INSERM U1163, Laboratoire des Maladies Rénales Héréditaires, Institut Imagine, Université Paris-Cité, France.
Laboratoires de biologie médicale SeqOIA, Paris, France.
Clin Genet. 2023 Jun;103(6):693-698. doi: 10.1111/cge.14305. Epub 2023 Feb 6.
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.
全基因组测序（WGS）现在可以识别非编码区域中的多个变体。然而，WGS 识别出的大量变体使得它们的解释变得复杂。通过鉴定第一个深内含子变体在 NPHS2 中，该变体编码 podocin，一种与常染色体隐性类固醇抵抗性肾病综合征（SRNS）相关的蛋白质，我们在此比较了三种不同的工具，包括新开发的靶向基于 NGS 的 RNA 测序，以探索内含子变异的剪接效应。WGS 最终确定了两个不同的 NPHS2 变体与疾病有关。通过 RT-PCR、外显子捕获 Minigene 测定和靶向 RNA 测序，我们能够从患者的肾脏组织中鉴定出 NPHS2 mRNA 的剪接缺陷。只有靶向 RNA-seq 同时分析了多个变体的影响，并提供了量化对剪接影响的机会。鉴定深内含子变体及其在疾病中的作用至关重要。通过计算机工具可以预测选择性剪接，但始终需要通过涉及组织的 RNA 分析进行功能测试来确认，这仍然是金标准。当 WGS 识别出多个具有潜在剪接影响的变体时，靶向 RNA 测序面板可能非常有价值。
