ABSTRACT
Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.
Subject(s)
DNA Copy Number Variations , Exome Sequencing , Exome , Rare Diseases , Humans , DNA Copy Number Variations/genetics , Rare Diseases/genetics , Rare Diseases/diagnosis , Exome/genetics , Male , Female , Cohort Studies , Genetic Testing/methodsABSTRACT
BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).
Subject(s)
Antigens, Neoplasm , Cell Cycle Proteins , Cytoskeletal Proteins , Gene Editing , Retinal Degeneration , Adolescent , Adult , Female , Humans , Male , Middle Aged , Young Adult , Antigens, Neoplasm/genetics , Cell Cycle Proteins/genetics , CRISPR-Cas Systems , Cytoskeletal Proteins/genetics , Genetic Therapy/adverse effects , Injections, Intraocular , Quality of Life , Retina , Retinal Degeneration/therapy , Retinal Degeneration/genetics , Visual AcuityABSTRACT
BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).
Subject(s)
Genetic Variation , Rare Diseases , Whole Genome Sequencing , Female , Humans , Male , Cohort Studies , Exome , Exome Sequencing , Genetic Diseases, Inborn/diagnosis , Genetic Diseases, Inborn/ethnology , Genetic Diseases, Inborn/genetics , Genetic Testing , Genome, Human , Phenotype , Rare Diseases/diagnosis , Rare Diseases/ethnology , Rare Diseases/genetics , Sequence Analysis, DNA , Child , Adolescent , Young Adult , AdultABSTRACT
EFEMP1 R345W is a dominant mutation causing Doyne honeycomb retinal dystrophy/malattia leventinese (DHRD/ML), a rare blinding disease with clinical pathology similar to age-related macular degeneration (AMD). Aged Efemp1 R345W/R345W knock-in mice (Efemp1ki/ki) develop microscopic deposits on the basal side of retinal pigment epithelial cells (RPE), an early feature in DHRD/ML and AMD. Here, we assessed the role of alternative complement pathway component factor B (FB) in the formation of these deposits. RNA-seq analysis of the posterior eyecups revealed increased unfolded protein response, decreased mitochondrial function in the neural retina (by 3 months of age) and increased inflammatory pathways in both neural retina and posterior eyecups (at 17 months of age) of Efemp1ki/ki mice compared with wild-type littermate controls. Proteomics analysis of eye lysates confirmed similar dysregulated pathways as detected by RNA-seq. Complement activation was increased in aged Efemp1ki/ki eyes with an approximately 2-fold elevation of complement breakdown products iC3b and Ba (P < 0.05). Deletion of the Cfb gene in female Efemp1ki/ki mice partially normalized the above dysregulated biological pathway changes and oral dosing of a small molecule FB inhibitor from 10 to 12 months of age reduced sub-RPE deposits by 65% (P = 0.029). In contrast, male Efemp1ki/ki mice had fewer sub-RPE deposits than age-matched females, no elevation of ocular complement activation and no effect of FB inhibition on sub-RPE deposits. The effects of FB deletion or inhibition on Efemp1ki/ki mice supports systemic inhibition of the alternative complement pathway as a potential treatment of dry AMD and DHRD/ML.
