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Despite increasing success in determining genetic diagnosis for patients with inherited retinal diseases (IRDs), mutations in about 30% of the IRD cases remain unclear or unsettled after targeted gene panel or whole exome sequencing. In this study, we aimed to investigate the contributions of structural variants (SVs) to settling the molecular diagnosis of IRD with whole-genome sequencing (WGS). A cohort of 755 IRD patients whose pathogenic mutations remain undefined were subjected to WGS. Four SV calling algorithms including include MANTA, DELLY, LUMPY and CNVnator were used to detect SVs throughout the genome. All SVs identified by any one of these four algorithms were included for further analysis. AnnotSV was used to annotate these SVs. SVs that overlap with known IRD-associated genes were examined with sequencing coverage, junction reads and discordant read pairs. Polymerase Chain Reaction (PCR) followed by Sanger sequencing was used to further confirm the SVs and identify the breakpoints. Segregation of the candidate pathogenic alleles with the disease was performed when possible. A total of 16 candidate pathogenic SVs were identified in 16 families, including deletions and inversions, representing 2.1% of patients with previously unsolved IRDs. Autosomal dominant, autosomal recessive and X-linked inheritance of disease-causing SVs were observed in 12 different genes. Among these, SVs in CLN3, EYS and PRPF31 were found in multiple families. Our study suggests that the contribution of SVs detected by short-read WGS is about 0.25% of our IRD patient cohort and is significantly lower than that of single nucleotide changes and small insertions and deletions.
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Doenças Retinianas , Humanos , Doenças Retinianas/genética , Mutação , Sequenciamento Completo do Genoma , Sequenciamento do Exoma , Alelos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Proteínas do Olho/genéticaRESUMO
Over 1,500 variants in the ABCA4 locus cause phenotypes ranging from severe, early-onset retinal degeneration to very late-onset maculopathies. The resulting ABCA4/Stargardt disease is the most prevalent Mendelian eye disorder, although its underlying clinical heterogeneity, including penetrance of many alleles, are not well-understood. We hypothesized that a share of this complexity is explained by trans-modifiers, i.e., variants in unlinked loci, which are currently unknown. We sought to identify these by performing exome sequencing in a large cohort for a rare disease of 622 cases and compared variation in seven genes known to clinically phenocopy ABCA4 disease to cohorts of ethnically matched controls. We identified a significant enrichment of variants in 2 out of the 7 genes. Moderately rare, likely functional, variants, at the minor allele frequency (MAF) <0.005 and CADD>25, were enriched in ROM1, where 1.3% of 622 patients harbored a ROM1 variant compared to 0.3% of 10,865 controls (p = 2.41E04; OR 3.81 95% CI [1.77; 8.22]). More importantly, analysis of common variants (MAF>0.1) identified a frequent haplotype in PRPH2, tagged by the p.Asp338 variant with MAF = 0.21 in the matched general population that was significantly increased in the patient cohort, MAF 0.25, p = 0.0014. Significant differences were also observed between ABCA4 disease subgroups. In the late-onset subgroup, defined by the hypomorphic p.Asn1868Ile variant and including c.4253+43G>A, the allele frequency for the PRPH2 p.Asp338 variant was 0.15 vs 0.27 in the remaining cohort, p = 0.00057. Known functional data allowed suggesting a mechanism by which the PRPH2 haplotype influences the ABCA4 disease penetrance. These associations were replicated in an independent cohort of 408 patients. The association was highly statistically significant in the combined cohorts of 1,030 cases, p = 4.00E-05 for all patients and p = 0.00014 for the hypomorph subgroup, suggesting a substantial trans-modifying role in ABCA4 disease for both rare and common variants in two unlinked loci.
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Transportadores de Cassetes de Ligação de ATP , Degeneração Macular , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas do Olho/genética , Frequência do Gene , Humanos , Degeneração Macular/genética , Mutação , Linhagem , Fenótipo , Doença de Stargardt/genética , Tetraspaninas/genéticaRESUMO
PURPOSE OF REVIEW: This review highlights the artificial intelligence, machine learning, and deep learning initiatives supported by the National Institutes of Health (NIH) and the National Eye Institute (NEI) and calls attention to activities and goals defined in the NEI Strategic Plan as well as opportunities for future activities and breakthroughs in ophthalmology. RECENT FINDINGS: Ophthalmology is at the forefront of artificial intelligence-based innovations in biomedical research that may lead to improvement in early detection and surveillance of ocular disease, prediction of progression, and improved quality of life. Technological advances have ushered in an era where unprecedented amounts of information can be linked that enable scientific discovery. However, there remains an unmet need to collect, harmonize, and share data in a machine actionable manner. Similarly, there is a need to ensure that efforts promote health and research equity by expanding diversity in the data and workforce. SUMMARY: The NIH/NEI has supported the development artificial intelligence-based innovations to advance biomedical research. The NIH/NEI has defined activities to achieve these goals in the NIH Strategic Plan for Data Science and the NEI Strategic Plan and have spearheaded initiatives to facilitate research in these areas.
