Autism spectrum disorder recurrence, resulting of germline mosaicism for a CHD2 gene missense variant.

Germline mosaicism for a novel missense variant p.Thr645Met located in the SNF2-related ATP dependent helicase domain of CHD2 in 2 affected siblings with autism spectrum disorder.

Refinement of cortical dysgeneses spectrum associated with TUBA1A mutations.

OBJECTIVE: We have recently shown that de novo mutations in the TUBA1A gene are responsible for a wide spectrum of neuronal migration disorders. To better define the range of these abnormalities, we searched for additional mutations in a cohort of 100 patients with lissencephaly spectrum for whom no mutation was identified in DCX, LIS1 and ARX genes and compared these data to five previously described patients with TUBA1A mutations. RESULTS: We detected de novo TUBA1A mutations in six patients and highlight the existence of a prominent form of TUBA1A related lissencephaly. In four patients, the mutations identified, c.1190T>C (p.L397P), c.1265G>A (p.R422H), c.1264C>T (p.R422C), c.1306G>T (p.G436R), have not been reported before and in two others, the mutation corresponds to a recurrent missense mutation, c.790C>T (p.R264C), likely to be a hot spot of mutation. All together, it emerges that the TUBA1A related lissencephaly spectrum ranges from perisylvian pachygyria, in the less severe form, to posteriorly predominant pachygyria in the most severe, associated with dysgenesis of the anterior limb of the internal capsule and mild to severe cerebellar hypoplasia. When compared with a large series of lissencephaly of other origins (ILS17, ILSX or unknown origin), these features appear to be specific to TUBA1A related lissencephaly. In addition, TUBA1A mutated patients share a common clinical phenotype that consists of congenital microcephaly, mental retardation and diplegia/tetraplegia. CONCLUSIONS: Our data highlight the presence of consistent and specific abnormalities that should allow the differentiation of TUBA1A related lissencephalies from those related to LIS1, DCX and ARX genes.

Mutations in the AP1S2 gene encoding the sigma 2 subunit of the adaptor protein 1 complex are associated with syndromic X-linked mental retardation with hydrocephalus and calcifications in basal ganglia.

Fried syndrome, first described in 1972, is a rare X-linked mental retardation that has been mapped by linkage to Xp22. Clinical characteristics include mental retardation, mild facial dysmorphism, calcifications of basal ganglia and hydrocephalus. A large four-generation family in which the affected males have striking clinical features of Fried syndrome were investigated for linkage to X-chromosome markers; the results showed that the gene for this condition lies within the interval DXS7109-DXS7593 in Xp22.2. In total, 60 candidate genes located in this region, including AP1S2, which was recently shown to be involved in mental retardation, were screened for mutations. A mutation in the third intron of AP1S2 was found in all affected male subjects in this large French family. The mutation resulted in skipping of exon 3, predicting a protein with three novel amino-acids and with termination at codon 64. In addition, the first known large Scottish family affected by Fried syndrome was reinvestigated, and a new nonsense mutation, p.Gln66X, was found in exon 3. Using CT, both affected patients from the French family who were analysed had marked calcifications of the basal ganglia, as previously observed in the first Scottish family, suggesting that the presence of distinctive basal ganglia calcification is an essential parameter to recognise this syndromic disorder. It may be possible to use this feature to identify families with X-linked mental retardation that should be screened for mutations in AP1S2.

[Epileptogenic brain malformations: radiological and clinical presentation and indications for genetic testing]. / Malformations cérébrales et épilepsie: présentations radiocliniques et implications pour le diagnostic génétique.

