Optical genome mapping and revisiting short-read genome sequencing data reveal previously overlooked structural variants disrupting retinal disease-associated genes.

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.

RPE-Directed Gene Therapy Improves Mitochondrial Function in Murine Dry AMD Models.

Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85-90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.

Using single molecule Molecular Inversion Probes as a cost-effective, high-throughput sequencing approach to target all genes and loci associated with macular diseases.

Macular degenerations (MDs) are a subgroup of retinal disorders characterized by central vision loss. Knowledge is still lacking on the extent of genetic and nongenetic factors influencing inherited MD (iMD) and age-related MD (AMD) expression. Single molecule Molecular Inversion Probes (smMIPs) have proven effective in sequencing the ABCA4 gene in patients with Stargardt disease to identify associated coding and noncoding variation, however many MD patients still remain genetically unexplained. We hypothesized that the missing heritability of MDs may be revealed by smMIPs-based sequencing of all MD-associated genes and risk factors. Using 17,394 smMIPs, we sequenced the coding regions of 105 iMD and AMD-associated genes and noncoding or regulatory loci, known pseudo-exons, and the mitochondrial genome in two test cohorts that were previously screened for variants in ABCA4. Following detailed sequencing analysis of 110 probands, a diagnostic yield of 38% was observed. This established an ''MD-smMIPs panel," enabling a genotype-first approach in a high-throughput and cost-effective manner, whilst achieving uniform and high coverage across targets. Further analysis will identify known and novel variants in MD-associated genes to offer an accurate clinical diagnosis to patients. Furthermore, this will reveal new genetic associations for MD and potential genetic overlaps between iMD and AMD.

Usher syndrome type IV: clinically and molecularly confirmed by novel ARSG variants.

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.

SARM1 Ablation Is Protective and Preserves Spatial Vision in an In Vivo Mouse Model of Retinal Ganglion Cell Degeneration.

The challenge of developing gene therapies for genetic forms of blindness is heightened by the heterogeneity of these conditions. However, mechanistic commonalities indicate key pathways that may be targeted in a gene-independent approach. Mitochondrial dysfunction and axon degeneration are common features of many neurodegenerative conditions including retinal degenerations. Here we explore the neuroprotective effect afforded by the absence of sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1), a prodegenerative NADase, in a rotenone-induced mouse model of retinal ganglion cell loss and visual dysfunction. Sarm1 knockout mice retain visual function after rotenone insult, displaying preservation of photopic negative response following rotenone treatment in addition to significantly higher optokinetic response measurements than wild type mice following rotenone. Protection of spatial vision is sustained over time in both sexes and is accompanied by increased RGC survival and additionally preservation of axonal density in optic nerves of Sarm1-/- mice insulted with rotenone. Primary fibroblasts extracted from Sarm1-/- mice demonstrate an increased oxygen consumption rate relative to those from wild type mice, with significantly higher basal, maximal and spare respiratory capacity. Collectively, our data indicate that Sarm1 ablation increases mitochondrial bioenergetics and confers histological and functional protection in vivo in the mouse retina against mitochondrial dysfunction, a hallmark of many neurodegenerative conditions including a variety of ocular disorders.

Clinical and Genetic Re-Evaluation of Inherited Retinal Degeneration Pedigrees following Initial Negative Findings on Panel-Based Next Generation Sequencing.

Although rare, inherited retinal degenerations (IRDs) are the most common reason for blind registration in the working age population. They are highly genetically heterogeneous (>300 known genetic loci), and confirmation of a molecular diagnosis is a prerequisite for many therapeutic clinical trials and approved treatments. First-tier genetic testing of IRDs with panel-based next-generation sequencing (pNGS) has a diagnostic yield of ≈70-80%, leaving the remaining more challenging cases to be resolved by second-tier testing methods. This study describes the phenotypic reassessment of patients with a negative result from first-tier pNGS and the rationale, outcomes, and cost of second-tier genetic testing approaches. Removing non-IRD cases from consideration and utilizing case-appropriate second-tier genetic testing techniques, we genetically resolved 56% of previously unresolved pedigrees, bringing the overall resolve rate to 92% (388/423). At present, pNGS remains the most cost-effective first-tier approach for the molecular assessment of diverse IRD populations Second-tier genetic testing should be guided by clinical (i.e., reassessment, multimodal imaging, electrophysiology), and genetic (i.e., single alleles in autosomal recessive disease) indications to achieve a genetic diagnosis in the most cost-effective manner.