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Inteligência Artificial , National Eye Institute (U.S.) , Promoção da Saúde , Humanos , National Institutes of Health (U.S.) , Qualidade de Vida , Estados UnidosRESUMO
BACKGROUND: There is a need to evaluate how the choice of time interval contributes to the lack of consistency of SDoH variables that appear as important to COVID-19 disease burden within an analysis for both case counts and death counts. METHODS: This study identified SDoH variables associated with U.S county-level COVID-19 cumulative case and death incidence for six different periods: the first 30, 60, 90, 120, 150, and 180 days since each county had COVID-19 one case per 10,000 residents. The set of SDoH variables were in the following domains: resource deprivation, access to care/health resources, population characteristics, traveling behavior, vulnerable populations, and health status. A generalized variance inflation factor (GVIF) analysis was used to identify variables with high multicollinearity. For each dependent variable, a separate model was built for each of the time periods. We used a mixed-effect generalized linear modeling of counts normalized per 100,000 population using negative binomial regression. We performed a Kolmogorov-Smirnov goodness of fit test, an outlier test, and a dispersion test for each model. Sensitivity analysis included altering the county start date to the day each county reached 10 COVID-19 cases per 10,000. RESULTS: Ninety-seven percent (3059/3140) of the counties were represented in the final analysis. Six features proved important for both the main and sensitivity analysis: adults-with-college-degree, days-sheltering-in-place-at-start, prior-seven-day-median-time-home, percent-black, percent-foreign-born, over-65-years-of-age, black-white-segregation, and days-since-pandemic-start. These variables belonged to the following categories: COVID-19 related, vulnerable populations, and population characteristics. Our diagnostic results show that across our outcomes, the models of the shorter time periods (30 days, 60 days, and 900 days) have a better fit. CONCLUSION: Our findings demonstrate that the set of SDoH features that are significant for COVID-19 outcomes varies based on the time from the start date of the pandemic and when COVID-19 was present in a county. These results could assist researchers with variable selection and inform decision makers when creating public health policy.
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COVID-19 , Segregação Social , Adulto , COVID-19/epidemiologia , Humanos , Políticas , SARS-CoV-2 , Determinantes Sociais da Saúde , Estados Unidos/epidemiologiaRESUMO
Molecular variant interpretation lacks disease gene-specific cohorts for determining variant enrichment in disease versus healthy populations. To address the molecular etiology of retinal degeneration, specifically the PRPH2-related retinopathies, we reviewed genotype and phenotype information obtained from 187 eyeGENE® participants from 161 families. Clinical details were provided by referring clinicians participating in the eyeGENE® Network. The cohort was sequenced for variants in PRPH2. Variant complementary DNA clusters and cohort frequency were compared to variants in public databases to help us to determine pathogenicity by current American College of Medical Genetics and Genomics/Association for Molecular Pathology interpretation criteria. The most frequent variant was c.828+3A>T, which affected 28 families (17.4%), and 25 of 79 (31.64%) variants were novel. The majority of missense variants clustered in the D2 intracellular loop of the peripherin-2 protein, constituting a hotspot. Disease enrichment was noted for 23 (29.1%) of the variants. Hotspot and disease-enrichment evidence modified variant classification for 16.5% of variants. The missense allele p.Arg172Trp was associated with a younger age of onset. To the best of our knowledge, this is the largest patient cohort review of PRPH2-related retinopathy. Large disease gene-specific cohorts permit gene modeling for hotspot and disease-enrichment analysis, providing novel variant classification evidence, including for novel missense variants.
