A novel COL4A5 splicing mutation causes alport syndrome in a Chinese family

Abstract

Background: Alport syndrome (AS) is characterised by haematuria, proteinuria, a gradual decline in kidney function, hearing loss, and eye abnormalities. The disease is caused by mutations in COL4An (n = 3, 4, 5) that encodes 3-5 chains of type IV collagen in the glomerular basement membrane. AS has three genetic models: X-linked, autosomal recessive, and autosomal dominant. The most common type of AS is X-linked AS, which is caused by COL4A5.

Methods: We enrolled children with renal insufficiency and a family history of kidney disorders. The proband was identified using whole-exome sequencing. Sanger sequencing was performed to verify the mutation site. Minigene technology was used to analyse the influence of mutant genes on pre-mRNA shearing, and the Iterative Threading ASSEmbly Refinement (I-TASSER) server was used to analyse the protein structure changes.

Results: The proband, together with her mother and younger brother, displayed microscopic haematuria and proteinuria, Pathological examination revealed mesangial hyperplasia and sclerosis. A novel mutation (NM_000495.5 c.4298-8G > A) in the intron of the COL4A5 gene in the proband was discovered, which was also present in the proband's mother, brother, and grandmother. In vitro minigene expression experiments verified that the c.4298-8G > A mutation caused abnormal splicing, leading to the retention of six base pairs at the end of intron 46. The I-TASSER software predicted that the mutation affected the hydrogen-bonding structure of COL4A5 and the electrostatic potential on the surface of the protein molecules.

Conclusions: Based on the patient's clinical history and genetic traits, we conclude that the mutation at the splicing site c.4298-8G > A of the COL4A5 gene is highly probable to be the underlying cause within this particular family. This discovery expands the genetic spectrum and deepens our understanding of the molecular mechanisms underlying AS.

Keywords: COL4A5 gene; Aberrant splicing; Alport syndrome; Minigene assay.

Fig. 1

Results of renal biopsy. (A, B) Electron microscopy shows irregular thickening and thinning of glomerular basement membrane. (C) Electron microscopy shows vacuolar degeneration in the renal tubular epithelium. (D, E, F) Masson staining shows infiltration of foamy cells in the interstitial

Fig. 2

Results of the mini-gene splicing assay. (A) Sanger sequence of minigene in wt and mut group (c. 4298-8 G > A). (B) The agarose gel electrophoresis of RT-PCR fragments showed that the product length was similar between wt and mut groups. (C) The Sanger sequences showed a 6 bp base (AAATAG) retention at the right of intron 46 in the variant group. (D) The schematic diagram showed the minigene comprising exon 46, exon 47, and intron 46 of the wt or mut (c. 4298-8 G > A). The variant c. 4298-8 G > A affected the normal splicing of COL4A5 mRNA resulting in 6 bp base retention at the right of intron 46. “*” represents variant location

Fig. 3

Family pedigree and confirmation of the variant of COL4A5. (A) The pedigree of the family. Filled symbols represent affected individuals (black: the 4298-8 G > A variant carriers). Arrow indicates the proband. (B) Identification of COLAA5 variant in the family through Sanger sequencing, the arrows represent sites of variant

Fig. 4

Analysis of COL4A5 mutation. (A) Evolutionary conservation of amino acid residues altered by p.(P1432_G1433insEI) across different species. NCBI accession numbers are Homo sapiens: NP_000486.1; Danio rerio: XP_021333076.1; Equus caballus: XP_005614463.1; Macaca mulatta: XP_014983488.2; Mus musculus: XP_006528759.1; Pan troglodytes: XP_016798385.2; Papio Anubis: XP_031516835.1. Asterisk (*) means Inserted amino acids. (B) Prediction of the change in the three-dimensional (3D) structure of hydrogen bond by COL4A5 p.( P1432_G1433insEI) variant. (C) Prediction of the change in the three-dimensional (3D) structure of surface electrostatic potential by COL4A5 p.( P1432_G1433insEI) variant