Clin Genet

Whole-genome sequencing (WGS) now allows identification of multiple variants in non-coding regions. The large number of variants identified by WGS however complicates their interpretation. Through identification of the first deep intronic variant in NPHS2, which encodes podocin, a protein implicated in autosomal recessive steroid resistant nephrotic syndrome (SRNS), we compare herein three different tools including a newly developed targeted NGS-based RNA-sequencing to explore the splicing effect of intronic variations. WGS identified two different variants in NPHS2 eventually involved in the disease. Through RT-PCR, exon-trapping Minigene assay and targeted RNA sequencing, we were able to identify the splicing defect in NPHS2 mRNA from patient kidney tissue. Only targeted RNA-seq simultaneously analyzed the effect of multiple variants and offered the opportunity to quantify consequences on splicing. Identifying deep intronic variants and their role in disease is of utmost importance. Alternative splicing can be predicted by in silico tools but always requires confirmation through functional testing with RNA analysis from the implicated tissue remaining the gold standard. When several variants with potential effects on splicing are identified by WGS, a targeted RNA sequencing panel could be of great value.