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Estudos de Associação Genética , Periferinas/genética , Doenças Retinianas/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Sistema de Registros , Adulto JovemRESUMO
Genetic testing in a multisite clinical trial network for inherited eye conditions is described in this retrospective review of data collected through eyeGENE®, the National Ophthalmic Disease Genotyping and Phenotyping Network. Participants in eyeGENE were enrolled through a network of clinical providers throughout the United States and Canada. Blood samples and clinical data were collected to establish a phenotype:genotype database, biorepository, and patient registry. Data and samples are available for research use, and participants are provided results of clinical genetic testing. eyeGENE utilized a unique, distributed clinical trial design to enroll 6,403 participants from 5,385 families diagnosed with over 30 different inherited eye conditions. The most common diagnoses given for participants were retinitis pigmentosa (RP), Stargardt disease, and choroideremia. Pathogenic variants were most frequently reported in ABCA4 (37%), USH2A (7%), RPGR (6%), CHM (5%), and PRPH2 (3%). Among the 5,552 participants with genetic testing, at least one pathogenic or likely pathogenic variant was observed in 3,448 participants (62.1%), and variants of uncertain significance in 1,712 participants (30.8%). Ten genes represent 68% of all pathogenic and likely pathogenic variants in eyeGENE. Cross-referencing current gene therapy clinical trials, over a thousand participants may be eligible, based on pathogenic variants in genes targeted by those therapies. This article is the first summary of genetic testing from thousands of participants tested through eyeGENE, including reports from 5,552 individuals. eyeGENE provides a launching point for inherited eye research, connects researchers with potential future study participants, and provides a valuable resource to the vision community.
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Coroideremia/genética , Oftalmopatias Hereditárias/genética , Retinose Pigmentar/genética , Doença de Stargardt/genética , Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Coroideremia/diagnóstico , Coroideremia/epidemiologia , Coroideremia/terapia , Proteínas da Matriz Extracelular/genética , Oftalmopatias Hereditárias/diagnóstico , Oftalmopatias Hereditárias/epidemiologia , Oftalmopatias Hereditárias/terapia , Proteínas do Olho/genética , Feminino , Testes Genéticos/tendências , Terapia Genética/tendências , Humanos , Masculino , Periferinas/genética , Retinose Pigmentar/diagnóstico , Retinose Pigmentar/epidemiologia , Retinose Pigmentar/terapia , Doença de Stargardt/diagnóstico , Doença de Stargardt/epidemiologia , Doença de Stargardt/terapiaRESUMO
The use of Common Data Elements can facilitate cross-study comparisons, data aggregation, and meta-analyses; simplify training and operations; improve overall efficiency; promote interoperability between different systems; and improve the quality of data collection. A Common Data Element is a combination of a precisely defined question (variable) paired with a specified set of responses to the question that is common to multiple datasets or used across different studies. Common Data Elements, especially when they conform to accepted standards, are identified by research communities from variable sets currently in use or are newly developed to address a designated data need. There are no formal international specifications governing the construction or use of Common Data Elements. Consequently, Common Data Elements tend to be made available by research communities on an empiric basis. Some limitations of Common Data Elements are that there may still be differences across studies in the interpretation and implementation of the Common Data Elements, variable validity in different populations, and inhibition by some existing research practices and the use of legacy data systems. Current National Institutes of Health efforts to support Common Data Element use are linked to the strengthening of National Institutes of Health Data Sharing policies and the investments in data repositories. Initiatives include cross-domain and domain-specific resources, construction of a Common Data Element Portal, and establishment of trans-National Institutes of Health working groups to address technical and implementation topics. The National Institutes of Health is seeking to lower the barriers to Common Data Element use through greater awareness and encourage the culture change necessary for their uptake and use. As National Institutes of Health, other agencies, professional societies, patient registries, and advocacy groups continue efforts to develop and promote the responsible use of Common Data Elements, particularly if linked to accepted data standards and terminologies, continued engagement with and feedback from the research community will remain important.
