A multi-cohort genome-wide association study in African ancestry individuals reveals risk loci for primary open-angle glaucoma.

Primary open-angle glaucoma (POAG), the leading cause of irreversible blindness worldwide, disproportionately affects individuals of African ancestry. We conducted a genome-wide association study (GWAS) for POAG in 11,275 individuals of African ancestry (6,003 cases; 5,272 controls). We detected 46 risk loci associated with POAG at genome-wide significance. Replication and post-GWAS analyses, including functionally informed fine-mapping, multiple trait co-localization, and in silico validation, implicated two previously undescribed variants (rs1666698 mapping to DBF4P2; rs34957764 mapping to ROCK1P1) and one previously associated variant (rs11824032 mapping to ARHGEF12) as likely causal. For individuals of African ancestry, a polygenic risk score (PRS) for POAG from our mega-analysis (African ancestry individuals) outperformed a PRS from summary statistics of a much larger GWAS derived from European ancestry individuals. This study quantifies the genetic architecture similarities and differences between African and non-African ancestry populations for this blinding disease.

Prevalence and factors associated with optic disc grey crescent in the Primary Open-Angle African Ancestry Glaucoma Genetics (POAAGG) Study.

AIM: To investigate the prevalence and factors associated with optic disc grey crescent (GC) in African Americans with glaucoma. METHODS: Stereo optic disc image features from subjects with glaucoma in the Primary Open-Angle African Ancestry Glaucoma Genetics Study were evaluated independently by non-physician graders and discrepancies adjudicated by an ophthalmologist. Risk factors for GC were evaluated by logistic regression models with intereye correlation accounted for by generalised estimating equations. Adjusted ORs (aORs) were generated. RESULTS: GC was present in 227 (15%) of 1491 glaucoma cases, with 57 (3.82%) bilateral and 170 (11.4%) unilateral. In multivariable analysis, factors associated with GC were younger age (aOR 1.27, 95% CI 1.11 to 1.43 for every decade younger in age, p=0.001), diabetes (aOR 1.46, 95% CI 1.09 to 1.96, p=0.01), optic disc tilt (aOR 1.84, 95% CI 1.36 to 2.48, p<0.0001), a sloping retinal region adjacent to the outer disc margin (aOR 2.37, 95% CI 1.74 to 3.32, p<0.0001) and beta peripapillary atrophy (aOR 2.32, 95% CI 1.60 to 3.37, p<0.0001). Subjects with GC had a lower mean (SD) value of the ancestral component q0 than those without GC (0.22 (0.15) vs 0.27 (0.20), p=0.001), consistent with higher degrees of African ancestry. CONCLUSIONS: More than 1 in 10 glaucoma cases with African ancestry have GC, occurring more frequently in younger subjects, higher degrees of African ancestry and diabetes. GC was associated with several ocular features, including optic disc tilt and beta peripapillary atrophy. These associations should be considered when evaluating black patients with primary open-angle glaucoma.

Risk factors for structural and functional progression of primary open-angle glaucoma in an African ancestry cohort.

BACKGROUND/AIMS: To investigate the rates of structural and functional progression of primary open-angle glaucoma in an African ancestry cohort and identify risk factors for progression. METHODS: This retrospective study included 1424 eyes from glaucoma cases in the Primary Open-Angle African American Glaucoma Genetics cohort, with ≥2 visits for retinal nerve fibre layer (RNFL) thickness and mean deviation (MD) measurements over ≥6-month follow-up. The rates of structural progression (change in RNFL thickness/year) and functional progression (change in MD/year) were calculated from linear mixed effects models, accounting for intereye correlation and longitudinal correlation. Eyes were categorised as slow, moderate or fast progressors. Risk factors for progression rates were assessed using univariable and multivariable regression models. RESULTS: The median (interquartile) rates of progression were -1.60 (-2.05 to -1.15) µm/year for RNFL thickness and -0.40 (-0.44 to -0.34) decibels/year for MD. Eyes were categorised as slow (structural: 19%, functional: 88%), moderate (structural: 54%, functional: 11%) and fast (structural: 27%, functional: 1%) progressors. In multivariable analysis, faster RNFL progression was independently associated with thicker baseline RNFL (p<0.0001), lower baseline MD (p=0.003) and beta peripapillary atrophy (p=0.03). Faster MD progression was independently associated with higher baseline MD (p<0.0001), larger cup-to-disc ratios (p=0.02) and lower body mass index (p=0.0004). CONCLUSION: The median rates of structural and functional progression in this African ancestry cohort were faster than the rates reported from previously published studies in other ethnic groups. Higher baseline RNFL thickness and MD values were associated with faster progression rates. Results highlight the importance of monitoring structural and functional glaucoma progression to provide timely treatment in early disease.