Malformations of cortical development (MCD) represent a major cause of developmental disabilities and severe epilepsy. Advances in imaging and genetics have improved the diagnosis and classification of these conditions. Up to now, eight genes have been involved in different types of MCD. Lissencephaly-pachygyria and subcortical band heterotopia (SBH) represent a malformative spectrum resulting from mutations of either LIS1 or DCX genes. LIS1 mutations cause a more severe malformation in the posterior brain regions. DCX mutations usually cause anteriorly predominant lissencephaly in males and SBH in female patients. Additional forms are X-linked lissencephaly with corpus callosum agenesis and ambiguous genitalia associated with mutations of the ARX gene. Lissencephaly with cerebellar hypoplasia (LCH) encompass heterogeneous disorders named LCH type a to d. LCHa are related with mutation in LIS1 or DCX, LCHb with mutation of RELN gene, and LCHd could be related with TUBA1A gene. Polymicrogyria encompass a wide range of clinical, aetiological and histological findings. Among several syndromes, recessive bilateral fronto-parietal polymicrogyria has been associated with mutations of the GPR56 gene. Bilateral perisylvian polymicrogyria showed a linkage to chromosome Xq28 in some pedigrees, and mutations in SRPX2 gene in others conditions. X-linked bilateral periventricular nodular heterotopia (BPNH) consists of BPNH with focal epilepsy in females and prenatal lethality in males. Filamin A (FLNA) mutations have been reported in some families and in sporadic patients. It is possible to infer the most likely causative gene by brain imaging studies and other clinical findings. Based on this experience, a detailed phenotype analysis is needed to develop the most efficient research on MCD in the future.

Differential effects of selenium on normal and neoplastic canine mammary cells.

Four different canine mammary tumor (CMT) cell lines and a nonneoplastic primary culture of mammary cells were examined for their in vitro responsiveness to selenium supplementation. These cell lines were found to vary in their metabolic response to increasing concentrations of selenium. Sensitivity to selenium, as sodium selenite, increased with increasing concentrations of this trace element in all of the neoplastic lines. These data also suggest that increasing the plating density of tumor cells further increases the sensitivity to selenium. A relatively selenium-sensitive cell line (CMT-13) and relatively insensitive cell line (CMT-11) were characterized on the basis of reduced growth resulting from selenium supplementation. Increasing the concentration of selenium to 0.75 microgram/ml depressed the growth of CMT-13 and CMT-11 cells by 75% and 11%, respectively, while no inhibition was observed in nonneoplastic cells. These cell lines also varied in their sensitivity to different forms of selenium. Selenodiglutathione was the most effective form of selenium examined that inhibited tumor cell growth. The sensitivity of the neoplastic lines was selenodiglutathione much greater than sodium selenite much greater than selenocystine greater than selenomethionine. None of the forms of selenium examined inhibited the growth of the nonneoplastic mammary cells in culture. Supplementation with sodium selenite (1 microgram Se per ml) for 60 min resulted in a dramatic depression in RNA biosynthesis in CMT-13, but not CMT-11 or nonneoplastic cells.

Five novel frameshift mutations in exon 3 and 4 of the MECP2 gene identified in Rett patients: Consequences for the molecular diagnosis strategy.

Rett syndrome (RTT) is a severe progressive neurological disorder that affects almost exclusively females. The gene responsible for this disorder, MECP2, was recently identified by candidate gene strategy. Mutations were detected in 70-85% of RTT cases. We report here five novel frameshift mutations (named 345delC, 895del202, 989ins18del8, 996insAG and 1124del53) in exon 3 and 4 of the MECP2 gene. To avoid the missing of few small deletions in RTT patients using classical mutation screening approaches, we suggest that screening of the mutations in the MECP2 gene in RTT girls should include at least a large PCR to amplify exon 4 entirely.

Inhibition of cholesterol ester transfer protein CGS 25159 and changes in lipoproteins in hamsters.

