基于表型驱动的全基因组测序分析鉴定出通过异常外显子化导致视网膜营养不良的深度内含子变异。
Phenotype Driven Analysis of Whole Genome Sequencing Identifies Deep Intronic Variants that Cause Retinal Dystrophies by Aberrant Exonization.
作者信息
Di Scipio Matteo, Tavares Erika, Deshmukh Shriya, Audo Isabelle, Green-Sanderson Kit, Zubak Yuliya, Zine-Eddine Fayçal, Pearson Alexander, Vig Anjali, Tang Chen Yu, Mollica Antonio, Karas Jonathan, Tumber Anupreet, Yu Caberry W, Billingsley Gail, Wilson Michael D, Zeitz Christina, Héon Elise, Vincent Ajoy
机构信息
Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Canada.
Sorbonne Université, INSERM, CNRS, Institut de la Vision, Paris, France.
出版信息
Invest Ophthalmol Vis Sci. 2020 Aug 3;61(10):36. doi: 10.1167/iovs.61.10.36.
PURPOSE
To demonstrate the effectiveness of combining retinal phenotyping and focused variant filtering from genome sequencing (GS) in identifying deep intronic disease causing variants in inherited retinal dystrophies.
METHODS
Affected members from three pedigrees with classical enhanced S-cone syndrome (ESCS; Pedigree 1), congenital stationary night blindness (CSNB; Pedigree 2), and achromatopsia (ACHM; Pedigree 3), respectively, underwent detailed ophthalmologic evaluation, optical coherence tomography, and electroretinography. The probands underwent panel-based genetic testing followed by GS analysis. Minigene constructs (NR2E3, GPR179 and CNGB3) and patient-derived cDNA experiments (NR2E3 and GPR179) were performed to assess the functional effect of the deep intronic variants.
RESULTS
The electrophysiological findings confirmed the clinical diagnosis of ESCS, CSNB, and ACHM in the respective pedigrees. Panel-based testing revealed heterozygous pathogenic variants in NR2E3 (NM_014249.3; c.119-2A>C; Pedigree 1) and CNGB3 (NM_019098.4; c.1148delC/p.Thr383Ilefs*13; Pedigree 3). The GS revealed heterozygous deep intronic variants in Pedigrees 1 (NR2E3; c.1100+1124G>A) and 3 (CNGB3; c.852+4751A>T), and a homozygous GPR179 variant in Pedigree 2 (NM_001004334.3; c.903+343G>A). The identified variants segregated with the phenotype in all pedigrees. All deep intronic variants were predicted to generate a splice acceptor gain causing aberrant exonization in NR2E3 [89 base pairs (bp)], GPR179 (197 bp), and CNGB3 (73 bp); splicing defects were validated through patient-derived cDNA experiments and/or minigene constructs and rescued by antisense oligonucleotide treatment.
CONCLUSIONS
Deep intronic mutations contribute to missing heritability in retinal dystrophies. Combining results from phenotype-directed gene panel testing, GS, and in silico splice prediction tools can help identify these difficult-to-detect pathogenic deep intronic variants.
目的
证明将视网膜表型分析与基因组测序（GS）中的聚焦变异筛选相结合，在识别遗传性视网膜营养不良中导致疾病的内含子深处变异方面的有效性。
方法
分别来自三个家系的受累成员，家系1患有典型性增强S-锥体综合征（ESCS），家系2患有先天性静止性夜盲（CSNB），家系3患有全色盲（ACHM），他们接受了详细的眼科评估、光学相干断层扫描和视网膜电图检查。先证者接受了基于基因 panel 的基因检测，随后进行 GS 分析。进行了小基因构建体（NR2E3、GPR179 和 CNGB3）和患者来源的 cDNA 实验（NR2E3 和 GPR179），以评估内含子深处变异的功能效应。
结果
电生理检查结果证实了各家族中 ESCS、CSNB 和 ACHM 的临床诊断。基于基因 panel 的检测在 NR2E3（NM_014249.3；c.119-2A>C；家系 1）和 CNGB3（NM_019098.4；c.1148delC/p.Thr383Ilefs*13；家系 3）中发现了杂合致病性变异。GS 在 家系 1（NR2E3；c.1100+1124G>A）和家系 3（CNGB3；c.852+4751A>T）中发现了杂合内含子深处变异，在 家系 2（NM_001004334.3；c.903+343G>A）中发现了纯合 GPR179 变异。在所有家系中，所识别的变异与表型共分离。所有内含子深处变异预计会产生一个剪接受体增益，导致 NR2E3（89 个碱基对（bp））、GPR179（197 bp）和 CNGB3（73 bp）中出现异常外显子化；通过患者来源的 cDNA 实验和/或小基因构建体验证了剪接缺陷，并通过反义寡核苷酸治疗得以挽救。
结论
内含子深处突变导致视网膜营养不良中存在遗传力缺失。将表型导向的基因 panel 检测、GS 和计算机剪接预测工具的结果相结合，有助于识别这些难以检测到的致病性内含子深处变异。
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