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TMPRSS3-related hearing loss presents challenges in correlating genotypic variants with clinical phenotypes due to the small sample sizes of previous studies. We conducted a cross-sectional genomics study coupled with retrospective clinical phenotype analysis on 127 individuals. These individuals were from 16 academic medical centers across 6 countries. Key findings revealed 47 unique TMPRSS3 variants with significant differences in hearing thresholds between those with missense variants versus those with loss-of-function genotypes. The hearing loss progression rate for the DFNB8 subtype was 0.3 dB/year. Post-cochlear implantation, an average word recognition score of 76% was observed. Of the 51 individuals with two missense variants, 10 had DFNB10 with profound hearing loss. These 10 all had at least one of 4 TMPRSS3 variants predicted by computational modeling to be damaging to TMPRSS3 structure and function. To our knowledge, this is the largest study of TMPRSS3 genotype-phenotype correlations. We find significant differences in hearing thresholds, hearing loss progression, and age of presentation, by TMPRSS3 genotype and protein domain affected. Most individuals with TMPRSS3 variants perform well on speech recognition tests after cochlear implant, however increased age at implant is associated with worse outcomes. These findings provide insight for genetic counseling and the on-going design of novel therapeutic approaches.
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Estudios de Asociación Genética , Pérdida Auditiva , Proteínas de la Membrana , Serina Endopeptidasas , Humanos , Femenino , Masculino , Serina Endopeptidasas/genética , Adulto , Proteínas de la Membrana/genética , Pérdida Auditiva/genética , Niño , Persona de Mediana Edad , Adolescente , Preescolar , Genotipo , Estudios de Cohortes , Fenotipo , Mutación Missense , Estudios Transversales , Adulto Joven , Estudios Retrospectivos , Anciano , Proteínas de NeoplasiasRESUMEN
BACKGROUND: A 12-nucleotide RIPOR2 in-frame deletion was recently identified as a relatively common and highly penetrant cause of autosomal dominant non-syndromic sensorineural hearing loss, type DFNA21, in the Netherlands. The associated hearing phenotype is variable. The allele frequency (AF) of 0.039% of this variant was determined in a local cohort, and the reported phenotype may be biased because studied families were identified based on index patients with hearing loss (HL). In this study, we determine the AF in a cohort from a different geographical region of the Netherlands. Additionally, we examine the hearing phenotype in individuals with the variant but not selected for HL. METHODS: The AF was determined in participants of the Rotterdam Study (RS), a large cohort study. The phenotype was characterised using individual clinical hearing data, including audiograms. RESULTS: The observed AF in the RS cohort was 0.072% and not statistically significantly different from the previously observed 0.039%. The AF in the two cohorts combined was 0.052%. Consistent with previous findings, we found a highly variable audiometric phenotype with non-penetrance of HL in 40% of subjects aged 55-81, which is higher than the 10% at age 50 previously observed. CONCLUSION: We found an overall higher AF and lower penetrance than previously reported, confirming that DFNA21 is relatively common in the Netherlands. This supports its potential suitability as a target for therapeutic development. Studying possible modifying factors is essential to explain the phenotypical variability and to identify patients eligible for such a therapy.
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Non-Syndromic Hereditary Hearing Loss (NSHHL) is a genetically heterogeneous sensory disorder with about 120 genes already associated. Through exome sequencing (ES) and data aggregation, we identified a family with six affected individuals and one unrelated NSHHL patient with predicted-to-be deleterious missense variants in USP48. We also uncovered an eighth patient presenting unilateral cochlear nerve aplasia and a de novo splice variant in the same gene. USP48 encodes a ubiquitin carboxyl-terminal hydrolase under evolutionary constraint. Pathogenicity of the variants is supported by in vitro assays that showed that the mutated proteins are unable to hydrolyze tetra-ubiquitin. Correspondingly, three-dimensional representation of the protein containing the familial missense variant is situated in a loop that might influence the binding to ubiquitin. Consistent with a contribution of USP48 to auditory function, immunohistology showed that the encoded protein is expressed in the developing human inner ear, specifically in the spiral ganglion neurons, outer sulcus, interdental cells of the spiral limbus, stria vascularis, Reissner's membrane and in the transient Kolliker's organ that is essential for auditory development. Engineered zebrafish knocked-down for usp48, the USP48 ortholog, presented with a delayed development of primary motor neurons, less developed statoacoustic neurons innervating the ears, decreased swimming velocity and circling swimming behavior indicative of vestibular dysfunction and hearing impairment. Corroboratingly, acoustic startle response assays revealed a significant decrease of auditory response of zebrafish lacking usp48 at 600 and 800 Hz wavelengths. In conclusion, we describe a novel autosomal dominant NSHHL gene through a multipronged approach combining ES, animal modeling, immunohistology and molecular assays.
