Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Minigene-Based Splice Assays Reveal the Effect of Non-Canonical Splice Site Variants in USH2A.

Non-canonical splice site variants are increasingly recognized as a relevant cause of the USH2A-associated diseases, non-syndromic autosomal recessive retinitis pigmentosa and Usher syndrome type 2. Many non-canonical splice site variants have been reported in public databases, but an effect on pre-mRNA splicing has only been functionally verified for a subset of these variants. In this study, we aimed to extend the knowledge regarding splicing events by assessing a selected set of USH2A non-canonical splice site variants and to study their potential pathogenicity. Eleven non-canonical splice site variants were selected based on four splice prediction tools. Ten different USH2A constructs were generated and minigene splice assays were performed in HEK293T cells. An effect on pre-mRNA splicing was observed for all 11 variants. Various events, such as exon skipping, dual exon skipping and partial exon skipping were observed and eight of the tested variants had a full effect on splicing as no conventionally spliced mRNA was detected. We demonstrated that non-canonical splice site variants in USH2A are an important contributor to the genetic etiology of the associated disorders. This type of variant generally should not be neglected in genetic screening, both in USH2A-associated disease as well as other hereditary disorders. In addition, cases with these specific variants may now receive a conclusive genetic diagnosis.

Scrutinizing pathogenicity of the USH2A c.2276 G > T; p.(Cys759Phe) variant.

The USH2A variant c.2276 G > T (p.(Cys759Phe)) has been described by many authors as a frequent cause of autosomal recessive retinitis pigmentosa (arRP). However, this is in contrast with the description of two asymptomatic individuals homozygous for this variant. We therefore assessed pathogenicity of the USH2A c.2276 G > T variant using extensive genetic and functional analyses. Whole genome sequencing and optical genome mapping were performed for three arRP cases homozygous for USH2A c.2276 G > T to exclude alternative genetic causes. A minigene splice assay was designed to investigate the effect of c.2276 G > T on pre-mRNA splicing, in presence or absence of the nearby c.2256 T > C variant. Moreover, an ush2ap.(Cys771Phe) zebrafish knock-in model mimicking human p.(Cys759Phe) was generated and characterized using functional and immunohistochemical analyses. Besides the homozygous c.2276 G > T USH2A variant, no alternative genetic causes were identified. Evaluation of the ush2ap.(Cys771Phe) zebrafish model revealed strongly reduced levels of usherin expression at the photoreceptor periciliary membrane, increased levels of rhodopsin localization in the photoreceptor cell body and decreased electroretinogram (ERG) b-wave amplitudes compared to wildtype controls. In conclusion, we confirmed pathogenicity of USH2A c.2276 G > T (p.(Cys759Phe)). Consequently, cases homozygous for c.2276 G > T can now receive a definite genetic diagnosis and can be considered eligible for receiving future QR-421a-mediated exon 13 skipping therapy.

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes.

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.

Molecular Inversion Probe-Based Sequencing of USH2A Exons and Splice Sites as a Cost-Effective Screening Tool in USH2 and arRP Cases.

A substantial proportion of subjects with autosomal recessive retinitis pigmentosa (arRP) or Usher syndrome type II (USH2) lacks a genetic diagnosis due to incomplete USH2A screening in the early days of genetic testing. These cases lack eligibility for optimal genetic counseling and future therapy. USH2A defects are the most frequent cause of USH2 and are also causative in individuals with arRP. Therefore, USH2A is an important target for genetic screening. The aim of this study was to assess unscreened or incompletely screened and unexplained USH2 and arRP cases for (likely) pathogenic USH2A variants. Molecular inversion probe (MIP)-based sequencing was performed for the USH2A exons and their flanking regions, as well as published deep-intronic variants. This was done to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in 29 unscreened or partially pre-screened USH2 and 11 partially pre-screened arRP subjects. In 29 out of these 40 cases, two (likely) pathogenic variants were successfully identified. Four of the identified SNVs and one CNV were novel. One previously identified synonymous variant was demonstrated to affect pre-mRNA splicing. In conclusion, genetic diagnoses were obtained for a majority of cases, which confirms that MIP-based sequencing is an effective screening tool for USH2A. Seven unexplained cases were selected for future analysis with whole genome sequencing.

Structural Variants Create New Topological-Associated Domains and Ectopic Retinal Enhancer-Gene Contact in Dominant Retinitis Pigmentosa.

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.

Maintenance of spatial gene expression by Polycomb-mediated repression after formation of a vertebrate body plan.

