NTBC treatment in tyrosinaemia type I: long-term outcome in French patients.

We describe a retrospective study of long-term outcome of 46 patients treated and regularly followed in France with 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3-cyclohexanedione (NTBC) for tyrosinaemia type I. Most had initial good response with normalization of liver function and metabolic parameters. Only one infant had no response to treatment and required liver transplantation. Among the 45 long-term treated patients, three underwent secondary liver transplantation: one for cirrhosis and two because of hepatocellular carcinoma. One of the latter died of transplantation complications, so that the overall survival rate was 97.5%. However, 17 of 45 showed persistent abnormal liver imaging (heterogeneous liver) and 6 had cirrhosis. Furthermore, 15 had persistently elevated levels of alpha-fetoprotein, highlighting the question of the persistent risk of carcinoma. Quality of life was usually good but compliance problems were frequent, mainly regarding the low phenylalanine-tyrosine diet. Few adverse effects were observed. A main concern was the high frequency of cognitive impairment causing schooling problems, which may be related to persistent chronic hypertyrosinaemia. In conclusion, this series confirms that NTBC treatment has clearly improved the vital prognosis and quality of life of tyrosinaemia type I patients but that many late complications persist. Long-term studies are necessary to determine whether this drug may prevent or only delay liver complications, andto survey the possible risks of the drug. A more restricted diet could be necessary to prevent the neurological impact of the disease.

[Late diagnosis of a McArdle disease's case (type V glycogenosis)]. / Diagnostic tardif d'une maladie de McArdle (glycogénose de type V).

McArdle's disease is a metabolic myopathy characterized by a myophosphorylase deficiency resulting in an inability to degrade glycogen stores. We report the case of a 48 years old patient who complained since adolescence of rest and exercise myalgias and presented a chronic increased plasma creatine kinase activity. First, a maximal exercise test was performed. This test demonstrated a quasi lack of rise of respiratory exchange ratio and of blood lactate, possibly due to a glycogenolytic/glycolytic pathway deficiency. Second, a biopsy of vastus lateralis muscle was performed using Bergström needle. As expected, the analysis of mitochondrial function was normal. The in vitro screening test of the glycogenolysis/glycolysis pathway showed a lack of lactate production in presence of glycogen substrate. The study of muscular metabolism of glycogen revealed a glycogen accumulation and a decrease of active and total phosphorylase activities. These data allowed us to diagnose a type V glycogenosis, or McArdle's disease. The patient appeared heterozygous for the most frequent mutation (p.R50X).

[Severe hyperhomocysteinemia revealing homocystinuria in two young adults with mild phenotype]. / Hyperhomocystéinémie sévère révélant une homocystinurie chez deux jeunes adultes présentant un phénotype peu marqué

INTRODUCTION: To the request of total plasma homocysteine determination in the investigation of vascular disease, diagnosis of homocystinuria in young adult patients with mild phenotype is not so rare. EXEGESIS: A 26-year-old man developed embolic cerebral infarction and a 22-year-old woman presented a right renal venous thrombosis one week after delivery. In each case, high concentration of total plasma homocysteine was first found and plasma and urinary amino acids analysis later on directed the diagnosis towards homocystinuria. Finally, reduced skin fibroblast cystathionine beta-synthase activity confirmed the diagnosis of homocystinuria. CONCLUSION: Total plasma homocysteine determination must be determined for screening for hyperhomocysteinemia in young adults with venous thromboembolism without characteristic phenotypic features of homocystinuria.

Identification of 5 novel mutations in the AGXT gene.

In order to identify additional genotypes in primary hyperoxaluria type 1, we sequenced the AGXT genes of 9 patients. We report 5 new mutations. Three are splice-site mutations situated at the end of intron 4 and 8 (647-1G>A, 969-1G>C, 969-3C>G), one is a missense mutation in exon 5 (D183N), and one is a short duplication in exon 2 (349ins7). Their consequence is always a lack of enzymatic activity of the Alanine-Glyoxylate Aminotransferase (AGT); for 4 of them, we were able to deduce that they were associated to the absence of AGT protein. These mutations are rare, as they have been found on one allele in our study (except 969-3C>G present in 2 unrelated families), and have not been previously reported.

Partial deletion of the AGXT gene (EX1_EX7del): A new genotype in hyperoxaluria type 1.

