D. J. Zack

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], ORu200a=u200a0.69 [95%CI 0.63–0.75], pu200a=u200a1.86×10−18), and the SIX1/SIX6 region on chromosome 14q23 (rs10483727 [A], ORu200a=u200a1.32 [95%CI 1.21–1.43], pu200a=u200a3.87×10−11). In sub-group analysis two loci were significantly associated with NPG: 9p21 containing the CDKN2BAS gene (rs2157719 [G], ORu200a=u200a0.58 [95% CI 0.50–0.67], pu200a=u200a1.17×10−12) and a probable regulatory region on 8q22 (rs284489 [G], ORu200a=u200a0.62 [95% CI 0.53–0.72], pu200a=u200a8.88×10−10). Both NPG loci were also nominally associated with a second type of glaucoma, exfoliation syndrome glaucoma (rs2157719 [G], ORu200a=u200a0.59 [95% CI 0.41–0.87], pu200a=u200a0.004 and rs284489 [G], ORu200a=u200a0.76 [95% CI 0.54–1.06], pu200a=u200a0.021), 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 pu200a=u200a0.009). 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, pxa0=xa08.0xa0×xa010−8). 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.

Discovery and functional annotation of SIX6 variants in primary open-angle glaucoma.

Glaucoma is a leading cause of blindness worldwide. Primary open-angle glaucoma (POAG) is the most common subtype and is a complex trait with multigenic inheritance. Genome-wide association studies have previously identified a significant association between POAG and the SIX6 locus (rs10483727, odds ratio (OR)u200a=u200a1.32, pu200a=u200a3.87×10−11). SIX6 plays a role in ocular development and has been associated with the morphology of the optic nerve. We sequenced the SIX6 coding and regulatory regions in 262 POAG cases and 256 controls and identified six nonsynonymous coding variants, including five rare and one common variant, Asn141His (rs33912345), which was associated significantly with POAG (ORu200a=u200a1.27, pu200a=u200a4.2×10−10) in the NEIGHBOR/GLAUGEN datasets. These variants were tested in an in vivo Danio rerio (zebrafish) complementation assay to evaluate ocular metrics such as eye size and optic nerve structure. Five variants, found primarily in POAG cases, were hypomorphic or null, while the sixth variant, found only in controls, was benign. One variant in the SIX6 enhancer increased expression of SIX6 and disrupted its regulation. Finally, to our knowledge for the first time, we have identified a clinical feature in POAG patients that appears to be dependent upon SIX6 genotype: patients who are homozygous for the SIX6 risk allele (His141) have a statistically thinner retinal nerve fiber layer than patients homozygous for the SIX6 non-risk allele (Asn141). Our results, in combination with previous SIX6 work, lead us to hypothesize that SIX6 risk variants disrupt the development of the neural retina, leading to a reduced number of retinal ganglion cells, thereby increasing the risk of glaucoma-associated vision loss.

The NEIGHBOR Consortium Primary Open Angle Glaucoma Genome-wide Association Study: Rationale, Study design and Clinical variables

Primary open-angle glaucoma (POAG) is a common disease with complex inheritance. The identification of genes predisposing to POAG is an important step toward the development of novel gene-based methods of diagnosis and treatment. Genome-wide association studies (GWAS) have successfully identified genes contributing to complex traits such as POAG however, such studies frequently require very large sample sizes, and thus, collaborations and consortia have been of critical importance for the GWAS approach. In this report we describe the formation of the NEIGHBOR consortium, the harmonized case control definitions used for a POAG GWAS, the clinical features of the cases and controls, and the rationale for the GWAS study design.

