背景:EYS中的双等位基因变异是某些人群中常染色体隐性遗传性色素性视网膜炎(arRP)的主要原因,一种可能导致法律失明的临床和遗传异质性疾病。EYS是视网膜中表达量最大的基因之一(~2Mb),其中结构变异(SVs)代表疾病的常见原因。然而,使用短读取测序(SRS)进行鉴定并不总是可行的.这里,我们在MinION测序平台(OxfordNanoporeTechnologies)上使用EYS的自适应采样进行了靶向长读测序(T-LRS),以明确诊断arRP家族,其受影响的个体(n=3)携带EYS基因中外显子32-33的杂合致病性缺失。由于这是在我们队列中另外三个家族中发现的复发性变异,我们还旨在表征核苷酸水平的已知缺失,以评估可能的创始人效应。
结果:家族A中的T-LRS揭示了EYS编码外显子43中的杂合AluYa5插入(chr6(GRCh37):g.6440524_64430525ins352),与先前鉴定的缺失以复合杂合性与疾病分离。使用IGV对先前的SRS比对进行的视觉检查显示了几个包含软剪切碱基的读数,伴随着Alu插入部位的覆盖率略有下降。这促使我们使用grep命令开发一个简化的程序,以根据SRS数据调查我们队列中这种变异的复发。此外,LRS还允许将CNV表征为跨越EYS的外显子32-33的〜56.4kb缺失(chr6(GRCh37):g.64764235_64820592del)。在四个家族中通过Sanger测序和连锁分析进一步表征的结果与创始人变体一致。
结论:据我们所知,这是关于将移动元素插入EYS编码序列的第一份报告,作为家庭中arRP的可能原因。我们的研究强调了LRS技术在表征和识别隐藏致病性SVs中的价值,如逆转录转座子插入,可能低估了其对罕见疾病病因的贡献。
BACKGROUND: Biallelic variants in EYS are the major cause of autosomal recessive retinitis pigmentosa (arRP) in certain populations, a clinically and genetically heterogeneous disease that may lead to legal blindness. EYS is one of the largest genes (~ 2 Mb) expressed in the retina, in which structural variants (SVs) represent a common cause of disease. However, their identification using short-read sequencing (SRS) is not always feasible. Here, we conducted targeted long-read sequencing (T-LRS) using adaptive sampling of EYS on the MinION sequencing platform (Oxford Nanopore Technologies) to definitively diagnose an arRP family, whose affected individuals (n = 3) carried the heterozygous pathogenic deletion of exons 32-33 in the EYS gene. As this was a recurrent variant identified in three additional families in our cohort, we also aimed to characterize the known deletion at the nucleotide level to assess a possible founder effect.
RESULTS: T-LRS in family A unveiled a heterozygous AluYa5 insertion in the coding exon 43 of EYS (chr6(GRCh37):g.64430524_64430525ins352), which segregated with the disease in compound heterozygosity with the previously identified deletion. Visual inspection of previous SRS alignments using IGV revealed several reads containing soft-clipped bases, accompanied by a slight drop in coverage at the Alu insertion site. This prompted us to develop a simplified program using grep command to investigate the recurrence of this variant in our cohort from SRS data. Moreover, LRS also allowed the characterization of the CNV as a ~ 56.4kb deletion spanning exons 32-33 of EYS (chr6(GRCh37):g.64764235_64820592del). The results of further characterization by Sanger sequencing and linkage analysis in the four families were consistent with a founder variant.
CONCLUSIONS: To our knowledge, this is the first report of a mobile element insertion into the coding sequence of EYS, as a likely cause of arRP in a family. Our study highlights the value of LRS technology in characterizing and identifying hidden pathogenic SVs, such as
retrotransposon insertions, whose contribution to the etiopathogenesis of rare diseases may be underestimated.