The RNA helicase and ATPase UPF1 affects transcriptome homeostasis and gene expression through nonsense-mediated RNA decay, splicing, and translation in Arabido

The contribution of splicing variants to molecular diagnostics of inherited diseases is reported to be less than 10%. This figure is likely an underestimation due to several factors including difficulty in predicting the effect of such variants, the need for functional assays, and the inability to detect them (depending on their locations and the sequencing technology used). The aim of this study was to assess the utility of Nanopore sequencing in characterizing and quantifying aberrant splicing events. For this purpose, we selected 19 candidate splicing variants that were identified in patients affected by inherited retinal dystrophies. Several in silico tools were deployed to predict the nature and estimate the magnitude of variant-induced aberrant splicing events. Minigene assay or whole blood-derived cDNA was used to functionally characterize the variants. PCR amplification of minigene-specific cDNA or the target gene in blood cDNA, combined with Nanopore sequencing, was used to identify the resulting transcripts. Thirteen out of nineteen variants caused aberrant splicing events, including cryptic splice site activation, exon skipping, pseudoexon inclusion, or a combination of these. Nanopore sequencing allowed for the identification of full-length transcripts and their precise quantification, which were often in accord with in silico predictions. The method detected reliably low-abundant transcripts, which would not be detected by conventional strategies, such as RT-PCR followed by Sanger sequencing.

Congenital cataracts (CCs) are a leading cause of preventable childhood blindness, with genetic factors playing a crucial role in their etiology. Nance–Horan syndrome (NHS) is a rare X-linked dominant disorder associated with CCs but is often underdiagnosed due to variable expressivity, particularly in female carriers. Objective: This study aimed to explore the genetic landscape of CCs in a Swiss cohort, focusing on two novel NHS and one novel GJA8 variants and their phenotypic presentation. Methods: Whole-exome sequencing (WES) was conducted on 20 unrelated Swiss families diagnosed with CCs. Variants were analyzed for pathogenicity using genetic databases, and segregation analysis was performed. Clinical data, including cataract phenotype and associated systemic anomalies, were assessed to establish genotype–phenotype correlations. Results: Potentially pathogenic DNA sequence variants were identified in 10 families, including three novel variants, one in GJA8 (c.584T>C) and two NHS variants (c.250_252insA and c.484del). Additional previously reported variants were detected in CRYBA1, CRYGC, CRYAA, MIP, EPHA2, and MAF, reflecting genetic heterogeneity in the cohort. Notably, NHS variants displayed significant phenotypic variability, suggesting dose-dependent effects and X-chromosome inactivation in female carriers. Conclusions: NHS remains underdiagnosed due to its variable expressivity and the late manifestation of systemic features, often leading to misclassification as isolated CC. This study highlights the importance of genetic testing in unexplained CC cases to improve early detection of syndromic forms. The identification of novel NHS and GJA8 variants provides new insights into the genetic complexity of CCs, emphasizing the need for further research on genotype–phenotype correlations.

Background/Objectives: Stargardt disease (STGD1) is an autosomal recessive disorder caused by pathogenic variants in ABCA4 that affects the retina and is characterised by progressive central vision loss. The onset of disease manifestations varies from childhood to early adulthood. Methods: Whole exome (WES), whole gene, and whole genome sequencing (WGS) were performed for a patient with STGD1. Results: WES revealed a heterozygous pathogenic missense variant in ABCA4, but no second pathogenic variant was found. ABCA4 whole-gene sequencing, subsequent WGS, and segregation analysis identified a complex deep-intronic allele (NM_000350.2(ABCA4):c.[1555-5882C>A;1555-5784C>G]) in trans to the missense variant. Minigene assays combined with nanopore sequencing were performed to characterise this deep-intronic complex allele in more detail. Surprisingly, the reference minigene revealed the existence of two pseudoexons in intron 11 of the ABCA4 gene that are included in low-abundance (<1%) transcripts. Both pseudoexons could be confirmed in cDNA derived from wildtype retinal organoids. Despite mild splicing predictions, the variant minigene revealed that the complex deep-intronic allele substantially increased the abundance of transcripts that included the pseudoexon overlapping with the variants. Two antisense oligonucleotides (AONs) were designed to rescue the aberrant splicing events. Both AONs increased the proportion of correctly spliced transcripts, and one of them rescued correct splicing to reference levels. Conclusions: Minigene assays combined with nanopore sequencing proved instrumental in identifying low-abundance transcripts including pseudoexons from wildtype ABCA4 intron 11, one of which was substantially increased by the complex allele.