ANGPTL7, a therapeutic target for increased intraocular pressure and glaucoma.

Glaucoma is a leading cause of blindness. Current glaucoma medications work by lowering intraocular pressure (IOP), a risk factor for glaucoma, but most treatments do not directly target the pathological changes leading to increased IOP, which can manifest as medication resistance as disease progresses. To identify physiological modulators of IOP, we performed genome- and exome-wide association analysis in >129,000 individuals with IOP measurements and extended these findings to an analysis of glaucoma risk. We report the identification and functional characterization of rare coding variants (including loss-of-function variants) in ANGPTL7 associated with reduction in IOP and glaucoma protection. We validated the human genetics findings in mice by establishing that Angptl7 knockout mice have lower (~2 mmHg) basal IOP compared to wild-type, with a trend towards lower IOP also in heterozygotes. Conversely, increasing murine Angptl7 levels via injection into mouse eyes increases the IOP. We also show that acute Angptl7 silencing in adult mice lowers the IOP (~2-4 mmHg), reproducing the observations in knockout mice. Collectively, our data suggest that ANGPTL7 is important for IOP homeostasis and is amenable to therapeutic modulation to help maintain a healthy IOP that can prevent onset or slow the progression of glaucoma.

Prevalence and Factors Associated with Optic Disc Tilt in the Primary Open-Angle African American Glaucoma Genetics Study.

PURPOSE: To investigate the prevalence and factors associated with optic disc tilt in the eyes of Black Americans with glaucoma. DESIGN: Cross-sectional. PARTICIPANTS: Subjects with glaucoma participating in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study. METHODS: Stereo pairs of optic disc images were assessed independently by POAAGG-certified nonphysician graders for quantitative features including maximum and minimum linear disc diameters, and qualitative features including gradeability of images, shape of the cup, rim plane position, ß-peripapillary atrophy, sloping region adjacent to the outer disc margin, and rim pallor. Discrepancies were adjudicated by an ophthalmologist. Descriptive statistics and P values were generated for associations of tilt with demographic and ocular characteristics. Stepwise multivariable analysis was performed with logistic regression using Generalized Estimating Equations (GEEs) to account for inter-eye correlation within subjects. MAIN OUTCOME MEASURES: Tilt Ovality Index (TOI) of >1.30 and Stereoscopically Identified optic disc Tilt (SIT). RESULTS: Among 1251 subjects with data on both eyes, 104 (8.3%) had TOI. Subjects with TOI were less likely to be male (adjusted odds ratio [aOR], 0.46, 95% confidence interval [CI], 0.29-0.74, P < 0.001). Eyes with TOI were less likely to have large cup disc ratios (aOR, 0.18, 95% CI, 0.06-0.53, P < 0.001) and less likely to have cylinder-shaped cups compared with conical-shaped cups (aOR, 0.31, 95% CI, 0.19-0.49, P < 0.001). Among 1007 subjects with data on both eyes, 254 (25.2%) had SIT. Subjects with SIT were younger (aOR, 0.95, 95% CI, 0.93-0.96, P < 0.001), and eyes with SIT were more likely to have oval-shaped discs compared with round discs (aOR, 1.82, 95% CI, 1.32-2.52, P < 0.001), more likely to have a sloping region adjacent to the outer disc margin instead of being flat (aOR, 3.26, 95% CI, 2.32-4.59, P < 0.001), and less likely to have cylinder-shaped cups compared with conical-shaped cups (aOR, 0.59, 95% CI, 0.41-0.85, P < 0.001). Both TOI and SIT were not associated with myopia. CONCLUSIONS: There are substantial numbers of tilted optic discs in glaucoma patients with African ancestry. They occur more frequently in female subjects and younger subjects and are associated with several ocular features but not with myopia.