As a result of screening, several isoflavans were identified to be antagonists of cholesterol ester transfer protein (CETP) activity. The present study evaluates CGS 25159, a synthetic isoflavan, as a putative inhibitor of CETP activity of human and hamster plasma. Determined by [3]CE transfer from HDL to VLDL + LDL fraction or by fluorescent-CE transfer assay, CGS 25159 inhibited CETP in both human plasma bottom fraction (d = 1.21 g/ml) and in plasma from Golden Syrian Hamsters with an IC50 < 10 microM. The compound also inhibited (IC 50 approximately equal to 15 microM) the reciprocal transfer of triglycerides in the incubated whole plasma from normal and hyperlipidemic hamsters. When orally administered to normolipidemic hamsters, CGS 25159 (10 mg/kg, 4 days) reduced plasma transfer activity by 35-60%. Treatment with CGS 25159 (10 and 30 mg/kg, p.o.) resulted in dose dependent and time dependent changes in CETP activity. After two weeks of treatment at 10 mg/kg, the changes in VLDL + LDL cholesterol, total triglycerides and HDL cholesterol were -22 +/- 4.6*, -23 +/- 7.5 and +10 +/- 2.8%, respectively. The corresponding changes at 30 mg/kg were -28 +/- 5.5*, -38 +/- 6.8* and +29 +/-4.4.*%, (*, P, 0.05; mean +/- S.E.M., n = 6). A single spin gradient density ultracentrifugation of plasma lipoproteins and treated animals showed an increase in HDL cholesterol and a redistribution to larger HDL particles. These data support the contention that pharmacological down regulation of CETP activity could result in favorable changes in lipoprotein profile.

Effect of a novel series of macrocyclic hypolipidemic agents on plasma lipid and lipoprotein levels of four non-primate species.

The ansamycins are structurally novel hypolipidemic agents derived from rifampicin, but lacking antibacterial activity. Oral or intravenous administration resulted in rapid lowering of plasma cholesterol in rats, hamsters, guinea pigs and dogs. In the chow-fed rat, three related compounds (CGP 43371, CGS 23810 and CGS 24565) exhibited ED50 values of 13.7, 3.1 and 0.18 mg/kg, respectively. A feature common to the lipid lowering documented in these four species was the concomitant reduction of low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. In the chow-fed rat, however, apolipoprotein AI (apo AI) levels were much less affected than were those of HDL cholesterol. CGP 43371 at 3 and 10 mg/kg, lowered HDL cholesterol by 20% and 39%, respectively, whereas plasma apo AI was reduced by only 1% and 12%. Similarly, in lipoprotein fractions separated by ultracentrifugation, apo AI was unchanged in the d = 1.019-1.21 g/ml fraction after treatment with 3 or 10 mg/kg of CGP 43371, but HDL cholesterol was reduced 12% and 26% in this fraction at the two dose levels. Plasma and lipoprotein apo B levels, on the other hand, were reduced to a level equivalent to that of the reduction in cholesterol. The ansamycins thus represent a new structural series which may possess a novel mechanism of action as well, involving differential effects on HDL cholesterol and protein.

Diaminoindanes as microsomal triglyceride transfer protein inhibitors.

The synthesis and biological activities of biarylamide-substituted diaminoindanes as microsomal triglyceride transfer protein (MTP) inhibitors are described. One of the more potent compounds, 8aR, inhibited both the secretion of apoB from Hep G2 cells and the MTP-mediated transfer of triglycerides between synthetic acceptor and donor liposomes with IC(50) values of 0.7 and 70 nM, respectively. In normolipidemic rats and dogs, oral administration of 8aR dose-dependently reduced both plasma triglycerides and total cholesterol. Moreover, in rats and dogs, 8aR also prevented the postprandial rise in plasma triglycerides following a bolus administration of a fat load. Because MTP inhibitors decrease very low density lipoprotein assembly in the liver, the potential for hepatic lipid accumulation was evaluated. In normolipidemic rats, hepatic cholesterol and triglyceride contents were dose-dependently increased by 8aR. However, hepatic lipid accumulation resulted in negligible change in total liver weight and was reversible after withdrawal of the compound.

DHPLC-based method for DNA methylation analysis of differential methylated regions from imprinted genes.

The bisulfite genomic sequencing method is one of the most widely used techniques for methylation analysis in heterogeneous unbiased PCR, amplifying for both methylated and unmethylated alleles simultaneously. However, it requires labor-intensive and time-consuming cloning and sequencing steps. In the current study, we used a denaturing high-performance liquid chromatography (DHPLC) procedure in a complementary way with the bisulfite genomic sequencing to analyze the methylation of differentially methylated regions (DMRs) of imprinted genes. We showed reliable and reproducible results in distinguishing overall methylation profiles of DMRs regions of human SNRPN, H19, MEST/PEG1, LIT1, IGF2, TSSC5, WT1 antisense, and mouse H19, Mest/Peg1, Igf2R imprinted genes. These DHPLC profiles were in accordance with bisulfite genomic sequencing data and may serve as a type of "fingerprint," revealing the overall methylation status of DMRs associated with sample heterogeneity. We conclude that DHPLC analysis could be used to increase the throughput efficiency of methylation pattern analysis of imprinted genes after the bisulfite conversion of genomic DNA and unbiased PCR amplification.

