Long-read RNA sequencing identifies a pathogenic retrotransposon insertion in NMNAT1 in Italian probands

Abstract

Purpose : Inherited retinal diseases (IRDs) are a genetically and clinically heterogeneous group of disorders leading to visual impairment. Due to limitations of current sequencing platforms, 30% of patients still lack a conclusive genetic diagnosis. Pathogenic intronic variants are part of this missing heritability and to test their potential effect on splicing, midigene assays have been used. Technical limitations, artefacts, and the time-consuming aspect of this technique hamper its employment in routine diagnostics. Here we report the use of targeted long-read RNA sequencing (lrRNA-seq) of whole blood. This enabled us to obtain full length transcript sequences to uncover splicing defects in persons with intronic variants detected by whole genome sequencing (WGS) or in persons with one recessive variant in a gene strongly correlated to their phenotypes.

Methods : We selected variants from three studies (PMID 34795310, 35047838 and 35608844) to validate this technique. Next, three monoallelic NMNAT1 cases were included for further studies. RNA was extracted from blood-derived samples and cDNA was synthesized using SuperScript IV. We performed PCR to amplify the entire (or portions of the) open reading frame of transcripts. After PacBio sequencing, resulting reads were bioinformatically processed to obtain exon junction information for IGV visualization.

Results : We validated our approach using known splicing variants in CHM, HGSNAT, and IMPG2 and detected the previously reported splicing defects. Thereafter, we used this method to identify potential splicing defects being missed with previous approaches in three Italian persons carrying a NMNAT1 pathogenic variant. With targeted lrRNA-seq, we identified the insertion of part of a SVA-F retrotransposon in intron two of NMNAT1 which is predicted to introduce a premature stop codon (p.Tyr41Serfs58*). Subsequent screening of 11 NMNAT1 Caucasian monoallelic cases yielded one additional case with this allele.

Conclusions : Despite recent advances, obtaining a genetic diagnosis for IRD patients remains challenging. With targeted lrRNA-seq, we established a straightforward method that allowed us to identify the genetic cause in four IRD cases. This approach is effective in increasing the diagnostic rate in IRD patients, either by validating splicing variants detected by WGS or by identifying pathogenic variants missed by short-read sequencing approaches.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.