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Pérdida Auditiva , Pez Cebra , Animales , Pérdida Auditiva/genética , Humanos , Hidrolasas , Reflejo de Sobresalto , Ubiquitina , Proteasas Ubiquitina-Específicas , Pez Cebra/genéticaRESUMEN
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.
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Cromosomas Humanos Par 17/química , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Retinitis Pigmentosa/genética , Factores de Transcripción/genética , Adulto , Secuencia de Aminoácidos , Diferenciación Celular , Reprogramación Celular , Niño , Mapeo Cromosómico , Estudios de Cohortes , Elementos de Facilitación Genéticos , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Expresión Génica , Genes Dominantes , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Proteínas Nucleares/metabolismo , Organoides/metabolismo , Organoides/patología , Hidrolasas Diéster Fosfóricas/metabolismo , Polimorfismo Genético , Cultivo Primario de Células , Células Fotorreceptoras Retinianas Conos/patología , Retinitis Pigmentosa/diagnóstico , Retinitis Pigmentosa/metabolismo , Retinitis Pigmentosa/patología , Factores de Transcripción/metabolismo , Secuenciación Completa del GenomaRESUMEN
PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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Genoma Humano , Enfermedades de la Retina , Humanos , Estudios Retrospectivos , Genoma Humano/genética , Mapeo Cromosómico , Análisis de Secuencia , Enfermedades de la Retina/genética , Variación Estructural del Genoma , Proteínas del Ojo/genéticaRESUMEN
Despite decades of research, there is much to be learned about the genetic landscape of sensorineural hearing loss. Novel genes for hearing loss remain to be identified while 'secrets' of the known genes need to be uncovered. These 'secrets' include regulatory mechanisms of gene activity and novel aspects of gene structure. To obtain a more complete picture of the genetics of hearing loss, the available experimental and bioinformatic tools need to be fully exploited. This is also true for data resources such as ENCODE. For the inner ear, however, such data resources and analytical tools need to be developed or extended. Collaborative studies provide opportunities to achieve this and to optimally use those tools and resources that are already available. This will accelerate the discoveries that are necessary for improving molecular genetic diagnostics and genetic counselling and for the development of therapeutic strategies.
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Sordera , Oído Interno , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Estudios de Asociación Genética , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/diagnóstico , Pérdida Auditiva Sensorineural/genética , HumanosRESUMEN
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.
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Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/genética , Humanos , Proteínas de Transporte de Membrana/genética , Mutación , Fenotipo , Transportadores de Sulfato/genética , Acueducto Vestibular/anomalíasRESUMEN
Usher syndrome (USH) is an autosomal recessively inherited disease characterized by sensorineural hearing loss (SNHL) and retinitis pigmentosa (RP) with or without vestibular dysfunction. It is highly heterogeneous both clinically and genetically. Recently, variants in the arylsulfatase G (ARSG) gene have been reported to underlie USH type IV. This distinct type of USH is characterized by late-onset RP with predominantly pericentral and macular changes, and late onset SNHL without vestibular dysfunction. In this study, we describe the USH type IV phenotype in three unrelated subjects. We identified three novel pathogenic variants, two novel likely pathogenic variants, and one previously described pathogenic variant in ARSG. Functional experiments indicated a loss of sulfatase activity of the mutant proteins. Our findings confirm that ARSG variants cause the newly defined USH type IV and support the proposed extension of the phenotypic USH classification.
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Retinitis Pigmentosa , Síndromes de Usher , Arilsulfatasas , Humanos , Proteínas Mutantes , Retinitis Pigmentosa/genética , Sulfatasas , Síndromes de Usher/genética , Síndromes de Usher/metabolismoRESUMEN
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.
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Proteínas de la Matriz Extracelular/metabolismo , Mutación , Oligonucleótidos Antisentido/administración & dosificación , Retinitis Pigmentosa/tratamiento farmacológico , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Exones , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Modelos Moleculares , Oligonucleótidos Antisentido/farmacología , Retina/metabolismo , Retinitis Pigmentosa/genética , Retinitis Pigmentosa/metabolismo , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Síndromes de Usher , Humanos , Síndromes de Usher/genética , Células HEK293 , Precursores del ARN , Proteínas de la Matriz Extracelular/genética , Mutación , Sitios de Empalme de ARN/genéticaRESUMEN
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.
