Determination of the pathogenicity of a novel COL4A5 missense variant by CRISPR-Cas9 in kidney podocytes

By conducting NGS and CRISPR/Cas9 gene-editing of podocytes, a novel COL4A5 missense variant, c.2641G>A (p. Gly881Arg), was confirmed to be the genetic defect of X-linked Alport syndrome in the Chinese family. Our findings extend the genetic spectrum of X-linked Alport syndrome with COL4A5 mutations and provide a method for evaluating the functional significance of novel COL4A5 missense variants in vitro.

Clinical data and detailed family history were collected. Targeted next-generation sequencing (NGS) was applied to examine potential pathogenic variants in COL4A3, COL4A4, COL4A5 genes in the proband, and then the variants were analyzed using bioinformatics tools and pedigree analysis. The CRISPR/Cas9 gene editing was used to knock in potential pathogenic variants in human podocytes, and then western blot analyses and immunofluorescence assays were used to measure COL4A5 protein expression.

The main purpose of this research was to investigate the influence of the novel COL4A5 missense mutation on collagen type IV.

Three patients (I: 2, II: 1 and II: 2) presented with microscopic hematuria and proteinuria, and the patient II: 1 progressed to abnormal renal function by age 14. A novel missense variant, c.2641G>A (p. Gly881Arg), located in exon 31 of COL4A5 gene, was chosen as a possible pathogenic variant. The variant significantly decreased collagen IV α5 chain expression in CRISPR/Cas9 gene edited podocytes.