Primary hyperoxaluria type 1 (PH1) is a rare autosomal (2q37.3) recessive metabolic disease caused by a deficiency of the hepatic peroxisomal enzyme alanine:glyoxylate amino transferase. Molecular heterogeneity is important in PH1 as most of the patients (if the parents are unrelated) are compound heterozygotes for rare mutations. We describe the first large deletion in the AGXT gene, removing exons 1 to 7 (EX1_EX7del) that was responsible for one case of severe PH1. This 10 kb deletion was identified by Southern blotting of genomic DNA digested by Xba I and hybridized with different exonic probes. Both parents (from Turkey) are first cousin and carry the deletion. It is of note that the presently reported patient did not exhibit any AGT catalytic activity and even so, he progressed towards end-stage renal disease only at 19 years old.

Erythrocyte ghost (Na+ + K+) ATPase activity in mice with hereditary muscular dystrophy (strain C57 BL/64J/dy).

Erythrocyte ghost (Na+ + K+) ATPase activity was studied in mice with hereditary muscular dystrophy (strain C 57 BL 6J/dy) and appropriate controls. No difference was observed in the enzymatic activity between dystrophic and any of the healthy genotypes. Ouabain 5 mM and 0.1 mM inhibited the enzymatic activity and no difference was observed between dystrophic and control animals. The results are discussed in the light of the literature.

Metabolic studies in twin brothers with 2-methylacetoacetyl-CoA thiolase deficiency.

We report clinical and biological investigations in two patients (twin brothers) with 2-methylacetoacetyl-CoA thiolase deficiency. Main clinical features included important staturo-ponderal delay, frequent infectious rhinopharyngitis episodes and an acute metabolic acidosis at the age of 4 years, this metabolic decompensation being adequately halted by bicarbonate supplementation. Since that age, patients developed rather favorably, however, with persistence of the staturo-ponderal delay. Organicaciduria typical of 2-methylacetoacetyl-CoA thiolase deficiency was recorded consisting of excessive excretion of tiglylglycine, 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-methylglutaconate, adipate and 2-methylacetoacetate. Blood carnitine levels were altered in patients with increased total and esterified carnitine concentrations and enhanced acyl/free carnitine ratios. Determination of acylcarnitine profiles showed that patients excreted excessive amounts of several acylcarnitines in urine including propionyl, butyryl, isobutyryl, isovaleryl, 2-methylbutyryl and tiglyl-carnitine, the latter acylcarnitine being prominent with, in one of the patients, occurrence of a previously undescribed isomer of this carnitine ester, possibly 2-ethylacrylyl-carnitine. Excretion of these acylcarnitines in urine was increased in response to L-carnitine although, as a whole, this therapy resulted in a less important stimulation of esterified carnitine removal in urine from patients than in the case of supplemented controls. Biochemical investigations on cultured skin fibroblasts confirmed 2-methylacetoacetyl-CoA thiolase deficiency. Through the present report on this rare disease in two siblings, we would like to underline that acylcarnitines can be used in the diagnosis of 2-methylacetoacetyl-CoA thiolase deficiency, a view supported by acylcarnitine profiles further determined in another patient with proven oxothiolase deficiency, adding this pathology to the list of beta-oxidation disorders that may be screened successfully through determination of acylcarnitine profiles in body fluids.

An atypical French form of pyruvate carboxylase deficiency.

A further case of pyruvate carboxylase deficiency, French type, with a particular clinical presentation and evolution is described. The initial neonatal symptoms started with respiratory distress, severe metabolic acidosis and a tendency to hypoglycemia. However, the clinical course was not rapidly deteriorating. At the age of 6 months he presented acute neurological symptoms, respiratory difficulty, lactic acidosis and hyperammonemia. Amino and organic acid abnormalities strongly suggested pyruvate carboxylase deficiency, which was confirmed by enzymatic studies in cultured fibroblasts and liver necropsy. Progressive deterioration and bronchopneumonia with cardiac failure and renal insufficiency led to death. Anatomic-pathologic studies revealed periventricular cysts and diffuse hypomyelination. Prenatal diagnosis of a further sibling was performed. The neonatal clinical presentation, biochemical abnormalities, and the presence of periventricular cysts suggested a French phenotype. However, the clinical course was less severe, suggesting a residual enzymatic activity and a possible milder mutation.

Mevalonic aciduria in 3 siblings: a new recognizable metabolic encephalopathy.

