Doppel and the prion protein (PrP) are two related proteins involved in different aspects of neuronal degeneration. While a structural modification of PrP is necessary and sufficient for its toxic effect, the neurotoxicity of Doppel in the Purkinje cells of the cerebellum relies solely on its overexpression. Understanding the Doppel-related neurotoxicity thus involves the analysis of its developmental and transcriptional regulation. Here we report for the first time that Doppel is expressed in the embryonic neurons of mice dorsal root ganglia and spinal cord and that the closely related Brn-3a and Brn-3b transcription factors are involved in its transcriptional regulation.
Abstract The analysis of individuals with ciliary chondrodysplasias can shed light on sensitive mechanisms controlling ciliogenesis and cell signalling that are essential to embryonic development and survival. Here we identify TCTEX1D2 mutations causing Jeune asphyxiating thoracic dystrophy with partially penetrant inheritance. Loss of TCTEX1D2 impairs retrograde intraflagellar transport (IFT) in humans and the protist Chlamydomonas , accompanied by destabilization of the retrograde IFT dynein motor. We thus define TCTEX1D2 as an integral component of the evolutionarily conserved retrograde IFT machinery. In complex with several IFT dynein light chains, it is required for correct vertebrate skeletal formation but may be functionally redundant under certain conditions.
Duchenne muscular Dystrophy (DMD) is an inherited disease caused by mutations in the dystrophin gene that disrupt the open reading frame, while in frame mutations result in Becker muscular dystrophy (BMD). Ullrich congenital muscular dystrophy (UCMD) is due to mutations affecting collagen VI genes. Specific muscle miRNAs (dystromirs) are potential non-invasive biomarkers for monitoring the outcome of therapeutic interventions and disease progression. We quantified miR-1, miR-133a,b, miR-206 and miR-31 in serum from patients with DMD, BMD, UCMD and healthy controls. MiR-1, miR-133a,b and miR-206 were upregulated in DMD, but unchanged in UCMD compared to controls. Milder DMD patients had higher levels of dystromirs than more severely affected patients. Patients with low forced vital capacity (FVC) values, indicating respiratory muscle weakness, had low levels of serum miR-1 and miR-133b. There was no significant difference in the level of the dystromirs in BMD compared to controls. We also assessed the effect of dystrophin restoration on the expression of the five dystromirs in serum of DMD patients treated systemically for 12 weeks with antisense oligomer eteplirsen that induces skipping of exon 51 in the dystrophin gene. The dystromirs were also analysed in muscle biopsies of DMD patients included in a single dose intramuscular eteplirsen clinical trial. Our analysis detected a trend towards normalization of these miRNA between the pre- and post-treatment samples of the systemic trial, which however failed to reach statistical significance. This could possibly be due to the small number of patients and the short duration of these clinical trials. Although longer term studies are needed to clarify the relationship between dystrophin restoration following therapeutic intervention and the level of circulating miRNAs, our results indicate that miR-1 and miR-133 can be considered as exploratory biomarkers for monitoring the progression of muscle weakness and indirectly the remaining muscle mass in DMD.
We show that the 3′UTR of the Brn‐3b mRNA does indeed contain regulatory sequences that mediate mRNA degradation upon serum starvation‐induced differentiation of ND7 neuroblastoma cells. The specific region mediating this effect has been characterized and two different microRNAs that potentially regulate the stability of Brn‐3b have been identified. Moreover we show that Dicer, one of the key enzymes in the production of microRNAs, is strongly up‐regulated in ND7 cells subjected to differentiation.
The use of Polymerase Chain Reaction (PCR) to incorporate mutated oligonucleotides into newly synthesized segments of DNA and the subsequent introduction of this DNA into competent cells is one of the most widely used methods of site-directed mutagenesis (Zoller et al.1984 DNA 3:479-488; Dalbodie-McFarland et al. 1982 Proc. Natl. Acad. Sci. USA 79:6409-6413). Several variations of this technique exist, but all require several and some time-consuming steps. Here we report the results of a rapid method of in vivo site-directed mutagenesis involving the direct transformation of Neurospora crassa spheroplasts with mutated oligonucleotides.
Imputing genotypes from reference panels created by whole-genome sequencing (WGS) provides a cost-effective strategy for augmenting the single-nucleotide polymorphism (SNP) content of genome-wide arrays. The UK10K Cohorts project has generated a data set of 3,781 whole genomes sequenced at low depth (average 7x), aiming to exhaustively characterize genetic variation down to 0.1% minor allele frequency in the British population. Here we demonstrate the value of this resource for improving imputation accuracy at rare and low-frequency variants in both a UK and an Italian population. We show that large increases in imputation accuracy can be achieved by re-phasing WGS reference panels after initial genotype calling. We also present a method for combining WGS panels to improve variant coverage and downstream imputation accuracy, which we illustrate by integrating 7,562 WGS haplotypes from the UK10K project with 2,184 haplotypes from the 1000 Genomes Project. Finally, we introduce a novel approximation that maintains speed without sacrificing imputation accuracy for rare variants.
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