Subject(s)
Macular Degeneration , Optic Disk Drusen , Male , Mice , Female , Animals , Complement Factor B/genetics , Macular Degeneration/genetics , Macular Degeneration/pathology , Optic Disk Drusen/pathology , Retina/pathology , Retinal Pigment Epithelium/pathologyABSTRACT
Mutations in NMNAT1, a key enzyme involved in the synthesis of NAD+ in the nucleus, lead to an early onset severe inherited retinal degeneration (IRD). We aimed to understand the role of nuclear NAD+ in the retina and to identify the molecular mechanisms underlying NMNAT1-associated disease, using a mouse model that harbors the p.V9M mutation in Nmnat1 (Nmnat1V9M/V9M). We identified temporal transcriptional reprogramming in the retinas of Nmnat1V9M/V9M mice prior to retinal degeneration, which begins at 4 weeks of age, with no significant alterations in gene expression at 2 weeks of age and over 2600 differentially expressed genes by 3 weeks of age. Expression of the primary consumer of NAD+ in the nucleus, PARP1, an enzyme involved in DNA damage repair and transcriptional regulation, as well as 7 other PARP family enzymes, was elevated in the retinas of Nmnat1V9M/V9M. This was associated with elevated levels of DNA damage, PARP-mediated NAD+ consumption and migration of Iba1+/CD45+ microglia/macrophages to the subretinal space in the retinas of Nmnat1V9M/V9M mice. These findings suggest that photoreceptor cells are especially sensitive to perturbation of genome homeostasis, and that PARP-mediated cell death may play a role in other genetic forms of IRDs, and potentially other forms of neurodegeneration.
Subject(s)
Nicotinamide-Nucleotide Adenylyltransferase , Retinal Degeneration , DNA Damage/genetics , Humans , NAD/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Poly(ADP-ribose) Polymerase Inhibitors , Retina/metabolism , Retinal Degeneration/genetics , Retinal Degeneration/metabolismABSTRACT
Nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1) is required for nuclear nicotinamide adenine mononucleotide (NAD+) biosynthesis in all nucleated cells, and despite its functional ubiquity, mutations in this gene lead to an isolated retinal degeneration. The mechanisms underlying how mutant NMNAT1 causes disease are not well understood, nor is the reason why the pathology is confined to the retina. Using a mouse model of NMNAT1-associated retinal degeneration that harbors the p.Val9Met mutation, we tested the hypothesis that decreased function of mutant NMNAT1 has a greater effect on the levels of NAD+ in the retina than elsewhere in the body. Measurements by liquid chromatography with tandem mass spectrometry showed an early and sustained decrease of NAD+ in mutant retinas that was not observed in other tissues. To understand how consumers of nuclear NAD+ are affected by the reduced availability of NAD+ in mutant retinas, poly(ADP-ribose) polymerase (PARP) and nuclear sirtuin activity were evaluated. PARP activity was elevated during disease progression, as evidenced by overproduction of poly(ADP-ribose) (PAR) in photoreceptors, whereas histone deacetylation activity of nuclear sirtuins was not altered. We hypothesized that PARP could be activated because of elevated levels of oxidative stress; however, we did not observe oxidative DNA damage, lipid peroxidation, or a low glutathione to oxidized glutathione ratio. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining revealed that photoreceptors appear to ultimately die by apoptosis, although the low NAD+ levels and overproduction of PAR suggest that cell death may include aspects of the parthanatos cell death pathway.
Subject(s)
Disease Models, Animal , Mutation , NAD/metabolism , Nicotinamide-Nucleotide Adenylyltransferase/genetics , Poly Adenosine Diphosphate Ribose/metabolism , Retina/metabolism , Retinal Degeneration/genetics , Animals , Apoptosis/genetics , Chromatography, Liquid , Humans , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Oxidative Stress , Poly (ADP-Ribose) Polymerase-1/genetics , Poly (ADP-Ribose) Polymerase-1/metabolism , Retinal Degeneration/metabolism , Sirtuins/metabolism , Tandem Mass SpectrometryABSTRACT
Kinesin-2 enables ciliary assembly and maintenance as an anterograde intraflagellar transport (IFT) motor. Molecular motor activity is driven by a heterotrimeric complex comprised of KIF3A and KIF3B or KIF3C plus one non-motor subunit, KIFAP3. Using exome sequencing, we identified heterozygous KIF3B variants in two unrelated families with hallmark ciliopathy phenotypes. In the first family, the proband presents with hepatic fibrosis, retinitis pigmentosa, and postaxial polydactyly; he harbors a de novo c.748G>C (p.Glu250Gln) variant affecting the kinesin motor domain encoded by KIF3B. The second family is a six-generation pedigree affected predominantly by retinitis pigmentosa. Affected individuals carry a heterozygous c.1568T>C (p.Leu523Pro) KIF3B variant segregating in an autosomal-dominant pattern. We observed a significant increase in primary cilia length in vitro in the context of either of the two mutations while variant KIF3B proteins retained stability indistinguishable from wild type. Furthermore, we tested the effects of KIF3B mutant mRNA expression in the developing zebrafish retina. In the presence of either missense variant, rhodopsin was sequestered to the photoreceptor rod inner segment layer with a concomitant increase in photoreceptor cilia length. Notably, impaired rhodopsin trafficking is also characteristic of recessive KIF3B models as exemplified by an early-onset, autosomal-recessive, progressive retinal degeneration in Bengal cats; we identified a c.1000G>A (p.Ala334Thr) KIF3B variant by genome-wide association study and whole-genome sequencing. Together, our genetic, cell-based, and in vivo modeling data delineate an autosomal-dominant syndromic retinal ciliopathy in humans and suggest that multiple KIF3B pathomechanisms can impair kinesin-driven ciliary transport in the photoreceptor.
