Abstract:
COL4A5 gene variant could induced the X-linked Alport syndrome 1, dominant inheritance (XLAS1; OMIM #301050). Based on HGMD, the splicing changes could be estimated to make up 22.8% of COL4A5, among which the intron variants contributed a lot. The splicing changes can be identified by targeted RNA sequencing, but it is difficult to obtain accessible patient samples and extracted COL4A5 transcripts is not amplified due to non-expression in Peripheral Blood Mononuclear Cells (PBMCs). In the study, we have induced the patient-derived PBMCs into iPSCs and lymphoblasts (phytohemagglutinin, PHA). By cDNA-Sanger sequencing, we identified the true pathogenic for two cis-intron variations in COL4A5 gene of the XLAS family in lymphoblasts and iPSCs from PBMCs, and the result also was verified by minigene assay in vitro. Through RNAseq and real-time PCR, we further verified the COL4A5 was expressed in lymphoblasts to sufficient to meet the sanger sequencing for splicing changes. In conclusion, we have developed a simple, inexpensive and robust method for identifying abnormal transcripts caused by pathogenic variants in the introns of the COL4A5 gene. Therefore, these variants account for a relatively high proportion of patients with XLAS who was missed with pathogenic variants through routine WES, and the c.4822-12T>C variant may become target for specific antisense oligonucleotide therapies.
Key word X-linked Alport syndrome 1, COL4A5, intronic variant, lymphoblast, splicing changes, case report.
