Multiple acyl-CoA dehydrogenase deficiency (MADD) as a cause of late-onset treatable metabolic disease.

INTRODUCTION: Late-onset multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare, treatable, beta-oxidation disorder responsible for neuromuscular symptoms in adults. This case series describes the clinical and biochemical features of 13 French patients with late-onset MADD. METHODS AND RESULTS: Thirteen ambulant patients (eight women, five men), with a median age at onset of 27 years, initially experienced exercise intolerance (n=9), isolated muscle weakness (n=1) and a multisystemic pattern with either central nervous system or hepatic dysfunction (n=3). During the worsening period, moderate rhabdomyolysis (n=5), a pseudomyasthenic pattern (n=5) and acute respiratory failure (n=1) have been observed. Weakness typically affected the proximal limbs and axial muscles, and there was sometimes facial asymmetry (n=3). Moderate respiratory insufficiency was noted in one case. Median baseline creatine kinase was 190IU/L. Lactacidemia was sometimes moderately increased at rest (3/10) and after exercise (1/3). The acylcarnitine profile was characteristic, with increases in all chain-length acylcarnitine species. Electromyography revealed a myogenic pattern, while muscle biopsy showed lipidosis, sometimes with COX-negative fibers (n=2). The mitochondrial respiratory chain was impaired in five cases, with coenzyme Q10 decreased in two cases. All patients harbored mutations in the ETFDH gene (four homozygous, seven compound heterozygous, two single heterozygous), with nine previously unidentified mutations. All patients were good responders to medical treatment, but exercise intolerance and/or muscular weakness persisted in 11 of them. CONCLUSION: Late-onset forms of MADD may present as atypical beta-oxidation disorders. Acylcarnitine profiling and muscle biopsy remain the most decisive investigations for assessing the diagnosis. These tests should thus probably be performed more widely, particularly in unexplained cases of neuromuscular and multisystemic disorders.

Molecular characterization of 82 patients with pyruvate dehydrogenase complex deficiency. Structural implications of novel amino acid substitutions in E1 protein.

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 E1ß (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.

Comprehensive cDNA study and quantitative analysis of mutant HADHA and HADHB transcripts in a French cohort of 52 patients with mitochondrial trifunctional protein deficiency.

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.

[Multiple acyl-CoA dehydrogenase deficiency (MADD): a curable cause of genetic muscular lipidosis]. / Déficit multiple en acyl-CoA déshydrogénases : une cause traitable de lipidose musculaire d'origine génétique.

INTRODUCTION: Multiple acyl-CoA dehydrogenase deficiency (MADD) is a rare genetic disease involving fatty acid oxidation. It is due to the deficiency of one of the two electron transporters: electron transfer flavoprotein (ETF) or electron transfer flavoprotein ubiquinone oxydoreductase (ETF-QO). Symptoms begin more often in childhood or in young adulthood with a multisystemic disease with encephalopathy or muscular weakness. CASE REPORTS: We report here two adult cases with ETF-QO deficiency, confirmed by mutation analysis (ETFDH gene), revealed by a muscular weakness associated with muscle lipidosis. One of our patients presented an acute encephalopathy with vomiting ten years before the onset of muscular symptoms. The second patient exhibited a slowly progressive pelvic girdle muscle weakness. Diagnosis was established by characteristic abnormalities of acylcarnitine profile by tandem mass spectrometry. For both patients, a dramatic clinical improvement was observed under treatment with riboflavine and L-carnitine. CONCLUSION: Since it is a treatable disorder, this diagnosis must be considered by performing an acylcarnitine profile in all patients presenting with an unexplained muscular weakness.

Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer.

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.

Carnitine-acylcarnitine translocase deficiency with severe hypoglycemia and auriculo ventricular block. Translocase assay in permeabilized fibroblasts.

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.

Arrhythmias and conduction defects as presenting symptoms of fatty acid oxidation disorders in children.

BACKGROUND: The clinical manifestations of inherited disorders of fatty acid oxidation vary according to the enzymatic defect. They may present as isolated cardiomyopathy, sudden death, progressive skeletal myopathy, or hepatic failure. Arrhythmia is an unusual presenting symptom of fatty acid oxidation deficiencies. METHODS AND RESULTS: Over a period of 25 years, 107 patients were diagnosed with an inherited fatty acid oxidation disorder. Arrhythmia was the predominant presenting symptom in 24 cases. These 24 cases included 15 ventricular tachycardias, 4 atrial tachycardias, 4 sinus node dysfunctions with episodes of atrial tachycardia, 6 atrioventricular blocks, and 4 left bundle-branch blocks in newborn infants. Conduction disorders and atrial tachycardias were observed in patients with defects of long-chain fatty acid transport across the inner mitochondrial membrane (carnitine palmitoyl transferase type II deficiency and carnitine acylcarnitine translocase deficiency) and in patients with trifunctional protein deficiency. Ventricular tachycardias were observed in patients with any type of fatty acid oxidation deficiency. Arrhythmias were absent in patients with primary carnitine carrier, carnitine palmitoyl transferase I, and medium chain acyl coenzyme A dehydrogenase deficiencies. CONCLUSIONS: The accumulation of arrhythmogenic intermediary metabolites of fatty acids, such as long-chain acylcarnitines, may be responsible for arrhythmias. Inborn errors of fatty acid oxidation should be considered in unexplained sudden death or near-miss in infants and in infants with conduction defects or ventricular tachycardia. Diagnosis can be easily ascertained by an acylcarnitine profile from blood spots on filter paper.

