期刊
CLINICAL GENETICS
卷 103, 期 6, 页码 693-698出版社
WILEY
DOI: 10.1111/cge.14305
关键词
deep intronic variants; NPHS2; RNAseq; splicing mutation; steroid resistant nephrotic syndrome; whole-genome sequencing
Whole-genome sequencing allows identification of multiple variants in non-coding regions, but the interpretation of these variants is complicated due to their large number. This study compares three different tools, including a newly developed targeted RNA sequencing, to explore the splicing effect of intronic variations in the NPHS2 gene, which is implicated in a genetic disorder.
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.
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