Next-Generation Sequencing Applications for Inherited Retinal Diseases.

Inherited retinal diseases (IRDs) represent a collection of phenotypically and genetically diverse conditions. IRDs phenotype(s) can be isolated to the eye or can involve multiple tissues. These conditions are associated with diverse forms of inheritance, and variants within the same gene often can be associated with multiple distinct phenotypes. Such aspects of the IRDs highlight the difficulty met when establishing a genetic diagnosis in patients. Here we provide an overview of cutting-edge next-generation sequencing techniques and strategies currently in use to maximise the effectivity of IRD gene screening. These techniques have helped researchers globally to find elusive causes of IRDs, including copy number variants, structural variants, new IRD genes and deep intronic variants, among others. Resolving a genetic diagnosis with thorough testing enables a more accurate diagnosis and more informed prognosis and should also provide information on inheritance patterns which may be of particular interest to patients of a child-bearing age. Given that IRDs are heritable conditions, genetic counselling may be offered to help inform family planning, carrier testing and prenatal screening. Additionally, a verified genetic diagnosis may enable access to appropriate clinical trials or approved medications that may be available for the condition.

Fibrotic Changes to Schlemm's Canal Endothelial Cells in Glaucoma.

Previous studies have shown that glaucomatous Schlemm's canal endothelial cells (gSCECs) are stiffer and associated with reduced porosity and increased extracellular matrix (ECM) material compared to SCECs from healthy individuals. We hypothesised that Schlemm's canal (SC) cell stiffening was a function of fibrotic changes occurring at the inner wall of SC in glaucoma. This study was performed in primary cell cultures isolated from the SC lumen of human donor eyes. RNA and protein quantification of both fibrotic and endothelial cell markers was carried out on both healthy and gSCECs. Functional assays to assess cell density, size, migration, proliferation, and mitochondrial function of these cells were also carried out. Indeed, we found that gSCECs deviate from typical endothelial cell characteristics and exhibit a more fibrotic phenotype. For example, gSCECs expressed significantly higher protein levels of the fibrotic markers α-SMA, collagen I-α1, and fibronectin, as well as significantly increased protein expression of TGFß-2, the main driver of fibrosis, compared to healthy SCECs. Interestingly, we observed a significant increase in protein expression of endothelial marker VE-cadherin in gSCECs, compared to healthy SCECs. gSCECs also appeared to be significantly larger, and surprisingly proliferate and migrate at a significantly higher rate, as well as showing significantly reduced mitochondrial activity, compared to healthy SCECs.

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.

Resolving the dark matter of ABCA4 for 1054 Stargardt disease probands through integrated genomics and transcriptomics.

PURPOSE: Missing heritability in human diseases represents a major challenge, and this is particularly true for ABCA4-associated Stargardt disease (STGD1). We aimed to elucidate the genomic and transcriptomic variation in 1054 unsolved STGD and STGD-like probands. METHODS: Sequencing of the complete 128-kb ABCA4 gene was performed using single-molecule molecular inversion probes (smMIPs), based on a semiautomated and cost-effective method. Structural variants (SVs) were identified using relative read coverage analyses and putative splice defects were studied using in vitro assays. RESULTS: In 448 biallelic probands 14 known and 13 novel deep-intronic variants were found, resulting in pseudoexon (PE) insertions or exon elongations in 105 alleles. Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions and flanking exon deletions. Eleven distinct large deletions were found, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. CONCLUSION: Deep sequencing of ABCA4 and midigene-based splice assays allowed the identification of SVs and causal deep-intronic variants in 25% of biallelic STGD1 cases, which represents a model study that can be applied to other inherited diseases.

Toward an elucidation of the molecular genetics of inherited retinal degenerations.

While individually classed as rare diseases, hereditary retinal degenerations (IRDs) are the major cause of registered visual handicap in the developed world. Given their hereditary nature, some degree of intergenic heterogeneity was expected, with genes segregating in autosomal dominant, recessive, X-linked recessive, and more rarely in digenic or mitochondrial modes. Today, it is recognized that IRDs, as a group, represent one of the most genetically diverse of hereditary conditions - at least 260 genes having been implicated, with 70 genes identified in the most common IRD, retinitis pigmentosa (RP). However, targeted sequencing studies of exons from known IRD genes have resulted in the identification of candidate mutations in only approximately 60% of IRD cases. Given recent advances in the development of gene-based medicines, characterization of IRD patient cohorts for known IRD genes and elucidation of the molecular pathologies of disease in those remaining unresolved cases has become an endeavor of the highest priority. Here, we provide an outline of progress in this area.

