The X-linked NLGN3 gene, encoding a postsynaptic cell adhesion molecule, was involved in a nonsyndromic monogenic form of autism spectrum disorder (ASD) by the description of one unique missense variant, p.Arg451Cys (Jamain et al. 2003). We investigated here the pathogenicity of additional missense variants identified in two multiplex families with intellectual disability (ID) and ASD: c.1789C>T, p.Arg597Trp, previously reported by our group (Redin et al. 2014) and present in three affected cousins and c.1540C>T, p.Pro514Ser, identified in two affected brothers. Overexpression experiments in HEK293 and HeLa cell lines revealed that both variants affect the level of the mature NLGN3 protein, its localization at the plasma membrane and its presence as a cleaved form in the extracellular environment, even more drastically than what was reported for the initial p.Arg451Cys mutation. The variants also induced an unfolded protein response, probably due to the retention of immature NLGN3 proteins in the endoplasmic reticulum. In comparison, the c.1894A>G, p.Ala632Thr and c.1022T>C, p.Val341Ala variants, present in males from the general population, have no effect. Our report of two missense variants affecting the normal localization of NLGN3 in a total of five affected individuals reinforces the involvement of the NLGN3 gene in a neurodevelopmental disorder characterized by ID and ASD.
A large French family including members affected by nonspecific X-linked mental retardation, with or without autism or pervasive developmental disorder in affected male patients, has been found to have a 2-base-pair deletion in the Neuroligin 4 gene (NLGN4) located at Xp22.33. This mutation leads to a premature stop codon in the middle of the sequence of the normal protein and is thought to suppress the transmembrane domain and sequences important for the dimerization of neuroligins that are required for proper cell-cell interaction through binding to beta-neurexins. As the neuroligins are mostly enriched at excitatory synapses, these results suggest that a defect in synaptogenesis may lead to deficits in cognitive development and communication processes. The fact that the deletion was present in both autistic and nonautistic mentally retarded males suggests that the NLGN4 gene is not only involved in autism, as previously described, but also in mental retardation, indicating that some types of autistic disorder and mental retardation may have common genetic origins.
In vitro folliculogenesis of cryopreserved ovarian tissue could be an effective method for insuring fertility for patients who receive gonadotoxic treatment. Although several culture systems have been described for growing female gametes in vitro, the production of competent oocytes for further development remains a considerable challenge. The purpose of our study was to determine whether maternal primary imprinting progresses normally during mouse oocyte growth in vitro. We analysed the DNA methylation status of differentially methylated regions of the imprinted genes H19, Mest/Peg1 and Igf2R using fully grown germinal vesicle-stage oocytes (fg oocytes) produced by in vitro folliculogenesis from early preantral follicles. When compared to fg oocytes removal from control females, we observed after in vitro development, a loss of methylation at the Igf2R locus in six out of seven independent experiments and Mest/Peg1 locus (one out of seven), and a gain of methylation at the H19 locus (one out of seven). These results provide insight into the dysregulation of the process of primary imprinting during oocyte growth in vitro and highlight the need for effective new biomarkers to identify complete nuclear reprogramming competence after in vitro folliculogenesis.
Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.
X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.
It has been shown that the dystrophin gene, which is defective in patients with Duchenne and Becker muscular dystrophy (reviewed in ref. 1), is transcribed in brain from a specific promoter that is different from the one used in muscle, and so the two types of transcripts differ at least in their first exon. We recently found that the dystrophin gene is expressed at a higher level in primary cultures of neuronal cells than in astro-glial cells derived from adult mouse brain. Here we investigate the use of two different promoters in each cell type. Our results demonstrate that the brain-type promoter of the dystrophin gene is highly specific to neurons, in which there is a significant increase in the amount of brain-specific messenger RNA during the course of in vitro maturation. By contrast, the muscle-type promoter is active in a wider range of cell types, including not only striated and smooth muscle, but also glial cells to a lesser extent, and probably neurons.
Title: Quantitative estimation of minor mRNAs by cDNA-polymerase chain reaction. Application to dystrophin mRNA in cultured myogenic and brain cells Chelly J, Montarras D, Pinset C, Berwald-Netter Y, Kaplan JC, Kahn A Ref: European Journal of Biochemistry, 187:691, 1990 : PubMed
Amplification of the mRNA polymerase chain reaction is a very sensitive technique to detect low-abundance transcripts. We describe in this paper conditions necessary to make this technique quantitative. Quantification is performed in the exponential phase of the amplification process and the results are standardized with respect to those obtained with an exogenous mRNA which is co-reverse-transcribed and co-amplified in the same reaction as the analyzed transcripts. The primers are chosen in different exons to distinguish the amplification of mRNA fragments from the amplification of contaminating DNA. Analysis of the kinetics of amplification and parameters influencing this kinetics shows that: (a) in the exponential phase of amplification, the amount of amplified fragments is proportional to the initial amount of transcripts; (b) in a certain range of length fragment, the yield of amplification is inversely proportional to the length of the amplified fragments. Using this method we have demonstrated that the dystrophin gene is already activated at the myoblastic stage. A quantitative estimation of the transcript showed that the expression of this gene increases strongly in the course of in vitro myogenesis. In primary culture of mouse brain cells, the dystrophin gene was found to be more expressed in neuronal than in glial cells.
