Hybrid minigene splicing assay verifies the pathogenicity of a novel splice site variant in the COL1A1 gene of a chinese patient with osteogenesis imperfecta type I

Abstract(#br)Background(#br)Osteogenesis imperfecta (OI) is a rare genetic bone disease associated with brittle bones and fractures. Among all known types, OI type I is the most common type and characterized by increased bone fragility, low bone mass, distinctly blue-gray sclera, and susceptibility to conductive hearing loss beginning in adolescence. Mutations in genes encoding type I collagen ( COL1A1 and COL1A2 ) contribute to the main pathogenic mechanism of OI.(#br)Methods(#br)Subtle mutation of the COL1A1 gene in the proband was detected by targeted next-generation sequencing (NGS) and confirmed by Sanger sequencing. We then assessed the effect of the mutation on the splicing of the COL1A1 gene by bioinformatics prediction and hybrid minigene splicing assay (HMSA).(#br)Results(#br)A novel splice site mutation c.1821+1 G > C was discovered in the proband by NGS and further confirmed by Sanger sequencing, which was also simultaneously identified from the proband’s mother and elder sister. Bioinformatics predicted that this mutation would result in a disappearance of the 5′ donor splice site in intron 26, thereby leading to abnormal splicing and generation of premature stop codon. The follow-up experimental data generated by HMSA was consistent with this prediction.(#br)Conclusion(#br)Our study identified a novel splice site mutation that caused OI type I in the proband by abnormal splicing and demonstrated that combined applications of NGS, bioinformatics and HMSA are comprehensive and effective methods for diagnosis and aberrant splicing study of OI

Human Metapneumovirus Glycoprotein G Inhibits Innate Immune Responses

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-ΔG) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-ΔG-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-ΔG enhanced activation of transcription factors belonging to the nuclear factor (NF)-κB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-ΔG also increased IRF- and NF-κB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor

Progress in Single-cell RNA Sequencing of Lung Adenocarcinoma

Lung adenocarcinoma (LUAD) is the most common subtype of lung cancer and one of the main causes of cancer-related deaths. In the past decade, with the widespread use of computed tomography (CT) in routine screening for lung cancer, the incidence of LUAD presenting as small pulmonary nodules radiologically, has increased remarkably. The mechanisms of the occurrence and progression of LUADs are complex, and the prognoses of patients with LUAD vary significantly. Although significant progress has been made in targeted therapy and immunotherapy for LUADs in recent years, the drug resistance of tumor cells has not been effectively overcome, which limits the benefits of patients. With the accomplishment of the Human Genome Project, sequencing-based genomic and transcriptomics have come into the field of clinical and scientific researches. Single-cell sequencing, as a new type of sequencing method that has captured increasing attention recently, can perform specific analysis of cell populations at single-cell level, which can reveal the unique changes of each cell type. Single-cell sequencing can also provide accurate assessment on heterogeneous stromal cells and cancer cells, which is helpful to reveal the complexity of molecular compositions and differences between non- and malignant tissues. To sum up, it is an urgent need for clinicians and basic scientists to deeply understand the pathogenesis and development of LUAD, the heterogeneity of tumor microenvironment (TME) and the mechanism of drug resistance formation through single-cell sequencing, so as to discover new therapeutic targets. In this paper, we reviewed and summarized the application and progress in single-cell sequencing of LUADs