Retinal Bioenergetics: New Insights for Therapeutics.

With 329 genes known to be involved in inherited retinal degenerations (IRDs), focus has shifted to generic targets for therapeutics, targets that could provide benefit irrespective of the underlying genetic condition. As one of the most energy-demanding tissues, the retina is acutely sensitive to dysfunction of its energy metabolism. Recent discoveries have shed light on the complex interconnectivity and interdependence of retinal cells on their choice metabolic pathways, highlighting a number of potential targets that could benefit cells in a mutation-independent manner. Some of the latest research on retinal metabolism and mitophagy in photoreceptors and retinal pigment epithelium is discussed, as is how these insights could potentially be used in the design of new therapies.

A Novel FLVCR1 Variant Implicated in Retinitis Pigmentosa.

Here we describe the identification and evaluation of a rare novel autosomal recessive mutation in FLVCR1 which is implicated solely in RP, with no evidence of posterior column ataxia in a number of affected patients. The mutation was detected as part of an ongoing target capture NGS study (Target 5000), aimed at identifying candidate variants in pedigrees with inherited retinal degenerations (IRDs) in Ireland. The mutation, FLVCR1 p.Tyr341Cys, was observed homozygously in seven affected patients across four pedigrees. FLVCR1 p.Tyr341Cys is a very rare mutation, with no previous reports of pathogenicity and no homozygous cases reported in online allele frequency databases. Our sequencing study identified seven homozygotes across multiple pedigrees, all with similar clinical presentations of RP without ataxia, a scenario extremely unlikely to occur by chance for a benign allele, particularly given the low population frequency of p.Tyr341Cys.

Mitochondrial disorders: aetiologies, models systems, and candidate therapies.

It has become evident that many human disorders are characterised by mitochondrial dysfunction either at a primary level, due to mutations in genes whose encoded products are involved in oxidative phosphorylation, or at a secondary level, due to the accumulation of mitochondrial DNA (mtDNA) mutations. This has prompted keen interest in the development of cell and animal models and in exploring innovative therapeutic strategies to modulate the mitochondrial deficiencies observed in these diseases. Key advances in these areas are outlined in this review, with a focus on Leber hereditary optic neuropathy (LHON). This exciting field is set to grow exponentially and yield many candidate therapies to treat this class of disease.

Gene expression changes during retinal development and rod specification.

PURPOSE: Retinitis pigmentosa (RP) typically results from individual mutations in any one of >70 genes that cause rod photoreceptor cells to degenerate prematurely, eventually resulting in blindness. Gene therapies targeting individual RP genes have shown efficacy at clinical trial; however, these therapies require the surviving photoreceptor cells to be viable and functional, and may be economically feasible for only the more commonly mutated genes. An alternative potential treatment strategy, particularly for late stage disease, may involve stem cell transplants into the photoreceptor layer of the retina. Rod progenitors from postnatal mouse retinas can be transplanted and can form photoreceptors in recipient adult retinas; optimal numbers of transplantable cells are obtained from postnatal day 3-5 (P3-5) retinas. These cells can also be expanded in culture; however, this results in the loss of photoreceptor potential. Gene expression differences between postnatal retinas, cultured retinal progenitor cells (RPCs), and rod photoreceptor precursors were investigated to identify gene expression patterns involved in the specification of rod photoreceptors. METHODS: Microarrays were used to investigate differences in gene expression between cultured RPCs that have lost photoreceptor potential, P1 retinas, and fresh P5 retinas that contain significant numbers of transplantable photoreceptors. Additionally, fluorescence-activated cell sorting (FACS) sorted Rho-eGFP-expressing rod photoreceptor precursors were compared with Rho-eGFP-negative cells from the same P5 retinas. Differential expression was confirmed with quantitative polymerase chain reaction (q-PCR). RESULTS: Analysis of the microarray data sets, including the use of t-distributed stochastic neighbor embedding (t-SNE) to identify expression pattern neighbors of key photoreceptor specific genes, resulted in the identification of 636 genes differentially regulated during rod specification. Forty-four of these genes when mutated have previously been found to cause retinal disease. Although gene function in other tissues may be known, the retinal function of approximately 61% of the gene list is as yet undetermined. Many of these genes' promoters contain binding sites for the key photoreceptor transcription factors Crx and Nr2e3; moreover, the genomic clustering of differentially regulated genes appears to be non-random. CONCLUSIONS: This study aids in understanding gene expression differences between rod photoreceptor progenitors versus cultured RPCs that have lost photoreceptor potential. The results provide insights into rod photoreceptor development and should expedite the development of cell-based treatments for RP. Furthermore, the data set includes a large number of retinopathy genes; less-well-characterized genes within this data set are a resource for those seeking to identify novel retinopathy genes in patients with RP (GEO accession: GSE59201).

