Person:

Wiggs, Janey

It is unclear if buccal cell samples contain sufficient human DNA with adequately sized fragments for high throughput genetic bioassays. Yet buccal cell sample collection is an attractive alternative to gathering blood samples for genetic epidemiologists engaged in large-scale genetic biomarker studies. We assessed the genotyping efficiency (GE) and genotyping concordance (GC) of buccal cell DNA samples compared to corresponding blood DNA samples, from 32 Nurses’ Health Study (NHS) participants using the Illumina Infinium 660W-Quad platform. We also assessed how GE and GC accuracy varied as a function of DNA concentration using serial dilutions of buccal DNA samples. Finally we determined the nature and genomic distribution of discordant genotypes in buccal DNA samples. The mean GE of undiluted buccal cell DNA samples was high (99.32%), as was the GC between the paired buccal and blood samples (99.29%). GC between the dilutions versus the undiluted buccal DNA was also very high (greater than 97%), though both GE and GC notably declined at DNA concentrations less than 5 ng/μl. Most (greater than 95%) genotype determinations in buccal cell samples were of the “missing call” variety (as opposed to the “alternative genotype call” variety) across the spectrum of buccal DNA concentrations studied. Finally, for buccal DNA concentration above 1.7 ng/ul, discordant genotyping calls did not cluster in any particular chromosome. Buccal cell-derived DNA represents a viable alternative to blood DNA for genotyping on a high-density platform.

Purpose: To evaluate genes involved in homocysteine metabolism as secondary risk factors for pseudoexfoliation syndrome (PXFS) and the associated glaucoma (PXFG). Methods: One hundred eighty-six unrelated patients with PXFS, including 140 patients with PXFG and 127 unrelated control subjects were recruited from the Massachusetts Eye and Ear Infirmary. All the patients and controls were Caucasian of European ancestry. Seventeen tag SNPs from 5 genes (methylenetetrahydrofolate reductase [MTHFR], methionine synthase [MTR], methionine synthase reductase [MTRR], methylenetetrahydrofolate dehydrogenase [MTHFD1], and cystathionine β-synthase [CBS]) were genotyped. Single-SNP association was analyzed using Fisher’s exact test (unconditional) or logistic regression after conditioning on the effects of age and three LOXL1 SNPs (rs1048661, rs3825942, and rs2165241). Interaction analysis was performed between the homocysteine and LOXL1 SNPs using logistic regression. Haplotype analysis and the set-based test were used to test for association of individual genes. Multiple comparisons were corrected using the Bonferroni method. Results: One SNP (rs8006686) in MTHFD1 showed a nominally significant association with PXFG (p=0.015, OR=2.23). None of the seventeen SNPs tested were significantly associated with PXFS or PXFG after correcting for multiple comparisons (Bonferroni corrected p>0.25). After controlling for the effects of age and three associated LOXL1 SNPs, none of the seventeen tested SNPs were associated with PXFS (p>0.12). No significant interaction effects on PXFS were identified between the homocysteine and LOXL1 SNPs (p>0.06). Haplotype analysis and the set-based test did not find significant association of individual genes with PXFS (p>0.23 and 0.20, respectively). Conclusions: Five genes that are critical components of the homocysteine metabolism pathway were evaluated as secondary factors for PXFS and PXFG. Our results suggest that these genes are not significant risk factors for the development of these conditions.

Purpose Mutations in the myocilin gene (MYOC) are associated with primary open-angle glaucoma (POAG) in many different populations. This study represents the first large survey of MYOC mutations in an African American population. Methods: We recruited 529 African American subjects with POAG and 270 African American control subjects in this study. A complete eye examination and blood collection was performed in all study subjects. Genomic DNA was extracted. The entire coding sequence of MYOC was amplified and sequenced using the Sanger method. Identified MYOC variants were compared with previously reported MYOC mutations. Results: We identified a total of 29 MYOC variants including six potential MYOC mutations. Two mutations (Thr209Asn and Leu215Gln) are novel and are found only in cases and no controls. We also identified four previously reported MYOC mutations in cases and no controls (Tyr453MetfsX11, Gln368X, Thr377Met, and Ser393Arg). The overall frequency of glaucoma-causing MYOC mutations in our African American population with POAG was 1.4%. Conclusions: We identified two novel probable glaucoma-causing MYOC mutations (Thr209Asn and Leu215Gln). This study indicates that, despite the high prevalence of POAG, MYOC mutations are rare in the African American population.

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(^{−10})). 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.

Background: Loss of vision in glaucoma is due to apoptotic retinal ganglion cell loss. While p53 modulates apoptosis, gene association studies between p53 variants and glaucoma have been inconsistent. In this study we evaluate the association between a p53 variant functionally known to influence apoptosis (codon 72 Pro/Arg) and the subset of primary open angle glaucoma (POAG) patients with early loss of central visual field. Methods: Genotypes for the p53 codon 72 polymorphism (Pro/Arg) were obtained for 264 POAG patients and 400 controls from the U.S. and in replication studies for 308 POAG patients and 178 controls from Australia (GIST). The glaucoma patients were divided into two groups according to location of initial visual field defect (either paracentral or peripheral). All cases and controls were Caucasian with European ancestry. Results: The p53-PRO/PRO genotype was more frequent in the U.S. POAG patients with early visual field defects in the paracentral regions compared with those in the peripheral regions or control group (p = 2.7×(10^{−5})). We replicated this finding in the GIST cohort (p = 7.3×(10^{−3}), and in the pooled sample (p = 6.6×(10^{−7})) and in a meta-analysis of both the US and GIST datasets (1.3×(10^{−6}), OR 2.17 (1.58–2.98 for the PRO allele). Conclusions: These results suggest that the p53 codon 72 PRO/PRO genotype is potentially associated with early paracentral visual field defects in primary open-angle glaucoma patients.