Polycomb group (PcG) proteins are transcriptional repressors that are important regulators of cell fate during embryonic development. Among them, Ezh2 is responsible for catalyzing the epigenetic repressive mark H3K27me3 and is essential for animal development. The ability of zebrafish embryos lacking both maternal and zygotic ezh2 to form a normal body plan provides a unique model for comprehensively studying Ezh2 function during early development in vertebrates. By using a multi-omics approach, we found that Ezh2 is required for the deposition of H3K27me3 and is essential for proper recruitment of Polycomb group protein Rnf2. However, despite the complete absence of PcG-associated epigenetic mark and proteins, only minor changes in H3K4me3 deposition and gene and protein expression occur. These changes were mainly due to local dysregulation of transcription factors outside their normal expression boundaries. Altogether, our results in zebrafish show that Polycomb-mediated gene repression is important immediately after the body plan is formed to maintain spatially restricted expression profiles of transcription factors, and we highlight the differences that exist in the timing of PcG protein action between vertebrate species.

Loss of the Polycomb group protein Rnf2 results in derepression of tbx-transcription factors and defects in embryonic and cardiac development.

The Polycomb group (PcG) protein family is a well-known group of epigenetic modifiers. We used zebrafish to investigate the role of Rnf2, the enzymatic subunit of PRC1. We found a positive correlation between loss of Rnf2 and upregulation of genes, especially of those whose promoter is normally bound by Rnf2. The heart of rnf2 mutants shows a tubular shaped morphology and to further understand the underlying mechanism, we studied gene expression of single wildtype and rnf2 mutant hearts. We detected the most pronounced differences at 3 dpf, including upregulation of heart transcription factors, such as tbx2a, tbx2b, and tbx3a. These tbx genes were decorated by broad PcG domains in wildtype whole embryo lysates. Chamber specific genes such as vmhc, myh6, and nppa showed downregulation in rnf2 mutant hearts. The marker of the working myocard, nppa, is negatively regulated by Tbx2 and Tbx3. Based on our findings and literature we postulate that loss of Rnf2-mediated repression results in upregulation and ectopic expression of tbx2/3, whose expression is normally restricted to the cardiac conductive system. This could lead to repression of chamber specific gene expression, a misbalance in cardiac cell types, and thereby to cardiac defects observed in rnf2 mutants.

The ezh2(sa1199) mutant zebrafish display no distinct phenotype.

Polycomb group (PcG) proteins are essential regulators of epigenetic gene silencing and development. The PcG protein enhancer of zeste homolog 2 (Ezh2) is a key component of the Polycomb Repressive Complex 2 and is responsible for placing the histone H3 lysine 27 trimethylation (H3K27me3) repressive mark on the genome through its methyltransferase domain. Ezh2 is highly conserved in vertebrates. We studied the role of ezh2 during development of zebrafish with the use of a mutant allele (ezh2(sa1199), R18STOP), which has a stop mutation in the second exon of the ezh2 gene. Two versions of the same line were used during this study. The first and original version of zygotic ezh2(sa1199) mutants unexpectedly retained ezh2 expression in brain, gut, branchial arches, and eyes at 3 days post-fertilization (dpf), as revealed by in-situ hybridization. Moreover, the expression pattern in homozygous mutants was identical to that of wild types, indicating that mutant ezh2 mRNA is not subject to nonsense mediated decay (NMD) as predicted. Both wild type and ezh2 mutant embryos presented edemas at 2 and 3 dpf. The line was renewed by selective breeding to counter select the non-specific phenotypes and survival was assessed. In contrast to earlier studies on ezh2 mutant zebrafish, ezh2(sa1199) mutants survived until adulthood. Interestingly, the ezh2 mRNA and Ezh2 protein were present during adulthood (70 dpf) in both wild type and ezh2(sa1199) mutant zebrafish. We conclude that the ezh2(sa1199) allele does not exhibit an ezh2 loss-of-function phenotype.

Normal formation of a vertebrate body plan and loss of tissue maintenance in the absence of ezh2.

Polycomb group (PcG) proteins are transcriptional repressors of numerous genes, many of which regulate cell cycle progression or developmental processes. We used zebrafish to study Enhancer of zeste homolog 2 (Ezh2), the PcG protein responsible for placing the transcriptional repressive H3K27me3 mark. We identified a nonsense mutant of ezh2 and generated maternal zygotic (MZ) ezh2 mutant embryos. In contrast to knockout mice for PcG proteins, MZezh2 mutant embryos gastrulate seemingly normal, but die around 2 days post fertilization displaying pleiotropic phenotypes. Expression analyses indicated that genes important for early development are not turned off properly, revealing a regulatory role for Ezh2 during zygotic gene expression. In addition, we suggest that Ezh2 regulates maternal mRNA loading of zygotes. Analyses of tissues arising later in development, such as heart, liver, and pancreas, indicated that Ezh2 is required for maintenance of differentiated cell fates. Our data imply that the primary role of Ezh2 is to maintain tissues after tissue specification. Furthermore, our work indicates that Ezh2 is essential to sustain tissue integrity and to set up proper maternal mRNA contribution, and presents a novel and powerful tool to study how PcG proteins contribute to early vertebrate development.