Gene therapies for inherited retinal disorders.

Significant advances have been made over the last decade or two in the elucidation of the molecular pathogenesis of inherited ocular disorders. In particular, remarkable successes have been achieved in exploration of gene-based medicines for these conditions, both in preclinical and in clinical studies. Progress in the development of gene therapies targeted toward correcting the primary genetic defect or focused on modulating secondary effects associated with retinal pathologies are discussed in the review. Likewise, the recent utilization of genes encoding light-sensing molecules to provide new functions to residual retinal cells in the degenerating retina is discussed. While a great deal has been learned over the last two decades, the next decade should result in an increasing number of preclinical studies progressing to human clinical trial, an exciting prospect for patients, those active in research and development and bystanders alike.

Representation of Women Among Individuals With Mild Variants in ABCA4-Associated Retinopathy: A Meta-Analysis.

Importance: Previous studies indicated that female sex might be a modifier in Stargardt disease, which is an ABCA4-associated retinopathy. Objective: To investigate whether women are overrepresented among individuals with ABCA4-associated retinopathy who are carrying at least 1 mild allele or carrying nonmild alleles. Data Sources: Literature data, data from 2 European centers, and a new study. Data from a Radboudumc database and from the Rotterdam Eye Hospital were used for exploratory hypothesis testing. Study Selection: Studies investigating the sex ratio in individuals with ABCA4-AR and data from centers that collected ABCA4 variant and sex data. The literature search was performed on February 1, 2023; data from the centers were from before 2023. Data Extraction and Synthesis: Random-effects meta-analyses were conducted to test whether the proportions of women among individuals with ABCA4-associated retinopathy with mild and nonmild variants differed from 0.5, including subgroup analyses for mild alleles. Sensitivity analyses were performed excluding data with possibly incomplete variant identification. χ2 Tests were conducted to compare the proportions of women in adult-onset autosomal non-ABCA4-associated retinopathy and adult-onset ABCA4-associated retinopathy and to investigate if women with suspected ABCA4-associated retinopathy are more likely to obtain a genetic diagnosis. Data analyses were performed from March to October 2023. Main Outcomes and Measures: Proportion of women per ABCA4-associated retinopathy group. The exploratory testing included sex ratio comparisons for individuals with ABCA4-associated retinopathy vs those with other autosomal retinopathies and for individuals with ABCA4-associated retinopathy who underwent genetic testing vs those who did not. Results: Women were significantly overrepresented in the mild variant group (proportion, 0.59; 95% CI, 0.56-0.62; P < .001) but not in the nonmild variant group (proportion, 0.50; 95% CI, 0.46-0.54; P = .89). Sensitivity analyses confirmed these results. Subgroup analyses on mild variants showed differences in the proportions of women. Furthermore, in the Radboudumc database, the proportion of adult women among individuals with ABCA4-associated retinopathy (652/1154 = 0.56) was 0.10 (95% CI, 0.05-0.15) higher than among individuals with other retinopathies (280/602 = 0.47). Conclusions and Relevance: This meta-analysis supports the likelihood that sex is a modifier in developing ABCA4-associated retinopathy for individuals with a mild ABCA4 allele. This finding may be relevant for prognosis predictions and recurrence risks for individuals with ABCA4-associated retinopathy. Future studies should further investigate whether the overrepresentation of women is caused by differences in the disease mechanism, by differences in health care-seeking behavior, or by health care discrimination between women and men with ABCA4-AR.

Towards Uncovering the Role of Incomplete Penetrance in Maculopathies through Sequencing of 105 Disease-Associated Genes.