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Pesquisa Biomédica , Elementos de Dados Comuns , Disseminação de Informação , Coleta de Dados , Humanos , National Institutes of Health (U.S.) , Estados UnidosRESUMO
PURPOSE: To characterize the incidence of kidney failure associated with intravitreal anti-VEGF exposure; and compare the risk of kidney failure in patients treated with ranibizumab, aflibercept, or bevacizumab. DESIGN: Retrospective cohort study across 12 databases in the Observational Health Data Sciences and Informatics (OHDSI) network. SUBJECTS: Subjects aged ≥ 18 years with ≥ 3 monthly intravitreal anti-VEGF medications for a blinding disease (diabetic retinopathy, diabetic macular edema, exudative age-related macular degeneration, or retinal vein occlusion). METHODS: The standardized incidence proportions and rates of kidney failure while on treatment with anti-VEGF were calculated. For each comparison (e.g., aflibercept versus ranibizumab), patients from each group were matched 1:1 using propensity scores. Cox proportional hazards models were used to estimate the risk of kidney failure while on treatment. A random effects meta-analysis was performed to combine each database's hazard ratio (HR) estimate into a single network-wide estimate. MAIN OUTCOME MEASURES: Incidence of kidney failure while on anti-VEGF treatment, and time from cohort entry to kidney failure. RESULTS: Of the 6.1 million patients with blinding diseases, 37 189 who received ranibizumab, 39 447 aflibercept, and 163 611 bevacizumab were included; the total treatment exposure time was 161 724 person-years. The average standardized incidence proportion of kidney failure was 678 per 100 000 persons (range, 0-2389), and incidence rate 742 per 100 000 person-years (range, 0-2661). The meta-analysis HR of kidney failure comparing aflibercept with ranibizumab was 1.01 (95% confidence interval [CI], 0.70-1.47; P = 0.45), ranibizumab with bevacizumab 0.95 (95% CI, 0.68-1.32; P = 0.62), and aflibercept with bevacizumab 0.95 (95% CI, 0.65-1.39; P = 0.60). CONCLUSIONS: There was no substantially different relative risk of kidney failure between those who received ranibizumab, bevacizumab, or aflibercept. Practicing ophthalmologists and nephrologists should be aware of the risk of kidney failure among patients receiving intravitreal anti-VEGF medications and that there is little empirical evidence to preferentially choose among the specific intravitreal anti-VEGF agents. FINANCIAL DISCLOSURES: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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Inibidores da Angiogênese , Bevacizumab , Injeções Intravítreas , Ranibizumab , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes de Fusão , Insuficiência Renal , Fator A de Crescimento do Endotélio Vascular , Humanos , Receptores de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/efeitos adversos , Ranibizumab/administração & dosagem , Ranibizumab/efeitos adversos , Bevacizumab/administração & dosagem , Bevacizumab/efeitos adversos , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/efeitos adversos , Estudos Retrospectivos , Masculino , Feminino , Insuficiência Renal/epidemiologia , Insuficiência Renal/complicações , Insuficiência Renal/induzido quimicamente , Incidência , Idoso , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/epidemiologia , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/complicações , Seguimentos , Fatores de Risco , Edema Macular/tratamento farmacológico , Edema Macular/epidemiologia , Edema Macular/diagnóstico , Oclusão da Veia Retiniana/tratamento farmacológico , Oclusão da Veia Retiniana/diagnóstico , Oclusão da Veia Retiniana/complicações , Oclusão da Veia Retiniana/epidemiologia , Cegueira/epidemiologia , Cegueira/induzido quimicamente , Cegueira/prevenção & controle , Cegueira/diagnóstico , Cegueira/etiologiaRESUMO
Purpose: Evaluate the degree of concept coverage of the general eye examination in one widely used electronic health record (EHR) system using the Observational Health Data Sciences and Informatics Observational Medical Outcomes Partnership (OMOP) common data model (CDM). Design: Study of data elements. Participants: Not applicable. Methods: Data elements (field names and predefined entry values) from the general eye examination in the Epic foundation system were mapped to OMOP concepts and analyzed. Each mapping was given a Health Level 7 equivalence designation-equal when the OMOP concept had the same meaning as the source EHR concept, wider when it was missing information, narrower when it was overly specific, and unmatched when there was no match. Initial mappings were reviewed by 2 graders. Intergrader agreement for equivalence designation was calculated using Cohen's kappa. Agreement on the mapped OMOP concept was calculated as a percentage of total mappable concepts. Discrepancies were discussed and a final consensus created. Quantitative analysis was performed on wider and unmatched concepts. Main Outcome Measures: Gaps in OMOP concept coverage of EHR elements and intergrader agreement of mapped OMOP concepts. Results: A total of 698 data elements (210 fields, 488 values) from the EHR were analyzed. The intergrader kappa on the equivalence designation was 0.88 (standard error 0.03, P < 0.001). There was a 96% agreement on the mapped OMOP concept. In the final consensus mapping, 25% (1% fields, 31% values) of the EHR to OMOP concept mappings were considered equal, 50% (27% fields, 60% values) wider, 4% (8% fields, 2% values) narrower, and 21% (52% fields, 8% values) unmatched. Of the wider mapped elements, 46% were missing the laterality specification, 24% had other missing attributes, and 30% had both issues. Wider and unmatched EHR elements could be found in all areas of the general eye examination. Conclusions: Most data elements in the general eye examination could not be represented precisely using the OMOP CDM. Our work suggests multiple ways to improve the incorporation of important ophthalmology concepts in OMOP, including adding laterality to existing concepts. There exists a strong need to improve the coverage of ophthalmic concepts in source vocabularies so that the OMOP CDM can better accommodate vision research. Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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Splice variants are known to cause diseases by utilizing alternative splice sites, potentially resulting in protein truncation or mRNA degradation by nonsense-mediated decay. Splice variants are verified when altered mature mRNA sequences are identified in RNA analyses or minigene assays. Using a quantitative minigene assay, qMini, we uncovered a previously overlooked class of disease-associated splice variants that did not alter mRNA sequence but decreased mature mRNA level, suggesting a potentially new pathogenic mechanism.