[Congenital galactosaemia: an unusual presentation]. / La galactosémie congénitale: une révélation singulière.

Congenital galactosaemia reveals usually in the second and third weeks of life with a severe liver dysfunction. We report on a case of congenital galactosaemia with, on the one hand, an early onset liver failure, without any free interval, and on the other hand, an hemophagocytic syndrome as a severe secondary outbreak with pulmonary haemorrhage. Appropriate diet led to normalisation of liver function. Hemophagocytosis, probably linked to an associated Klebsiella Pneumoniae sepsis, had a favourable outcome after antibiotic and corticosteroid therapy.

Genotype/phenotype correlation in carnitine palmitoyl transferase II deficiency: lessons from a compound heterozygous patient.

Carnitine palmitoyl transferase II deficiency, an inherited disorder of long-chain fatty acid oxidation, may result in either a mild form (muscle disease in adults) or a severe form (hepatocardiomuscular syndrome in infants). The difference in severity between these two forms is related to a difference in levels of residual carnitine palmitoyl transferase II activity and long-chain fatty acid oxidation and in genotypes. Few data are, however, available regarding compound heterozygotes for a 'mild' and a 'severe' carnitine palmitoyl transferase II mutation. We report on such a patient carrying both the 'mild' S113L substitution and the 'severe' Y628S mutation. The patient's clinical picture (cardiac arrest at 6 years) was markedly more serious than usually observed in S113L homozygotes, and suggested that 'mild'/'severe' compound heterozygosity makes patients at risk from life-threatening events. Palmitate oxidation and carnitine palmitoyl transferase II activity were lower in lymphocytes from the S113L/Y628S patient than in those from a S113L homozygote. Thus, assessment of carnitine palmitoyl transferase II mutations, long-chain fatty acid oxidation, and carnitine palmitoyl transferase II activity, may help in predicting the potential severity of the muscular form of carnitine palmitoyl transferase II deficiency.

Enhancement of normal polymorphonuclear cells respiratory burst in ascitic fluid by fibronectin. Comparison between cirrhotic and malignant ascitic fluids.

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.

The Philadelphia variant of galactokinase in human erythrocytes: physicochemical and catalytic properties.

The Philadelphia variant of galactokinase (GALKP) is responsible for an asymptomatic disorder of galactose metabolism. Individuals with GALKP phenotype are common among black people. They exhibit reduced galactokinase (GALK) activity in their red blood cells but normal activity in their white blood cells. We explored the biochemical characteristics of hemolysates from individuals with the GALKP phenotype and from controls. In mixed hemolysates from a control and a proband, the GALK activity measured did not suggest the presence of an inhibitor. We observed that the catalytic properties, pI and thermolability in hemolysates from controls and GALKP individuals were identical. Thus, the Philadelphia variant of galactokinase seems not to alter biochemical properties of the red blood cell enzyme. A silent amino acid substitution, or the dysfunction of a regulatory gene might be likely suggested to explain the reduced enzyme activity.

The Philadelphia variant of galactokinase: impaired [1-14C]galactose oxidation by intact erythrocytes.

Probands with the Philadelphia variant of galactokinase (GALKP) are black people who exhibit reduced galactokinase (GALK) activity in their red blood cells (RBC), but normal activity in their white blood cells (WBC). This reduced RBC GALK was demonstrated in disrupted erythrocytes. To investigate the possibility of a missing cofactor in hemolysates from individuals with GALKP phenotype, we compared [1-14C]galactose oxidation by intact erythrocytes, with the direct GALK assay in disrupted erythrocytes. The rate of [1-14C]glucose oxidation was also measured in order to differentiate an impaired galactose metabolism from a defect further along the pentose phosphate pathway. A good correlation (p less than 0.001) was found between the direct GALK assay and [1-14C]galactose oxidation in control subjects, which indicates that this method can be used effectively for the detection of GALK defects. This was further supported by studies on samples from heterozygotes and homozygotes for the GALKG deficient gene. For all the probands with a GALKP phenotype, diminished CO2 production from galactose was observed in the absence of impaired glucose metabolism. This allowed us to confirm the existence of a GALK deficiency in intact erythrocytes due to the GALKP variant. Further studies of RBC GALK catalytic properties are needed to investigate the molecular basis of this GALK deficiency.

Mitochondrial very-long-chain acyl-coenzyme A dehydrogenase deficiency: clinical characteristics and diagnostic considerations in 30 patients.

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.

Rapid diagnosis of long chain and medium chain fatty acid oxidation disorders using lymphocytes.