Purpose: One approach to identify genes that contribute to common complex ocular disorders such as primary open angle glaucoma (POAG) is to study the genetic determinates of endophenotypes that are defined by underlying pre-disposing heritable quantitative traits such as central corneal thickness (CCT). Collagen VIII is a major component of Descemet’s membrane and studies in mice have indicated that targeted inactivation of the genes encoding the collagen type 8 alpha1 (Col8a1) and collagen type 8 alpha2 (Col8a2) subunits (COL8A1 and COL8A2) results in thinning of the corneal stroma and of Descemet’s membrane. The purpose of this study is to evaluate COL8A1 and COL8A2 as candidate genes for thin CCT in human POAG patients. Methods: 100 Caucasian POAG patients were enrolled in this study. The entire COL8A1 and COL8A2 coding sequence was determined in 8 patients with CCT<513 µm (one standard deviation (36 microns) below the mean (550 microns) and 8 patients with CCT>586 µm (one standard deviation above the mean). Selected COL8A2 exons containing variants of interest were sequenced in the full POAG cohort. Association and quantitative trait analyses were performed. Results: Three patients with CCT less than 513 µm and advanced POAG were found to have missense changes in COL8A2; two patients had a previously identified mutation, R155Q and one had a novel change, P678L (p=0.0035, Fisher’s exact test). Missense changes were not found in any of the patients with CCT>513 µm and missense changes in the COL8A1 gene were not found in any patient. One common COL8A2 SNP, rs274754 was also statistically associated with CCT (p=0.018). Conclusions: In this study we have identified COL8A2 missense changes in a group of Caucasian patients with very thin CCT and advanced POAG. These results suggest that DNA sequence variants in the COL8A2 gene may be associated with thin corneas in some glaucoma patients. Further study of COL8A2 variants in other patient populations, especially those with thinner CCT such as African-Americans would provide further support for a role of COL8A2 in corneal thickness and in glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the (α_1) subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle. The optic neuropathy associated with soluble guanylate cyclase (α_1)–deficiency was accompanied by modestly increased intraocular pressure and retinal vascular dysfunction. Moreover, data from a candidate gene association study suggests that a variant in the locus containing the genes encoding for the (α_1) and (β_1) subunits of soluble guanylate cyclase is associated with POAG in patients presenting with initial paracentral vision loss, a disease subtype thought to be associated with vascular dysregulation. These findings provide new insights into the pathogenesis and genetics of POAG and suggest new therapeutic strategies for POAG.

Background: Pseudoexfoliation syndrome is a major risk factor for glaucoma in many populations throughout the world. Using a U.S. clinic-based case control sample with broad ethnic diversity, we show that three common SNPs in LOXL1 previously associated with pseudoexfoliation in Nordic populations are significantly associated with pseudoexfoliation syndrome and pseudoexfoliation glaucoma. Methods: Three LOXL1 SNPs were genotyped in a patient sample (206 pseudoexfoliation, 331 primary open angle glaucoma, and 88 controls) from the Glaucoma Consultation Service at the Massachusetts Eye and Ear Infirmary. The SNPs were evaluation for association with pseudeoexfoliation syndrome, pseudoexfoliation glaucoma, and primary open angle glaucoma. Results: The strongest association was found for the G allele of marker rs3825942 (G153D) with a frequency of 99% in pseudoexfoliation patients (with and without glaucoma) compared with 79% in controls ((p = 1.6 × 10^{-15}); OR = 20.93, 95%CI: 8.06, 54.39). The homozygous GG genotype is also associated with pseudoexfoliation when compared to controls ((p = 1.2 × 10^{-12}); OR = 23.57, 95%CI: 7.95, 69.85). None of the SNPs were significantly associated with primary open angle glaucoma. Conclusion: The pseudoexfoliation syndrome is a common cause of glaucoma. These results indicate that the G153D LOXL1 variant is significantly associated with an increased risk of pseudoexfoliation and pseudoexfoliation glaucoma in an ethnically diverse patient population from the Northeastern United States. Given the high prevalence of pseudooexfoliation in this geographic region, these results also indicate that the G153D LOXL1 variant is a significant risk factor for adult-onset glaucoma in this clinic based population.

Glaucoma is characterized by irreversible optic nerve degeneration and is the most frequent cause of irreversible blindness worldwide. Here, the International Glaucoma Genetics Consortium conducts a meta-analysis of genome-wide association studies of vertical cup-disc ratio (VCDR), an important disease-related optic nerve parameter. In 21,094 individuals of European ancestry and 6,784 individuals of Asian ancestry, we identify 10 new loci associated with variation in VCDR. In a separate risk-score analysis of five case-control studies, Caucasians in the highest quintile have a 2.5-fold increased risk of primary open-angle glaucoma as compared with those in the lowest quintile. This study has more than doubled the known loci associated with optic disc cupping and will allow greater understanding of mechanisms involved in this common blinding condition.