Recruitment strategies and lessons learned from a large genetic study of African Americans.

Genetic studies must enroll large numbers of participants to obtain adequate statistical power. Data are needed on how researchers can best use limited financial and practical resources to achieve these targets, especially in under-represented populations. This paper provides a retrospective analysis of the recruitment strategies for a large glaucoma genetics study in African Americans. The Primary Open-Angle African American Glaucoma Genetics study enrolled 10,192 African American subjects from the Philadelphia region. Major recruitment approaches included clinic enrollment from University of Pennsylvania (UPenn) sites, clinic enrollment from external sites, sampling of Penn Medicine Biobank (PMBB), and community outreach. We calculated the enrollment yield, cost per subject, and seasonal trends of these approaches. The majority (65%) of subject were enrolled from UPenn sites with an average cost of $133/subject. Over time, monthly case enrollment declined as the pool of eligible subjects was depleted. Expanding to external sites boosted case numbers ($129/subject) and the biobank provided additional controls at low cost ($5/subject), in large part due to the generosity of PMBB providing samples free of cost. Community outreach was costly with low return on enrollment ($978/subject for 220 subjects). Summer months (Jun-Aug) produced the highest recruitment yields (p<0.001). Genetic studies will benefit from a multi-pronged and culturally sensitive recruitment approach. In our experience, the biobank was most cost-effective for control enrollment, while recruitment from clinics (including expansion to new sites) was necessary to recruit fully phenotyped cases.

A Precise Method to Evaluate 360 Degree Measures of Optic Cup and Disc Morphology in an African American Cohort and Its Genetic Applications.

(1) Background: Vertical cup-to-disc ratio (CDR) is an important measure for evaluating damage to the optic nerve head (ONH) in glaucoma patients. However, this measure often does not fully capture the irregular cupping observed in glaucomatous nerves. We developed and evaluated a method to measure cup-to-disc ratio (CDR) at all 360 degrees of the ONH. (2) Methods: Non-physician graders from the Scheie Reading Center outlined the cup and disc on digital stereo color disc images from African American patients enrolled in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study. After converting the resultant coordinates into polar representation, the CDR at each 360-degree location of the ONH was obtained. We compared grader VCDR values with clinical VCDR values, using Spearman correlation analysis, and validated significant genetic associations with clinical VCDR, using grader VCDR values. (3) Results: Graders delineated outlines of the cup contour and disc boundaries twice in each of 1815 stereo disc images. For both cases and controls, the mean CDR was highest at the horizontal bisector, particularly in the temporal region, as compared to other degree locations. There was a good correlation between grader CDR at the vertical bisector and clinical VCDR (Spearman Correlation OD: r = 0.78 [95% CI: 0.76-0.79]). An SNP in the MPDZ gene, associated with clinical VCDR in a prior genome-wide association study, showed a significant association with grader VCDR (p = 0.01) and grader CDR area ratio (p = 0.02). (4) Conclusions: The CDR of both glaucomatous and non-glaucomatous eyes varies by degree location, with the highest measurements in the temporal region of the eye. This method can be useful for capturing innate eccentric ONH morphology, tracking disease progression, and identifying genetic associations.

