Splicing defects and CRISPR-Cas9 correction in isogenic homozygous photoreceptor precursors harboring clustered deep-intronic ABCA4 variants.

Splicing defects from deep-intronic variants significantly contribute to the mutational spectrum in ABCA4-associated inherited retinal diseases, necessitating functional validation for their pathological classification. Typically, minigene assays in HEK293(T) can qualitatively assess splicing defects, yet they often fail to quantitatively reproduce the resulting mis-splicing patterns, leaving uncertainty on severity and pathogenicity. As a potential cellular model derived from patient cells, photoreceptor precursor cells (PPCs) play a pivotal role in assessing the severity of specific splicing mutations. Nevertheless, the accessibility of biosamples is commonly constrained, and their establishment is costly and laborious. In this study, we combined and investigated the use of a minigene assay and isogenic PPCs, as superior qualitative and more accessible cellular models for the assessment of splicing defects. Specifically, we focused on the clustered c.5196+1013A>G, c.5196+1056A>G, and c.5196+1216C>A deep-intronic variants in intron 36 of ABCA4, comparing their resulting (mis)splicing patterns in minigene-transfected cells and isogenic CRISPR-Cas9-knocked-in PPCs harboring these pathogenic variants in homozygous state. Moreover, we demonstrate the successful correction of these three splicing defects in homozygous mutant PPCs using a single pair of guide RNAs to target Cas9 cleavage, thereby identifying an efficient gene editing strategy for therapeutic applications.

Diagnostic genome sequencing improves diagnostic yield: a prospective single-centre study in 1000 patients with inherited eye diseases.

PURPOSE: Genome sequencing (GS) is expected to reduce the diagnostic gap in rare disease genetics. We aimed to evaluate a scalable framework for genome-based analyses 'beyond the exome' in regular care of patients with inherited retinal degeneration (IRD) or inherited optic neuropathy (ION). METHODS: PCR-free short-read GS was performed on 1000 consecutive probands with IRD/ION in routine diagnostics. Complementary whole-blood RNA-sequencing (RNA-seq) was done in a subset of 74 patients. An open-source bioinformatics analysis pipeline was optimised for structural variant (SV) calling and combined RNA/DNA variation interpretation. RESULTS: A definite genetic diagnosis was established in 57.4% of cases. For another 16.7%, variants of uncertain significance were identified in known IRD/ION genes, while the underlying genetic cause remained unresolved in 25.9%. SVs or alterations in non-coding genomic regions made up for 12.7% of the observed variants. The RNA-seq studies supported the classification of two unclear variants. CONCLUSION: GS is feasible in clinical practice and reliably identifies causal variants in a substantial proportion of individuals. GS extends the diagnostic yield to rare non-coding variants and enables precise determination of SVs. The added diagnostic value of RNA-seq is limited by low expression levels of the major IRD disease genes in blood.

KCNV2-associated retinopathy: genotype-phenotype correlations - KCNV2 study group report 3.

BACKGROUND/AIMS: To investigate genotype-phenotype associations in patients with KCNV2 retinopathy. METHODS: Review of clinical notes, best-corrected visual acuity (BCVA), molecular variants, electroretinography (ERG) and retinal imaging. Subjects were grouped according to the combination of KCNV2 variants-two loss-of-function (TLOF), two missense (TM) or one of each (MLOF)-and parameters were compared. RESULTS: Ninety-two patients were included. The mean age of onset (mean±SD) in TLOF (n=55), TM (n=23) and MLOF (n=14) groups was 3.51±0.58, 4.07±2.76 and 5.54±3.38 years, respectively. The mean LogMAR BCVA (±SD) at baseline in TLOF, TM and MLOF groups was 0.89±0.25, 0.67±0.38 and 0.81±0.35 for right, and 0.88±0.26, 0.69±0.33 and 0.78±0.33 for left eyes, respectively. The difference in BCVA between groups at baseline was significant in right (p=0.03) and left eyes (p=0.035). Mean outer nuclear layer thickness (±SD) at baseline in TLOF, MLOF and TM groups was 37.07±15.20 µm, 40.67±12.53 and 40.38±18.67, respectively, which was not significantly different (p=0.85). The mean ellipsoid zone width (EZW) loss (±SD) was 2051 µm (±1318) for patients in the TLOF, and 1314 µm (±965) for MLOF. Only one patient in the TM group had EZW loss at presentation. There was considerable overlap in ERG findings, although the largest DA 10 ERG b-waves were associated with TLOF and the smallest with TM variants. CONCLUSIONS: Patients with missense alterations had better BCVA and greater structural integrity. This is important for patient prognostication and counselling, as well as stratification for future gene therapy trials.