Subject(s)
Ciliopathies/genetics , Ciliopathies/pathology , Genes, Dominant/genetics , Kinesins/genetics , Mutation , Retina/pathology , Amino Acid Sequence , Animals , Cats , Child, Preschool , Cilia/pathology , Female , Genome-Wide Association Study , Heterozygote , Humans , Kinesins/chemistry , Kinesins/metabolism , Larva , Male , Middle Aged , Pedigree , Phenotype , Photoreceptor Cells/metabolism , Retina/cytology , Retina/growth & development , Retina/metabolism , Rhodopsin/metabolism , Young Adult , Zebrafish/genetics , Zebrafish/growth & developmentABSTRACT
Inherited retinal degenerations (IRDs) are at the focus of current genetic therapeutic advancements. For a genetic treatment such as gene therapy to be successful, an accurate genetic diagnostic is required. Genetic diagnostics relies on the assessment of the probability that a given DNA variant is pathogenic. Non-coding variants present a unique challenge for such assessments as compared to coding variants. For one, non-coding variants are present at much higher number in the genome than coding variants. In addition, our understanding of the rules that govern the non-coding regions of the genome is less complete than our understanding of the coding regions. Methods that allow for both the identification of candidate non-coding pathogenic variants and their functional validation may help overcome these caveats allowing for a greater number of patients to benefit from advancements in genetic therapeutics. We present here an unbiased approach combining whole genome sequencing (WGS) with patient-induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) transcriptome analysis. With this approach, we identified and functionally validated a novel pathogenic non-coding variant in a small family with a previously unresolved genetic diagnosis.
Subject(s)
Genetic Markers , Genetic Variation , Genome, Human , RNA-Seq/methods , Retinal Degeneration/genetics , Retinal Degeneration/pathology , Whole Genome Sequencing/methods , Child , Female , Gene Expression Profiling , Humans , Male , Pedigree , Exome SequencingABSTRACT
PURPOSE: In Mendelian disease diagnosis, variant analysis is a repetitive, error-prone, and time consuming process. To address this, we have developed the Mendelian Analysis Toolkit (MATK), a configurable, automated variant ranking program. METHODS: MATK aggregates variant information from multiple annotation sources and uses expert-designed rules with parameterized weights to produce a ranked list of potentially causal solutions. MATK performance was measured by a comparison between MATK-aided and human-domain expert analyses of 1060 families with inherited retinal degeneration (IRD), analyzed using an IRD-specific gene panel (589 individuals) and exome sequencing (471 families). RESULTS: When comparing MATK-assisted analysis with expert curation in both the IRD-specific gene panel and exome sequencing (1060 subjects), 97.3% of potential solutions found by experts were also identified by the MATK-assisted analysis (541 solutions identified with MATK of 556 solutions found by conventional analysis). Furthermore, MATK-assisted analysis identified 114 additional potential solutions from the 504 cases unsolved by conventional analysis. CONCLUSION: MATK expedites the process of identification of likely solving variants in Mendelian traits, and reduces variability stemming from human error and researcher bias. MATK facilitates data reanalysis to keep up with the constantly improving annotation sources and next-generation sequencing processing pipelines. The software is open source and available at https://gitlab.com/matthew_maher/mendelanalysis.
