Kidney Transcriptome Sequencing Improves Molecular Diagnosis and Reveals Splicing Complexity Across the Alport...

Hereditary glomerular basement membrane (GBM) disorders caused by pathogenic variants in COL4A 3/A4/A5 are increasingly recognized as Alport spectrum. Disease severity varies widely and is influenced by variant type, allelic dosage, sex, and genetic modifiers. DNA-based testing incompletely captures this heterogeneity, particularly splice-altering and regulatory variants, limiting precise molecular diagnosis and prognostic stratification.

We performed whole-transcriptome sequencing on 93 kidney biopsies from 93 patients (90 families) with ultrastructural GBM abnormalities. RNA-derived variant detection, splicing analysis, and gene expression profiling were integrated with histopathology, electron microscopy, and clinical data. Aberrant splicing events were quantified, and differential gene expression and microRNA (miRNA) motif enrichment analyses assessed molecular changes associated with kidney function decline.

Transcriptome sequencing identified 64 pathogenic or likely pathogenic variants, including 14 splice-altering variants (22%), often involving noncanonical events, whose functional consequences are not readily resolved by routine DNA sequencing alone. Pathogenic or likely pathogenic variants were detected in 52 of 93 patients (56%), including novel missense and splice-altering variants in COL4A3, COL4A4, COL4A5, and CLCN5. Quantitative splicing revealed heterogeneous exon 27 skipping in COL4A4, indicating regulated exon usage. Gene expression profiling showed progressive estimated glomerular filtration rate (eGFR)-associated remodeling with activation of inflammatory and profibrotic pathways and suppression of renal transporter genes. miRNA enrichment implicated miR-335-5p, miR-325-3p, and miR-874-3p as potential regulators.

Kidney transcriptome sequencing improves molecular diagnosis across the Alport spectrum, enables interpretation of splice-altering variants, and captures signatures linked to progression. Integrating RNA-based analysis may refine classification, enhance prognostic assessment, and support precision medicine in hereditary nephropathies.