Genetic background modulates phenotypic expressivity in OPA1 mutated mice, relevance to DOA pathogenesis.

Dominant optic atrophy (DOA) is mainly caused by OPA1 mutations and is characterized by the degeneration of retinal ganglion cells (RGCs), whose axons form the optic nerve. The penetrance of DOA is incomplete and the disease is marked by highly variable expressivity, ranging from asymptomatic patients to some who are totally blind or who suffer from multisystemic effects. No clear genotype-phenotype correlation has been established to date. Taken together, these observations point toward the existence of modifying genetic and/or environmental factors that modulate disease severity. Here, we investigated the influence of genetic background on DOA expressivity by switching the previously described DOA mouse model bearing the c.1065 + 5G → A Opa1 mutation from mixed C3H; C57BL/6 J to a pure C57BL/6 J background. We no longer observed retinal and optic nerve abnormalities; the findings indicated no degeneration, but rather a sex-dependent negative effect on RGC connectivity. This highlights the fact that RGC synaptic alteration might precede neuronal death, as has been proposed in other neurodegenerative diseases, providing new clinical considerations for early diagnosis as well as a new therapeutic window for DOA. Furthermore, our results demonstrate the importance of secondary genetic factors in the variability of DOA expressivity and offer a model for screening for aggravating environmental and genetic factors.

Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia.

PURPOSE: CNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential. METHODS: We established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology-based variant re-classification. RESULTS: We provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology-based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%. CONCLUSION: Our aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.

In Vivo Efficacy and Safety Evaluations of Therapeutic Splicing Correction Using U1 snRNA in the Mouse Retina.

Efficacy and safety considerations constitute essential steps during development of in vivo gene therapies. Herein, we evaluated efficacy and safety of splice factor-based treatments to correct mutation-induced splice defects in an Opa1 mutant mouse line. We applied adeno-associated viruses to the retina. The viruses transduced retinal cells with an engineered U1 snRNA splice factor designed to correct the Opa1 splice defect. We found the treatment to be efficient in increasing wild-type Opa1 transcripts. Correspondingly, Opa1 protein levels increased significantly in treated eyes. Measurements of retinal morphology and function did not reveal therapy-related side-effects supporting the short-term safety of the treatment. Alterations of potential off-target genes were not detected. Our data suggest that treatments of splice defects applying engineered U1 snRNAs represent a promising in vivo therapeutic approach. The therapy increased wild-type Opa1 transcripts and protein levels without detectable morphological, functional or genetic side-effects in the mouse eye. The U1 snRNA-based therapy can be tailored to specific disease gene mutations, hence, raising the possibility of a wider applicability of this promising technology towards treatment of different inherited retinal diseases.

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Extra-viral DNA in adeno-associated viral vector preparations induces TLR9-dependent innate immune responses in human plasmacytoid dendritic cells.

Adeno-associated viral (AAV) vector suspensions produced in either human derived HEK cells or in Spodoptera frugiperda (Sf9) insect cells differ in terms of residual host cell components as well as species-specific post-translational modifications displayed on the AAV capsid proteins. Here we analysed the impact of these differences on the immunogenic properties of the vector. We stimulated human plasmacytoid dendritic cells with various lots of HEK cell-produced and Sf9 cell-produced AAV-CMV-eGFP vectors derived from different manufacturers. We found that AAV8-CMV-eGFP as well as AAV2-CMV-eGFP vectors induced lot-specific but not production platform-specific or manufacturer-specific inflammatory cytokine responses. These could be reduced or abolished by blocking toll-like receptor 9 signalling or by enzymatically reducing DNA in the vector lots using DNase. Successful HEK cell transduction by DNase-treated AAV lots and DNA analyses demonstrated that DNase did not affect the integrity of the vector but degraded extra-viral DNA. We conclude that both HEK- and Sf9-cell derived AAV preparations can contain immunogenic extra-viral DNA components which can trigger lot-specific inflammatory immune responses. This suggests that improved strategies to remove extra-viral DNA impurities may be instrumental in reducing the immunogenic properties of AAV vector preparations.