Subject(s)
Retinal Degeneration , Automation , High-Throughput Nucleotide Sequencing , Humans , Retinal Degeneration/diagnosis , Retinal Degeneration/genetics , Software , Exome SequencingABSTRACT
PURPOSE: To assess the safety of the subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human choroideremia (CHM)-encoding cDNA in CHM. DESIGN: Prospective, open-label, nonrandomized, dose-escalation, phase I/II clinical trial. PARTICIPANTS: Fifteen CHM patients (ages 20-57 years at dosing). METHODS: Patients received uniocular subfoveal injections of low-dose (up to 5 × 1010 vector genome [vg] per eye, n = 5) or high-dose (up to 1 × 1011 vg per eye, n = 10) of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human CHM-encoding cDNA (AAV2-hCHM). Patients were evaluated preoperatively and postoperatively for 2 years with ophthalmic examinations, multimodal retinal imaging, and psychophysical testing. MAIN OUTCOME MEASURES: Visual acuity, perimetry (10-2 protocol), spectral-domain OCT (SD-OCT), and short-wavelength fundus autofluorescence (SW-FAF). RESULTS: We detected no vector-related or systemic toxicities. Visual acuity returned to within 15 letters of baseline in all but 2 patients (1 developed acute foveal thinning, and 1 developed a macular hole); the rest showed no gross changes in foveal structure at 2 years. There were no significant differences between intervention and control eyes in mean light-adapted sensitivity by perimetry or in the lateral extent of retinal pigment epithelium relative preservation by SD-OCT and SW-FAF. Microperimetry showed nonsignificant (< 3 standard deviations of the intervisit variability) gains in sensitivity in some locations and participants in the intervention eye. There were no obvious dose-dependent relationships. CONCLUSIONS: Visual acuity was within 15 letters of baseline after the subfoveal AAV2-hCHM injections in 13 of 15 patients. Acute foveal thinning with unchanged perifoveal function in 1 patient and macular hole in 1 patient suggest foveal vulnerability to the subretinal injections. Longer observation intervals will help establish the significance of the minor differences in sensitivities and rate of disease progression observed between intervention and control eyes.
Subject(s)
Choroideremia , Retinal Perforations , Adult , Choroideremia/diagnosis , Choroideremia/genetics , Choroideremia/therapy , DNA, Complementary , Dependovirus/genetics , Fluorescein Angiography , Genetic Therapy/methods , Humans , Middle Aged , Prospective Studies , Retinal Perforations/therapy , Serogroup , Tomography, Optical Coherence , Young AdultABSTRACT
Rod-cone dystrophy (RCD), also called retinitis pigmentosa, is characterized by rod followed by cone photoreceptor degeneration, leading to gradual visual loss. Mutations in over 65 genes have been associated with non-syndromic RCD explaining 60% to 70% of cases, with novel gene defects possibly accounting for the unsolved cases. Homozygosity mapping and whole-exome sequencing applied to a case of autosomal recessive non-syndromic RCD from a consanguineous union identified a homozygous variant in WDR34. Mutations in WDR34 have been previously associated with severe ciliopathy syndromes possibly associated with a retinal dystrophy. This is the first report of a homozygous mutation in WDR34 associated with non-syndromic RCD.