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Despite increasing success in determining genetic diagnosis for patients with inherited retinal diseases (IRDs), mutations in about 30% of the IRD cases remain unclear or unsettled after targeted gene panel or whole exome sequencing. In this study, we aimed to investigate the contributions of structural variants (SVs) to settling the molecular diagnosis of IRD with whole-genome sequencing (WGS). A cohort of 755 IRD patients whose pathogenic mutations remain undefined was subjected to WGS. Four SV calling algorithms including include MANTA, DELLY, LUMPY, and CNVnator were used to detect SVs throughout the genome. All SVs identified by any one of these four algorithms were included for further analysis. AnnotSV was used to annotate these SVs. SVs that overlap with known IRD-associated genes were examined with sequencing coverage, junction reads, and discordant read pairs. PCR followed by Sanger sequencing was used to further confirm the SVs and identify the breakpoints. Segregation of the candidate pathogenic alleles with the disease was performed when possible. In total, sixteen candidate pathogenic SVs were identified in sixteen families, including deletions and inversions, representing 2.1% of patients with previously unsolved IRDs. Autosomal dominant, autosomal recessive, and X-linked inheritance of disease-causing SVs were observed in 12 different genes. Among these, SVs in CLN3, EYS, PRPF31 were found in multiple families. Our study suggests that the contribution of SVs detected by short-read WGS is about 0.25% of our IRD patient cohort and is significantly lower than that of single nucleotide changes and small insertions and deletions.
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PURPOSE OF REVIEW: Molecular genetics is revolutionizing the diagnosis and treatment of inherited eye diseases. The National Eye Institute of the National Institutes of Health (NIH), in an effort to facilitate future basic and clinical research in inherited eye disease, created The National Ophthalmic Disease Genotyping and Phenotyping Network (eyeGENE). This review describes the process and utility of the eyeGENE program as it relates to ophthalmic clinical practice. RECENT FINDINGS: Over the last few years, genetic testing of specific genes associated with inherited eye conditions is becoming the standard practice. Vision research and human clinical trials relying on molecular genetic testing of individuals with inherited eye conditions are becoming more common. Eye healthcare professionals must consider the options to assist patients in obtaining genetic testing results and locating trials or studies that may have benefit. SUMMARY: eyeGENE is a DNA repository and patient registry for inherited eye diseases coupled to phenotypic descriptors and molecular genetic information. Through eyeGENE, healthcare professionals throughout the United States and Canada can obtain Clinical Laboratory Improvement Amendments-certified clinical molecular genetic results on their patients. Researchers may request access to a de-identified database of phenotype and genotype information about eyeGENE participants and DNA aliquots for their research studies. eyeGENE also offers participants the option of being included in a patient registry, whereby they may be re-contacted if an approved clinical study for which they might qualify is offered.
Assuntos
Oftalmopatias Hereditárias/genética , Bases de Dados de Ácidos Nucleicos , Estudos de Associação Genética , Pesquisa em Genética , Testes Genéticos , Humanos , Sistema de RegistrosRESUMO
Genes required for ergot alkaloid biosynthesis are clustered in the genomes of several fungi. Several conserved ergot cluster genes have been hypothesized, and in some cases demonstrated, to encode early steps of the pathway shared among fungi that ultimately make different ergot alkaloid end products. The deduced amino acid sequence of one of these conserved genes (easC) indicates a catalase as the product, but a role for a catalase in the ergot alkaloid pathway has not been established. We disrupted easC of Aspergillus fumigatus by homologous recombination with a truncated copy of that gene. The resulting mutant (ΔeasC) failed to produce the ergot alkaloids typically observed in A. fumigatus, including chanoclavine-I, festuclavine, and fumigaclavines B, A, and C. The ΔeasC mutant instead accumulated N-methyl-4-dimethylallyltryptophan (N-Me-DMAT), an intermediate recently shown to accumulate in Claviceps purpurea strains mutated at ccsA (called easE in A. fumigatus) (Lorenz et al. Appl Environ Microbiol 76:1822-1830, 2010). A ΔeasE disruption mutant of A. fumigatus also failed to accumulate chanoclavine-I and downstream ergot alkaloids and, instead, accumulated N-Me-DMAT. Feeding chanoclavine-I to the ΔeasC mutant restored ergot alkaloid production. Complementation of either ΔeasC or ΔeasE mutants with the respective wild-type allele also restored ergot alkaloid production. The easC gene was expressed in Escherichia coli, and the protein product displayed in vitro catalase activity with H(2)O(2) but did not act, in isolation, on N-Me-DMAT as substrate. The data indicate that the products of both easC (catalase) and easE (FAD-dependent oxidoreductase) are required for conversion of N-Me-DMAT to chanoclavine-I.