Color Vision in Blue Cone Monochromacy: Outcome Measures for a Clinical Trial.

Purpose: Blue cone monochromacy (BCM) is an X-linked retinopathy due to mutations in the OPN1LW/OPN1MW gene cluster. Symptoms include reduced visual acuity and disturbed color vision. We studied BCM color vision to determine outcome measures for future clinical trials. Methods: Patients with BCM and normal-vision participants were examined with Farnsworth-Munsell (FM) arrangement tests and the Color Assessment and Diagnosis (CAD) test. A retrospective case series in 36 patients with BCM (ages 6-70) was performed with the FM D-15 test. A subset of six patients also had Roth-28 Hue and CAD tests. Results: All patients with BCM had abnormal results for D-15, Roth-28, and CAD tests. With D-15, there was protan-deutan confusion and no bimodal tendency. Roth-28 results reinforced that finding. There was symmetry in color vision metrics between the two eyes and coherence between sessions with the arrangement tests and CAD. Severe abnormalities in red-green sensitivity with CAD were expected. Unexpected were different levels of yellow-blue results with two patterns of abnormal thresholds: moderate elevation in two younger patients and severe elevation in four patients ≥35 years. Coefficients of repeatability and intersession means were tabulated for all test modalities. Conclusions: Given understanding of advantages, disadvantages, and complexities of interpretation of results, both an arrangement test and CAD should be useful monitors of color vision through a clinical trial in BCM. Translational Relevance: Our pilot studies in BCM of arrangement and CAD tests indicated both were clinically feasible and interpretable in the context of this cone gene disease.

Effective splicing restoration of a deep-intronic ABCA4 variant in cone photoreceptor precursor cells by CRISPR/SpCas9 approaches.

Stargardt disease is an autosomal recessively inherited retinal disorder commonly caused by pathogenic variants in the ABCA4 gene encoding the ATP-binding cassette subfamily A member 4 (ABCA4) protein. Several deep-intronic variants in ABCA4 have been classified as disease causing. By strengthening a cryptic splice site, deep-intronic variant c.5197-557G>T induces the inclusion of a 188-bp intronic sequence in the mature mRNA, resulting in a premature termination codon. Here, we report the design and evaluation of three CRISPR-Cas9 approaches implementing Streptococcus pyogenes Cas9 (single and dual guide RNA) or Streptococcus pyogenes Cas9 nickase (dual guide RNA) for their potential to correct c.5197-557G>T-induced aberrant splicing in minigene splicing assays and patient-derived cone photoreceptor precursor cells. The different strategies were able to rescue correct splicing by up to 83% and increase the overall correctly spliced transcripts by 1.8-fold, demonstrating the successful CRISPR-Cas9-mediated rescue in patient-derived photoreceptor precursor cells of an ABCA4 splicing defect. The results provide initial evidence of possible permanent splicing correction for Stargardt disease, expanding the therapeutic toolbox to counteract deep-intronic pathogenic variants in ABCA4.

Novel OPN1LW/OPN1MW Exon 3 Haplotype-Associated Splicing Defect in Patients with X-Linked Cone Dysfunction.

