DDX58(RIG-I)-related disease is associated with tissue-specific interferon pathway activation.

BACKGROUND: Singleton-Merten syndrome (SGMRT) is a rare immunogenetic disorder that variably features juvenile open-angle glaucoma (JOAG), psoriasiform skin rash, aortic calcifications and skeletal and dental dysplasia. Few families have been described and the genotypic and phenotypic spectrum is poorly defined, with variants in DDX58 (DExD/H-box helicase 58) being one of two identified causes, classified as SGMRT2. METHODS: Families underwent deep systemic phenotyping and exome sequencing. Functional characterisation with in vitro luciferase assays and in vivo interferon signature using bulk and single cell RNA sequencing was performed. RESULTS: We have identified a novel DDX58 variant c.1529A>T p.(Glu510Val) that segregates with disease in two families with SGMRT2. Patients in these families have widely variable phenotypic features and different ethnic background, with some being severely affected by systemic features and others solely with glaucoma. JOAG was present in all individuals affected with the syndrome. Furthermore, detailed evaluation of skin rash in one patient revealed sparse inflammatory infiltrates in a unique distribution. Functional analysis showed that the DDX58 variant is a dominant gain-of-function activator of interferon pathways in the absence of exogenous RNA ligands. Single cell RNA sequencing of patient lesional skin revealed a cellular activation of interferon-stimulated gene expression in keratinocytes and fibroblasts but not in neighbouring healthy skin. CONCLUSIONS: These results expand the genotypic spectrum of DDX58-associated disease, provide the first detailed description of ocular and dermatological phenotypes, expand our understanding of the molecular pathogenesis of this condition and provide a platform for testing response to therapy.

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Precision medicine to prevent glaucoma-related blindness.

PURPOSE OF REVIEW: Approximately 10% of patients become blind despite using evidence-based guidelines developed from clinical trials and epidemiology studies. Our purpose is to review opportunities to decrease glaucoma-related blindness using the emerging principles of precision medicine. RECENT FINDINGS: The current review focuses on three topics: first, candidate biomarkers for angle-based surgeries, second, head-mounted display (HMD) technology for vision and testing, and third, glaucoma risk alleles discovered by genome-wide association studies. First, in angle-based surgeries, tracers injected into the anterior chamber or Schlemm's canal have allowed visualization of aqueous veins. We describe an innovative use of optical coherence tomography angiography to visualize aqueous veins in a case with 6-year successful outcome following catheter-based trabeculotomy. Second, HMD technology can augment perceived vision and can be used for perimetry testing. Third, developing genetic risk scores that characterize patients who are at highest risk for blindness is a priority. Such biomarker risk scores will integrate genome-wide association study-based risk alleles for glaucoma along with well known demographic and clinical risk factors. SUMMARY: As we gain more knowledge, precision medicine will enable clinicians to decrease glaucoma-related blindness by providing more timely interventions to those patients who are at highest risk for progression to blindness. VIDEO ABSTRACT: http://links.lww.com/COOP/A29.

Common variants at 9p21 and 8q22 are associated with increased susceptibility to optic nerve degeneration in glaucoma.

Optic nerve degeneration caused by glaucoma is a leading cause of blindness worldwide. Patients affected by the normal-pressure form of glaucoma are more likely to harbor risk alleles for glaucoma-related optic nerve disease. We have performed a meta-analysis of two independent genome-wide association studies for primary open angle glaucoma (POAG) followed by a normal-pressure glaucoma (NPG, defined by intraocular pressure (IOP) less than 22 mmHg) subgroup analysis. The single-nucleotide polymorphisms that showed the most significant associations were tested for association with a second form of glaucoma, exfoliation-syndrome glaucoma. The overall meta-analysis of the GLAUGEN and NEIGHBOR dataset results (3,146 cases and 3,487 controls) identified significant associations between two loci and POAG: the CDKN2BAS region on 9p21 (rs2157719 [G], OR = 0.69 [95%CI 0.63-0.75], p = 1.86×10⁻¹8), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], OR = 1.32 [95%CI 1.21-1.43], p = 3.87×10⁻¹¹). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], OR = 0.58 [95% CI 0.50-0.67], p = 1.17×10⁻¹²) and a probable regulatory region on 8q22 (rs284489 [G], OR = 0.62 [95% CI 0.53-0.72], p = 8.88×10⁻¹°). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], OR = 0.59 [95% CI 0.41-0.87], p = 0.004 and rs284489 [G], OR = 0.76 [95% CI 0.54-1.06], p = 0.021), suggesting that these loci might contribute more generally to optic nerve degeneration in glaucoma. Because both loci influence transforming growth factor beta (TGF-beta) signaling, we performed a genomic pathway analysis that showed an association between the TGF-beta pathway and NPG (permuted p = 0.009). These results suggest that neuro-protective therapies targeting TGF-beta signaling could be effective for multiple forms of glaucoma.