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Aspergillus fumigatus/metabolismo , Catalase , Ergolinas/metabolismo , Alcaloides de Claviceps/biossíntese , Proteínas Fúngicas/metabolismo , Oxirredutases/metabolismo , Proteínas Recombinantes/metabolismo , Compostos Alílicos/metabolismo , Aspergillus fumigatus/genética , Catalase/genética , Catalase/metabolismo , Claviceps/genética , Claviceps/metabolismo , Clonagem Molecular , Ergonovina/metabolismo , Alcaloides de Claviceps/metabolismo , Escherichia coli , Proteínas Fúngicas/genética , Peróxido de Hidrogênio/metabolismo , Alcaloides Indólicos/metabolismo , Família Multigênica , Oxirredutases/genética , Proteínas Recombinantes/genética , Recombinação Genética , Deleção de Sequência , Triptofano/análogos & derivados , Triptofano/metabolismoRESUMO
High throughput sequencing technologies have revolutionized the identification of mutations responsible for a diverse set of Mendelian disorders, including inherited retinal disorders (IRDs). However, the causal mutations remain elusive for a significant proportion of patients. This may be partially due to pathogenic mutations located in non-coding regions, which are largely missed by capture sequencing targeting the coding regions. The advent of whole-genome sequencing (WGS) allows us to systematically detect non-coding variations. However, the interpretation of these variations remains a significant bottleneck. In this study, we investigated the contribution of deep-intronic splice variants to IRDs. WGS was performed for a cohort of 571 IRD patients who lack a confident molecular diagnosis, and potential deep intronic variants that affect proper splicing were identified using SpliceAI. A total of six deleterious deep intronic variants were identified in eight patients. An in vitro minigene system was applied to further validate the effect of these variants on the splicing pattern of the associated genes. The prediction scores assigned to splice-site disruption positively correlated with the impact of mutations on splicing, as those with lower prediction scores demonstrated partial splicing. Through this study, we estimated the contribution of deep-intronic splice mutations to unassigned IRD patients and leveraged in silico and in vitro methods to establish a framework for prioritizing deep intronic variant candidates for mechanistic and functional analyses.
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Inherited retinal dystrophy (IRD) is a heterogenous blinding eye disease and affects more than 200,000 Americans and millions worldwide. By far, 270 protein-coding genes have been identified to cause IRD when defective. However, only one microRNA (miRNA), miR-204, has been reported to be responsible for IRD when a point-mutation occurs in its seed sequence. Previously, we identified that a conserved, polycistronic, paralogous miRNA cluster, the miR-183/96/182 cluster, is highly specifically expressed in all photoreceptors and other sensory organs; inactivation of this cluster in mice resulted in syndromic IRD with multi-sensory defects. We hypothesized that mutations in the miR-183/96/182 cluster in human cause IRD. To test this hypothesis, we perform mutation screening in the pre-miR-183, -96, -182 in >1000 peripheral blood DNA samples of patients with various forms of IRD. We identified six sequence variants, three in pre-miR-182 and three in pre-miR-96. These variants are in the pre-miRNA-182 or -96, but not in the mature miRNAs, and are unlikely to be the cause of the IRD in these patients. In spite of this, the nature and location of these sequence variants in the pre-miRNAs suggest that some may have impact on the biogenesis and maturation of miR-182 or miR-96 and potential roles in the susceptibility to diseases. Although reporting on negative results so far, our study established a system for mutation screening in the miR-183/96/182 cluster in human for a continued effort to unravel and provides deeper insight into the potential roles of miR-183/96/182 cluster in human diseases.