Single Cell Sequencing of Induced Pluripotent Stem Cell Derived Retinal Ganglion Cells (iPSC-RGC) Reveals Distinct Molecular Signatures and RGC Subtypes.

We intend to identify marker genes with differential gene expression (DEG) and RGC subtypes in cultures of human-induced pluripotent stem cell (iPSC)-derived retinal ganglion cells. Single-cell sequencing was performed on mature and functional iPSC-RGCs at day 40 using Chromium Single Cell 3' V3 protocols (10X Genomics). Sequencing libraries were run on Illumina Novaseq to generate 150 PE reads. Demultiplexed FASTQ files were mapped to the hg38 reference genome using the STAR package, and cluster analyses were performed using a cell ranger and BBrowser2 software. QC analysis was performed by removing the reads corresponding to ribosomal and mitochondrial genes, as well as cells that had less than 1X mean absolute deviation (MAD), resulting in 4705 cells that were used for further analyses. Cells were separated into clusters based on the gene expression normalization via PCA and TSNE analyses using the Seurat tool and/or Louvain clustering when using BBrowser2 software. DEG analysis identified subsets of RGCs with markers like MAP2, RBPMS, TUJ1, BRN3A, SOX4, TUBB3, SNCG, PAX6 and NRN1 in iPSC-RGCs. Differential expression analysis between separate clusters identified significant DEG transcripts associated with cell cycle, neuron regulatory networks, protein kinases, calcium signaling, growth factor hormones, and homeobox transcription factors. Further cluster refinement identified RGC diversity and subtype specification within iPSC-RGCs. DEGs can be used as biomarkers for RGC subtype classification, which will allow screening model systems that represent a spectrum of diseases with RGC pathology.

Association of the SNP rs112369934 near TRIM66 Gene with POAG Endophenotypes in African Americans.

We investigated the association of the single nucleotide polymorphism (SNP) rs112369934 near the TRIM66 gene with qualitative and quantitative phenotypes of primary open-angle glaucoma (POAG) in African Americans (AA). AA subjects over 35 years old were recruited for the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study in Philadelphia, PA. Glaucoma cases were evaluated for phenotypes associated with POAG pathogenesis, and the associations between rs112369934 and phenotypes were investigated by logistic regression analysis and in gender-stratified case cohorts: The SNP rs112369934 was found to have a suggestive association with retinal nerve fiber layer (RNFL) thickness and cup-to-disc ratio (CDR) in 1087 male AA POAG cases, individuals with the TC genotype having thinner RNFL (95% CI 0.85 to 6.61, p = 0.01) and larger CDR (95% CI -0.07 to -0.01, p = 0.02) than those with wildtype TT. No other significant associations were found. In conclusion SNP rs112369934 may play a role in POAG pathogenesis in male AA individuals. However, this SNP has been implicated in higher POAG risk in both male and female AA POAG cases.

LMX1B Locus Associated with Low-Risk Baseline Glaucomatous Features in the POAAGG Study.

Primary open-angle glaucoma (POAG) is the leading cause of irreversible blindness worldwide and has been associated with multiple genetic risk factors. The LMX1B gene is a genetic susceptibility factor for POAG, and several single-nucleotide polymorphisms (SNPs) were shown to be associated with POAG in our own prior Primary Open-Angle African American Glaucoma Genetics (POAAGG) study genome-wide association study (GWAS). This study evaluated the association of the LMX1B locus with baseline optic disc and clinical phenotypic characteristics of glaucoma patients from our African American cohort. Compared to the GG genotype in SNP rs187699205, the GC genotype in this SNP was found to be significantly associated with a smaller cup-to-disc ratio (CDR) and increased (better) visual field mean deviation (MD) in glaucoma cases. None of the glaucoma cases with the GC genotype had disc hemorrhages, disc notching, or beanpot disc appearance. In conclusion, glaucoma phenotypes differed significantly by LMX1B variant in African American patients with POAG, and a SNP variant was associated with certain disease features considered lower risk.

