Detection of Pathogenic Intronic Variants for COL4A5 Gene in X-Linked Alport Syndrome: Developing a Novel Methodology

Abstract

COL4A5 gene variants could result in the X-linked Alport syndrome 1, dominant inheritance (XLAS1) (Online Mendelian Inheritance in Man (OMIM) #301050). The splicing changes can be identified through targeted RNA sequencing (RNA seq), but obtaining accessible patient samples from urine cells and skin fibroblasts is challenging. Moreover, extracted COL4A5 transcripts cannot be amplified due to their nonexpression in peripheral blood mononuclear cells (PBMCs). In the study, we induced patient-derived PBMCs to differentiate into induced pluripotent stem cells (iPSCs) and lymphoblasts (using phytohemagglutinin (PHA)). Through cDNA Sanger sequencing, we identified the true pathogenicity of two cis-intron variants of the COL4A5 gene in lymphoblasts and iPSCs. Furthermore, the results were also verified by minigene assay in vitro. Through RNA seq and real-time PCR, we further confirmed that the expression level of COL4A5 in lymphoblasts is sufficient for Sanger sequencing to detect splicing changes. In conclusion, we have devised a straightforward, cost-effective, noninvasive, and robust method for detecting abnormal transcripts resulting from pathogenic variants in the COL4A5 gene. Furthermore, this method is equally suitable for genes that are expressed in lymphoblasts but not in PBMCs. Finally, splicing changes account for a relatively high proportion of patients with XLAS who were overlooked through routine whole-exome sequencing (WES), and the c.4822-12T>C variant may become a target for specific antisense oligonucleotide therapies.