Genome-wide association study and meta-analysis of intraocular pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma and is influenced by genetic and environmental factors. Recent genome-wide association studies (GWAS) reported associations with IOP at TMCO1 and GAS7, and with primary open-angle glaucoma (POAG) at CDKN2B-AS1, CAV1/CAV2, and SIX1/SIX6. To identify novel genetic variants and replicate the published findings, we performed GWAS and meta-analysis of IOP in >6,000 subjects of European ancestry collected in three datasets: the NEI Glaucoma Human genetics collaBORation, GLAUcoma Genes and ENvironment study, and a subset of the Age-related Macular Degeneration-Michigan, Mayo, AREDS and Pennsylvania study. While no signal achieved genome-wide significance in individual datasets, a meta-analysis identified significant associations with IOP at TMCO1 (rs7518099-G, p = 8.0 × 10(-8)). Focused analyses of five loci previously reported for IOP and/or POAG, i.e., TMCO1, CDKN2B-AS1, GAS7, CAV1/CAV2, and SIX1/SIX6, revealed associations with IOP that were largely consistent across our three datasets, and replicated the previously reported associations in both effect size and direction. These results confirm the involvement of common variants in multiple genomic regions in regulating IOP and/or glaucoma risk.

Hypothesis-independent pathway analysis implicates GABA and acetyl-CoA metabolism in primary open-angle glaucoma and normal-pressure glaucoma.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. Using genome-wide association single-nucleotide polymorphism data from the Glaucoma Genes and Environment study and National Eye Institute Glaucoma Human Genetics Collaboration comprising 3,108 cases and 3,430 controls, we assessed biologic pathways as annotated in the KEGG database for association with risk of POAG. After correction for genic overlap among pathways, we found 4 pathways, butanoate metabolism (hsa00650), hematopoietic cell lineage (hsa04640), lysine degradation (hsa00310) and basal transcription factors (hsa03022) related to POAG with permuted p < 0.001. In addition, the human leukocyte antigen (HLA) gene family was significantly associated with POAG (p < 0.001). In the POAG subset with normal-pressure glaucoma (NPG), the butanoate metabolism pathway was also significantly associated (p < 0.001) as well as the MAPK and Hedgehog signaling pathways (hsa04010 and hsa04340), glycosaminoglycan biosynthesis-heparan sulfate pathway (hsa00534) and the phenylalanine, tyrosine and tryptophan biosynthesis pathway (hsa0400). The butanoate metabolism pathway overall, and specifically the aspects of the pathway that contribute to GABA and acetyl-CoA metabolism, was the only pathway significantly associated with both POAG and NPG. Collectively these results implicate GABA and acetyl-CoA metabolism in glaucoma pathogenesis, and suggest new potential therapeutic targets.

Congenital cataracts: de novo gene conversion event in CRYBB2.

