背景:5'非翻译区(5'UTR)是蛋白质翻译的必需调节剂。预测5个UTR变种的影响具有挑战性,很少在常规诊断中执行。这里,我们提出了一种综合优先排序策略和功能测定的组合方法,以评估两个大型遗传性视网膜疾病(IRD)患者队列中的5'UTR变异.
方法:我们对视网膜RNA-seq数据进行了同工型水平的再分析,以鉴定在视网膜中表达最高的378个IRD基因的蛋白质编码转录本。我们通过不同的全外显子组测序(WES)试剂盒评估了其5'UTR的覆盖率。在来自100,000个基因组项目(n=2397WGS)和内部数据库(n=1682WES)的IRD子队列的全基因组测序(WGS)和WES数据中分析了选定的5个UTR,分别。对鉴定的变异体进行5个UTR相关特征的注释,并根据其预测的功能结果分为7类。我们通过整合人口频率,每个类别的具体标准,以及家族和表型数据。使用多种方法对候选变体的选择进行功能验证。
结果:视网膜基因表达的同种型水平重新定量显示,76个IRD基因具有非规范的视网膜富集同种型,其中20个与其典型同种型相比显示出完全不同的5'UTR。根据探头设计,3-20%的IRD基因有5个UTR被WES完全捕获。在分析了两个队列中的这些区域之后,我们优先考虑了10个基因中的11个(可能)致病变异(ARL3,MERTK,NDP,NMNAT1,NPHP4,PAX6,PRPF31,PRPF4,RDH12,RD3),其中7个是小说。功能分析进一步支持三种变体的致病性。已证明PRPF31:c的错误剪接。-9+1G>T变体。MERTK:c。-125G>一种变体,重叠转录起始位点,显示显著降低荧光素酶mRNA水平和活性。RDH12:c.在顺式中发现-123C>T变体,具有低态RDH12:c.701G>A(p。Arg234His)在11例患者中变异。这5个UTR变体,预测将引入上游开放阅读框架,显示导致RDH12蛋白降低,但mRNA水平未改变。
结论:这项研究证明了IRD中涉及的5个UTR变异的重要性,并提供了一种适用于其他遗传性疾病的5个UTR注释和验证的系统方法。
5\' untranslated regions (5\'UTRs) are essential modulators of protein translation. Predicting the impact of 5\'UTR variants is challenging and rarely performed in routine diagnostics. Here, we present a combined approach of a comprehensive prioritization strategy and functional assays to evaluate 5\'UTR variation in two large cohorts of patients with inherited retinal diseases (IRDs).
We performed an isoform-level re-analysis of retinal RNA-seq data to identify the protein-coding transcripts of 378 IRD genes with highest expression in retina. We evaluated the coverage of their 5\'UTRs by different whole exome sequencing (WES) kits. The selected 5\'UTRs were analyzed in whole genome sequencing (WGS) and WES data from IRD sub-cohorts from the 100,000 Genomes Project (n = 2397 WGS) and an in-house database (n = 1682 WES), respectively. Identified variants were annotated for 5\'UTR-relevant features and classified into seven categories based on their predicted functional consequence. We developed a variant prioritization strategy by integrating population frequency, specific criteria for each category, and family and phenotypic data. A selection of candidate variants underwent functional validation using diverse approaches.
Isoform-level re-quantification of retinal gene expression revealed 76 IRD genes with a non-canonical retina-enriched isoform, of which 20 display a fully distinct 5\'UTR compared to that of their canonical isoform. Depending on the probe design, 3-20% of IRD genes have 5\'UTRs fully captured by WES. After analyzing these regions in both cohorts, we prioritized 11 (likely) pathogenic variants in 10 genes (ARL3, MERTK, NDP, NMNAT1, NPHP4, PAX6, PRPF31, PRPF4, RDH12, RD3), of which 7 were novel. Functional analyses further supported the pathogenicity of three variants. Mis-splicing was demonstrated for the PRPF31:c.-9+1G>T variant. The MERTK:c.-125G>A variant, overlapping a transcriptional start site, was shown to significantly reduce both luciferase mRNA levels and activity. The RDH12:c.-123C>T variant was found in cis with the hypomorphic RDH12:c.701G>A (p.Arg234His) variant in 11 patients. This 5\'UTR variant, predicted to introduce an upstream open reading frame, was shown to result in reduced RDH12 protein but unaltered mRNA levels.
This study demonstrates the importance of 5\'UTR variants implicated in IRDs and provides a systematic approach for 5\'UTR annotation and validation that is applicable to other inherited diseases.