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Moléculas de Adhesión Celular/genética , Células Ciliadas Auditivas/patología , Pérdida Auditiva Sensorineural/genética , Audición/genética , Animales , Adhesión Celular/genética , Cóclea/patología , Sordera/genética , Epitelio/patología , Femenino , Homocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Mutación/genética , Neuronas/patología , Ganglio Espiral de la Cóclea/patologíaRESUMEN
PURPOSE: The ClinGen Variant Curation Expert Panels (VCEPs) provide disease-specific rules for accurate variant interpretation. Using the hearing loss-specific American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) guidelines, the Hearing Loss VCEP (HL VCEP) illustrates the utility of expert specifications in variant interpretation. METHODS: A total of 157 variants across nine HL genes, previously submitted to ClinVar, were curated by the HL VCEP. The curation process involved collecting published and unpublished data for each variant by biocurators, followed by bimonthly meetings of an expert curation subgroup that reviewed all evidence and applied the HL-specific ACMG/AMP guidelines to reach a final classification. RESULTS: Before expert curation, 75% (117/157) of variants had single or multiple variants of uncertain significance (VUS) submissions (17/157) or had conflicting interpretations in ClinVar (100/157). After applying the HL-specific ACMG/AMP guidelines, 24% (4/17) of VUS and 69% (69/100) of discordant variants were resolved into benign (B), likely benign (LB), likely pathogenic (LP), or pathogenic (P). Overall, 70% (109/157) variants had unambiguous classifications (B, LB, LP, P). We quantify the contribution of the HL-specified ACMG/AMP codes to variant classification. CONCLUSION: Expert specification and application of the HL-specific ACMG/AMP guidelines effectively resolved discordant interpretations in ClinVar. This study highlights the utility of ClinGen VCEPs in supporting more consistent clinical variant interpretation.
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Genoma Humano , Pérdida Auditiva , Humanos , Pruebas Genéticas , Variación Genética/genética , Pérdida Auditiva/diagnóstico , Pérdida Auditiva/genéticaRESUMEN
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.
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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.
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Proteínas del Ojo/genética , Pérdida Auditiva/diagnóstico , Patología Molecular , Distrofias Retinianas/diagnóstico , Heterogeneidad Genética , Pérdida Auditiva/genética , Pérdida Auditiva/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación/genética , Distrofias Retinianas/genética , Distrofias Retinianas/patologíaRESUMEN
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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Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Edición Génica , Técnicas de Sustitución del Gen/métodos , Enfermedades Genéticas Congénitas/genética , Ingeniería Genética/métodos , Proteínas de Pez Cebra/genética , Animales , Mutagénesis , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Pez Cebra , Proteínas de Pez Cebra/antagonistas & inhibidores , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Retinitis Pigmentosa/genética , Proteínas de Pez Cebra/genética , Animales , Sistemas CRISPR-Cas , Exones , Proteínas del Ojo/química , Proteínas del Ojo/metabolismo , Terapia Genética/métodos , Fenotipo , Dominios Proteicos , Retinitis Pigmentosa/patología , Retinitis Pigmentosa/terapia , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
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Análisis Costo-Beneficio , Exones/genética , Proteínas de la Matriz Extracelular/genética , Sondas Moleculares/metabolismo , Sitios de Empalme de ARN/genética , Retinitis Pigmentosa/genética , Análisis de Secuencia de ADN , Síndromes de Usher/genética , Secuencia de Bases , Variaciones en el Número de Copia de ADN/genética , Eliminación de Gen , Humanos , Polimorfismo de Nucleótido Simple/genética , Retinitis Pigmentosa/economía , Síndromes de Usher/economíaRESUMEN
ATP2B2 encodes the PMCA2 Ca2+ pump that plays an important role in maintaining ion homeostasis in hair cells among others by extrusion of Ca2+ from the stereocilia to the endolymph. Several mouse models have been described for this gene; mice heterozygous for loss-of-function defects display a rapidly progressive high-frequency hearing impairment. Up to now ATP2B2 has only been reported as a modifier, or in a digenic mechanism with CDH23 for hearing impairment in humans. Whole exome sequencing in hearing impaired index cases of Dutch and Polish origins revealed five novel heterozygous (predicted to be) loss-of-function variants of ATP2B2. Two variants, c.1963G>T (p.Glu655*) and c.955delG (p.Ala319fs), occurred de novo. Three variants c.397+1G>A (p.?), c.1998C>A (p.Cys666*), and c.2329C>T (p.Arg777*), were identified in families with an autosomal dominant inheritance pattern of hearing impairment. After normal newborn hearing screening, a rapidly progressive high-frequency hearing impairment was diagnosed at the age of about 3-6 years. Subjects had no balance complaints and vestibular testing did not yield abnormalities. There was no evidence for retrocochlear pathology or structural inner ear abnormalities. Although a digenic inheritance pattern of hearing impairment has been reported for heterozygous missense variants of ATP2B2 and CDH23, our findings indicate a monogenic cause of hearing impairment in cases with loss-of-function variants of ATP2B2.