Subject(s)
Carrier Proteins/genetics , Cone-Rod Dystrophies/genetics , Adult , Genetic Association Studies , Humans , Male , Pedigree , WD40 RepeatsABSTRACT
The design of efficient therapies for age-related macular degeneration (AMD) is limited by our understanding of the pathogenesis of basal deposits, which form between retinal pigment epithelium (RPE) and Bruch's membrane (BrM) early in disease, and involve activation of the complement system. To investigate the roles of BrM, RPE and complement in an AMD, we generated abnormal extracellular matrix (ECM) using CRISPR-edited ARPE-19 cells. We introduced to these cells the p.R345W mutation in EFEMP1, which causes early-onset macular degeneration. The abnormal ECM binds active complement C3 and causes the formation of basal deposits by normal human fetal (hf)RPE cells. Human fetal RPE (hfRPE) cells grown on abnormal ECM or BrM explants from AMD donors show chronic activation of the alternative complement pathway by excessive deposition of C3b. This process is exacerbated by impaired ECM turnover via increased matrix metalloproteinase-2 activity. The local cleavage of C3 via convertase-independent mechanisms can be a new therapeutic target for early AMD.
Subject(s)
Complement Pathway, Alternative/physiology , Macular Degeneration/genetics , Retinal Pigment Epithelium/metabolism , Bruch Membrane/pathology , Cell Line , Cells, Cultured , Complement C3/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/genetics , Extracellular Matrix Proteins/metabolism , Fetus , Humans , Macular Degeneration/pathology , MutationABSTRACT
Intraflagellar transport (IFT) is a bidirectional transport process that occurs along primary cilia and specialized sensory cilia, such as photoreceptor outersegments. Genes coding for various IFT components are associated with ciliopathies. Mutations in IFT172 lead to diseases ranging from isolated retinal degeneration to severe syndromic ciliopathies. In this study, we created a mouse model of IFT172-associated retinal degeneration to investigate the ocular disease mechanism. We found that depletion of IFT172 in rod photoreceptors leads to a rapid degeneration of the retina, with severely reduced electroretinography (ERG) responses by 1 month and complete outer-nuclear layer (ONL) degeneration by 2 months. We investigated molecular mechanisms of degeneration and show that IFT172 protein reduction leads to mislocalization of specific photoreceptor outersegment (OS) proteins (RHO, RP1, IFT139), aberrant light-driven translocation of alpha transducin and altered localization of glioma-associated oncogene family member 1 (GLI1). This mouse model exhibits key features of the retinal phenotype observed in patients with IFT172-associated blindness and can be used for in vivo testing of ciliopathy therapies.
Subject(s)
Carrier Proteins/genetics , Cilia/genetics , Intracellular Signaling Peptides and Proteins/genetics , Retinal Degeneration/genetics , Adaptor Proteins, Signal Transducing , Animals , Cilia/pathology , Cytoskeletal Proteins , Disease Models, Animal , Electroretinography , Humans , Mice , Mice, Knockout , Mutation , Retina/diagnostic imaging , Retina/pathology , Retinal Degeneration/diagnostic imaging , Retinal Degeneration/pathology , Retinal Rod Photoreceptor Cells/pathology , Zinc Finger Protein GLI1/geneticsABSTRACT
Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.
Subject(s)
Leigh Disease/genetics , Mitochondria/genetics , Mitochondrial Diseases/genetics , Mitochondrial Proton-Translocating ATPases/genetics , Adenosine Triphosphate/biosynthesis , Child , Child, Preschool , Dimerization , Exons/genetics , Founder Effect , Gene Frequency , Haplotypes , Humans , Infant , Infant, Newborn , Jews/genetics , Leigh Disease/metabolism , Leigh Disease/pathology , Male , Mitochondria/metabolism , Mitochondria/pathology , Mitochondrial Diseases/metabolism , Mitochondrial Diseases/pathology , Mutation , Oxidative Phosphorylation , RNA Splice Sites/genetics , Exome SequencingABSTRACT
PURPOSE: Current sequencing strategies can genetically solve 55-60% of inherited retinal degeneration (IRD) cases, despite recent progress in sequencing. This can partially be attributed to elusive pathogenic variants (PVs) in known IRD genes, including copy-number variations (CNVs), which have been shown as major contributors to unsolved IRD cases. METHODS: Five hundred IRD patients were analyzed with targeted next-generation sequencing (NGS). The NGS data were used to detect CNVs with ExomeDepth and gCNV and the results were compared with CNV detection with a single-nucleotide polymorphism (SNP) array. Likely causal CNV predictions were validated by quantitative polymerase chain reaction (qPCR). RESULTS: Likely disease-causing single-nucleotide variants (SNVs) and small indels were found in 55.6% of subjects. PVs in USH2A (11.6%), RPGR (4%), and EYS (4%) were the most common. Likely causal CNVs were found in an additional 8.8% of patients. Of the three CNV detection methods, gCNV showed the highest accuracy. Approximately 30% of unsolved subjects had a single likely PV in a recessive IRD gene. CONCLUSION: CNV detection using NGS-based algorithms is a reliable method that greatly increases the genetic diagnostic rate of IRDs. Experimentally validating CNVs helps estimate the rate at which IRDs might be solved by a CNV plus a more elusive variant.