The Role of Genetic Ancestry as a Risk Factor for Primary Open-angle Glaucoma in African Americans.

Purpose: POAG is the leading cause of irreversible blindness in African Americans. In this study, we quantitatively assess the association of autosomal ancestry with POAG risk in a large cohort of self-identified African Americans. Methods: Subjects recruited to the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study were classified as glaucoma cases or controls by fellowship-trained glaucoma specialists. POAAGG subjects were genotyped using the MEGA Ex array (discovery cohort, n = 3830; replication cohort, n = 2135). Population structure was interrogated using principal component analysis in the context of the 1000 Genomes Project superpopulations. Results: The majority of POAAGG samples lie on an axis between African and European superpopulations, with great variation in admixture. Cases had a significantly lower mean value of the ancestral component q0 than controls for both cohorts (P = 6.14-4; P = 3-6), consistent with higher degree of African ancestry. Among POAG cases, higher African ancestry was also associated with thinner central corneal thickness (P = 2-4). Admixture mapping showed that local genetic ancestry was not a significant risk factor for POAG. A polygenic risk score, comprised of 23 glaucoma-associated single nucleotide polymorphisms from the NHGRI-EBI genome-wide association study catalog, was significant in both cohorts (P < 0.001), suggesting that both known POAG single nucleotide polymorphisms and an omnigenic ancestry effect influence POAG risk. Conclusions: In sum, the POAAGG study population is very admixed, with a higher degree of African ancestry associated with an increased POAG risk. Further analyses should consider social and environmental factors as possible confounding factors for disease predisposition.

Evaluating TNF-α and Interleukin-2 (IL-2) Levels in African American Primary Open-Angle Glaucoma Patients.

PURPOSE: To establish if SNPs in TNF-α and IL-2 genes are associated with Primary Open-Angle Glaucoma (POAG) in African Americans (AA). We also determined whether plasma TNF-α and IL-2 levels could serve as biomarkers for POAG in African Americans using sandwich enzyme-linked immunosorbent assay. METHODS: A single SNP association analysis was performed to investigate the association between potential gene variants in TNF-α and IL-2 genes and POAG in the AA population. Plasma samples from 190 African Americans (72 from normal subjects and 118 POAG cases) were obtained for TNF- α studies and 367 samples (135 from normal subjects and 232 from POAG cases) were obtained for IL-2 studies. TNF-α levels and IL-2 levels were measured by sandwich enzyme-linked immunosorbent assays (ELISA) and analyzed to see if they reached significance in cases with POAG and endophenotypes when compared to normal subjects. RESULTS: The SNP, rs1800630, in TNF-α gene was found to be marginally associated with POAG. SNPs in IL-2 gene were not associated with POAG in the case-control analysis. No significant difference was found between TNF-α levels and IL-2 levels in normal and POAG case subjects in our study. IL-2 levels were inversely correlated with high IOP in POAG cases. CONCLUSIONS: Although we found a marginal SNP association of TNF-α, assessing the expression levels of TNF-α and IL-2 may serve as promising biomarkers for African American POAG. Further investigation is needed to determine if POAG can be subdivided into more specified cohorts of the disease, which may affect plasma cytokine levels differently.

Molecular Genetics and Functional Analysis Implicate CDKN2BAS1-CDKN2B Involvement in POAG Pathogenesis.