Mevalonic aciduria, due to mevalonate kinase deficiency, is the first recognized defect in the biosynthesis of cholesterol and isoprenoids. Very few patients with this disorder have been reported. Three siblings born from consanguineous parents are reported. Several clinical signs were present in all 3 children, including failure-to-thrive, susceptibility to infections, hepatosplenomegaly, cataract, and psychomotor retardation. Dysmorphic features were more apparent in the two older siblings. Urinary organic acid analysis by gas chromatography/mass spectrometry invariably revealed a high urinary excretion rate of mevalonic acid. Mevalonate kinase activity assayed in fibroblasts was very low. Diagnosis of this very rare disease may be suspected simply on clinical evidence; it is confirmed by abnormal excretion of mevalonic acid.

Common resistance mechanisms to nucleoside analogues in variants of the human erythroleukemic line K562.

Variants of the human K562 were developed against the nucleoside analogues cytosine arabinoside, 2 chlorodeoxyadenosine, fludarabine and gemcitabine. The resistant lines displayed a high degree of cross-resistance to all nucleoside analogues, with little or no cross resistance to other agents. There was a profound accumulation defect of the different nucleoside analogues in all of the variants. There was a strong overexpression of 5'nucleotidase, measured by rt-PCR and enzyme activity, in all resistant variants. There was a two fold increase of ribonucleotide reductase in the fludarabine resistant line and increased expression of purine nucleoside phosphorylase in the 2 chlorodeoxyadenosine selected line. Karyotypic analysis revealed the loss of a 6(q16;q22) deletion present in the parental line in all of the resistant lines. This portion of chromosome 6 has been shown to contain the gene for 5'nucleotidase. Early events in the transport and metabolism appear to be involved in the resistance mechanisms to nucleoside analogues and are responsible for broad cross resistance to this family of compounds.

[Hyperglycinemia without ketosis. Biochemical and enzymatic study]. / Hyperglycinémie sans cétose. Etude biochimique et enzymatique.

We report a biochemical and enzymatic study of two neonatal cases of non ketotic hyperglycinemia. We report the comparative evolution of glycine level in plasma and CSF during a restrictive diet excluding glycine and serine. The high levels of glycine found in CSF and brain are likely to reflect the brain damage. After autopsy, the glycine synthase activity determination shows a significative partial deficiency in the liver and a total deficiency in the brain. Glycine synthase affinity for glycine is similar for controls and patients and this lead use to think that the deficiency is due to a diminished biosynthesis of the enzyme molecule.

[Delayed diagnosis of homocystinuria by major deficiency in cystathionine beta synthase]. / Diagnostic tardif d'homocystinurie par déficit majeur en cystathionine beta synthase.

INTRODUCTION: Homocystinuria due to cystathionine beta synthase (CBS) deficiency is a special type of hyperhomocysteinemia because of its clinical expression (thrombotic events, ectopic lens and mental retardation). It's a rare, hereditary recessive autosomic disease generally diagnosed during childhood. EXEGESIS: Thrombophilia examination in a 50-year-old man found a dramatically increase homocysteinemia. Homocystinuria, profile of plasmatic amino acids and reduced CBS activity, (0.05 microkat/kg protein; N = 1.5 +/- 0.8) confirmed homocystinuria's diagnosis. Family study demonstrates that three siblings suffer from homocystinuria. Vitamin enriched diet with pyridoxin, vitamin B12 and folates induced reducing hyperhomocysteinemia and homocystinuria. CONCLUSION: This case report, original because of the diagnosis age, suggests a hyperhomocysteinemia's screening in patients with recurrent thrombotic events.

[Late onset 3-HMG-CoA lyase deficiency: a rare but treatable disorder]. / Déficit en 3-HMG-CoA lyase à révélation tardive : savoir reconnaître une maladie rare mais traitable.

3-Hydroxy-3-methylglutaric aciduria is a rare autosomal recessive genetic disorder due to a deficiency of the 3-hydroxy-3-methylglutarylCoA lyase (HMG-CoA lyase), a mitochondrial enzyme involved in ketogenesis and in the final step of l-leucine catabolism. HMG-CoA lyase deficiency can lead, in particular circumstances, such as fever, prolonged fasting or digestive disorders, to brutal and severe hypoglycemia with metabolic acidosis and sometimes fatal coma. We report on a new case of 3-hydroxy-3-methylglutaric aciduria particular by its late onset in a 3-year-old patient. Molecular investigation identified two new sequence modifications in the HMGCL gene: c.494G>A (p.Arg165Gln) and c.820G>A (p.Gly274Arg). We remind about this case report that the therapeutical is mainly preventive and allows a very good prognosis for this disease. Long-term treatment consists in limited fasting time, continuous low protein diet and l-carnitine supplementation. Preventive measures are essential: prevention of fasting and emergency treatment during intercurrent infections.

