Generation and Characterization of a Zebrafish Model for ADGRV1-Associated Retinal Dysfunction Using CRISPR/Cas9 Genome Editing Technology.

Worldwide, around 40,000 people progressively lose their eyesight as a consequence of retinitis pigmentosa (RP) caused by pathogenic variants in the ADGRV1 gene, for which currently no treatment options exist. A model organism that mimics the human phenotype is essential to unravel the exact pathophysiological mechanism underlying ADGRV1-associated RP, and to evaluate future therapeutic strategies. The introduction of CRISPR/Cas-based genome editing technologies significantly improved the possibilities of generating mutant models in a time- and cost-effective manner. Zebrafish have been recognized as a suitable model to study Usher syndrome-associated retinal dysfunction. Using CRISPR/Cas9 technology we introduced a 4bp deletion in adgrv1 exon 9 (adgrv1rmc22). Immunohistochemical analysis showed that Adgrv1 was absent from the region of the photoreceptor connecting cilium in the adgrv1rmc22 zebrafish retina. Here, the absence of Adgrv1 also resulted in reduced levels of the USH2 complex members usherin and Whrnb, suggesting that Adgrv1 interacts with usherin and Whrnb in zebrafish photoreceptors. When comparing adgrv1rmc22 zebrafish with wild-type controls, we furthermore observed increased levels of aberrantly localized rhodopsin in the photoreceptor cell body, and decreased electroretinogram (ERG) B-wave amplitudes which indicate that the absence of Adgrv1 results in impaired retinal function. Based on these findings we present the adgrv1rmc22 zebrafish as the first ADGRV1 mutant model that displays an early retinal dysfunction. Moreover, the observed phenotypic changes can be used as quantifiable outcome measures when evaluating the efficacy of future novel therapeutic strategies for ADGRV1-associated RP.

A protein domain-oriented approach to expand the opportunities of therapeutic exon skipping for USH2A-associated retinitis pigmentosa.

Loss-of-function mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). We previously presented skipping of USH2A exon 13 as a promising treatment paradigm for USH2A-associated RP. However, RP-associated mutations are often private, and evenly distributed along the USH2A gene. In order to broaden the group of patients that could benefit from therapeutic exon skipping strategies, we expanded our approach to other USH2A exons in which unique loss-of-function mutations have been reported by implementing a protein domain-oriented dual exon skipping strategy. We first generated zebrafish mutants carrying a genomic deletion of the orthologous exons of the frequently mutated human USH2A exons 30-31 or 39-40 using CRISPR-Cas9. Excision of these in-frame combinations of exons restored usherin expression in the zebrafish retina and rescued the photopigment mislocalization typically observed in ush2a mutants. To translate these findings into a future treatment in humans, we employed in vitro assays to identify and validate antisense oligonucleotides (ASOs) with a high potency for sequence-specific dual exon skipping. Together, the in vitro and in vivo data demonstrate protein domain-oriented ASO-induced dual exon skipping to be a highly promising treatment option for RP caused by mutations in USH2A.

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.

Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eysΔexon40-44 transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, EysΔexon40-44 protein expression could not be detected. Visual motor response experiments revealed that eysΔexon40-44 larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations.

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Double Porphyrin Cage Compounds.

The synthesis and characterization of double porphyrin cage compounds are described. They consist of two porphyrins that are each attached to a diphenylglycoluril-based clip molecule via four ethyleneoxy spacers, and are linked together by a single alkyl chain using "click"-chemistry. Following a newly developed multistep synthesis procedure we report three of these double porphyrin cages, linked by spacers of different lengths, i.e. 3, 5, and 11 carbon atoms. The structures of the double porphyrin cages were fully characterized by NMR, which revealed that they consist of mixtures of two diastereoisomers. Their zinc derivatives are capable of forming sandwich-like complexes with the ditopic ligand 1,4-diazabicyclo[2,2,2]octane (dabco).

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature.

Purpose: Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR. Methods: Using CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked. Results: Intriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf. Conclusions: Together, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina.

Methods in Experimental Work Break Research: A Scoping Review.