The genes in the 9p21 locus (CDKN2B-AS1 & CDKN2B) are widely associated with Primary open-angle glaucoma (POAG). However, the functional importance of this locus in POAG pathogenesis is still unexplored. This study investigated the role of CDKN2BAS1-CDKN2B axis in POAG. We observed significant association of CDKN2B-AS1 SNP rs4977756 with POAG and its endophenotypic traits (vertical cup-disc ratio (p = 0.033) and central corneal thickness (p = 0.008)) by screening African American POAG cases (n = 1567) and controls (n = 1600). A luciferase reporter assay in Human embryonic kidney 293T (HEK293T) cells revealed that the region surrounding rs4977756 likely serves as a transcriptional repressor. siRNA-mediated knockdown of CDKN2B-AS1 in HEK293T cells and trabecular meshwork (TM) cells resulted in significantly increased expression of CDKN2B, which was also observed in human POAG ocular tissues. Pathway focused qRT-PCR gene expression analysis showed increased cellular senescence, TGFß signaling and ECM deposition in TM cells after CDKN2B-AS1 suppression. In conclusion, we report that CDKN2B-AS1 may act as a regulator, and it could function by modulating the expression of CDKN2B. In addition, increase in CDKN2B levels due to CDKN2B-AS1 suppression may result in the senescence of trabecular meshwork cells leading to POAG pathogenesis.

Next-Generation Technologies and Strategies for the Management of Retinoblastoma.

Retinoblastoma (RB) is an inherited retinal disorder (IRD) caused by the mutation in the RB1 gene or, rarely, by alterations in the MYCN gene. In recent years, new treatment advances have increased ocular and visual preservation in the developed world. The management of RB has improved significantly in recent decades, from the use of external beam radiation to recently, more localized treatments. Determining the underlying genetic cause of RB is critical for timely management decisions. The advent of next-generation sequencing technologies have assisted in understanding the molecular pathology of RB. Liquid biopsy of the aqueous humor has also had significant potential implications for tumor management. Currently, patients' genotypic information, along with RB phenotypic presentation, are considered carefully when making treatment decisions aimed at globe preservation. Advances in molecular testing that improve our understanding of the molecular pathology of RB, together with multiple directed treatment options, are critical for developing precision medicine strategies to treat this disease.

Primary Open-Angle African American Glaucoma Genetics (POAAGG) Study: gender and risk of POAG in African Americans.

The purpose of this study was to investigate the association between gender and primary open-angle glaucoma (POAG) among African Americans and to assess demographic, systemic, and behavioral factors that may contribute to differences between genders. The Primary Open-Angle African American Glaucoma Genetics (POAAGG) study had a case-control design and included African Americans 35 years and older, recruited from the greater Philadelphia, Pennsylvania. Diagnosis of POAG was based on evidence of both glaucomatous optic nerve damage and characteristic visual field loss. Demographic and behavioral information, history of systemic diseases and anthropometric measurements were obtained at study enrollment. Gender differences in risk of POAG were examined using multivariate logistic regression. A total of 2,290 POAG cases and 2,538 controls were included in the study. The percentage of men among cases was higher than among controls (38.6% vs 30.3%, P<0.001). The subjects' mean age at enrollment was significantly higher for cases compared to controls (70.2±11.3 vs. 61.6±11.8 years, P<0.003). Cases had lower rates of diabetes (40% vs. 46%, P<0.001), higher rates of systemic hypertension (80% vs. 72%, P<0.001), and lower body mass index (BMI) (29.7±6.7 vs. 31.9±7.4, P<0.001) than controls. In the final multivariable model, male gender was significantly associated with POAG risk (OR, 1.64; 95% CI, 1.44-1.87; P<0.001), after adjusting for age, systemic hypertension, diabetes, and BMI. Within the POAAGG study, men were at higher risk of having POAG than women. Pending genetic results from this study will be used to better understand the underlying genetic variations that may account for these differences.

Mitochondrial haplogroup L1c2 is associated with increased disease severity in African American patients with primary open-angle glaucoma.

