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

Pathogenic variants in COL4A3, COL4A4 and COL4A5, including noncanonical splice-altering variants, with heterogeneous skipping of COL4A4 exon 27 indicating regulated exon usage.

Hereditary glomerular basement membrane disorders caused by pathogenic variants in COL4A3, COL4A4 and COL4A5 form the Alport spectrum, whose severity depends on variant type, allelic dosage, sex and genetic modifiers. The authors performed whole-transcriptome sequencing on 93 kidney biopsies from 93 patients (90 families) with ultrastructural glomerular basement membrane abnormalities, integrating RNA-derived variant detection, splicing analysis and expression profiling with histopathology, electron microscopy and clinical data. Transcriptome sequencing identified 64 pathogenic or likely pathogenic variants, including 14 splice-altering variants (22%) that frequently involved noncanonical events not readily resolved by routine DNA sequencing; a pathogenic or likely pathogenic variant was found in 52 of 93 patients (56%), including novel missense and splice-altering variants in COL4A3, COL4A4, COL4A5 and CLCN5. Quantitative splicing analysis revealed heterogeneous skipping of COL4A4 exon 27, pointing to regulated exon usage. Expression profiling showed progressive remodelling associated with estimated glomerular filtration rate, with activation of inflammatory and profibrotic pathways, suppression of renal transporter genes, and motif enrichment for miR-335-5p, miR-325-3p and miR-874-3p.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

The practical gain is resolving, directly in the affected tissue, splice-altering variants that DNA testing alone leaves as variants of uncertain significance, and they are not marginal: 22% of the variants retained. The trade-off is invasive material and a retrospective series of biopsies already performed, which restricts use to patients already investigated histologically, and 41 of 93 patients remain without a molecular diagnosis. Before this becomes a routine step, one would want to know what an accessible tissue would yield, and to confirm prospectively that the expression signatures genuinely predict decline in kidney function.

Analysis by Dr Thibaut Benquey