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Background/Objectives : Neural retina leucine zipper (NRL) is a transcription factor involved in the differentiation of rod photoreceptors. Pathogenic variants in the gene encoding NRL have been associated with autosomal dominant retinitis pigmentosa and autosomal recessive clumped pigmentary retinal degeneration. Only a dozen unrelated families affected by recessive NRL -related retinal dystrophy have been described. The purpose of this study was to expand the genotypic spectrum of this disease by reporting clinical and genetic findings of two unrelated families. Methods : Index patients affected by retinal dystrophy were genetically tested by whole-exome sequencing (WES) and whole-genome sequencing (WGS). Segregation analysis within the families was performed for candidate variants. A minigene assay was performed to functionally characterize a variant suspected to affect splicing. Results : Variant filtering revealed homozygous NRL variants in both families. The variant in patient A was a small deletion encompassing the donor splice site of exon 1 of transcript NM_006177.3. The minigene assay revealed that this variant led to two aberrant transcripts that used alternative cryptic donor splice sites located in intron 1. In patient B, a stop-gain variant was identified in the last exon of NRL in a homozygous state due to maternal uniparental disomy of chromosome 14. Conclusions : Our study expands the genotypic spectrum of autosomal recessive NRL -related retinal dystrophy. Moreover, it underscores the importance of actively maintaining bioinformatic pipelines for variant detection and the utility of minigene assays in functionally characterizing candidate splicing variants.

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&#95;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.

X-linked retinitis pigmentosa (XLRP) caused by mutations in the RPGR gene is one of the most severe forms of RP due to its early onset and intractable progression. Most cases have been associated with genetic variants within the purine-rich exon ORF15 region of this gene. RPGR retinal gene therapy is currently being investigated in several clinical trials. Therefore, it is crucial to report and functionally characterize (all novel) potentially pathogenic DNA sequence variants. Whole-exome sequencing (WES) was performed for the index patient. The splicing effects of a non-canonical splice variant were tested on cDNA from whole blood and a minigene assay. WES revealed a rare, non-canonical splice site variant predicted to disrupt the wildtype splice acceptor and create a novel acceptor site 8 nucleotides upstream of RPGR exon 12. Reverse-transcription PCR analyses confirmed the disruption of the correct splicing pattern, leading to the insertion of eight additional nucleotides in the variant transcript. Transcript analyses with minigene assays and cDNA from peripheral blood are useful tools for the characterization of splicing defects due to variants in the RPGR and may increase the diagnostic yield in RP. The functional analysis of non-canonical splice variants is required to classify those variants as pathogenic according to the ACMG's criteria.

Coloboma and microphthalmia (C/M) are related congenital eye malformations, which can cause significant visual impairment. Molecular diagnosis is challenging as the genes associated to date with C/M account for only a small percentage of cases. Overall, the genetic cause remains unknown in up to 80% of patients. High throughput DNA sequencing technologies, including whole-exome sequencing (WES), are therefore a useful and efficient tool for genetic screening and identification of new mutations and novel genes in C/M. In this study, we analyzed the DNA of 19 patients with C/M from 15 unrelated families using singleton WES and data analysis for 307 genes of interest. We identified seven novel and one recurrent potentially disease-causing variants in CRIM1 , CHD7 , FAT1 , PTCH1 , PUF60 , BRPF1 , and TGFB2 in 47% of our families, three of which occurred de novo. The detection rate in patients with ocular and extraocular manifestations (67%) was higher than in patients with an isolated ocular phenotype (46%). Our study highlights the significant genetic heterogeneity in C/M cohorts and emphasizes the diagnostic power of WES for the screening of patients and families with C/M.