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The predominant cause of Fuchs endothelial corneal dystrophy (FECD) is a CTG repeat expansion (termed CTG18.1) situated within an intron of the transcription factor encoding gene,TCF4. Here we use a primary FECD case-derived corneal endothelial cell (CEC) system to enhance our understanding of multiple pathogenic processes underlying CTG18.1-mediated FECD. We define differential gene expression and alternative splice events using long- and short-read RNA-seq datasets generated from 15 biologically independent primary CEC lines, comprehensively characterizing aberrant splicing-related aspects of the disease. Further targeted analysis of these data, alongside a complementary spatial transcriptomics approach, reveals a unique and distinctive pattern ofTCF4-specific dysregulation that underpins expansion-positive FECD and isolates downstream consequences of this shift as an important pathogenic component of disease. To explore whetherTCF4dysregulation, irrespective of CTG18.1 expansions, could cause FECD we also interrogated exome data generated from a large (n=141) genetically refined cohort of CTG18.1 expansion-negative FECD cases. We identify four rare and predicted deleterious (minor allele frequency 15)TCF4variants all encompassed by only a small fraction (10/93) of totalTCF4transcripts suggesting that, in rare instances, disruption of a specific subset of TCF4 isoforms may also confer CEC-specific disease independent of CTG18.1 expansions. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity andTCF4isoform-specific dysregulation, underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.Graphical abstract

North Carolina macular dystrophy (NCMD) is a rare autosomal dominant disease affecting macular development. The disease is caused by non-coding single nucleotide variants (SNVs) in two hotspot regions near PRDM13 and by duplications in two distinct chromosomal loci, overlapping DNase I hypersensitive sites near either PRDM13 or IRX1.To unravel the mechanisms by which these variants cause disease, we first established a genome-wide multi-omics retinal database, RegRet. Integration of UMI-4C profiles we generated on adult human retina then allowed fine-mapping of the interactions of the PRDM13 and IRX1 gene promoters, and the identification of eighteen candidate cis-regulatory elements (cCREs), the activity of which was investigated by luciferase and Xenopus enhancer assays.Next, luciferase assays showed that the non-coding SNVs located in the two hotspot regions of PRDM13 affect cCRE activity, including two novel NCMD-associated non-coding SNVs that we identified. Interestingly, the cCRE containing one of these SNVs was shown to interact with the PRDM13 promoter, demonstrated in vivo activity in Xenopus, and is active at the developmental stage when progenitor cells of the central retina exit mitosis, putting forward this region as a PRDM13 enhancer.Finally, mining of single-cell transcriptional data of embryonic and adult retina revealed the highest expression of PRDM13 and IRX1 when amacrine cells start to synapse with retinal ganglion cells, supporting the hypothesis that altered PRDM13 or IRX1 expression impairs interactions between these cells during retinogenesis.Overall, this study gained insight into the cis-regulatory mechanisms of NCMD and supports that this condition is a retinal enhanceropathy.Graphical abstract

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.