Deficiency of the minor spliceosome component U4atac snRNA secondarily results in ciliary defects in human and zebrafish.

In the human genome, about 750 genes contain one intron excised by the minor spliceosome. This spliceosome comprises its own set of snRNAs, among which U4atac. Its noncoding gene, RNU4ATAC, has been found mutated in Taybi-Linder (TALS/microcephalic osteodysplastic primordial dwarfism type 1), Roifman (RFMN), and Lowry-Wood (LWS) syndromes. These rare developmental disorders, whose physiopathological mechanisms remain unsolved, associate ante- and post-natal growth retardation, microcephaly, skeletal dysplasia, intellectual disability, retinal dystrophy, and immunodeficiency. Here, we report bi-allelic RNU4ATAC mutations in five patients presenting with traits suggestive of the Joubert syndrome (JBTS), a well-characterized ciliopathy. These patients also present with traits typical of TALS/RFMN/LWS, thus widening the clinical spectrum of RNU4ATAC-associated disorders and indicating ciliary dysfunction as a mechanism downstream of minor splicing defects. Intriguingly, all five patients carry the n.16G>A mutation, in the Stem II domain, either at the homozygous or compound heterozygous state. A gene ontology term enrichment analysis on minor intron-containing genes reveals that the cilium assembly process is over-represented, with no less than 86 cilium-related genes containing at least one minor intron, among which there are 23 ciliopathy-related genes. The link between RNU4ATAC mutations and ciliopathy traits is supported by alterations of primary cilium function in TALS and JBTS-like patient fibroblasts, as well as by u4atac zebrafish model, which exhibits ciliopathy-related phenotypes and ciliary defects. These phenotypes could be rescued by WT but not by pathogenic variants-carrying human U4atac. Altogether, our data indicate that alteration of cilium biogenesis is part of the physiopathological mechanisms of TALS/RFMN/LWS, secondarily to defects of minor intron splicing.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

Sex chromosome and sex locus characterization in goldfish, Carassius auratus (Linnaeus, 1758).

BACKGROUND: Goldfish is an important model for various areas of research, including neural development and behavior and a species of significant importance in aquaculture, especially as an ornamental species. It has a male heterogametic (XX/XY) sex determination system that relies on both genetic and environmental factors, with high temperatures being able to produce female-to-male sex reversal. Little, however, is currently known on the molecular basis of genetic sex determination in this important cyprinid model. Here we used sequencing approaches to better characterize sex determination and sex-chromosomes in an experimental strain of goldfish. RESULTS: Our results confirmed that sex determination in goldfish is a mix of environmental and genetic factors and that its sex determination system is male heterogametic (XX/XY). Using reduced representation (RAD-seq) and whole genome (pool-seq) approaches, we characterized sex-linked polymorphisms and developed male specific genetic markers. These male specific markers were used to distinguish sex-reversed XX neomales from XY males and to demonstrate that XX female-to-male sex reversal could even occur at a relatively low rearing temperature (18 °C), for which sex reversal has been previously shown to be close to zero. We also characterized a relatively large non-recombining region (~ 11.7 Mb) on goldfish linkage group 22 (LG22) that contained a high-density of male-biased genetic polymorphisms. This large LG22 region harbors 373 genes, including a single candidate as a potential master sex gene, i.e., the anti-Mullerian hormone gene (amh). However, no sex-linked polymorphisms were detected in the coding DNA sequence of the goldfish amh gene. CONCLUSIONS: These results show that our goldfish strain has a relatively large sex locus on LG22, which is likely the Y chromosome of this experimental population. The presence of a few XX males even at low temperature also suggests that other environmental factors in addition to temperature could trigger female-to-male sex reversal. Finally, we also developed sex-linked genetic markers, which will be important tools for future research on sex determination in our experimental goldfish population. However, additional work would be needed to explore whether this sex locus is conserved in other populations of goldfish.

Clinical interpretation of variants identified in RNU4ATAC, a non-coding spliceosomal gene.

Biallelic variants in RNU4ATAC, a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.