Use of RNA‑sequencing to detect abnormal transcription of the collagen α‑2 (VI) chain gene that can lead to Bethlem myopathy

Abstract

Bethlem myopathy (BM) is an autosomal dominant or autosomal recessive disorder and is usually associated with mutations in the collagen VI genes. In the present study, the pathogenicity of a novel splice-site mutation was explored using RNA-sequencing in a family with suspected BM, and a myopathy panel was performed in the proband. The genetic status of all family members was confirmed using Sanger sequencing. Clinical data and magnetic resonance imaging (MRI) features were also documented. In silico analysis was performed to predict the effects of the splice mutation. RNA-sequencing and reverse transcription (RT)-PCR were used to assess aberrant splicing. Immunocytochemistry was conducted to measure collagen VI protein levels within the gastrocnemius and in cultured skin fibroblasts. The results revealed that three patients in the family shared a similar classic BM presentation. MRI revealed distinct patterns of fatty infiltration in the lower extremities. A novel splicing mutation c.736-1G>C in the collagen α-2 (VI) chain ( COL6A2 ) gene was found in all three patients. In silico analysis predicted that the mutation would destroy the normal splice acceptor site. RNA-sequencing detected two abnormal splicing variants adjacent to the mutation site, and RT-PCR confirmed the RNA-sequencing findings. Furthermore, a defect in the collagen protein within cultured fibroblasts was detected using immunocytochemistry. The mutation c.736-1G>C in the COL6A2 gene caused aberrant splicing and led to premature termination of protein translation. In conclusion, these findings may improve our knowledge of mutations of the COL6A2 gene associated with BM and demonstrated that RNA-sequencing can be a powerful tool for finding the underlying mechanism of a disease-causing mutations at a splice site.

Comprehensive Transcriptomic Profiling of m6A Modification in Age-Related Hearing Loss

“…However, the m6 A modification of EGFR mRNA in ARHL is still poorly understood and its role in ARHL is the next step in our research plan. High-throughput sequencing platforms may produce sequencing errors originating from the sequencing technology itself [54]. We validated differentially expressed genes in the combined MeRIP-Seq and mRNA-Seq analyses by MeRIP-qPCR and qRT-PCR, respectively, the results of which were consistent with those of high-throughput sequencing, further indicating the reliability of high-throughput sequencing in this study.…”