Structural variants (SVs) play an important role in inherited retinal diseases (IRD). Although the identification of SVs significantly improved upon the availability of genome sequencing, it is expected that involvement of SVs in IRDs is higher than anticipated. We revisited short-read genome sequencing data to enhance the identification of gene-disruptive SVs. Optical genome mapping was performed to improve SV detection in short-read genome sequencing-negative cases. In addition, reanalysis of short-read genome sequencing data was performed to improve the interpretation of SVs and to re-establish SV prioritization criteria. In a monoallelic USH2A case, optical genome mapping identified a pericentric inversion (173 megabase), with 1 breakpoint disrupting USH2A. Retrospectively, the variant could be observed in genome sequencing data but was previously deemed false positive. Reanalysis of short-read genome sequencing data (427 IRD cases) was performed which yielded 30 pathogenic SVs affecting, among other genes, USH2A (n = 15), PRPF31 (n = 3), and EYS (n = 2). Eight of these (>25%) were overlooked during previous analyses. Critical evaluation of our findings allowed us to re-establish and improve our SV prioritization and interpretation guidelines, which will prevent missing pathogenic events in future analyses. Our data suggest that more attention should be paid to SV interpretation and the current contribution of SVs in IRDs is still underestimated.

CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models.

Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.

Background: Hearing loss is one of the most prevalent disabilities worldwide, and has a significant impact on quality of life. The adult-onset type of the condition is highly heritable but the genetic causes are largely unknown, which is in contrast to childhood-onset hearing loss. Methods: Family and cohort studies included exome sequencing and characterisation of the hearing phenotype. Ex vivo protein expression addressed the functional effect of a DNA variant. Results: An in-frame deletion of 12 nucleotides in RIPOR2 was identified as a highly penetrant cause of adult-onset progressive hearing loss that segregated as an autosomal dominant trait in 12 families from the Netherlands. Hearing loss associated with the deletion in 63 subjects displayed variable audiometric characteristics and an average (SD) age of onset of 30.6 (14.9) years (range 0–70 years). A functional effect of the RIPOR2 variant was demonstrated by aberrant localisation of the mutant RIPOR2 in the stereocilia of cochlear hair cells and failure to rescue morphological defects in RIPOR2-deficient hair cells, in contrast to the wild-type protein. Strikingly, the RIPOR2 variant is present in 18 of 22 952 individuals not selected for hearing loss in the Southeast Netherlands. Conclusion: Collectively, the presented data demonstrate that an inherited form of adult-onset hearing loss is relatively common, with potentially thousands of individuals at risk in the Netherlands and beyond, which makes it an attractive target for developing a (genetic) therapy.

The identification of pathogenic variants in monogenic diseases has been of interest to researchers and clinicians for several decades. However, for inherited diseases with extremely high genetic heterogeneity, such as hearing loss and retinal dystrophies, establishing a molecular diagnosis requires an enormous effort. In this review, we use these two genetic conditions as examples to describe the initial molecular genetic identification approaches, as performed since the early 90s, and subsequent improvements and refinements introduced over the years. Next, the history of DNA sequencing from conventional Sanger sequencing to high-throughput massive parallel sequencing, a.k.a. next-generation sequencing, is outlined, including their advantages and limitations and their impact on identifying the remaining genetic defects. Moreover, the development of recent technologies, also coined “third-generation” sequencing, is reviewed, which holds the promise to overcome these limitations. Furthermore, we outline the importance and complexity of variant interpretation in clinical diagnostic settings concerning the massive number of different variants identified by these methods. Finally, we briefly mention the development of novel approaches such as optical mapping and multiomics, which can help to further identify genetic defects in the near future.

Pathogenic variants in SLC26A4 have been associated with autosomal recessive hearing loss (arHL) and a unilateral or bilateral enlarged vestibular aqueduct (EVA). SLC26A4 is the second most frequently mutated gene in arHL. Despite the strong genotype–phenotype correlation, a significant part of cases remains genetically unresolved. In this study, we investigated a cohort of 28 Dutch index cases diagnosed with HL in combination with an EVA but without (M0) or with a single (M1) pathogenic variant in SLC26A4. To explore the missing heritability, we first determined the presence of the previously described EVA-associated haplotype (Caucasian EVA (CEVA)), characterized by 12 single nucleotide variants located upstream of SLC26A4. We found this haplotype and a delimited V1-CEVA haplotype to be significantly enriched in our M1 patient cohort (10/16 cases). The CEVA haplotype was also present in two M0 cases (2/12). Short- and long-read whole genome sequencing and optical genome mapping could not prioritize any of the variants present within the CEVA haplotype as the likely pathogenic defect. Short-read whole-genome sequencing of the six M1 cases without this haplotype and the two M0/CEVA cases only revealed previously overlooked or misinterpreted splice-altering SLC26A4 variants in two cases, who are now genetically explained. No deep-intronic or structural variants were identified in any of the M1 subjects. With this study, we have provided important insights that will pave the way for elucidating the missing heritability in M0 and M1 SLC26A4 cases. For pinpointing the pathogenic effect of the CEVA haplotype, additional analyses are required addressing defect(s) at the RNA, protein, or epigenetic level.