The number of studies on work breaks and the importance of this subject is growing rapidly, with research showing that work breaks increase employees' wellbeing and performance and workplace safety. However, comparing the results of work break research is difficult since the study designs and methods are heterogeneous and there is no standard theoretical model for work breaks. Based on a systematic literature search, this scoping review included a total of 93 studies on experimental work break research conducted over the last 30 years. This scoping review provides a first structured evaluation regarding the underlying theoretical framework, the variables investigated, and the measurement methods applied. Studies using a combination of measurement methods from the categories "self-report measures," "performance measures," and "physiological measures" are most common and to be preferred in work break research. This overview supplies important information for ergonomics researchers allowing them to design work break studies with a more structured and stronger theory-based approach. A standard theoretical model for work breaks is needed in order to further increase the comparability of studies in the field of experimental work break research in the future.

Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G Mutation in USH2A.

The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% ± 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.

MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse.

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.Ile24Metfs∗22). 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.

Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish.

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

C2orf71a/pcare1 is important for photoreceptor outer segment morphogenesis and visual function in zebrafish.

Mutations in C2orf71 are causative for autosomal recessive retinitis pigmentosa and occasionally cone-rod dystrophy. We have recently discovered that the protein encoded by this gene is important for modulation of the ciliary membrane through the recruitment of an actin assembly module, and have therefore renamed the gene to PCARE (photoreceptor cilium actin regulator). Here, we report on the identification of two copies of the c2orf71/pcare gene in zebrafish, pcare1 and pcare2. To study the role of the gene most similar to human PCARE, pcare1, we have generated a stable pcare1 mutant zebrafish model (designated pcare1 rmc100/rmc100 ) in which the coding sequence was disrupted using CRISPR/Cas9 technology. Retinas of both embryonic (5 dpf) and adult (6 mpf) pcare1 rmc100/rmc100 zebrafish display a clear disorganization of photoreceptor outer segments, resembling the phenotype observed in Pcare-/- mice. Optokinetic response and visual motor response measurements indicated visual impairment in pcare1 rmc100/rmc100 zebrafish larvae at 5 dpf. In addition, electroretinogram measurements showed decreased b-wave amplitudes in pcare1 rmc100/rmc100 zebrafish as compared to age- and strain-matched wild-type larvae, indicating a defect in the transretinal current. Altogether, our data show that lack of pcare1 causes a retinal phenotype in zebrafish and indicate that the function of the PCARE gene is conserved across species.

Usherin defects lead to early-onset retinal dysfunction in zebrafish.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

Functional work breaks in a high-demanding work environment: an experimental field study.

Work breaks are known to have positive effects on employees' health, performance and safety. Using a sample of twelve employees working in a stressful and cognitively demanding working environment, this experimental field study examined how different types of work breaks (boxing, deep relaxation and usual breaks) affect participants' mood, cognitive performance and neurophysiological state compared to a control condition without any break. In a repeated measures experimental design, cognitive performance was assessed using an auditory oddball test and a Movement Detection Test. Brain cortical activity was recorded using electroencephalography. Individual's mood was analysed using a profile of mood state. Although neurophysiological data showed improved relaxation of cortical state after boxing (vs. 'no break' and 'deep relaxation'), neither performance nor mood assessment showed similar results. It remains questionable whether there is a universal work break type that has beneficial effects for all individuals. Practitioner Summary: Research on work breaks and their positive effects on employees' health and performance often disregards break activities. This experimental field study in a stressful working environment investigated the effect of different work break activities. A universal work break type that is beneficial for this workplace could not be identified.

Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation.

Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

Short Bouts of Intensive Exercise During the Workday Have a Positive Effect on Neuro-cognitive Performance.

Beside its positive impact on physical health, exercise is indicated to positively affect cognitive performance based on a relocation of cortical activity. This study examined the influence of different types of breaks on cognitive performance and related cortical activity in office-based employees. Breaks were filled with exercise, resting or a usual break and a control condition where employees continued working without any break. Cognitive performance was assessed using the d2-R test and two commercially available cognitive tasks. Brain cortical activity was recorded using electroencephalography before and after breaks. Individual's mood was analysed using a profile of mood state. Results indicate a positive effect of a 3-min boxing intervention on cognitive performance, mirrored by a decrease in prefrontal cortex activity. Although perceived psychological state was increased after the usual break, this is reflected in neither cortical activity nor cognitive performance. With respect to the fact that also bike activity resulted an increase in prefrontal alpha-2 activity, a positive effect of exercise on neuro-cognitive performance can be stated. Health and economic benefits may result from brief physical activity breaks and help to maintain workplace performance and job satisfaction. Copyright © 2015 John Wiley & Sons, Ltd.

NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish.

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking.

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.

Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy.

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors.

Unexpected CEP290 mRNA splicing in a humanized knock-in mouse model for Leber congenital amaurosis.

Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.