Certain combinations of common variants in exon 3 of OPN1LW and OPN1MW, the genes encoding the apo-protein of the long- and middle-wavelength sensitive cone photoreceptor visual pigments in humans, induce splicing defects and have been associated with dyschromatopsia and cone dysfunction syndromes. Here we report the identification of a novel exon 3 haplotype, G-C-G-A-T-T-G-G (referring to nucleotide variants at cDNA positions c.453, c.457, c.465, c.511, c.513, c.521, c.532, and c.538) deduced to encode a pigment with the amino acid residues L-I-V-V-A at positions p.153, p.171, p.174, p.178, and p.180, in OPN1LW or OPN1MW or both in a series of seven patients from four families with cone dysfunction. Applying minigene assays for all observed exon 3 haplotypes in the patients, we demonstrated that the novel exon 3 haplotype L-I-V-V-A induces a strong but incomplete splicing defect with 3-5% of residual correctly spliced transcripts. Minigene splicing outcomes were similar in HEK293 cells and the human retinoblastoma cell line WERI-Rb1, the latter retaining a cone photoreceptor expression profile including endogenous OPN1LW and OPN1MW gene expression. Patients carrying the novel L-I-V-V-A haplotype presented with a mild form of Blue Cone Monochromacy or Bornholm Eye Disease-like phenotype with reduced visual acuity, reduced cone electroretinography responses, red-green color vision defects, and frequently with severe myopia.

The landscape of submicroscopic structural variants at the OPN1LW/OPN1MW gene cluster on Xq28 underlying blue cone monochromacy.

Blue cone monochromacy (BCM) is an X-linked retinal disorder characterized by low vision, photoaversion, and poor color discrimination. BCM is due to the lack of long-wavelength-sensitive and middle-wavelength-sensitive cone photoreceptor function and caused by mutations in the OPN1LW/OPN1MW gene cluster on Xq28. Here, we investigated the prevalence and the landscape of submicroscopic structural variants (SVs) at single-base resolution in BCM patients. We found that about one-third (n = 73) of the 213 molecularly confirmed BCM families carry an SV, most commonly deletions restricted to the OPN1LW/OPN1MW gene cluster. The structure and precise breakpoints of the SVs were resolved in all but one of the 73 families. Twenty-two families-all from the United States-showed the same SV, and we confirmed a common ancestry of this mutation. In total, 42 distinct SVs were identified, including 40 previously unreported SVs, thereby quadrupling the number of precisely mapped SVs underlying BCM. Notably, there was no "region of overlap" among these SVs. However, 90% of SVs encompass the upstream locus control region, an essential enhancer element. Its minimal functional extent based on deletion mapping in patients was refined to 358 bp. Breakpoint analyses suggest diverse mechanisms underlying SV formation as well as in one case the gene conversion-based exchange of a 142-bp deletion between opsin genes. Using parsimonious assumptions, we reconstructed the composition and copy number of the OPN1LW/OPN1MW gene cluster prior to the mutation event and found evidence that large gene arrays may be predisposed to the occurrence of SVs at this locus.

Central Visual Function and Genotype-Phenotype Correlations in PDE6A-Associated Retinitis Pigmentosa.

Purpose: Autosomal recessive retinitis pigmentosa (arRP) can be caused by mutations in the phosphodiesterase 6A (PDE6A) gene. Here, we describe the natural course of disease progression with respect to central retinal function (i.e., visual acuity, contrast sensitivity, and color vision) and establish a detailed genotype--phenotype correlation. Methods: Forty-four patients (26 females; mean age ± SD, 43 ± 13 years) with a confirmed genetic diagnosis of PDE6A-associated arRP underwent comprehensive ophthalmological examinations including best-corrected visual acuity (BCVA) with Early Treatment Diabetic Retinopathy Study charts, contrast sensitivity (CS) with Pelli-Robson charts at distances of 3 m and 1 m, and color vision testing using Roth 28-Hue and Panel D-15 saturated color cups. Results: The most frequently observed variants were c.998+1G>A/p.?, c.304C>A/p.R102S, and c.2053G>A/p.V685M. Central retinal function in patients homozygous for variant c.304C>A/p.R102S was better when compared to patients homozygous for variant c.998+1G>A/p.?, although the former were older at baseline. Central retinal function was similar in patients homozygous for variant c.304C>A/p.R102S and patients heterozygous for variants c.304C>A/p.R102S and c.2053G>A/p.V685M, although the latter were younger at baseline. Annual decline rates in central retinal function were small. Conclusions: We conclude that the severity of the different disease-causing PDE6A mutations in humans with respect to central visual function may be ranked as follows: c.2053G>A/p.V685M in homozygous state (most severe) > c.998+1G>A/p.? in homozygous state > c.304C>A/p.R102S and c.2053G>A/p.V685M in compound-heterozygous state > c.304C>A/p.R102S in homozygous state (mildest). The assessment of treatment efficacy in interventional trials will remain challenging due to small annual decline rates in central retinal function.

