Summary The cause of autosomal-dominant retinitis pigmentosa (adRP), which leads to loss of vision and blindness, was investigated in families lacking a molecular diagnosis. A refined locus for adRP on Chr17q22 (RP17) was delineated through genotyping and genome sequencing, leading to the identification of structural variants (SVs) that segregate with disease. Eight different complex SVs were characterized in 22 adRP-affected families with >300 affected individuals. All RP17 SVs had breakpoints within a genomic region spanning YPEL2 to LINC01476. To investigate the mechanism of disease, we reprogrammed fibroblasts from affected individuals and controls into induced pluripotent stem cells (iPSCs) and differentiated them into photoreceptor precursor cells (PPCs) or retinal organoids (ROs). Hi-C was performed on ROs, and differential expression of regional genes and a retinal enhancer RNA at this locus was assessed by qPCR. The epigenetic landscape of the region, and Hi-C RO data, showed that YPEL2 sits within its own topologically associating domain (TAD), rich in enhancers with binding sites for retinal transcription factors. The Hi-C map of RP17 ROs revealed creation of a neo-TAD with ectopic contacts between GDPD1 and retinal enhancers, and modeling of all RP17 SVs was consistent with neo-TADs leading to ectopic retinal-specific enhancer- GDPD1 accessibility. qPCR confirmed increased expression of GDPD1 and increased expression of the retinal enhancer that enters the neo-TAD. Altered TAD structure resulting in increased retinal expression of GDPD1 is the likely convergent mechanism of disease, consistent with a dominant gain of function. Our study highlights the importance of SVs as a genomic mechanism in unsolved Mendelian diseases.

In a Dutch consanguineous family with recessively inherited nonsyndromic hearing impairment (HI), homozygosity mapping combined with whole-exome sequencing revealed a MPZL2 homozygous truncating variant, c.72del (p.Ile24Metfs22). By screening a cohort of phenotype-matched subjects and a cohort of HI subjects in whom WES had been performed previously, we identified two additional families with biallelic truncating variants of MPZL2. Affected individuals demonstrated symmetric, progressive, mild to moderate sensorineural HI. Onset of HI was in the first decade, and high-frequency hearing was more severely affected. There was no vestibular involvement. MPZL2 encodes myelin protein zero-like 2, an adhesion molecule that mediates epithelial cell-cell interactions in several (developing) tissues. Involvement of MPZL2 in hearing was confirmed by audiometric evaluation of Mpzl2-mutant mice. These displayed early-onset progressive sensorineural HI that was more pronounced in the high frequencies. Histological analysis of adult mutant mice demonstrated an altered organization of outer hair cells and supporting cells and degeneration of the organ of Corti. In addition, we observed mild degeneration of spiral ganglion neurons, and this degeneration was most pronounced at the cochlear base. Although MPZL2 is known to function in cell adhesion in several tissues, no phenotypes other than HI were found to be associated with MPZL2 defects. This indicates that MPZL2 has a unique function in the inner ear. The present study suggests that deleterious variants of Mplz2/MPZL2 affect adhesion of the inner-ear epithelium and result in loss of structural integrity of the organ of Corti and progressive degeneration of hair cells, supporting cells, and spiral ganglion neurons.

BackgroundHearing 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.MethodsFamily and cohort studies included exome sequencing and characterisation of the hearing phenotype. Ex vivo protein expression addressed the functional effect of a DNA variant.ResultsAn 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.ConclusionCollectively, 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.

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.

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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.

In conclusion, our results underscore the causality of segregating variants in for autosomal recessive RP. Moreover, our data indicate that KIAA1549 plays a role in photoreceptor function.

Introduction: Retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA) are two groups of inherited retinal diseases (IRDs) where the rod photoreceptors degenerate followed by the cone photoreceptors of the retina. A genetic diagnosis for IRDs is challenging since >280 genes are associated with these conditions. While whole exome sequencing (WES) is commonly used by diagnostic facilities, the costs and required infrastructure prevent its global applicability. Previous studies have shown the cost-effectiveness of sequence analysis using single molecule Molecular Inversion Probes (smMIPs) in a cohort of patients diagnosed with Stargardt disease and other maculopathies.Methods: Here, we introduce a smMIPs panel that targets the exons and splice sites of all currently known genes associated with RP and LCA, the entire RPE65 gene, known causative deep-intronic variants leading to pseudo-exons, and part of the RP17 region associated with autosomal dominant RP, by using a total of 16,812 smMIPs. The RP-LCA smMIPs panel was used to screen 1,192 probands from an international cohort of predominantly RP and LCA cases.Results and discussion: After genetic analysis, a diagnostic yield of 56% was obtained which is on par with results from WES analysis. The effectiveness and the reduced costs compared to WES renders the RP-LCA smMIPs panel a competitive approach to provide IRD patients with a genetic diagnosis, especially in countries with restricted access to genetic testing.