Differential reactivity of putative genotype 2 hepatitis C virus F protein between chronic and recovered infections

To date, all studies regarding hepatitis C virus (HCV) F protein have been based on expression in vitro/in vivo of recombinant protein or monoclonal antibodies derived from genotype 1a or 1b sequences, but not from other genotypes. The objective of this study was to prepare a putative genotype 2 recombinant F protein and evaluate its reactivity in plasma from individuals with chronic HCV infection or who had recovered from infection. One genotype 2 strain was selected for F protein (F-2) and core expression in bacterial culture. An ELISA was developed and applied to samples from patients with chronic infection or recovered infection of various genotypes. The anti-F-2 response in 117 samples showed a significantly higher reactivity in chronic than in recovered HCV-infected blood donors (

A de novo variant in ADGRL2 suggests a novel mechanism underlying the previously undescribed association of extreme microcephaly with severely reduced sulcation and rhombencephalosynapsis

International audienceExtreme microcephaly and rhombencephalosynapsis represent unusual pathological conditions, each of which occurs in isolation or in association with various other cerebral and or extracerebral anomalies. Unlike microcephaly for which several disease-causing genes have been identified with different modes of inheritance, the molecular bases of rhombencephalosynapsis remain unknown and rhombencephalosynapsis presents mainly as a sporadic condition consistent with de novo dominant variations. We report for the first time the association of extreme microcephaly with almost no sulcation and rhombencephalosynapsis in a fœtus for which comparative patient-parent exome sequencing strategy revealed a heterozygous de novo missense variant in the ADGRL2 gene. ADGRL2 encodes latrophilin 2, an adhesion G-protein-coupled receptor whose exogenous ligand is α-latrotoxin. Adgrl2 immunohistochemistry and in situ hybridization revealed expression in the telencephalon, mesencephalon and rhombencephalon of mouse and chicken embryos. In human brain embryos and fœtuses, Adgrl2 immunoreactivity was observed in the hemispheric and cerebellar germinal zones, the cortical plate, basal ganglia, pons and cerebellar cortex. Microfluorimetry experiments evaluating intracellular calcium release in response to α-latrotoxin binding showed significantly reduced cytosolic calcium release in the fœtus amniocytes vs amniocytes from age-matched control fœtuses and in HeLa cells transfected with mutant ADGRL2 cDNA vs wild-type construct. Embryonic lethality was also observed in constitutive Adgrl2 mice. In Adgrl2 mice, MRI studies revealed microcephaly and vermis hypoplasia. Cell adhesion and wound healing assays demonstrated that the variation increased cell adhesion properties and reduced cell motility. Furthermore, HeLa cells overexpressing mutant ADGRL2 displayed a highly developed cytoplasmic F-actin network related to cytoskeletal dynamic modulation. ADGRL2 is the first gene identified as being responsible for extreme microcephaly with rhombencephalosynapsis. Increased cell adhesion, reduced cell motility and cytoskeletal dynamic alterations induced by the variant therefore represent a new mechanism responsible for microcephaly

Metabolic causes of nonimmune hydrops fetalis: A next-generation sequencing panel as a first-line investigation.

International audienceHydrops fetalis is a life-threatening fetal condition, and 85% of all cases are classified as nonimmune hydrops fetalis (NIHF). Up to 15% of NIHF cases may be due to inborn errors of metabolism (IEM), but a large proportion of cases linked to metabolic disorders remains undiagnosed. This lack of diagnosis may be related to the limitations of conventional biological procedures, which involve sequential investigations and require multiple samples and steps. In addition, this approach is time consuming. We have developed a next-generation sequencing (NGS) panel to investigate metabolic causes of NIHF, ascites, and polyhydramnios associated to another fetal abnormality

Robust hepatitis C virus infection in vitro

The absence of a robust cell culture model of hepatitis C virus (HCV) infection has severely limited analysis of the HCV life cycle and the development of effective antivirals and vaccines. Here we report the establishment of a simple yet robust HCV cell culture infection system based on the HCV JFH-1 molecular clone and Huh-7-derived cell lines that allows the production of virus that can be efficiently propagated in tissue culture. This system provides a powerful tool for the analysis of host-virus interactions that should facilitate the discovery of antiviral drugs and vaccines for this important human pathogen

Antiviral Stilbene 1,2-Diamines Prevent Initiation of Hepatitis C Virus RNA Replication at the Outset of Infection▿

The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ∼300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a >1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen

Mutations in CNTNAP1 and ADCY6 are responsible for severe arthrogryposis multiplex congenita with axoglial defects

International audienceNon-syndromic arthrogryposis multiplex congenita (AMC) is characterized by multiple congenital contractures resulting from reduced fetal mobility. Genetic mapping and whole exome sequencing (WES) were performed in 31 multiplex and/or consanguineous undiagnosed AMC families. Although this approach identified known AMC genes, we here report pathogenic mutations in two new genes. Homozygous frameshift mutations in CNTNAP1 were found in four unrelated families. Patients showed a marked reduction in motor nerve conduction velocity (\textless10 m/s) and transmission electron microscopy (TEM) of sciatic nerve in the index cases revealed severe abnormalities of both nodes of Ranvier width and myelinated axons. CNTNAP1 encodes CASPR, an essential component of node of Ranvier domains which underlies saltatory conduction of action potentials along the myelinated axons, an important process for neuronal function. A homozygous missense mutation in adenylate cyclase 6 gene (ADCY6) was found in another family characterized by a lack of myelin in the peripheral nervous system (PNS) as determined by TEM. Morpholino knockdown of the zebrafish orthologs led to severe and specific defects in peripheral myelin in spite of the presence of Schwann cells. ADCY6 encodes a protein that belongs to the adenylate cyclase family responsible for the synthesis of cAMP. Elevation of cAMP can mimic axonal contact in vitro and upregulates myelinating signals. Our data indicate an essential and so far unknown role of ADCY6 in PNS myelination likely through the cAMP pathway. Mutations of genes encoding proteins of Ranvier domains or involved in myelination of Schwann cells are responsible for novel and severe human axoglial diseases

Phenotypic spectrum and genomics of undiagnosed arthrogryposis multiplex congenita

Arthrogryposis multiplex congenita (AMC) is characterised by congenital joint contractures in two or more body areas. AMC exhibits wide phenotypic and genetic heterogeneity. Our goals were to improve the genetic diagnosis rates of AMC, to evaluate the added value of whole exome sequencing (WES) compared with targeted exome sequencing (TES) and to identify new genes in 315 unrelated undiagnosed AMC families