Relatively mild blue cone monochromacy phenotype caused by various haplotypes in the L- and M-cone opsin genes.

Purpose: Blue cone monochromacy (BCM) is an X-linked retinopathy caused by mutations in the red and green cone opsin genes. The aim of this study was to establish the clinical, genetic, and electrophysiological characteristics of a specific form of BCM. Methods: Patients harboring mutations in the OPN1LW/OPN1MW genes underwent a full clinical examination, including ocular examination, color vision, full-field electroretinography, color fundus and autofluorescence photography, and optical coherence tomography. Genetic analysis was performed using whole-exome sequencing, duplex PCR, PCR/restriction fragment length polymorphism, and Sanger sequencing. IBM SPSS Statistics v. 21.0 was used for the data analysis. Results: Twenty-five patients harboring various haplotypes in exon 3 of the OPN1LW/OPN1MW genes were recruited. They showed a milder incomplete phenotype of BCM than the typical BCM control group. They presented significantly better visual acuity (logarithm of the minimum angle of resolution [logMAR] 0.48 ± 0.26 vs. 1.10 ± 0.54; p < 0.0001) and a highly myopic refraction (-7.81 ± 5.81 D vs. -4.78 ± 5.27 D; p = 0.0222) compared with the BCM control group. The study group had higher 30-Hz cone flicker responses (28.60 ± 15.02 µv; n = 24), whereas the BCM group had none (0.66 ± 2.12 µv; n = 21; p < 0.0001). The Lanthony 15-HUE desaturated test was variable for the exon 3 haplotype group, with a tendency toward the deutan-protan axis. Conclusions: The present study included genetic and clinical data from the largest cohort of patients with exon 3 haplotypes that were previously shown to cause missplicing of the OPN1LW and OPN1MW genes. Analysis of the clinical data revealed better best-corrected visual acuity, more severe myopia, and higher 30-Hz cone flicker responses in the patients with exon 3 haplotypes than in those with typical BCM.

Comprehensive variant spectrum of the CNGA3 gene in patients affected by achromatopsia.

Achromatopsia (ACHM) is a congenital cone photoreceptor disorder characterized by impaired color discrimination, low visual acuity, photosensitivity, and nystagmus. To date, six genes have been associated with ACHM (CNGA3, CNGB3, GNAT2, PDE6C, PDE6H, and ATF6), the majority of these being implicated in the cone phototransduction cascade. CNGA3 encodes the CNGA3 subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors and is one of the major disease-associated genes for ACHM. Herein, we provide a comprehensive overview of the CNGA3 variant spectrum in a cohort of 1060 genetically confirmed ACHM patients, 385 (36.3%) of these carrying "likely disease-causing" variants in CNGA3. Compiling our own genetic data with those reported in the literature and in public databases, we further extend the CNGA3 variant spectrum to a total of 316 variants, 244 of which we interpreted as "likely disease-causing" according to ACMG/AMP criteria. We report 48 novel "likely disease-causing" variants, 24 of which are missense substitutions underlining the predominant role of this mutation class in the CNGA3 variant spectrum. In addition, we provide extensive in silico analyses and summarize reported functional data of previously analyzed missense, nonsense and splicing variants to further advance the pathogenicity assessment of the identified variants.

Biallelic Loss-of-Function NDUFA12 Variants Cause a Wide Phenotypic Spectrum from Leigh/Leigh-Like Syndrome to Isolated Optic Atrophy.