Diagnostic value of serum immunoglobulinaemia D level in patients with a clinical suspicion of hyper IgD syndrome.

OBJECTIVE: The hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) was originally defined by the presence of a high serum level of immunoglobulin D associated with recurrent fever. Since the discovery of the mevalonate kinase gene (MVK) gene encoding the mevalonate kinase enzyme, most patients with a clinical diagnostic of HIDS are now found to have a mevalonate kinase deficiency based on metabolic and genetic data. We aimed to asses the value of a high IgD serum level for the diagnosis of HIDS in a cohort of patients with a phenotype of recurrent fever, and to characterize patients with a high IgD serum level without mevalonate kinase mutation. METHODS: Main clinical and biological data of 50 patients who presented with clinical signs compatible with HIDS have been prospectively registered on a standard form. Clinical data have been analysed according the IgD serum level and the presence of MVK mutation. RESULTS: The metabolic and genetic data establishing the diagnosis of HIDS correlated in all cases. In this series of 50 patients, the sensitivity of a high IgD value for the diagnosis of HIDS is 0.79. In five patients with MVK mutation, IgD levels were found to be in the normal range. Likelihood ratios indicate that IgD measurement is not relevant for the diagnostic of HIDS. Most patients with a high serum IgD level and no MVK mutation have no definite diagnosis. CONCLUSION: The clinical relevance of the IgD measurement for the diagnosis of MKD in our population appears as poor, as reflected by likelihood ratios which are both close to 1.

Genetic heterogeneity of the GLDC gene in 28 unrelated patients with glycine encephalopathy.

Glycine encephalopathy, or nonketotic hyperglycinaemia (NKH; Mckusick 238300) is a severe autosomal recessive disease due to a defect in the glycine cleavage system (GCS), which is a complex of four subunits: P-, T-, H- and L-proteins. A P-protein (glycine decarboxylase or GLDC) deficiency was reported in about 80% of NKH patients. We performed mutation analysis of the complete coding sequence of the GLDC gene in 28 unrelated patients with neonatal NKH using denaturing high-performance liquid chromatography (DHPLC) and sequencing. Forty different gene alterations were identified, confirming the large molecular heterogeneity of the GLDC gene. Eighteen alterations were clearly disease-causing: two large deletions, four one-base deletions (c.28delC, c.1175delC, c.2186delC, c.2422delA), one 1-base insertion (c.1002_1003insT), one 4-base insertion (c.1285_1286insCAAA), one insertion/deletion (c.2153_2155delinsTCCTGGTTTA), five nonsense mutations (p.E153X, p.R236X, p.E270X, p.R337X, p.R424X) and four splice site mutations (c.861+1G > T, c.1402-1C > G, c.2316-1G > A, c.2919+1G > A). Additionally, we identified one intronic mutation outside the consensus splice sites (c.2838+5G > A) and 21 nucleotide substitutions leading to amino acid change (including three previously described mutations: p.T269M, p.R461Q, p.G771R), the pathogenicity of which should be confirmed by expression studies (p.S132W, p.Y138F, p.G171A, p.T187K, p.R212K, p.T269M, p.R373W, p.I440N, p.R461Q, p.N533Y, p.C644F, p.H651R, p.V705M, p.N732K, p.G771R, p.H775R, p.T830M, p.A841P, p.D880V, p.S957P and p.R966G). Mutation analysis allowed us to identify sequence alterations in both alleles for 19 patients and in one allele for 7 patients One patient was carrying three mutations (p.Y138F, p.T269M and p.E153X) and one patient was carrying two amino acid substitutions on the same allele (p.V705M and p.R212K) and an unidentified mutation on the other allele. No mutation could be found in two patients, suggesting possible defects in the H-protein or gene alterations that could not be identified by our technique. The potential use of genotype determination for prenatal diagnosis is emphasized.