PURPOSE: To identify the cause of congenital cataracts in a consanguineous family of Ashkenazi Jewish ancestry. METHODS: We performed genome-wide linkage analysis and whole-exome sequencing for the initial discovery of variants, and we confirmed the variants using gene-specific primers and Sanger sequencing. RESULTS: We found significant evidence of linkage to chromosome 22, under an autosomal dominant inheritance model, with a maximum logarithm of the odds (LOD) score of 3.91 (16.918 to 25.641 Mb). Exome sequencing identified three nonsynonymous changes in the CRYBB2 exon 5 coding sequence that are consistent with the sequence of the corresponding region of the pseudogene CRYBB2P1. The identification of these changes was complicated by possible mismapping of some mutated CRYBB2 sequences to CRYBB2P1. Sequencing with gene-specific primers confirmed that the changes--rs2330991, c.433 C>T (p.R145W); rs2330992, c.440A>G (p.Q147R); and rs4049504, c.449C>T (p.T150M)--present in all ten affected family members are located in CRYBB2 and are not artifacts of cross-reaction with CRYBB2P1. We did not find these changes in six unaffected family members, including the unaffected grandfather who contributed the affected haplotype, nor did we find them in the 100 Ashkenazi Jewish controls. CONCLUSIONS: Our data are consistent with a de novo gene conversion event, transferring 270 base pairs at most from CRYBB2P1 to exon 5 of CRYBB2. This study highlights how linkage mapping can be complicated by de novo mutation events, as well as how sequence-analysis pipeline mapping of short reads from next-generation sequencing can be complicated by the existence of pseudogenes or other highly homologous sequences.

Association of CAV1/CAV2 genomic variants with primary open-angle glaucoma overall and by gender and pattern of visual field loss.

PURPOSE: The CAV1/CAV2 (caveolin 1 and caveolin 2) genomic region previously was associated with primary open-angle glaucoma (POAG), although replication among independent studies has been variable. The aim of this study was to assess the association between CAV1/CAV2 single nucleotide polymorphisms (SNPs) and POAG in a large case-control dataset and to explore associations by gender and pattern of visual field (VF) loss further. DESIGN: Case-control study. PARTICIPANTS: We analyzed 2 large POAG data sets: the Glaucoma Genes and Environment (GLAUGEN) study (976 cases, 1140 controls) and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium (2132 cases, 2290 controls). METHODS: We studied the association between 70 SNPs located within the CAV1/CAV2 genomic region in the GLAUGEN and NEIGHBOR studies, both genotyped on the Illumina Human 660WQuadv1C BeadChip array and imputed with the Markov Chain Haplotyping algorithm using the HapMap 3 reference panel. We used logistic regression models of POAG in the overall population and separated by gender, as well as by POAG subtypes defined by type of VF defect (peripheral or paracentral). Results from GLAUGEN and NEIGHBOR were meta-analyzed, and a Bonferroni-corrected significance level of 7.7 × 10(-4) was used to account for multiple comparisons. MAIN OUTCOME MEASURES: Overall POAG, overall POAG by gender, and POAG subtypes defined by pattern of early VF loss. RESULTS: We found significant associations between 10 CAV1/CAV2 SNPs and POAG (top SNP, rs4236601; pooled P = 2.61 × 10(-7)). Of these, 9 were significant only in women (top SNP, rs4236601; pooled P = 1.59 × 10(-5)). Five of the 10 CAV1/CAV2 SNPs were associated with POAG with early paracentral VF (top SNP, rs17588172; pooled P = 1.07 × 10(-4)), and none of the 10 were associated with POAG with peripheral VF loss only or POAG among men. CONCLUSIONS: CAV1/CAV2 SNPs were associated significantly with POAG overall, particularly among women. Furthermore, we found an association between CAV1/CAV2 SNPs and POAG with paracentral VF defects. These data support a role for caveolin 1, caveolin 2, or both in POAG and suggest that the caveolins particularly may affect POAG pathogenesis in women and in patients with early paracentral VF defects.

Estrogen pathway polymorphisms in relation to primary open angle glaucoma: an analysis accounting for gender from the United States.

