PDF(Pubmed)
Abstract：
Biallelic variants in USH2A are associated with retinitis pigmentosa (RP) and Type 2 Usher Syndrome (USH2), leading to impaired vision and, additionally, hearing loss in the latter. Although the introduction of next-generation sequencing into clinical diagnostics has led to a significant uplift in molecular diagnostic rates, many patients remain molecularly unsolved. It is thought that non-coding variants or variants of uncertain significance contribute significantly to this diagnostic gap. This study aims to demonstrate the clinical utility of the reverse transcription-polymerase chain reaction (RT-PCR)-Oxford Nanopore Technology (ONT) sequencing of USH2A mRNA transcripts from nasal epithelial cells to determine the splice-altering effect of candidate variants. Five affected individuals with USH2 or non-syndromic RP who had undergone whole genome sequencing were recruited for further investigation. All individuals had uncertain genotypes in USH2A, including deep intronic rare variants, c.8682-654C>G, c.9055+389G>A, and c.9959-2971C>T; a synonymous variant of uncertain significance, c.2139C>T; p.(Gly713=); and a predicted loss of function duplication spanning an intron/exon boundary, c.3812-3_3837dup p.(Met1280Ter). In silico assessment using SpliceAI provided splice-altering predictions for all candidate variants which were investigated using ONT sequencing. All predictions were found to be accurate; however, in the case of c.3812-3_3837dup, the outcome was a complex cryptic splicing pattern with predominant in-frame exon 18 skipping and a low level of exon 18 inclusion leading to the predicted stop gain. This study detected and functionally characterised simple and complex mis-splicing patterns in USH2A arising from previously unknown deep intronic variants and previously reported variants of uncertain significance, confirming the pathogenicity of the variants.
摘要：
USH2A的双等位基因变异与色素性视网膜炎（RP）和2型Usher综合征（USH2）有关，导致视力受损，此外，听力损失在后者。尽管将下一代测序引入临床诊断中已导致分子诊断率的显着提高，许多患者的分子仍未溶解。认为非编码变体或具有不确定意义的变体显著地促成该诊断缺口。这项研究旨在证明逆转录聚合酶链反应（RT-PCR）-牛津纳米孔技术（ONT）测序的临床实用性来自鼻上皮细胞的USH2AmRNA转录本，以确定候选变体的剪接改变作用。招募了5名接受全基因组测序的USH2或非综合征性RP患者进行进一步研究。所有个体在USH2A中都有不确定的基因型，包括深内含子的罕见变异，c.8682-654C>G，c.9055+389G>A，和c.9959-2971C>T；意义不确定的同义词变体，c.2139C>T；p。（Gly713=）；以及跨内含子/外显子边界的功能重复预测损失，c.3812-3_3837页（Met1280Ter）。使用SpleeAI的计算机模拟评估提供了使用ONT测序研究的所有候选变体的剪接改变预测。所有的预测都是准确的；然而，在c.3812-3_3837dup的情况下，结果是复杂的隐匿性剪接模式,主要的帧内外显子18跳跃和低水平的外显子18包含导致预测的停止增益.这项研究检测并在功能上表征了USH2A中的简单和复杂的错误剪接模式，这些模式是由先前未知的深层内含子变体和先前报道的不确定意义的变体引起的。确认变体的致病性。