False-positive results in neonatal screening for cystic fibrosis based on a three-stage protocol (IRT/DNA/IRT): Should we adjust IRT cut-off to ethnic origin?

Since 1979, newborn screening for cystic fibrosis (CF) has been possible by measuring immunoreactive tryspinogen (IRT) in blood spots. In France, a programme based on a three-stage strategy (IRT/DNA/IRT) started in 2002. In the Rhône-Alpes area, the positive screening rate (i.e. the proportion of samples sent for genotyping) observed after the first IRT measurement was higher than the expected rate (0.65% versus 0.50%), without a greater CF incidence. We hypothesized that the IRT reference range could differ according to the ethnic origin of the newborns. 35 141 newborns were studied and divided into two groups: European ethnic group 26 324 (75%) and North African ethnic group 8817 (25%). 243 positive newborns were identified: 146 (60%) in the European ethnic group and 97 (40%) in the North African ethnic group. Three CF patients and 11 unaffected heterozygotes were found in the European group, but no mutations were found in the North African group. Mean IRT values and the percentage of IRT values over the cut-off were significantly higher in the North African group than in the European group (mean IRT = 21.17 microg/L and 19.74 microg/L, p < 0.0001; %IRT > cut-off = 1.1% and 0.5%, respectively). For the positive screened newborns, term and IRT mean were comparable, whereas birth weight was higher in the North African ethnic group. These results lead us to conclude that (i) newborns from families of North African origin have higher IRT values and (ii) most of the positive screened newborns in this population could be considered as 'false positives'. These conclusions could explain, in part, the large variations seen in the positive screening rate in the French CF neonatal screening and raise the question whether it is relevant to adapt cut-off to ethnic origin of the newborns.

First-trimester enzymatic and molecular prenatal diagnosis of mevalonic aciduria.

Prenatal diagnosis was offered to a family at risk of mevalonic aciduria. A chorionic villus sample was obtained and both mevalonate kinase activity and mutation analysis were done. An affected fetus was diagnosed.

Atypical MRI findings in Canavan disease: a patient with a mild course.

Canavan disease is a severe, progressive leukodystrophy with an autosomal recessive inheritance, caused by aspartoacylase (ASPA) deficiency. The characteristic MRI features include diffuse, symmetrical white matter degeneration in the subcortical areas, with bilateral involvement of the globus pallidus. Proton magnetic resonance spectroscopy of the brain shows an increase in the concentration of N-acetylaspartic acid (NAA). The altered NAA metabolism has been traced to mutations in the gene encoding ASPA, located on chromosome 17 (17p13-ter). We present here a patient with a mild form of Canavan disease confirmed with the absent ASPA activity, atypical MRI findings, related to compound heterozygosity for a missense mutation, p.Tyr288Cys, and the known pan-European mutation, the p.Ala305Glu.

Fetal type IV glycogen storage disease: clinical, enzymatic, and genetic data of a pure muscular form with variable and early antenatal manifestations in the same family.

We report on a family of three consecutive fetuses affected by type IV glycogen storage disease (GSD IV). In all cases, cervical cystic hygroma was observed on the 12-week-ultrasound examination. During the second trimester, fetal hydrops developed in the first pregnancy whereas fetal akinesia appeared in the second pregnancy. The diagnosis was suggested by microscopic examination of fetal tissues showing characteristic inclusions exclusively in striated fibers, then confirmed by enzymatic studies on frozen muscle. Antenatal diagnosis was performed on the third and fourth pregnancies: cervical cystic hygroma and low glycogen branching enzyme (GBE) activity on chorionic villi sample (CVS) were detected in the third pregnancy whereas ultrasound findings were normal and GBE activity within normal range on CVS in the fourth pregnancy. Molecular analysis showed that the mother was heterozygous for a c.1471G > C mutation in exon 12, leading to the replacement of an alanine by a tyrosine at codon 491 (p.A491T); the father was heterozygous for a c.895G > T mutation in exon 7, leading to the creation of a stop codon at position 299 (p.G299X). GSD IV has to be considered in a context of cervical cystic hygroma with normal karyotype, particularly when second trimester hydrops or akinesia develop. Enzymatic analysis of GBE must be performed on CVS or amniotic cells to confirm the diagnosis. Characteristic intracellular inclusions are specific to the disease and should be recognized, even in macerated tissues after fetal death. Genetic analysis of the GBE gene may help to shed some light on the puzzling diversity of GSD IV phenotypes.