Changes in H3 influenza A virus receptor specificity during replication in humans.

Influenza A viruses of the H3 subtype caused the 1968 Hong Kong pandemic, the hemagglutinin (HA) gene being introduced into humans following a reassortment event with an avian virus. Receptor specificity and serum inhibitor sensitivity of the HA of influenza A viruses are linked to the host species. Human H3 viruses preferentially recognize N-acetyl sialic acid linked to galactose by alpha2,6 linkages (Neu5Acalpha2,6Gal) and are sensitive to serum inhibitors, whereas avian and equine viruses preferentially recognize Neu5Acalpha2,3Gal linkages and are resistant to serum inhibitors. We have examined the receptor specificity and serum inhibitor sensitivity of H3 human influenza A viruses from the time they were introduced into the human population to gain insight into the mechanism of viral molecular evolution and host tropism. All of the viruses were sensitive to neutralization and hemagglutination inhibition by horse serum. Early H3 viruses were resistant to pig and rabbit serum inhibitors. Viruses isolated after 1977 were uniformly sensitive to inhibition by pig and rabbit sera. The recognition of Neu5Acalpha2,3Gal or Neu5Acalpha2,6Gal linkages was not correlated with the serum sensitivity. These data showed that the receptor specificity of HA, measured as inhibitor sensitivity, has changed during replication in humans since its introduction from an avian virus.

Inhibition of human breast cancer cells by selenium.

The viability of human breast cancer cells (cell lines MCF-7 and MDA-MB 231) was inhibited in vitro in a dose-dependent manner by selenium supplementation. However, a normal diploid human cell line (MRC-5) was relatively resistant to selenium supplementation. The presence of selenium as Na2SeO3 at 1.1 X 10(-6) M reduced cancer cell viability by approximately 50%, whereas non-cancerous cells were not affected. Parenteral administration of sodium selenite also significantly inhibited the growth of the cancerous cell lines transplanted into nude mice. Selenium administration at 0.8 micrograms/g body wt resulted in an 80-93% reduction in the rate of tumor growth without apparent ill effects on the host.

Uncertainties in the reference dose for methylmercury.

This paper critically examines the National Academy of Sciences and the National Research Council report on the toxicological effects of methyl mercury and the recently published US Environmental Protection Agency Reference Dose (RfD) for Methylmercury. Particular scrutiny is placed on the choice of the critical study and the underlining assumptions utilized in the selection of specific uncertainty factors (UFs) and the rationale for using a less-than-default factor of 10. The UFs that were utilized or considered by other agencies and organizations are also critically examined, explained and compared to one another. Based on these analyses, the authors suggest research that could be performed that would ameliorate the uncertainty of choosing a more precise partial UFor that may even provide completeness of database to allow for selecting of a UF for unity, thus improving the precision of the current published RfD.

Mathematical modelling and quantitative methods.

The present review reports on the mathematical methods and statistical techniques presently available for hazard characterisation. The state of the art of mathematical modelling and quantitative methods used currently for regulatory decision-making in Europe and additional potential methods for risk assessment of chemicals in food and diet are described. Existing practices of JECFA, FDA, EPA, etc., are examined for their similarities and differences. A framework is established for the development of new and improved quantitative methodologies. Areas for refinement, improvement and increase of efficiency of each method are identified in a gap analysis. Based on this critical evaluation, needs for future research are defined. It is concluded from our work that mathematical modelling of the dose-response relationship would improve the risk assessment process. An adequate characterisation of the dose-response relationship by mathematical modelling clearly requires the use of a sufficient number of dose groups to achieve a range of different response levels. This need not necessarily lead to an increase in the total number of animals in the study if an appropriate design is used. Chemical-specific data relating to the mode or mechanism of action and/or the toxicokinetics of the chemical should be used for dose-response characterisation whenever possible. It is concluded that a single method of hazard characterisation would not be suitable for all kinds of risk assessments, and that a range of different approaches is necessary so that the method used is the most appropriate for the data available and for the risk characterisation issue. Future refinements to dose-response characterisation should incorporate more clearly the extent of uncertainty and variability in the resulting output.

