated a corresponding decrease in immunoreactivity of the alpha and beta subunits of the PDH complex. In addition to the normal spliced mRNA product of the E1alpha PDH gene, patient samples contained significant levels of an aberrantly spliced mRNA with the first 45 nucleotides of intron 7 inserted in-frame between exons 7 and 8. The genomic DNA analysis found no mutation in the coding regions but revealed a hemizygous intronic G to A substitution 26 nucleotides downstream from the normal exon 7 5'-splice site. Splicing experiments in COS-7 cells demonstrated that this point mutation at intron 7 position 26 is responsible for the aberrant splicing phenotype, which involves a switch from the use of the normal 5'-splice site (intron 7 position 1) to the cryptic 5'-splice site downstream of the mutation (intron 7 position 45). The intronic mutation is unusual in that it generates a consensus binding motif for the splicing factor, SC35, which normally binds to exonic enhancer elements resulting in increased exon inclusion. Thus, the aberrant splicing phenotype is most likely explained by the generation of a de novo splicing enhancer motif, which activates the downstream cryptic 5'-splice site. The mutation documented here is a novel case of intron retention responsible for a human genetic disease.