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Biomarcadores/análisis , Predisposición Genética a la Enfermedad , Pérdida Auditiva/genética , Mutación , ATPasas Transportadoras de Calcio de la Membrana Plasmática/genética , Adolescente , Adulto , Anciano , Niño , Preescolar , Femenino , Estudios de Seguimiento , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , Pronóstico , Adulto JovenRESUMEN
PURPOSE: To characterize new molecular factors implicated in a hereditary congenital facial paresis (HCFP) family and otosclerosis. METHODS: We performed exome sequencing in a four-generation family presenting nonprogressive HCFP and mixed hearing loss (HL). MEPE was analyzed using either Sanger sequencing or molecular inversion probes combined with massive parallel sequencing in 89 otosclerosis families, 1604 unrelated affected subjects, and 1538 unscreened controls. RESULTS: Exome sequencing in the HCFP family led to the identification of a rare segregating heterozygous frameshift variant p.(Gln425Lysfs*38) in MEPE. As the HL phenotype in this family resembled otosclerosis, we performed variant burden and variance components analyses in a large otosclerosis cohort and demonstrated that nonsense and frameshift MEPE variants were significantly enriched in affected subjects (p = 0.0006-0.0060). CONCLUSION: MEPE exerts its function in bone homeostasis by two domains, an RGD and an acidic serine aspartate-rich MEPE-associated (ASARM) motif inhibiting respectively bone resorption and mineralization. All variants associated with otosclerosis are predicted to result in nonsense mediated decay or an ASARM-and-RGD-truncated MEPE. The HCFP variant is predicted to produce an ASARM-truncated MEPE with an intact RGD motif. This difference in effect on the protein corresponds with the presumed pathophysiology of both diseases, and provides a plausible molecular explanation for the distinct phenotypic outcome.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Parálisis Facial/congénito , Glicoproteínas/genética , Otosclerosis/genética , Fosfoproteínas/genética , Adulto , Huesos/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Parálisis Facial/etiología , Parálisis Facial/genética , Parálisis Facial/metabolismo , Familia , Femenino , Enfermedades Genéticas Ligadas al Cromosoma X/genética , Variación Genética/genética , Glicoproteínas/metabolismo , Pérdida Auditiva/genética , Heterocigoto , Humanos , Masculino , Linaje , Fenotipo , Fosfoproteínas/metabolismo , Secuenciación del Exoma/métodosRESUMEN
BACKGROUND: Retinitis pigmentosa (RP) shows substantial genetic heterogeneity. It has been estimated that in approximately 60%-80% of RP cases, the genetic diagnosis can be found using whole exome sequencing (WES). In this study, the purpose was to identify causative variants in individuals with genetically unexplained retinal disease, which included one consanguineous family with two affected siblings and one case with RP. METHODS: To identify the genetic defect, WES was performed in both probands, and clinical analysis was performed. To obtain insight into the function of KIAA1549 in photoreceptors, mRNA expression, knockdown and protein localisation studies were performed. RESULTS: Through analysis of WES data, based on population allele frequencies, and in silico prediction tools, we identified a homozygous missense variant and a homozygous frameshift variant in KIAA1549 that segregate in two unrelated families. Kiaa1549 was found to localise at the connecting cilium of the photoreceptor cells and the synapses of the mouse retina. Both variants affect the long transcript of KIAA1549, which encodes a 1950 amino acid protein and shows prominent brain expression. The shorter transcript encodes a 734 amino acid protein with a high retinal expression and is affected by the identified missense variant. Strikingly, knockdown of the long transcript also leads to decreased expression of the short transcript likely explaining the non-syndromic retinal phenotype caused by the two variants targeting different transcripts. CONCLUSION: In conclusion, our results underscore the causality of segregating variants in KIAA1549 for autosomal recessive RP. Moreover, our data indicate that KIAA1549 plays a role in photoreceptor function.