The effect of various seleno-compounds on ehrlich ascites tumor cells.

The effect of various concentrations and forms of selenium on in vitro viability of Ehrlich Ascites Tumor Cells (EATC) was investigated. Sodium selenite, selenium dioxide, seleno-DL-cystine, and seleno-DL-methionine, dramatically decreased EATC viability as measured by dye exclusion. Sodium selenate only marginally decreased EATC viability. Cell viabilities decreased with increasing selenium in the incubation media and as a function of time. Viabilities determined by dye exclusion did not correlate with the inhibition of tumor growth observed after treatment with selenium. Intraperitoneal injections of selenite in mice previously inoculated with EATC significantly inhibited tumor development. Delaying intraperitoneal injections of selenite to 5 and 7 days after inoculation of mice with EATC reduced the effectiveness of this nutrient on the inhibition of EATC growth. Incubation of EATC in vitro with supplemental selenium prior to injection of mice completely inhibited EATC development in vivo before any appreciable alteration in cell viability was observed.

Boron tolerable intake: re-evaluation of toxicokinetics for data-derived uncertainty factors.

Boron, which is ubiquitous in the environment, causes developmental and reproductive effects in experimental animals. This observation has led to efforts to establish a Tolerable Intake value for boron. Although risk assessors agree on the use of fetal weight decreases observed in rats as an appropriate critical effect, consensus on the adequacy of toxicokinetic data as a basis for replacement of default uncertainty factors remains to be reached. A critical analysis of the existing data on boron toxicokinetics was conducted to clarify the appropriateness of replacing default uncertainty factors (10-fold for interspecies differences and 10-fold for intraspecies differences) with data-derived values. The default uncertainty factor for variability in response from animals to humans of 10-fold (default values of 4-fold for kinetics and 2.5-fold for dynamics) was recommended, since clearance of boron is 3- to 4-fold higher in rats than in humans and data on dynamic differences--in order to modify the default value--are unavailable. A data-derived adjustment of 6-fold (1.8 for kinetics and 3.1 for dynamics) rather than the default uncertainty factor of 10-fold was considered appropriate for intrahuman variability, based on variability in glomerular filtration rate during pregnancy in humans and the lack of available data on dynamic differences. Additional studies to investigate the toxicokinetics of boron in rats would be useful to provide a stronger basis for replacement of default uncertainty factors for interspecies variation.

Post-traumatic cutaneous mucormycosis in diabetes mellitus. Short-term antifungal therapy.

Mucormycosis is infrequently encountered in the pediatric population in any of its forms (nasopharyngeal, disseminated, pulmonary, or cutaneous) and generally is associated with the immunocompromised host. We present an adolescent with poorly controlled diabetes mellitus who developed a progressive skin lesion 3 weeks after a motor vehicle accident. Rhizopus species was isolated from the lesion, and the biopsy revealed a fungal vasculopathy. Control of her diabetes, aggressive surgical intervention and a 10-day course of antifungal therapy (amphotericin B) resulted in a favorable outcome. This article illustrates the importance of considering cutaneous fungal infections, especially those in the class zygomycetes, in the diabetic patient with unusual, severe or persistent skin lesions. Early recognition is essential in order to avoid morbidity and mortality from this unusual opportunistic infection.

[RNA isolation and purification methods]. / Les méthodes d'extraction et de purification des ARN.

Recent advances in human, bacterial and viral genome projects and the development of quantitative real-time reverse transcription-polymerase chain reaction methods offer the possibility of analysing a large number of gene transcripts. These molecular developments represent an important advancein the field of genetics, cancer, virology, bacteriology and hematology. A limiting step remains the isolation of high quality mRNA purified from biological samples. This review describes the different methods used to isolate mRNA from biological samples and to verify RNA integrity and gives precise details about RNA storage conditions.