OBJECTIVE: The purpose of this study is to evaluate the role mitochondrial inheritance plays in primary open-angle glaucoma (POAG) characteristics in African Americans. METHODS: POAG cases from the L1c2 and L1b mitochondrial haplogroups were compared in a retrospective case-case study. Twenty-six pairs of self-identified African American POAG cases from L1c2 and L1b mitochondrial haplogroups matched on age (mean [SD] = 71.2 [9.6] and 71.3 [9.6] years, respectively; p = 0.97), sex (21 female and 5 male pairs), and family history of glaucoma (positive in 15/26 [58%] pairs) were included. RESULTS: L1c2 subjects displayed higher vertical cup-to-disc ratio (0.75 [0.12] and 0.67 [0.16], respectively; p = 0.01, Bonferroni-corrected p = 0.08), worse pattern standard deviation on visual field (VF) testing (5.5 [3.5] and 3.5 [2.7]; p = 0.005, Bonferroni-corrected p = 0.02), and more severe glaucoma based on American Glaucoma Society staging criteria (p = 0.04, Bonferroni-corrected p = 0.32) compared to L1b subjects. L1c2 also trended towards worse mean deviation on VF compared to L1b (-8.2 [7.6] and -5.8 [6.8], respectively, p = 0.17). Best corrected visual acuity, central corneal thickness, maximum intraocular pressure (IOP), and cataract severity were comparable between L1c2 and L1b haplogroups (p ≥ 0.49), as was retinal nerve fiber layer thickness on optical coherence tomography (75.1 [14.1] and 75.1 [13.0]; p = 0.99). CONCLUSION: Results demonstrated worse glaucomatous cupping and more severe VF loss in the L1c2 compared to the L1b haplogroup despite comparable IOP. Findings implicate mitochondrial inheritance as a factor affecting POAG severity and may ultimately contribute to stratifying POAG patients into phenotypically and genotypically distinct subgroups.

SNP located in an AluJb repeat downstream of TMCO1, rs4657473, is protective for POAG in African Americans.

AIMS: To determine the association of single nucleotide polymorphisms (SNPs) downstream from the TMCO1 gene with primary open-angle glaucoma (POAG) in African Americans (AA). METHODS: AA subjects were recruited for the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study from the Scheie Eye Institute and its satellite sites in Philadelphia. A region containing an AluJb repeat and seven SNPs, including rs4656461 near the TMCO1 gene, were PCR-Sanger sequenced from POAAGG cases (n=1537) and controls (n=1570). Association between POAG and SNPs near TMCO1 was investigated by logistic regression analysis. Phenotypic trait associations with these SNPs were assessed by analysis of variance. Electrophoretic mobility shift assay (EMSA) was performed to assess the affinity of human T-box 5 (TBX5) protein for a predicted binding motif in the TMCO1 region. Dual Luciferase assays were performed by transfecting recombinant plasmids containing the region surrounding the above SNPs in HEK293T and trabecular meshwork cells. RESULTS: The SNP rs4657473 (C>T) was associated with POAG; the TT genotype was protective (OR 0.20, 95% CI 0.09 to 0.42; p<0.001). No significant associations were found between the TMCO1 variants and phenotypic traits. EMSA confirmed the affinity of TBX5 for a predicted binding motif containing TMCO1 SNP rs4657475. Luciferase assays demonstrated a regulatory function for the genomic region around SNP rs4656561, located within AluJb repeat. CONCLUSION: Our results demonstrate that a SNP downstream of TMCO1, rs4657473, is associated with POAG in an AA population. Our studies suggest a regulatory role for the previously POAG-associated locus near the TMCO1 gene that may affect gene expression.

The association of mitochondrial DNA haplogroups with POAG in African Americans.

