Exon skipping is a promising strategy for Duchenne muscular dystrophy (DMD) disease-modifying therapy. To make this approach safe, ensuring that excluding one or more exons will restore the reading frame and that the resulting protein will retain critical functions of the full-length dystrophin protein is necessary. However, in vivo testing of the consequences of skipping exons that encode the N-terminal actin-binding domain (ABD) has been confounded by the absence of a relevant animal model. We created a mouse model of the disease recapitulating a novel human mutation, a large de novo deletion of exons 8-34 of the DMD gene, found in a Russian DMD patient. This mutation was achieved by deleting exons 8-34 of the X-linked mouse Dmd gene using CRISPR/Cas9 genome editing, which led to a reading frame shift and the absence of functional dystrophin production. Male mice carrying this deletion display several important signs of muscular dystrophy, including a gradual age-dependent decrease in muscle strength, increased creatine kinase, muscle fibrosis and central nucleation. The degrees of these changes are comparable to those observed in mdx mice, a standard laboratory model of DMD. This new model of DMD will be useful for validating therapies based on skipping exons that encode the N-terminal ABD and for improving our understanding of the role of the N-terminal domain and central rod domain in the biological function of dystrophin. Simultaneous skipping of exons 6 and 7 should restore the gene reading frame and lead to the production of a protein that might retain functionality despite the partial deletion of the ABD.

Bipedalism, speech, and intellect are the most prominent traits that emerged in the evolution of Homo sapiens. Here, we describe a novel genetic cause of an “involution” phenotype in four patients, who are characterized by quadrupedal locomotion, intellectual impairment, the absence of speech, small stature, and hirsutism, observed in a consanguineous Brazilian family. Using whole-genome sequencing analysis and homozygous genetic mapping, we identified genes bearing homozygous genetic variants and found a homozygous 36.2 kb deletion in the gene of glutamate receptor delta 2 (GRID2) in the patients, resulting in the lack of a coding region from the fifth to the seventh exons. The GRID2 gene is highly expressed in the cerebellum cortex from prenatal development to adulthood, specifically in Purkinje neurons. Deletion in this gene leads to the loss of the alpha chain in the extracellular amino-terminal protein domain (ATD), essential in protein folding and transport from the endoplasmic reticulum (ER) to the cell surface. Then, we studied the evolutionary trajectories of the GRID2 gene. There was no sign of strong selection of the highly conservative GRID2 gene in ancient hominids (Neanderthals and Denisovans) or modern humans; however, according to in silico tests using the Mfold tool, the GRID2 gene possibly gained human-specific mutations that increased the stability of GRID2 mRNA.

Background and Clinical Significance: The oocyte zona pellucida (ZP) is an extracellular glycoprotein matrix with essential roles in oogenesis, fertilization, and early embryonic development. Abnormal ZP morphology is associated with female infertility and adverse outcomes after assisted reproductive technology (ART), but its molecular basis remains incompletely understood. Case Presentation: We present three patients with female infertility, abnormal oocyte ZP morphology, and adverse embryological outcomes, including fertilization failure and early developmental arrest. Whole-genome sequencing identified an extremely rare heterozygous missense variant in the ZP4 gene (rs1254095560) in one patient, resulting in the p.Leu342Pro substitution at a conserved amino acid position within the functionally important ZP-C subdomain of the protein. In silico modeling showed that the p.Leu342Pro substitution may alter the spatial folding of the ZP4 protein. Conclusions: These results and the known role of ZP4 in the organization of the zona pellucida allow this variant to be considered a candidate genetic factor potentially associated with disruption of the zona pellucida structure. The absence of comparable, apparently pathogenic coding variants in ZP genes in two other patients with a similar zona pellucida phenotype suggests genetic heterogeneity of this phenotype.