Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes. In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease. DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays. Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio) for “homozygous” DNA from healthy individuals. “Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected. Genotyping by this method was therefore reliable. This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis

Focus on 1,25-Dihydroxyvitamin D3 in the Peripheral Nervous System

In this review, we draw attention to the roles of calcitriol (1,25-dihydroxyvitamin D3) in the trophicity of the peripheral nervous system. Calcitriol has long been known to be crucial in phosphocalcium homeostasis. However, recent discoveries concerning its involvement in the immune system, anti-cancer defenses, and central nervous system development suggest a more pleiotropic role than previously thought. Several studies have highlighted the impact of calcitriol deficiency as a promoting factor of various central neurological diseases, such as multiple sclerosis, amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease. Based on these findings and recent publications, a greater role for calcitriol may be envisioned in the peripheral nervous system. Indeed, calcitriol is involved in myelination, axonal homogeneity of peripheral nerves, and neuronal-cell differentiation. This may have useful clinical consequences, as calcitriol supplementation may be a simple means to avoid the onset and/or development of peripheral nervous-system disorders

Effect of Leucovorin (Folinic Acid) on the Developmental Quotient of Children with Down's Syndrome (Trisomy 21) and Influence of Thyroid Status

International audienceBACKGROUND: Seven genes involved in folate metabolism are located on chromosome 21. Previous studies have shown that folate deficiency may contribute to mental retardation in Down's syndrome (DS). METHODOLOGY: We investigated the effect of oral folate supplementation (daily dose of 1.0+/-0.3 mg/kg) on cognitive functions in DS children, aged from 3 to 30 months. They received 1 mg/kg leucovorin or placebo daily, for 12 months, in a single-centre, randomised, double-blind study. Folinic acid (leucovorin, LV) was preferred to folic acid as its bioavailability is higher. The developmental age (DA) of the patients was assessed on the Brunet-Lezine scale, from baseline to the end of treatment. RESULTS: The intent-to-treat analysis (113 patients) did not show a positive effect of leucovorin treatment. However, it identified important factors influencing treatment effect, such as age, sex, and concomitant treatments, including thyroid treatment in particular. A per protocol analysis was carried out on patients evaluated by the same examiner at the beginning and end of the treatment period. This analysis of 87 patients (43 LV-treated vs. 44 patients on placebo) revealed a positive effect of leucovorin on developmental age (DA). DA was 53.1% the normal value with leucovorin and only 44.1% with placebo (p<0.05). This positive effect of leucovorin was particularly strong in patients receiving concomitant thyroxin treatment (59.5% vs. 41.8%, p<0.05). No adverse event related to leucovorin was observed. CONCLUSION: These results suggest that leucovorin improves the psychomotor development of children with Down's syndrome, at least in some subgroups of the DS population, particularly those on thyroxin treatment. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00294593

Etude de la pertinence du diagnostic biologique de la maladie d'Alzheimer à partir de l'expérience du CHU Dupuytren (apport du dosage du peptide Ab1-40)

LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

Mécanismes physiopathologiques responsables des troubles de la motilité intestinale étudiés sur 2 modèles expérimentaux d'atrésie intestinales chez le rat

L atrésie intestinale est une malformation congénitale du tube digestif qui nécessite une intervention rapide. Les suites opératoires sont grevées par des troubles fonctionnels de l alimentation nécessitant un support nutritionnel parentéral avec une longue hospitalisation et une morbidité élevée conduisant parfois au décès de ces patients. Des anomalies du développement du système nerveux entérique et de l épithélium ont été pressenties comme étant à l origine de ces troubles fonctionnels. Ces anomalies seraient prédominantes pour certains soit en amont de l obstacle, soit, pour d autres, en aval de l atrésie. Pour préciser cela, nous avons développé deux modèles animaux originaux de création d obstruction du tube digestif chez le rat qui nous ont permis d effectuer des études immunohistochimiques à l aide de marqueurs de la différenciation du SNE et de l épithélium. Nous avons montré que des anomalies de développement du plexus myentérique prédominaient en aval de l obstacle intestinal généré dans ce modèle par une ligature intestinale. Nous avons également pu reproduire un type d atrésies survenant chez l homme, les atrésies intestinales multiples en utilisant un second modèle tératogénique consistant à injecter de l adriamycine par voie intra-péritonéale à des rates gestantes. L étude des fragments atrétiques a confirmé l existence d anomalies du développement du SNE post-atrétique et a permis de mettre en évidence le rôle des facteurs endocrines dans la maturation du SNE dans ce contexte pathologique.Small bowel atresia remains a challenging problem among neonatal digestive disorders. In all cases, the treatment is based on surgical intervention consisting of resection of the obstructed part of the bowel followed by a primary anastomosis. A successful surgical correction and an adequate length of small intestine are not always sufficient to obtain a full recovery of the bowel. In fact, in about 20% of treated newborns, small bowel function remains not optimal, making it impossible to give these children a normal oral feed. Disturbances in enteric nervous system (ENS) and in epithelium development have been suspected in the pathogenesis of these disorders. A part of the authors consider that these abnormalities are mainly observed in the upper part of the obstruction. For the others, such disturbances are mainly observed in the lower part. Thus, we developed 2 animal models of intestinal obstruction in the rat using immunohistochemical reagents involved in epithelium and enteric nervous system differentiation. We demonstrated in the lower part of a surgical induced obstruction, a delay in the maturation of the ENS. Using a teratogenic model (adriamycin), we also reproduced multiple gastrointestinal atresia, a rare kind of human intestinal atresia with a poor prognosis. We also demonstrated the major role of amniotic fluid digestion and mesenteric blood supply in the ENS maturation.LIMOGES-BU Médecine pharmacie (870852108) / SudocSudocFranceF

Myenteric plexus alterations downstream from a prenatal intestinal obstruction in a rat model.