PURPOSE: Circulating estrogen levels are relevant in glaucoma phenotypic traits. We assessed the association between an estrogen metabolism single nucleotide polymorphism (SNP) panel in relation to primary open angle glaucoma (POAG), accounting for gender. METHODS: We included 3,108 POAG cases and 3,430 controls of both genders from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) consortium genotyped on the Illumina 660W-Quad platform. We assessed the relation between the SNP panels representative of estrogen metabolism and POAG using pathway- and gene-based approaches with the Pathway Analysis by Randomization Incorporating Structure (PARIS) software. PARIS executes a permutation algorithm to assess statistical significance relative to the pathways and genes of comparable genetic architecture. These analyses were performed using the meta-analyzed results from the GLAUGEN and NEIGHBOR data sets. We evaluated POAG overall as well as two subtypes of POAG defined as intraocular pressure (IOP) ≥22 mmHg (high-pressure glaucoma [HPG]) or IOP <22 mmHg (normal pressure glaucoma [NPG]) at diagnosis. We conducted these analyses for each gender separately and then jointly in men and women. RESULTS: Among women, the estrogen SNP pathway was associated with POAG overall (permuted p=0.006) and HPG (permuted p<0.001) but not NPG (permuted p=0.09). Interestingly, there was no relation between the estrogen SNP pathway and POAG when men were considered alone (permuted p>0.99). Among women, gene-based analyses revealed that the catechol-O-methyltransferase gene showed strong associations with HTG (permuted gene p≤0.001) and NPG (permuted gene p=0.01). CONCLUSIONS: The estrogen SNP pathway was associated with POAG among women.

Novel and known MYOC exon 3 mutations in an admixed Peruvian primary open-angle glaucoma population.

PURPOSE: The aim of this study was to characterize a representative sample of the Peruvian population suffering open-angle glaucoma (OAG) with respect to the myocilin gene (MYOC) mutations, glaucoma phenotype, and ancestry for future glaucoma risk assessment. METHODS: DNA samples from 414 unrelated Peruvian subjects, including 205 open-angle glaucoma cases (10 juvenile glaucoma [JOAG], 19 normal-tension glaucoma [NTG], and 176 POAG) and 209 randomly sampled controls, were screened for nucleotide changes in MYOC exon 3 by conformational sensitive gel electrophoresis (CSGE) and mutation screening. RESULTS: We identified a probable causative novel MYOC missense mutation, Gly326Ser, in one POAG case and found a consistent genotype-phenotype correlation in eight of his relatives. We also found the known causative MYOC mutation Trp286Arg in one JOAG case and one POAG case. A known causative single base MYOC deletion, T1357, was found in one POAG case. Two previously reported silent polymorphisms, Thr325Thr and Tyr347Tyr, were found in both the case and the control populations. A novel missense variant, Met476Arg, was identified in two unrelated controls. CONCLUSIONS: The screening of exon 3 of MYOC in a representative sample of 205 independent POAG patients from Peru and 209 matched controls identified novel and previously reported mutations (both pathogenic and nonpathogenic) from other global regions. These results reflect the complex admixture of Amerindian and Old World ancestry in urban populations of Latin America, in general, and in Peru, in particular. It will be important to gather information about the ancestral origin of MYOC and other POAG gene mutations to develop screening panels and risk assessment for POAG in Peru.

The relationship between statin use and open-angle glaucoma.

