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c.6480-35A>G,一种与斯塔加特病相关的新型分支点变异。

c.6480-35A>G, a novel branchpoint variant associated with Stargardt disease.

作者信息

Rodríguez-Hidalgo María, de Bruijn Suzanne E, Corradi Zelia, Rodenburg Kim, Lara-López Araceli, Valverde-Megías Alicia, Ávila-Fernández Almudena, Fernandez-Caballero Lidia, Del Pozo-Valero Marta, Corominas Jordi, Gilissen Christian, Irigoyen Cristina, Cremers Frans P M, Ayuso Carmen, Ruiz-Ederra Javier, Roosing Susanne

机构信息

Department of Neuroscience, Biodonostia Health Research Institute, Donostia-San Sebastián, Spain.

Department of Genetic, Physical Anthropology and Animal Physiology, University of the Basque Country UPV/EHU, Leioa, Spain.

出版信息

Front Genet. 2023 Sep 7;14:1234032. doi: 10.3389/fgene.2023.1234032. eCollection 2023.

Abstract

Inherited retinal dystrophies (IRDs) can be caused by variants in more than 280 genes. The ATP-binding cassette transporter type A4 () gene is one of these genes and has been linked to Stargardt disease type 1 (STGD1), fundus flavimaculatus, cone-rod dystrophy (CRD), and pan-retinal CRD. Approximately 25% of the reported variants affect RNA splicing. In most cases, it is necessary to perform a functional assay to determine the effect of these variants. Whole genome sequencing (WGS) was performed in one Spanish proband with Stargardt disease. The putative pathogenicity of c.6480-35A>G on splicing was investigated both and . The approach was based on the deep-learning tool SpliceAI. For the approach we used a midigene splice assay in HEK293T cells, based on a previously established wild-type midigene (BA29) containing exons 46 to 48. Through the analysis of WGS data, we identified two candidate variants in in one proband: a previously described deletion, c.699_768+342del (p.(Gln234Phefs*5)), and a novel branchpoint variant, c.6480-35A>G. Segregation analysis confirmed that the variants were in . For the branchpoint variant, SpliceAI predicted an acceptor gain with a high score (0.47) at position c.6480-47. A midigene splice assay in HEK293T cells revealed the inclusion of the last 47 nucleotides of intron 47 creating a premature stop codon and allowed to categorize the variant as moderately severe. Subsequent analysis revealed the presence of this variant as a second allele besides c.1958G>A p.(Arg653His) in an additional Spanish proband in a large cohort of IRD cases. A splice-altering effect of the branchpoint variant, confirmed by the midigene splice assay, along with the identification of this variant in a second unrelated individual affected with STGD, provides sufficient evidence to classify the variant as likely pathogenic. In addition, this research highlights the importance of studying non-coding regions and performing functional assays to provide a conclusive molecular diagnosis.

摘要

遗传性视网膜营养不良(IRDs)可由280多个基因的变异引起。ATP结合盒转运蛋白A4()基因就是其中之一,与1型斯塔加特病(STGD1)、黄斑黄素沉着症、锥杆营养不良(CRD)和全视网膜CRD有关。在已报道的变异中,约25%影响RNA剪接。在大多数情况下,有必要进行功能测定以确定这些变异的影响。对一名患有斯塔加特病的西班牙先证者进行了全基因组测序(WGS)。从和两方面研究了c.6480-35A>G对剪接的潜在致病性。方法基于深度学习工具SpliceAI。对于方法,我们在HEK293T细胞中使用了一个中基因剪接试验,该试验基于一个先前建立的包含外显子46至48的野生型中基因(BA29)。通过对WGS数据的分析,我们在一名先证者的基因中鉴定出两个候选变异:一个先前描述的缺失,c.699_768+342del(p.(Gln234Phefs*5)),以及一个新的分支点变异,c.6480-35A>G。分离分析证实这些变异是。对于分支点变异,SpliceAI预测在c.6480-47位置有一个高分(0.47)的受体增益。在HEK293T细胞中进行的中基因剪接试验显示内含子47的最后47个核苷酸被包含进来,产生了一个提前终止密码子,并可将该变异分类为中度严重。随后的分析显示,在一大群IRD病例中的另一名西班牙先证者中,除了c.1958G>A p.(Arg653His)外,该变异作为第二个等位基因存在。中基因剪接试验证实的分支点变异的剪接改变效应,以及在另一名受STGD影响的无关个体中鉴定出该变异,为将该变异分类为可能致病提供了充分证据。此外,本研究强调了研究非编码区和进行功能测定以提供确定性分子诊断的重要性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4adb/10539688/05968d416eb5/fgene-14-1234032-g001.jpg

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