The challenge of regenerative therapies for the optic nerve in glaucoma

ABSTRACT This review arose from a discussion of regenerative therapies to treat optic nerve degeneration in glaucoma at the 2015 Lasker/IRRF Initiative on Astrocytes and Glaucomatous Neurodegeneration. In addition to the authors, participants included Jonathan Crowston, Andrew Huberman, Elaine Johnson, Richard Lu, Hemai Phatnami, Rebecca Sappington, and Don Zack. Glaucoma is a neurodegenerative disease of the optic nerve, and is the leading cause of irreversible blindness worldwide. The disease progresses as sensitivity to intraocular pressure (IOP) is conveyed through the optic nerve head to distal retinal ganglion cell (RGC) projections. Because the nerve and retina are components of the central nervous system (CNS), their intrinsic regenerative capacity is limited. However, recent research in regenerative therapies has resulted in multiple breakthroughs that may unlock the optic nerves regenerative potential. Increasing levels of Schwann‐cell derived trophic factors and reducing potent cell‐intrinsic suppressors of regeneration have resulted in axonal regeneration even beyond the optic chiasm. Despite this success, many challenges remain. RGC axons must be able to form new connections with their appropriate targets in central brain regions and these connections must be retinotopically correct. Furthermore, for new axons penetrating the optic projection, oligodendrocyte glia must provide myelination. Additionally, reactive gliosis and inflammation that increase the regenerative capacity must be outweigh pro‐apoptotic processes to create an environment within which maximal regeneration can occur. HIGHLIGHTSGlaucoma is detected late in progression, necessitating regenerative therapies.Regeneration occurs when tropic factors and suppressors are properly modulated.Once regenerated, axons must still terminate upon appropriate targets.Inflammation must be balanced with cell death to create a regenerative environment.

Vascular tone pathway polymorphisms in relation to primary open-angle glaucoma

AimsVascular perfusion may be impaired in primary open-angle glaucoma (POAG); thus, we evaluated a panel of markers in vascular tone-regulating genes in relation to POAG.MethodsWe used Illumina 660W-Quad array genotype data and pooled P-values from 3108 POAG cases and 3430 controls from the combined National Eye Institute Glaucoma Human Genetics Collaboration consortium and Glaucoma Genes and Environment studies. Using information from previous literature and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, we compiled single-nucleotide polymorphisms (SNPs) in 186 vascular tone-regulating genes. We used the ‘Pathway Analysis by Randomization Incorporating Structure’ analysis software, which performed 1000 permutations to compare the overall pathway and selected genes with comparable randomly generated pathways and genes in their association with POAG.ResultsThe vascular tone pathway was not associated with POAG overall or POAG subtypes, defined by the type of visual field loss (early paracentral loss (n=224 cases) or only peripheral loss (n=993 cases)) (permuted P≥0.20). In gene-based analyses, eight were associated with POAG overall at permuted P<0.001: PRKAA1, CAV1, ITPR3, EDNRB, GNB2, DNM2, HFE, and MYL9. Notably, six of these eight (the first six listed) code for factors involved in the endothelial nitric oxide synthase activity, and three of these six (CAV1, ITPR3, and EDNRB) were also associated with early paracentral loss at P<0.001, whereas none of the six genes reached P<0.001 for peripheral loss only.DiscussionAlthough the assembled vascular tone SNP set was not associated with POAG, genes that code for local factors involved in setting vascular tone were associated with POAG.

Systems genetics identifies a role for Cacna2d1 regulation in elevated intraocular pressure and glaucoma susceptibility

Glaucoma is a multi-factorial blinding disease in which genetic factors play an important role. Elevated intraocular pressure is a highly heritable risk factor for primary open angle glaucoma and currently the only target for glaucoma therapy. Our study helps to better understand underlying genetic and molecular mechanisms that regulate intraocular pressure, and identifies a new candidate gene, Cacna2d1, that modulates intraocular pressure and a promising therapeutic, pregabalin, which binds to CACNA2D1 protein and lowers intraocular pressure significantly. Because our study utilizes a genetically diverse population of mice with known sequence variants, we are able to determine that the intraocular pressure-lowering effect of pregabalin is dependent on the Cacna2d1 haplotype. Using human genome-wide association study (GWAS) data, evidence for association of a CACNA2D1 single-nucleotide polymorphism and primary open angle glaucoma is found. Importantly, these results demonstrate that our systems genetics approach represents an efficient method to identify genetic variation that can guide the selection of therapeutic targets.Elevated intraocular pressure (IOP) is a heritable risk factor for primary open angle glaucoma. Using forward mouse genetics, cell biology, pharmacology and human genetic data, the authors identify CACNA2D1 as an IOP risk gene that can be therapeutically targeted by the drug pregabalin in animal models.

grID: A CRISPR-Cas9 guide RNA Database and Resource for Genome-Editing

CRISPR-Cas9 genome-editing is a revolutionary technology that is transforming biological research. The explosive growth and advances in CRISPR research over the last few years, coupled with the potential for clinical applications and therapeutics, is heralding a new era for genome engineering. To further support this technology platform and to provide a universal CRISPR annotation system, we introduce the grID database (http://crispr.technology), an extensive compilation of gRNA properties including sequence and variations, thermodynamic parameters, off-target analyses, and alternative PAM sites, among others. To aid in the design of optimal gRNAs, the website is integrated with other prominent databases, providing a wealth of additional resources to guide users from in silico analysis through experimental CRISPR targeting. Here, we make available all the tools, protocols, and plasmids that are needed for successful CRISPR-based genome targeting.

