Increasing the Genetic Diagnosis Yield in Inherited Retinal Dystrophies: Assigning Pathogenicity to Novel Non-canonical Splice Site Variants

Abstract

Aims: We aimed to validate the pathogenicity of genetic variants identified in inherited retinal dystrophy (IRD) patients, which were located in non-canonical splice sites (NCSS). Methods: After next generation sequencing (NGS) analysis (target gene panels or whole exome sequencing (WES)), NCSS variants were prioritized according to in silico predictions. In vivo and in vitro functional tests were used to validate their pathogenicity. Results: Four novel NCSS variants have been identified. They are located in intron 33 and 34 of ABCA4 (c.4774-9G>A and c.4849-8C>G, respectively), intron 2 of POC1B (c.101-3T>G) and intron 3 of RP2 (c.884-14G>A). Functional analysis detected different aberrant splicing events, including intron retention, exon skipping and intronic nucleotide addition, whose molecular effect was either the disruption or the elongation of the open reading frame of the corresponding gene. Conclusions: Our data increase the genetic diagnostic yield of IRD patients and expand the landscape of pathogenic variants, which will have an impact on the genotype–phenotype correlations and allow patients to opt for the emerging gene and cell therapies.

The Alter Retina: Alternative Splicing of Retinal Genes in Health and Disease

“…One of the current challenges in the genetic diagnosis of IRDs is the detection and functional validation of variants that have not been previously reported and whose functional significance remains unclear. New cis-acting mutations causing retina-specific splicing defects are usually tested in HEK293T cells using in vitro mini- and midi-genes splice assays because IRD genes are not commonly expressed in accessible human tissues [ 88 , 89 , 90 ]. However, HEK293T cells do not reproduce retinal cell conditions since they do not express retina-specific splicing factors and adjuvant proteins.…”

“…Therefore, occasionally, they result in disease expression, particularly in those patients who carry only a pathogenic allele, thus explaining why some variants are found to be pathogenic in some individuals but not in others. Another example is the NCSS variant c.4849-8C>G, which has also been proved to be pathogenic since it lowers the value of the Pyrimidine tract upstream of the 3′ splice site of intron 34, thus producing transcripts with intron retention that leads to premature protein truncation ( Figure 4 B) [ 88 ].…”

Identification of Deep-Intronic Splice Mutations in a Large Cohort of Patients With Inherited Retinal Diseases

“…One alternative to circumvent these obstacles is to use in vitro minigenes, which directly determine whether single nucleotide polymorphisms (SNPs) disrupt splicing regulation. Many previous studies have validated the minigene assay as a viable approach to evaluate splicing alterations ( Spickett et al, 2016 ; Zernant et al, 2018 ; Bauwens et al, 2019 ; Toulis et al, 2020 ).…”

Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

“…Due to the multitude of loci associated with IRDs (almost 300 loci, ), molecular diagnostics often relies on targeted enrichment and high-throughput sequencing, either whole-exome (WES) [ 3 , 4 , 5 , 6 ] or gene panels [ 5 , 6 , 7 , 8 , 9 , 10 ]. Studies have reported that diagnostic yield when using these standard methods (WES/gene panels) is typically between 50% and 76%, depending, amongst other factors, on the specific clinical subtype being investigated [ 6 , 7 , 8 , 9 , 11 , 12 , 13 , 14 ].…”