Mitochondrial dysfunction has been implicated in the pathogenesis of primary open-angle glaucoma (POAG). However, the potential significance of mitochondrial DNA (mtDNA) haplogroups to POAG has not been evaluated in the overaffected African American population. To investigate the association of mtDNA haplogroups with POAG and its phenotypic characteristics, genotyping data from 4081 African American subjects (1919 cases and 2162 controls) was analyzed using 1293 positions on mtDNA. The overall frequency of mtDNA haplogroups in the Primary Open-Angle African American Glaucoma Genetics (POAAGG) study cohort was 37% L3, 29% L2, 21% L1, 4% L0, and 10% non-African haplogroups (non-L). When all haplogroups (L0, L1, L2, and non-L) were compared against theL3 reference group, after adjusting by age and principal component of ancestry, the non-L3 haplogroups showed higher risk of POAG (OR-1.19, p = 0.02), with a particularly strong association among males (OR = 1.41, p = 0.003). More specifically the non-L group was associated with higher POAG risk than the L3 haplogroup (OR = 1.77, p = 0.007, Bonferroni adjusted p = 0.027) and to the L3e (n = 256, OR = 1.92, p = 0.007, Bonferroni adjusted p = 0.029). No significant association was found when genders were analyzed together or in female only analysis. There were no significant differences in various POAG endophenotypes across mtDNA haplogroups. This study expands our knowledge of mitochondrial genetics and mtDNA haplogroup associations in African American POAG. Further work is needed to better understand the functional role of mtDNA polymorphisms and their interactions with nuclear genes that affect POAG.

The MT-CO1 V83I Polymorphism is a Risk Factor for Primary Open-Angle Glaucoma in African American Men.

Purpose: We investigate the function of the V83I polymorphism (m.6150G>A, rs879053914) in the mitochondrial cytochrome c oxidase subunit 1 (MT-CO1) gene and its role in African American (AA) primary open-angle glaucoma (POAG). Methods: This study used Sanger sequencing (1339 cases, 850 controls), phenotypic characterization of Primary Open-Angle African American Glaucoma Genetics study (POAAGG) cases, a masked chart review of CO1 missense cases (V83I plus M117T, n = 29) versus wild type cases (n = 29), a yeast 2-hybrid (Y2H) cDNA library screen, and quantification of protein-protein interactions by Y2H and ELISA. Results: The association of V83I with POAG in AA was highly significant for men (odds ratio [OR] 6.5; 95% confidence interval [CI] 2.0-21.3, P = 0.0001), but not for women (OR 1.1; 95% CI, 0.62-2.00, P = 0.78). POAG cases having CO1 double missense mutation (V83I + M117T, L1c2 haplogroup) had a higher cup-to-disc ratio (0.77 vs. 0.71, P = 0.04) and significantly worse visual function (average pattern standard deviation, 6.5 vs. 4.3, P = 0.009; average mean deviation -10.4 vs. -4.5, P = 0.006) when compared to matched wild type cases (L1b haplogroup). Interaction of the V83I region of CO1 with amyloid beta peptide (Aß) was confirmed by ELISA assay, and this interaction was abrogated by V83I. A Y2H screen of an adult human brain cDNA library with the V83 region of CO1 as bait retrieved the UBQLN1 gene. Conclusions: The V83I polymorphism was associated strongly with POAG in AA men and disrupts Aß-binding to CO1. This region also interacts with a neuroprotective protein, UBQLN1.

Complete Transcriptome Profiling of Normal and Age-Related Macular Degeneration Eye Tissues Reveals Dysregulation of Anti-Sense Transcription.

Age-related macular degeneration (AMD) predominantly affects the retina and retinal pigment epithelium in the posterior eye. While there are numerous studies investigating the non-coding transcriptome of retina and RPE, few significant differences between AMD and normal tissues have been reported. Strand specific RNA sequencing of both peripheral retina (PR) and RPE-Choroid-Sclera (PRCS), in both AMD and matched normal controls were generated. The transcriptome analysis reveals a highly significant and consistent impact on anti-sense transcription as well as moderate changes in the regulation of non-coding (sense) RNA. Hundreds of genes that do not express anti-sense transcripts in normal PR and PRCS demonstrate significant anti-sense expression in AMD in all patient samples. Several pathways are highly enriched in the upregulated anti-sense transcripts-in particular the EIF2 signaling pathway. These results call for a deeper exploration into anti-sense and noncoding RNA regulation in AMD and their potential as therapeutic targets.