BACKGROUND: Biallelic loss-of-function NDUFA12 variants have hitherto been linked to mitochondrial complex I deficiency presenting with heterogeneous clinical and radiological features in nine cases only. OBJECTIVES: To fully characterize, both phenotypically and genotypically, NDUFA12-related mitochondrial disease. METHODS: We collected data from cases identified by screening genetic databases of several laboratories worldwide and systematically reviewed the literature. RESULTS: Nine unreported NDUFA12 cases from six pedigrees were identified, with presentation ranging from movement disorder phenotypes (dystonia and/or spasticity) to isolated optic atrophy. MRI showed basal ganglia abnormalities (n = 6), optic atrophy (n = 2), or was unremarkable (n = 1). All carried homozygous truncating NDUFA12 variants, three of which are novel. CONCLUSIONS: Our case series expands phenotype-genotype correlations in NDUFA12-associated mitochondrial disease, providing evidence of intra- and inter-familial clinical heterogeneity for the same variant. It confirms NDUFA12 variants should be included in the diagnostic workup of Leigh/Leigh-like syndromes - particularly with dystonia - as well as isolated optic atrophy.

DNAJC30 disease-causing gene variants in a large Central European cohort of patients with suspected Leber's hereditary optic neuropathy and optic atrophy.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) has been considered a prototypical mitochondriopathy and a textbook example for maternal inheritance linked to certain disease-causing variants in the mitochondrial genome. Recently, an autosomal recessive form of LHON (arLHON) has been described, caused by disease-causing variants in the nuclear encoded gene DNAJC30. METHODS AND RESULTS: In this study, we screened the DNAJC30 gene in a large Central European cohort of patients with a clinical diagnosis of LHON or other autosomal inherited optic atrophies (OA). We identified likely pathogenic variants in 35/1202 patients, corresponding to a detection rate of 2.9%. The previously described missense variant c.152A>G;p.(Tyr51Cys) accounts for 90% of disease-associated alleles in our cohort and we confirmed a strong founder effect. Furthermore, we identified two novel pathogenic variants in DNAJC30: the nonsense variant c.610G>T;p.(Glu204*) and the in-frame deletion c.230_232del;p.(His77del). Clinical investigation of the patients with arLHON revealed a younger age of onset, a more frequent bilateral onset and an increased clinically relevant recovery compared with LHON associated with disease-causing variants in the mitochondrial DNA. CONCLUSION: This study expands previous findings on arLHON and emphasises the importance of DNAJC30 in the genetic diagnostics of LHON and OA in European patients.

Genome-Wide Association Study Identifies Two Common Loci Associated with Pigment Dispersion Syndrome/Pigmentary Glaucoma and Implicates Myopia in its Development.

PURPOSE: To identify genetic variants associated with pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) in unrelated patients and to further understand the genetic and potentially causal relationships between PDS and associated risk factors. DESIGN: A 2-stage genome-wide association meta-analysis with replication and subsequent in silico analyses including Mendelian randomization. PARTICIPANTS: A total of 574 cases with PG or PDS and 52 627 controls of European descent. METHODS: Genome-wide association analyses were performed in 4 cohorts and meta-analyzed in 3 stages: (1) a discovery meta-analysis was performed in 3 cohorts, (2) replication was performed in the fourth cohort, and (3) all 4 cohorts were meta-analyzed to increase statistical power. Two-sample Mendelian randomization was used to determine whether refractive error and intraocular pressure exert causal effects over PDS. MAIN OUTCOME MEASURES: The association of genetic variants with PDS and whether myopia exerts causal effects over PDS. RESULTS: Significant association was present at 2 novel loci for PDS/PG. These loci and follow-up analyses implicate the genes gamma secretase activator protein (GSAP) (lead single nucleotide polymorphism [SNP]: rs9641220, P = 6.0×10-10) and glutamate metabotropic receptor 5 (GRM5)/TYR (lead SNP: rs661177, P = 3.9×10-9) as important factors in disease risk. Mendelian randomization showed significant evidence that negative refractive error (myopia) exerts a direct causal effect over PDS (P = 8.86×10-7). CONCLUSIONS: Common SNPs relating to the GSAP and GRM5/TYR genes are associated risk factors for the development of PDS and PG. Although myopia is a known risk factor, this study uses genetic data to demonstrate that myopia is, in part, a cause of PDS and PG.