PURPOSE: To determine whether 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) affect the risk of developing open-angle glaucoma (OAG) in persons with hyperlipidemia. DESIGN: Retrospective, longitudinal cohort analysis. PARTICIPANTS: Individuals aged ≥60 years with hyperlipidemia enrolled in a national United States managed care network between 2001 and 2009. METHODS: Multivariable Cox regression analyses were performed to assess the relationship between statin use and the development of OAG (from no prior OAG diagnosis), progression from a prior diagnosis of glaucoma suspect to a diagnosis of OAG, and need for medical or operative interventions for OAG. Regression models were adjusted for sociodemographic factors and medical and ocular comorbidities. MAIN OUTCOME MEASURES: Hazard ratios (HRs) with 95% confidence intervals (CIs). RESULTS: Of the 524 109 individuals with hyperlipidemia, 316 182 (60%) had ≥1 outpatient prescription for statins. The hazard of developing OAG decreased 0.3% (adjusted HR, 0.997; 95% CI 0.994-0.999) for every additional month of statin consumption. Individuals with hyperlipidemia who took statins continuously for 2 years had an 8% (adjusted HR, 0.922; 95% CI, 0.870-0.976) decreased OAG risk relative to those who received no statin therapy. The hazard of progressing from a diagnosis of glaucoma suspect to OAG decreased 0.4% (adjusted HR, 0.996; 95% CI, 0.993-0.999) for every additional month of statin exposure. Individuals who took statins continuously for 2 years had a 9% (adjusted HR, 0.907; 95% CI, 0.846-0.973) decreased risk of progressing from glaucoma suspect to OAG relative to those who received no statin therapy. The hazard of requiring medical treatment for OAG decreased 0.4% (adjusted HR, 0.996; 95% CI, 0.993-0.998) for every additional month of statin exposure. No differences in need for glaucoma surgery were noted among those with OAG who were and were not taking statins (adjusted HR, 1.002; 95% CI, 0.994-1.010). CONCLUSIONS: Statin use was associated with a significant reduction in the risk of OAG among persons with hyperlipidemia. Given the mounting evidence of statin protection against OAG including both basic science and observational clinical studies, an interventional prospective study might provide additional insights into the role of statins in the prevention of early OAG.

Differences in rates of glaucoma among Asian Americans and other racial groups, and among various Asian ethnic groups.

PURPOSE: To determine the incidence and prevalence of different glaucoma types among Asian Americans and other races, and evaluate the hazard for glaucoma among different races and Asian ethnicities. DESIGN: Retrospective, longitudinal, cohort study. PARTICIPANTS: A group of 2,259,061 eye care recipients, aged ≥ 40, who were enrolled in a US managed-care network in 2001-2007. METHODS: Incidence and prevalence rates of open-angle glaucoma (OAG), narrow-angle glaucoma (NAG), and normal-tension glaucoma (NTG) were calculated and stratified by race and Asian ethnicity. Cox regression was performed to assess the hazard of developing OAG, NAG, and NTG for Asian Americans and other races, and among different Asian ethnicities, with adjustment for potentially confounding variables. MAIN OUTCOME MEASURES: Multivariable adjusted hazard of OAG, NAG, and NTG among different races and Asian ethnicities. RESULTS: The OAG prevalence rate for Asian Americans, 6.52%, was similar to that of Latinos (6.40%) and higher than that of non-Hispanic whites (5.59%). The NAG and NTG prevalence rates were considerably higher among Asian Americans (3.01% and 0.73%, respectively) relative to other races. After adjustment for potential confounding factors, Asian Americans had a 51% increased hazard of OAG (adjusted hazard ratio [HR], 1.51 [95% confidence interval (CI), 1.42-1.60]), a 123% increased hazard of NAG (adjusted HR, 2.23; CI, 2.07-2.41), and a 159% increased hazard of NTG (adjusted HR, 2.59; CI, 2.22-3.02) compared with non-Hispanic whites. Vietnamese Americans (adjusted HR, 3.78; CI, 3.19-4.48), Pakistani Americans (adjusted HR, 2.45, CI 1.50-4.01), and Chinese Americans (adjusted HR, 2.31, CI 2.06-2.59) had considerably higher hazards of NAG, whereas Japanese Americans (adjusted HR, 4.37, CI 3.24-5.89) had a substantially higher hazard of NTG, compared with non-Asian Americans. CONCLUSIONS: Given the rapid rise in the number of Asian Americans in the US population, resources should be devoted to identifying and treating glaucoma in these patients. Eye-care providers should be aware of the increased risk for OAG, NAG, and NTG among Asian Americans relative to other races. Knowing Asian-American patients' ancestral country of origin may permit more precise estimation of their risks for OAG, NAG, and NTG. FINANCIAL DISCLOSURE(S): The authors have no proprietary or commercial interest in any materials discussed in this article.

Novel TMEM98, MFRP, PRSS56 variants in a large United States high hyperopia and nanophthalmos cohort.