A method based on the release of tritiated water from [9,10(n)-3H] palmitic and myristic acids previously described for fibroblasts, was adapted for lymphocytes for the rapid diagnosis of fatty acid oxidation disorders. Optimal concentrations for both substrates and linearity of the assay were established. Normal values were established in control subjects of different age groups (58 children and 117 adults) and 16 patients with known fatty acid oxidation disorders were tested. Tritiated water production from patients' lymphocytes was expressed as a ratio between residual oxidations of palmitate and myristate and the results show that this method allows good differentiation between long chain and medium chain fatty acid oxidation defects.

[Occurrence and nature of hyperamylasemia in chronic alcoholics]. / Prévalence et nature de l'hyperamylasémie chez les alcooliques chroniques.

The purpose of this work was: a) the prospective study of the prevalence of hyperamylasemia in 100 patients with chronic alcoholism; b) the determination of the serum isoamylase distribution in patients with hyperamylasemia by an inhibitor assay; c) the search of the origin of elevated serum isoamylase S. Moderate hyperamylasemia was found in 15 patients. The importance of alcohol abuse, the prevalences of cirrhosis and smokers were not statistically different from those observed in normoamylasemic patients. After one week of hospitalization, serum amylase was still elevated in 11 of 14 alcoholic patients. Hyperamylasemia was due to an increase in the isoamylase P in 5 cases, in the isoamylase S in 7 cases, and in both forms in 3 cases. Activities of serum lipase and isoamylase P were roughly parallel. Only two out of 8 patients with elevated isoamylase P had chronic pancreatitis. The salivary origin of elevated isoamylase S was suspected in only one out of 10 patients. This work shows that the origin of moderate hyperamylasemia, observed in alcoholic patients, is often extrapancreatic. It is suggested that the dosage of serum lipase simpler than that of isoamylases, may be routinely used in chronic alcoholic patients for diagnostic purposes.

[Late neurologic complications of galactosemia: study of 3 cases]. / Complications neurologiques tardives de la galactosémie: étude de trois cas.

Galactosemia is an autosomal recessive, inborn error of galactose metabolism due to the deficiency of galactose-I-phosphate uridyl transferase. Late-onset neurologic complications may develop despite Galactose restriction. Three adult patients are reported. They suffered from mental retardation. Two of them developed progressive cerebellar ataxia, spastic gait and postural tremor. The magnetic resonance imaging revealed moderate cortical atrophy, multifocal areas of increased signal in the periventricular white matter on T2-weighted images, and in one case, abnormal myelination. The Fluoro-2-deoxy-D-glucose position emission tomography showed different patterns of regional hypometabolism.

[Hereditary abnormalities of galactose metabolism: diagnosis and biochemical supervision (author's transl)]. / Anomalies héréditaires du métabolisme du galactose: diagnostic et surveillance biochimiques.

The authors define the main stages of the biochemical study of hereditary abnormalities of galactose metabolism. They review laboratory examinations for detection, enzyme examinations which provide the diagnostic proof, further examinations which permit one to follow the course and efficacy of a galactose-free diet, the demonstration of genetic variants, the technics of antenatal diagnosis and routine neonatal detection.

[Severe Reye syndrome: report of 14 cases managed in a pediatric intensive care unit over 11 years]. / Syndrome de Reye sévère: à propos de 14 cas pris en charge dans une unité de réanimation pédiatrique pendant 11 ans.

UNLABELLED: Idiopathic Reye syndrome is a rare disease revealed by unexplained encephalopathy and microvesicular liver steatosis. Some clinical and epidemiological studies mainly performed in English speaking countries questioned the reality of Reye syndrome because numerous know inherited metabolic diseases, and some of them unrecognized, could mimick this disorder. We focused in our study on severe forms of Reye syndrome admitted to a pediatric intensive care unit. METHODS: Retrospective study over the last eleven years (1991-2001) included all the pediatric patients admitted to our tertiary referral center with the classical American Reye syndrome criteria (e.g. CDC). Extensive metabolic screening was performed in all cases, except for the ultimately dead patients. RESULT: Fourteen patients (mean age 52 months) were included. Fever always occurred before their admission and aspirin (n = 12) or acetaminophen (n = 7) was prescribed. Median Glasgow scale was 7 on admission. Mean amoniac plasma level was 320 mumol/L and alanine-aminotransferase peak plasma level 1475 +/- 1387 IU/L. Mechanical ventilation was started in ten children and six of them underwent continuous venovenous hemofiltration. Three patients ultimately died and 11 survived with a mean five years follow-up without relapses or neurological impairment. Any of them demonstrated inherited metabolic disease except for one infant with hereditary fructose intolerance. CONCLUSION: Unlike widespread opinion, severe Reye syndrome without identified metabolic disorders seems to not disappear in our country. Reye syndrome remains a potentially life threatening disease and raises for aggressive treatment of brain edema. If aspirin and Reye syndrome association are not formally documented in France, cautiousness must be kept in mind and all the aspirin adverse effects notifications should be addressed to the public drugs survey network.