Identification and pathogenicity analysis of a novel intronic COL4A5 variant in a Chinese family

X-linked Alport syndrome (XLAS) is a disorder of type IV collagen structure caused by pathogenic variants of the COL4A5 gene and characterized by progressive kidney disease, hearing loss, and ocular abnormalities. Although mutation screening is commonly performed for AS-associated genes, transcriptional analysis is not a standard test for patients with XLAS, and the functional consequences of splicing abnormalities caused by intronic variants are rarely studied.

In this study, nine family members from a three-generation pedigree in Gansu Province, China, were investigated. We used targeted next-generation sequencing to identify genetic variants in family members, confirmed the mutation site by Sanger sequencing, and predicted the pathogenicity of the variant using bioinformatics software. The splicing effects of the mutation were analyzed using minigene testing with HEK 293T cells, and further confirmed by in vivo transcript analysis using RNA extracted from the proband's skin cells.

The proband was a 5-year-old male child who presented with microhematuria at the age of 4.5 years. Four relatives in this pedigree had a family history of kidney disease, all presenting with microhematuria with or without proteinuria. The proband's elder brother and maternal uncle had progressed to kidney failure. Immunofluorescence staining of the proband's epidermal basement membrane showed negative expression of the α5 chain. A novel splice variant (c.1587+4A>G) was identified in intron 23 of COL4A5 among the affected family members. Sanger sequencing confirmed that this variant co-segregated with the disease phenotype. In silico analysis predicted that this variant may cause abnormal splicing. Transcript analysis of the proband's skin tissue and minigene assay demonstrated that the variant causes abnormal mRNA splicing and exon 23 skipping, leading to a frameshift, premature termination codon, and predicted nonsense-mediated mRNA decay.

The study identified a novel intronic variant (c.1587+4A>G) in the COL4A5 gene in a Chinese family with XLAS, and functional experiments verified that this variant induces aberrant splicing. This study highlights the importance of transcript analysis for intronic variants and further expands the mutational spectrum of COL4A5 in XLAS.