Nanophthalmos is a rare condition defined by a small, structurally normal eye with resultant high hyperopia. While six genes have been implicated in this hereditary condition (MFRP, PRSS56, MYRF, TMEM98, CRB1,VMD2/BEST1), the relative contribution of these to nanophthalmos or to less severe high hyperopia (≥ + 5.50 spherical equivalent) has not been fully elucidated. We collected probands and families (n = 56) with high hyperopia or nanophthalmos (≤ 21.0 mm axial length). Of 53 families that passed quality control, plausible genetic diagnoses were identified in 10/53 (18.8%) by high-throughput panel or pooled exome sequencing. These include 1 TMEM98 family (1.9%), 5 MFRP families (9.4%), and 4 PRSS56 families (7.5%), with 4 additional families having single allelic hits in MFRP or PRSS56 (7.5%). A novel deleterious TMEM98 variant (NM_015544.3, c.602G>C, p.(Arg201Pro)) segregated with disease in 4 affected members of a family. Multiple novel missense and frameshift variants in MFRP and PRSS56 were identified. PRSS56 families were more likely to have choroidal folds than other solved families, while MFRP families were more likely to have retinal degeneration. Together, this study defines the prevalence of nanophthalmos gene variants in high hyperopia and nanophthalmos and indicates that a large fraction of cases remain outside of single gene coding sequences.

Association of a Primary Open-Angle Glaucoma Genetic Risk Score With Earlier Age at Diagnosis.

IMPORTANCE: Genetic variants associated with primary open-angle glaucoma (POAG) are known to influence disease risk. However, the clinical effect of associated variants individually or in aggregate is not known. Genetic risk scores (GRS) examine the cumulative genetic load by combining individual genetic variants into a single measure, which is assumed to have a larger effect and increased power to detect relevant disease-related associations. OBJECTIVE: To investigate if a GRS that comprised 12 POAG genetic risk variants is associated with age at disease diagnosis. DESIGN, SETTING, AND PARTICIPANTS: A cross-sectional study included individuals with POAG and controls from the Glaucoma Genes and Environment (GLAUGEN) study and the National Eye Institute Glaucoma Human Genetics Collaboration (NEIGHBOR) study. A GRS was formulated using 12 variants known to be associated with POAG, and the alleles associated with increasing risk of POAG were aligned in the case-control sets. In case-only analyses, the association of the GRS with age at diagnosis was analyzed as an estimate of disease onset. Results from cohort-specific analyses were combined with meta-analysis. Data collection started in August 2012 for the NEIGHBOR cohort and in July 2008 for the GLAUGEN cohort and were analyzed starting in March 2018. MAIN OUTCOMES AND MEASURES: Association of a 12 single-nucleotide polymorphism POAG GRS with age at diagnosis in individuals with POAG using linear regression. RESULTS: The GLAUGEN study included 976 individuals with POAG and 1140 controls. The NEIGHBOR study included 2132 individuals with POAG and 2290 controls. For individuals with POAG, the mean (SD) age at diagnosis was 63.6 (9.8) years in the GLAUGEN cohort and 66.0 (13.7) years in the NEIGHBOR cohort. For controls, the mean (SD) age at enrollment was 65.5 (9.2) years in the GLAUGEN cohort and 68.9 (11.4) years in the NEIGHBOR cohort. All study participants were European white. The GRS was strongly associated with POAG risk in case-control analysis (odds ratio per 1-point increase in score = 1.24; 95% CI, 1.21-1.27; P = 3.4 × 10-66). In case-only analyses, each higher GRS unit was associated with a 0.36-year earlier age at diagnosis (ß = -0.36; 95% CI, -0.56 to -0.16; P = 4.0 × 10-4). Individuals in the top 5% of the GRS had a mean (SD) age at diagnosis of 5.2 (12.8) years earlier than those in the bottom 5% GRS (61.4 [12.7] vs 66.6 [12.9] years; P = 5.0 × 10-4). CONCLUSIONS AND RELEVANCE: A higher dose of POAG risk alleles was associated with an earlier age at glaucoma diagnosis. On average, individuals with POAG with the highest GRS had 5.2-year earlier age at diagnosis of disease. These results suggest that a GRS that comprised genetic variants associated with POAG could help identify patients with risk of earlier disease onset impacting screening and therapeutic strategies.