Inherited macular dystrophies (iMDs) are a group of genetic disorders, which affect the central region of the retina. To investigate the genetic basis of iMDs, we used single-molecule Molecular Inversion Probes to sequence 105 maculopathy-associated genes in 1352 patients diagnosed with iMDs. Within this cohort, 39.8% of patients were considered genetically explained by 460 different variants in 49 distinct genes of which 73 were novel variants, with some affecting splicing. The top five most frequent causative genes were ABCA4 (37.2%), PRPH2 (6.7%), CDHR1 (6.1%), PROM1 (4.3%) and RP1L1 (3.1%). Interestingly, variants with incomplete penetrance were revealed in almost one-third of patients considered solved (28.1%), and therefore, a proportion of patients may not be explained solely by the variants reported. This includes eight previously reported variants with incomplete penetrance in addition to CDHR1:c.783G>A and CNGB3:c.1208G>A. Notably, segregation analysis was not routinely performed for variant phasing-a limitation, which may also impact the overall diagnostic yield. The relatively high proportion of probands without any putative causal variant (60.2%) highlights the need to explore variants with incomplete penetrance, the potential modifiers of disease and the genetic overlap between iMDs and age-related macular degeneration. Our results provide valuable insights into the genetic landscape of iMDs and warrant future exploration to determine the involvement of other maculopathy genes.

Optimisation of AAV-NDI1 Significantly Enhances Its Therapeutic Value for Correcting Retinal Mitochondrial Dysfunction.

AAV gene therapy for ocular disease has become a reality with the market authorisation of LuxturnaTM for RPE65-linked inherited retinal degenerations and many AAV gene therapies currently undergoing phase III clinical trials. Many ocular disorders have a mitochondrial involvement from primary mitochondrial disorders such as Leber hereditary optic neuropathy (LHON), predominantly due to mutations in genes encoding subunits of complex I, to Mendelian and multifactorial ocular conditions such as dominant optic atrophy, glaucoma and age-related macular degeneration. In this study, we have optimised the nuclear yeast gene, NADH-quinone oxidoreductase (NDI1), which encodes a single subunit complex I equivalent, creating a candidate gene therapy to improve mitochondrial function, independent of the genetic mutation driving disease. Optimisation of NDI1 (ophNdi1) substantially increased expression in vivo, protected RGCs and increased visual function, as assessed by optokinetic and photonegative response, in a rotenone-induced murine model. In addition, ophNdi1 increased cellular oxidative phosphorylation and ATP production and protected cells from rotenone insult to a significantly greater extent than wild type NDI1. Significantly, ophNdi1 treatment of complex I deficient patient-derived fibroblasts increased oxygen consumption and ATP production rates, demonstrating the potential of ophNdi1 as a candidate therapy for ocular disorders where mitochondrial deficits comprise an important feature.

Usher Syndrome on the Island of Ireland: A Genotype-Phenotype Review.

Purpose: Usher syndrome (USH) is a genetically heterogeneous group of autosomal recessive (AR) syndromic inherited retinal degenerations (IRDs) representing 50% of deaf-blindness. All subtypes include retinitis pigmentosa, sensorineural hearing loss, and vestibular abnormalities. Thorough phenotyping may facilitate genetic diagnosis and intervention. Here we report the clinical/genetic features of an Irish USH cohort. Methods: USH patients were selected from the Irish IRD registry (Target 5000). Patients were examined clinically (deep-phenotyping) and genetically using a 254 IRD-associated gene target capture sequencing panel, USH2A exon, and whole genome sequencing. Results: The study identified 145 patients (24.1% USH1 [n = 35], 73.8% USH2 [n = 107], 1.4% USH3 [n = 2], and 0.7% USH4 [n = 1]). A genetic diagnosis was reached in 82.1%, the majority (80.7%) being MYO7A or USH2A genotypes. Mean visual acuity and visual field (VF) were 0.47 ± 0.58 LogMAR and 31.3° ± 32.8°, respectively, at a mean age of 43 years. Legal blindness criteria were met in 40.7%. Cataract was present in 77.4%. ADGRV1 genotypes had the most VF loss, whereas USH2A patients had greater myopia and CDH23 had the most astigmatism. Variants absent from gnomAD non-Finnish Europeans and ClinVar represented more than 20% of the variants identified and were detected in ADGRV1, ARSG, CDH23, MYO7A, and USH2A. Conclusions: USH is a genetically diverse group of AR IRDs that have a profound impact on affected individuals and their families. The prevalence and phenotype/genotype characteristics of USH in Ireland have, as yet, gone unreported. Understanding the genotype of Irish USH patients may guide clinical and genetic characterization facilitating access to existing/novel therapeutics.