Minigene-based splicing analysis uncovers pathogenic splice-altering effects of PAX2.

BACKGROUND: Missense variants in disease-associated genes cause aberrant pre-mRNA splicing, carrying profound implications for molecular diagnostics, pathogenic mechanism elucidation, and personalized therapy development. The distinct genotype-phenotype correlation observed in PAX2-related disorders underscores the need to evaluate how identified PAX2 gene variants impact pre-mRNA splicing. This study aimed to investigate the effects of PAX2 missense variants localized to the first or last nucleotides of an exon on pre-mRNA splicing. METHODS: Four PAX2 variants located at the last nucleotide of exons [NM_000278.5:c.212G > A (p.Arg71Lys), c.212G > T (p.Arg71Met), c.212G > C (p.Arg71Thr), and c.919G > A (p.Gly307Ser)] obtained from the Human Gene Mutation Database, along with one variant identified in our cohort [c.427G > C (p.Val143Leu)], were analyzed. A minigene splicing assay was performed to evaluate their functional effects on pre-mRNA splicing. Comprehensive clinical data were collected from the patient harboring the c.427G > C variant. RESULTS: Three bioinformatic prediction tools indicated that aforementioned five candidate variants were likely to affect RNA splicing. The in vitro minigene assay demonstrated that all these variants consistently induced the skipping of target exons. For the patient with the PAX2 c.427G > C variant, renal dysfunction was observed and postulated to be linked to renal hypoplasia; concomitantly, the patient presented with proteinuria, a complication primarily ascribed to obesity-related focal segmental glomerulosclerosis. Following three years of clinical management, the patient achieved weight normalization, accompanied by stable renal function and proteinuria. CONCLUSIONS: Transcript-level RNA analysis reclassified PAX2 missense variants located at the last nucleotide of exons as splice-altering variants, which highlights the indispensable role of transcript-level assessment in characterizing exonic point variants of disease-causing genes to advance pathogenic mechanism research and personalized therapy.