Madeline 2.0 PDE: a new program for local and web-based pedigree drawing.

UNLABELLED: The Madeline 2.0 Pedigree Drawing Engine (PDE) is a pedigree drawing program for use in linkage and family-based association studies. The program is designed to handle large and complex pedigrees with an emphasis on readability and aesthetics. For complex pedigrees, we use a hybrid algorithm in which consanguinous loops are drawn as cyclic graphs whenever possible, but we resort to acyclic graphs when matings can no longer be connected without line crossings. A similar hybrid approach is used to avoid line crossings for matings between distant descendants of different founding groups. Written in object-oriented C++ and released under the GNU General Public License (GPL), Madeline 2.0 PDE reads input files specified on the command line and generates pedigree drawings without user interaction. Pedigree output in scalable vector graphics (SVG) format can be viewed in browsers with native SVG rendering support or in vector graphics editors. We provide an easy-to-use public web service, which is experimental and still under development. AVAILABILITY: http://kellogg.umich.edu/madeline.

Effects of timolol on MYOC, OPTN, and WDR36 RNA levels.

OBJECTIVES: To evaluate if timolol affects expression of 3 open-angle glaucoma genes and to study its ability to modulate dexamethasone-induced up-regulation of MYOC. METHODS: We used quantitative polymerase chain reaction assay of glaucoma gene transcript levels from human trabecular meshwork (HTM) cultures exposed to 3 different doses of timolol. Three HTM cell cultures were grown with or without 1 of 3 timolol doses in the presence or absence of dexamethasone. RESULTS: All 3 concentrations of timolol reduced MYOC RNA levels in 1 HTM culture compared with an untreated control and showed negligible effects in the other 2 cultures. Timolol had no effect on dexamethasone-induced MYOC transcript levels in any of the 3 cultures. Timolol, dexamethasone, and dexamethasone plus timolol had a negligible effect on OPTN and WDR36 RNA levels. CONCLUSIONS: Timolol can reduce MYOC RNA levels in HTM cultures from some individuals. Timolol does not alter OPTN or WDR36 levels or ameliorate MYOC induction by dexamethasone in vitro. CLINICAL RELEVANCE: It remains to be determined whether timolol could reduce production of misfolded myocilin protein by HTM cells in individuals with MYOC missense mutations. A broader survey of interindividual variation in response to timolol as well as mechanistic studies are still needed before potential therapeutic implications can be explored.

Recurrent Myocilin Asn480Lys glaucoma causative mutation arises de novo in a family of Andean descent.

PURPOSE: To search for MYOC mutations in Peruvian primary open angle glaucoma (POAG) families. PATIENTS AND METHODS: Two patients from each of the 11 POAG Peruvian families were screened for sequence variants in MYOC coding exons by conformational sensitive gel electrophoresis and sequencing was performed on the samples indicating probable sequence changes. RESULTS: We detected 2 families bearing distortions of conformational sensitive gel electrophoresis indicating mutations. Sequencing of these samples revealed coding sequence changes. A native Andean descent family presented with a MYOC mutation, Asn480Lys (C-->G at nucleotide 1440). This is different from the previously reported C-->A change at nucleotide 1440 that causes Asn480Lys in 2 unrelated French and Dutch families with glaucoma of variable expressivity, and indicates a third independent event. A second family of admixed origin showed the presence of the known Arg76Lys polymorphism. CONCLUSIONS: In the study of MYOC variants in 11 POAG Peruvian families, we have found a family of ethnically admixed origin with polymorphism Arg76Lys and a family of Andean descent bearing a third event of the Asn480Lys, the MYOC mutation that has been reported in the highest number of POAG patients (>80 cases). Analysis of this family could contribute with information about disease manifestation, progression, and treatment response in the context of a distinct genetic background and also climatic, altitude, and socioeconomical conditions.