494. Development of a pH Sensor to Probe Endosomal Buffering of Polymeric Nanoparticles Effective for Gene Delivery

Introduction: Polymeric gene delivery suffers from low efficacy compared to viral gene delivery with one of the primary barriers to successful transfection being efficient endosomal escape. Cationic polymers have been hypothesized to facilitate endosomal escape via the proton sponge mechanism by buffering hydrogen ions in the endosomal compartment. Here we have created a nucleic acid based pH sensor and applied it using flow cytometry and confocal microscopy to investigate endosomal buffering of synthetic biodegradable cationic polymers for gene delivery, correlating the pH of delivered DNA with transfection. Methods: We created a nucleic acid pH sensor by conjugating pH sensitive (FITC, OG) and insensitive (Cy5) fluorophores to plasmid DNA. The fluorescence ratio of the sensor was calibrated to pH using flow cytometry and confocal microscopy following electroporation into cells. Cells were transfected with the plasmid pH sensor complexed with cationic polymers including poly(beta-amino ester)s (PBAEs) of variable transfection efficacy to investigate endosomal buffering. Additionally, confocal microscopy was used to assess colocalization of the plasmid pH sensor with a lysosomal dye. Results: PBAEs were demonstrated to effectively buffer endosomes and avoid lysosomal fate, whereas the negative controls poly-L-lysine (PLL) and polyethylenimine (PEI) were shown to accumulate in lysosomes by 24h post-transfection. The polymer molecular weight and weight-weight ratio to plasmid DNA was shown to have an effect on endosomal buffering as well as transfection efficacy in the case of PBAEs. From confocal microscopy analysis of endosomes, local pH was observed to be higher at the endosomal membrane than in the center, which may be indicative of polymer enrichment along the membrane due to excess soluble polymer. Conclusions: The constructed DNA sensor gave a linear relationship with intracellular pH. When investigating PBAE-induced buffering, results were consistent with the hypothesized proton sponge mechanism. These cationic polymers were demonstrated to affect endosomal pH and DNA lysosomal fate and lead to successful transfection.Fig. 1Confocal microscopy was used to assess the compartmental pH of endosomes containing nanoparticles formed with the pH sensor. Lysosome colocalization is shown in merged thresholded images with lysosomes (blue), DNA non-colocalized with lysosomes (pink) and DNA colocalized with lysosomes (yellow). Scattergrams show DNA fluorescence (horizontal) and lysosome fluorescence (vertical). Scale bar 10 µm.View Large Image | Download PowerPoint SlideFig. 2Confocal microscopy images were analyzed at (A) 1 hour and (B) 24 hours post-transfection using Pearsons correlation coefficient M1 to assess the fraction of DNA colocalized with lysosomal stain. When delivered via PBAE 447, the fraction of DNA colocalized with lysosomes was significantly different than both bPEI and PLL at both time points.View Large Image | Download PowerPoint Slide

Retinal ganglion cells: Development, function, and disease

Thirteenth Annual Vision Research Conference n nApril 30-May 1-2, 2010 n nConvention Center n nFt. Lauderdale, FL n nThe 13th Annual Vision Research Conference, “Retinal ganglion cells: development, function, and disease” was held at the Convention Center at Ft. Lauderdale, Florida, April 30-May 1, 2010. The meeting was very well attended (294 registrations), and 39 invited speakers reviewed the latest developments in pathogenesis and neuroprotection for ganglion cells, optic nerve damage and regeneration, ganglion cell development and axonal targeting, signal processing, ganglion cell death, and the latest on photosensitive ganglion cells. n nThis special issue contains 11 excellent contributions from speakers and poster presenters of the meeting. The contributions focus mainly on three sessions, pathogenesis/optic nerve damage, ganglion cell development, and photosensitive ganglion cells. Outstanding contributions are a mini review on dominant optic atrophy in a mouse model (Dr. Marcela Votruba), a review on how retinal progenitors specify retinal ganglion cell fate (Dr. William H. Klein), an analysis of mutant and wild-type ganglion cell using genetically-directed sparse labeling (Dr. Tudor Badea), and a review on visual responses in the lateral geniculate (Dr. Rob Lucas).