Long-read sequencing improves the genetic diagnosis of retinitis pigmentosa by identifying an Alu retrotransposon insertion in the EYS gene

• Published in: Mobile DNA 2024-05, Vol.15 (1), p.9-9, Article 9
• Main Authors: Fernández-Suárez, Elena, González-Del Pozo, María, Méndez-Vidal, Cristina, Martín-Sánchez, Marta, Mena, Marcela, de la Morena-Barrio, Belén, Corral, Javier, Borrego, Salud, Antiñolo, Guillermo
• Format: Article
• Language: English
• Subjects: Alu insertion; Analysis; Blindness; CNV; DNA sequencing; EYS; Family; Genes; Inherited retinal diseases; Long-read sequencing; Nanopore sequencing; Nucleotide sequencing; Retinitis pigmentosa
• ISSN: 1759-8753; 1759-8753
• DOI: 10.1186/s13100-024-00320-1

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. 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. 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.