Phenotypic Variation in a Four-Generation Family with Aniridia Carrying a Novel PAX6 Mutation.

Aniridia is a congenital disease that affects almost all eye structures and is primarily caused by loss-of-function mutations in the PAX6 gene. The degree of vision loss in aniridia varies and is dependent on the extent of foveal, iris, and optic nerve hypoplasia and the presence of glaucoma, cataracts, and corneal opacification. Here, we describe a 4-generation family in which 7 individuals across 2 generations carry a novel disease-causing frameshift mutation (NM_000280.4(PAX6):c.565TC>T) in PAX6. This mutation results in an early stop codon in exon 8, which is predicted to cause nonsense-mediated decay of the truncated mRNA and a functionally null PAX6 allele. Family members with aniridia showed differences in multiple eye phenotypes including iris and optic nerve hypoplasia, congenital and acquired corneal opacification, glaucoma, and strabismus. Visual acuity ranged from 20/100 to less than 20/800. Patients who required surgical intervention for glaucoma or corneal opacification had worse visual outcomes. Our results show that family members carrying a novel PAX6 frameshift mutation have variable expressivity, leading to different ocular comorbidities and visual outcomes.

Testosterone Pathway Genetic Polymorphisms in Relation to Primary Open-Angle Glaucoma: An Analysis in Two Large Datasets.

Purpose: Sex hormones may be associated with primary open-angle glaucoma (POAG), although the mechanisms are unclear. We previously observed that gene variants involved with estrogen metabolism were collectively associated with POAG in women but not men; here we assessed gene variants related to testosterone metabolism collectively and POAG risk. Methods: We used two datasets: one from the United States (3853 cases and 33,480 controls) and another from Australia (1155 cases and 1992 controls). Both datasets contained densely called genotypes imputed to the 1000 Genomes reference panel. We used pathway- and gene-based approaches with Pathway Analysis by Randomization Incorporating Structure (PARIS) software to assess the overall association between a panel of single nucleotide polymorphisms (SNPs) in testosterone metabolism genes and POAG. In sex-stratified analyses, we evaluated POAG overall and POAG subtypes defined by maximum IOP (high-tension [HTG] or normal tension glaucoma [NTG]). Results: In the US dataset, the SNP panel was not associated with POAG (permuted P = 0.77), although there was an association in the Australian sample (permuted P = 0.018). In both datasets, the SNP panel was associated with POAG in men (permuted P ≤ 0.033) and not women (permuted P ≥ 0.42), but in gene-based analyses, there was no consistency on the main genes responsible for these findings. In both datasets, the testosterone pathway association with HTG was significant (permuted P ≤ 0.011), but again, gene-based analyses showed no consistent driver gene associations. Conclusions: Collectively, testosterone metabolism pathway SNPs were consistently associated with the high-tension subtype of POAG in two datasets.

Investigation of founder effects for the Thr377Met Myocilin mutation in glaucoma families from differing ethnic backgrounds.

PURPOSE: The aim of this study was to determine if there is a common founder for the Thr377Met myocilin mutation in primary open angle glaucoma (POAG) families with various ethnic backgrounds. METHODS: Genomic DNA of 24 POAG-affected individuals from nine pedigrees with the Thr377Met mutation and 104 unaffected family members was genotyped with six microsatellite markers and four single nucleotide polymorphisms. The families were from Greece, India, Finland, the USA, and Australia. To assess the degree of linkage disequilibrium across MYOC in the general population we also investigated data generated from the HapMap consortium. RESULTS: Three distinct haplotypes associated with the Thr377Met myocilin mutation were identified. The families from the USA and Greece, as well as the three Australian families originating from Greece and the former Yugoslavian Republic of Macedonia had one common haplotype. Interestingly, however, HapMap data suggest that linkage disequilibrium across MYOC was not strong. CONCLUSIONS: The Thr377Met myocilin mutation has arisen at least three separate times. Evidence for genetic founder effects in this prevalent age-related, yet heterogeneous, disease has important implications for future gene identification strategies.