The COL4A3 c.3418+1G>T variant causes exon 39 skipping in minigene assays: functional and exploratory structural analyses

Abstract

Background Canonical splice-site variants are an important cause of Alport syndrome, but the transcript consequences of many heterozygous COL4A3 variants remain experimentally unverified. We characterized COL4A3 c.3418 + 1G>T in a 38-year-old woman with persistent isolated microscopic hematuria. Notably, the proband’s mother also had microscopic hematuria but did not carry c.3418 + 1G>T, precluding supportive co-segregation. Methods Whole-exome sequencing identified the variant, which was subsequently confirmed by Sanger sequencing. Splicing effects were assessed using SpliceAI, Pangolin, and MaxEntScan. Minigene assays were performed in HEK293T, HK-2, and human glomerular endothelial cells. Protein consequences were examined using AlphaFold-derived models, PyMOL visualization, and exploratory 5-ns molecular dynamics simulations of local α3α4α5(IV) trimer models, with additional independently initialized 20-ns replicate simulations performed as a supplementary reproducibility analysis. Results Computational tools consistently predicted loss of the canonical donor splice site. Minigene assays demonstrated complete exon 39 skipping in all 3 cell lines, resulting in an 81-bp in-frame deletion of 27 amino acids within the α3(IV) collagenous domain. Sequence examination showed that the resulting junction preserved the Gly-X-Y register, although its protein-level structural consequences remain undetermined. Exploratory molecular dynamics analysis showed descriptive differences between wild-type and mutant models. The additional 20-ns simulations showed broadly similar RMSD ranges across independent replicates, although RMSD values varied across prespecified time blocks, indicating limited short-timescale sampling consistency. These findings were therefore considered hypothesis-generating rather than evidence of full convergence, altered stability, or impaired interchain interactions. The patient exhibited persistent isolated microscopic hematuria without proteinuria, renal dysfunction, or extrarenal manifestations, compatible with a mild autosomal dominant Alport syndrome phenotype. Conclusion COL4A3 c.3418 + 1G>T causes complete exon 39 skipping in the tested minigene systems and a 27-amino-acid in-frame deletion. Under a conservative author-level application of the ACMG/AMP framework, PVS1_Moderate and PM2_Supporting yield a primary classification of Variant of Uncertain Significance (VUS); PP4 was not applied in the primary classification because isolated microscopic hematuria has limited specificity and the maternal phenotype was discordant. A Likely Pathogenic classification is retained only as a conditional author-level alternative if PVS1 (RNA) is calibrated to Strong and PP4 is applied.