Subject(s)
Retinal Degeneration , DNA Copy Number Variations/genetics , Eye Proteins/genetics , Genes, Recessive , High-Throughput Nucleotide Sequencing , Humans , Retinal Degeneration/diagnosis , Retinal Degeneration/genetics , VirulenceABSTRACT
Purpose: To evaluate the phenotypic spectrum of autosomal recessive RP1-associated retinal dystrophies and assess genotypic associations. Methods: A retrospective multicenter study was performed of patients with biallelic RP1-associated retinal dystrophies. Data including presenting symptoms and age, visual acuity, kinetic perimetry, full field electroretinogram, fundus examination, multimodal retinal imaging, and RP1 genotype were evaluated. Results: Nineteen eligible patients from 17 families were identified and ranged in age from 10 to 56 years at the most recent evaluation. Ten of the 21 unique RP1 variants identified were novel, and mutations within exon 2 accounted for nearly half of alleles across the cohort. Patients had clinical diagnoses of retinitis pigmentosa (13), cone-rod dystrophy (3), Leber congenital amaurosis (1), early-onset severe retinal dystrophy (1), and macular dystrophy (1). Macular atrophy was a common feature across the cohort. Symptom onset occurred between 4 and 30 years of age (mean 14.9 years, median 13 years), but there were clusters of onset age that correlated with the effects of RP1 mutations at a protein level. Patients with later-onset disease, including retinitis pigmentosa, had at least one missense variant in an exon 2 DCX domain. Conclusions: Biallelic RP1 mutations cause a broad spectrum of retinal disease. Exon 2 missense mutations are a significant contributor to disease and can be associated with a considerably later onset of retinitis pigmentosa than that typically associated with biallelic RP1 mutations.
Subject(s)
Microtubule-Associated Proteins/genetics , Retinal Dystrophies/genetics , Adolescent , Adult , Alleles , Child , Cohort Studies , Cone-Rod Dystrophies/genetics , DNA Mutational Analysis , Electroretinography , Eye Diseases, Hereditary/genetics , Female , Genotype , Humans , Leber Congenital Amaurosis/genetics , Macular Degeneration/genetics , Male , Middle Aged , Mutation , Mutation, Missense , Phenotype , Retinal Dystrophies/diagnostic imaging , Retinal Dystrophies/physiopathology , Retinitis Pigmentosa/genetics , Retrospective Studies , Visual AcuityABSTRACT
Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r2 = 0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders.