International audienceThe enteric nervous system maturation occurs during embryonic life and continues after birth. Some prenatal events on the digestive tract such as intestinal atresia have been shown to dramatically alter this maturation. Thus, we developed a fetal rat model of intestinal atresia by surgically obstructing the small bowel at embryonic day E18. Fetuses were removed at day E21, and small bowels sections were examined by immuno-histochemistry. Synaptophysin and smooth muscle actin staining was used to define the cellular aspect. Labeling revealed marked alterations of the myenteric plexus in the lower extremity of the occluded small bowel. At day E21, the myenteric plexus of the lower part and the 2 muscle layers surrounding it, retained the staining pattern observed at day E17. This cellular pattern was classified as: immature (aspect at day E17) vs. mature (aspect of day E21) by 3 pathologists not familiar with the study. The number of samples with an immature cellular pattern at the lower end of the occluded bowel was different from that observed for the upper end (Mac Nemar test, p<0.008). Our study suggests that a prenatal obstruction induces a maturation delay of the myenteric plexus downstream of the obstruction. This might be important for pediatric purposes

High metabolic level in patients with familial amyotrophic lateral sclerosis.

International audienceAn abnormally elevated level of resting energy expenditure (REE, measured by indirect calorimetry) has been reported in a subset of patients with sporadic amyotrophic lateral sclerosis (SALS). Hypermetabolism (measured REE/calculated REE (cREE) >/= 1.1, or 110%) has also been observed in transgenic mice harbouring ALS-causing mutations in the SOD1 gene. By contrast, the REE of patients with familial amyotrophic lateral sclerosis (FALS) has never been assessed. Our objective was to evaluate the metabolic and nutritional parameters of FALS patients and to compare them with those of SALS patients, and search for correlations with clinical parameters. Eleven patients with FALS (from 10 different families, none carrying a SOD1 mutation) were evaluated by indirect calorimetry in our centre. As a control group, we used a sample of 33 patients with SALS, matched for age and sex with the FALS patients. 11/11 (100%) patients with FALS were hypermetabolic, compared to 17/33 (52%) patients with SALS (p =0.009). Measured REE (mREE) and mREE/cREE (metabolic level) were significantly higher in FALS patients than in SALS patients (p =0.03 and p =0.0008, respectively). No correlation was found between metabolic measures and neurological or respiratory parameters. In conclusion, hypermetabolism appears to be a common feature of subjects with FALS, suggesting that this impairment of energy homeostasis may be genetically driven. The high metabolic level of FALS patients should be taken into account for their nutritional management (need for a high-energy diet to prevent malnutrition)

[Ultrastructural lesions of axonal mitochondria in patients with childhood-onset Charcot-Marie-Tooth disease due to MFN2 mutations] : Anomalies ultrastructurales des mitonchondries axonales chez des patients atteints de formes précoces de maladie de Charcot-Marie-Tooth dues à des mutations de la mitofusine 2

National audienceWe present neuropathological findings based on sural nerve biopsy in six children with mutations of the mitofusin 2 gene (MFN2). All six children had severe axonal neuropathies (mild or severe hereditary motor and sensory neuropathy, HMSN), with onset in early childhood. All had a marked decrease in the density of mainly large myelinated fibers. Although neurophysiological findings were suggestive of axonal degeneration, some onion bulbs were present in each case. Unequivocal mitochondrial changes were apparent only on longitudinal sections. Many axonal mitochondria appeared smaller than normal and round or spherical instead of tubular. These mitochondria were abnormally aggregated, accumulating primarily at the axon periphery. This peripheral distribution was clearest in residual large myelinated fibers. The inner and outer mitochondrial membranes were irregular, and the cristae were quite often disrupted. These changes were observed in both myelinated and unmyelinated fibers. Mitofusin 2 is a large mitochondrial transmembrane GTPase, with two coiled coil domains and two transmembrane spans. It is targeted to the outer mitochondrial membrane, where it interacts with mitofusin 1 to regulate the mitochondrial network architecture by stimulating mitochondrialfusion. The mitochondrial changes we observed could thus result from abnormal mitochondrial fusion and fission. Neuropathologic abnormalities can be sufficiently characteristic to suggest the genetic basis of some hereditary neuropathies such as those associated with mutations in MPZ, GJB1, GDAP1, MTMR2, SH3TC2, PRX, FGD4 and LMNA. This may also be true of MFN2-related neuropathies

Contribution of electron microscopy to the study of neuropathies associated with an IgG monoclonal paraproteinemia.

A typical monoclonal IgG dysglobulinemia whether benign (monoclonal gammopathy of undetermined significance, MGUS) or malignant can give rise to peripheral neuropathy by damaging nerves. At first, neurotoxicity of the chemotherapy if the patient is treated must be ruled out in such cases. Indeed, a variety of other mechanisms have been described: endoneurial deposits of immunoglobulin, infiltration of the immunoglobulin within myelin sheaths, POEMS syndrome, deposits of amyloid, chronic inflammatory demyelinating polyradiculoneuropathy and infiltration of malignant cells. Ultrastructural examination of a nerve biopsy can be decisive in combination with routine histological and immunopathological examinations. Characterization of the mechanism of the neuropathy in a dysglobulinemic context is important as it governs therapeutic options, which in certain cases are particularly beneficial