Subject(s)
Genetic Variation , High-Throughput Nucleotide Sequencing/methods , Retinal Diseases/genetics , Rhodopsin/genetics , Gene Expression Regulation , Gene Library , Genetic Predisposition to Disease , Genomics , HEK293 Cells , Humans , Models, Biological , Rhodopsin/metabolism , Sequence Analysis, DNAABSTRACT
PURPOSE: With the advent of gene therapies for inherited retinal degenerations (IRDs), genetic diagnostics will have an increasing role in clinical decision-making. Yet the genetic cause of disease cannot be identified using exon-based sequencing for a significant portion of patients. We hypothesized that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and evaluated patients with single coding pathogenic variants in RPGRIP1 to test this hypothesis. METHODS: IRD families underwent targeted panel sequencing. Unsolved cases were explored by exome and genome sequencing looking for additional pathogenic variants. Candidate pathogenic variants were then validated by Sanger sequencing, quantitative polymerase chain reaction, and in vitro splicing assays in two cell lines analyzed through amplicon sequencing. RESULTS: Among 1722 families, 3 had biallelic loss-of-function pathogenic variants in RPGRIP1 while 7 had a single disruptive coding pathogenic variants. Exome and genome sequencing revealed potential noncoding pathogenic variants in these 7 families. In 6, the noncoding pathogenic variants were shown to lead to loss of function in vitro. CONCLUSION: Noncoding pathogenic variants were identified in 6 of 7 families with single coding pathogenic variants in RPGRIP1. The results suggest that noncoding pathogenic variants contribute significantly to the genetic causality of IRDs and RPGRIP1-mediated IRDs are more common than previously thought.
Subject(s)
DNA, Intergenic/genetics , Proteins/genetics , Retinal Degeneration/genetics , Adult , Chromosome Mapping , Cytoskeletal Proteins , DNA Mutational Analysis/methods , DNA, Intergenic/physiology , Female , HEK293 Cells , Humans , Male , Mutation , Pedigree , Proteins/physiology , Retinal Degeneration/etiology , Exome Sequencing/methods , Whole Genome Sequencing/methodsABSTRACT
Parthanatos is a programmed cell death pathway mediated by the effects of pathogenically high levels of poly(ADP-ribose) polymerase 1 (PARP1) activity. This process underlies a broad range of diseases affecting many tissues and organs across the body, including the retina. This chapter reviews mechanisms that are currently understood to drive parthanatos in the context of retinal diseases associated with this form of cell death. Toxicity of upregulated poly(ADP-ribose) (PAR) content, NAD+ and ATP depletion, translocation of apoptosis-inducing factor (AIF) to the nucleus, and loss of glycolytic function are discussed. Since therapies that preserve vulnerable cells remain elusive for the vast majority of retinal diseases, pharmacologically blocking parthanatos may be an effective treatment strategy for cases in which this process contributes to pathogenesis.
Subject(s)
Parthanatos , Retinal Diseases/pathology , Apoptosis Inducing Factor , Humans , Poly (ADP-Ribose) Polymerase-1/genetics , Poly Adenosine Diphosphate Ribose , Protein TransportABSTRACT
Mutations in USH2A gene account for most cases of Usher syndrome type II (USH2), characterized by a combination of congenital hearing loss and progressive vision loss. In particular, approximately 30% of USH2A patients harbor a single base pair deletion, c.2299delG, in exon 13 that creates a frameshift and premature stop codon, leading to a nonfunctional USH2A protein. The USH2A protein, also known as usherin, is an extremely large transmembrane protein (5202 aa), which limits the use of conventional AAV-mediated gene therapy; thus development of alternative approaches is required for the treatment of USH2A patients. As usherin contains multiple repetitive domains, we hypothesize that removal of one or more of those domains encoded by mutant exon(s) in the USH2A gene may reconstitute the reading frame and restore the production of a shortened yet adequately functional protein. In this study, we deleted the exon 12 of mouse Ush2a gene (corresponding to exon 13 of human USH2A) using CRISPR/Cas9-based exon-skipping approach and revealed that a shortened form of Ush2a that lacks exon 12 (Ush2a-∆Ex12) is produced and localized correctly in the cochlea. When the Ush2a-∆Ex12 allele is expressed on an Ush2a null background, the Ush2a-∆Ex12 protein can successfully restore the impaired hair cell structure and the auditory function in the Ush2a-/- mice. These results demonstrate that CRISPR/Cas9-based exon-skipping strategy holds a great therapeutic potential for the treatment of USH2A patients.