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Biochemical studies have suggested that elevated serum levels of homocysteine may be associated with primary open-angle glaucoma (POAG)1, 2 and with pseudoexfoliation glaucoma (PEXG).3 More recentl...

Thirty-one members of a family affected with X-linked ocular albinism (OA1) were studied to characterize the clinical phenotype and identify the disease-causing mutation. The family members were examined with ophthalmoscopy, electroretinography, and Goldmann perimetry. Linkage analysis was performed with markers from the OA1 locus. Exons 2 and 8 of the OA1 gene were assayed with the polymerase chain reaction (PCR). The six affected males had visual acuities ranging from 20/40 to 20/200. All had nystagmus, iris transillumination, and foveal hypoplasia. The eldest affected male had 20/40 vision and was asymptomatic. The level of the visual acuity of the affected males was not related to the degree of retinal pigmentation. All seven female carriers had normal visual function but were found to have iris transillumination defects and variable retinal pigmentary appearance ranging from minimal pigmentary disturbance, patchy and diffuse hypopigmentation, to classic 'mud-splattered' appearance. Linkage analysis was consistent with a disease-causing mutation at the OA1 locus. PCR analysis revealed a deletion which includes at least the portion of the OA1 gene between exons 2 and 8. Affected males with X-linked ocular albinism can have a visual disability that ranges from almost none to legal blindness, and the female carriers can have variable retinal pigmentary appearance. Mutation screening of the OA1 gene can be used to confirm the diagnosis in isolated males of some families, and genetic linkage analysis can be used to accurately identify carriers even when the specific mutation cannot be identified.

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.

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

We identified a pattern of concentric circular transillumination defects (TIDs) in a few patients with exfoliation syndrome (XFS) using an infrared detection system. This pattern of iris abnormality has also been observed in a mouse model of XFS. The objective of the current study is to determine whether concentric iris TIDs are specific to XFS and may have some diagnostic utility for identifying early cases of disease.A total of 68 volunteers from the University of Iowa Glaucoma Clinic with normal eyes (n=21) or diagnoses of either XFS (n=12), pigment dispersion syndrome (PDS) (n=8), or primary open-angle glaucoma (POAG) (n=27) were enrolled in the study. The irides of these subjects were each examined by 4 ophthalmologists masked to their diagnosis, using infrared videography. The presence of concentric, circular TIDs on the videos was graded as none (grade 0), possible (grade 1), definite (grade 2), or prominent (grade 3) by 4 examiners. We searched for an association between the presence of concentric bands of transillumination and the diagnosis of XFS after removing the effect of different raters was evaluated using the Cochran-Mentel-Haenszel test. We performed the same analysis for PDS and for POAG.The presence of any concentric, circular iris TIDs (grades 1 to 3) was detected in a mean of 38% normal subjects, 35% POAG patients, 53% PDS patients, and 77% of XFS patients. When the frequency of concentric, circular iris transillumination (grades 1 to 3 pooled) was compared between each of the patient groups and normal controls, a significant difference was detected between XFS patients and controls (P=0.000019). No significant difference was detected between POAG and controls (P=0.64) or between PDS and controls (P=0.20). Furthermore, prominent concentric, circular iris transillumination (grade 3) was only observed in XFS.Detection of concentric, circular iris TIDs with an infrared system is easy, inexpensive, rapid, and relatively specific in XFS. Future larger studies will be needed to confirm the findings of this small pilot study. Furthermore, this examination technique has the potential to help physicians to make earlier diagnoses of XFS and to better plan for future surgeries to minimize risk of complication.

Optic nerve head (ONH) structure is an important biological feature of the eye used by clinicians to diagnose and monitor progression of diseases such as glaucoma. ONH structure is commonly examined using stereo fundus imaging or optical coherence tomography. Stereo fundus imaging provides stereo views of the ONH that retain 3D information useful for characterizing structure. In order to quantify 3D ONH structure, we applied a stereo correspondence algorithm to a set of stereo fundus images. Using these quantitative 3D ONH structure measurements, eigen structures were derived using principal component analysis from stereo images of 565 subjects from the Ocular Hypertension Treatment Study (OHTS). To evaluate the usefulness of the eigen structures, we explored associations with the demographic variables age, gender, and race. Using regression analysis, the eigen structures were found to have significant (p < 0.05) associations with both age and race after Bonferroni correction. In addition, classifiers were constructed to predict the demographic variables based solely on the eigen structures. These classifiers achieved an area under receiver operating characteristic curve of 0.62 in predicting a binary age variable, 0.52 in predicting gender, and 0.67 in predicting race. The use of objective, quantitative features or eigen structures can reveal hidden relationships between ONH structure and demographics. The use of these features could similarly allow specific aspects of ONH structure to be isolated and associated with the diagnosis of glaucoma, disease progression and outcomes, and genetic factors.

The gene that causes normal tension glaucoma (NTG) in a large pedigree was recently mapped to a region of chromosome 12q14 (GLC1P) that contains the genes TBK1, XPOT, RASSF3, and GNS. We sought to investigate the structure of the chromosome 12q14 duplication and explore the ocular expression of GLC1P locus genes.The location of the chromosome 12q14 duplication in this pedigree was examined with fluorescent in situ hybridization (FISH) using probes for TBK1 and GNS. The expression pattern of XPOT, TBK1, RASSF3, and GNS was investigated with immunohistochemistry of human eyes.The karyotype of an NTG patient from pedigree GGO-414 was normal and FISH studies demonstrated that the duplicated DNA is organized as a tandem repeat on chromosome 12q14. Of the genes in or near the chromosome 12q14 duplication, TBK1 showed expression in the retina that is specific to the retinal ganglion cells and the retinal nerve fiber layer. Expression of RASSF3 and XPOT was relatively uniform throughout the retina, while GNS expression was expressed in a pattern consistent with Müller cells.Previous studies demonstrated that chromosome 12q14 duplications are associated with NTG inherited as an autosomal dominant trait. FISH studies now demonstrate that the duplicated segments are tandemly organized on chromosome 12q14 in close proximity. The specific expression of TBK1 in human retinal ganglion cells compared to the widespread pattern of expression of neighboring genes provides additional evidence that TBK1 is the glaucoma gene in the chromosome 12q14 duplication within the GLC1P locus.

Copy number variations (duplications) of TANK binding kinase 1 (TBK1) have been associated with normal tension glaucoma (NTG), a common cause of blindness worldwide. Mutations in other genes involved in autophagy (TLR4 and OPTN) have been associated with NTG. Here we report searching for additional proteins involved in autophagy that may also have roles in NTG.HEK-293T cells were transfected to produce synthetic TBK1 protein with FLAG and S tags. Proteins that associate with TBK1 were isolated from HEK-293T lysates using tandem affinity purification (TAP) and polyacrylamide gel electrophoresis (PAGE). Isolated proteins were identified with mass spectrometry. A cohort of 148 NTG patients and 77 controls from Iowa were tested for glaucoma-causing mutations in genes that encode identified proteins that interact with TBK1 using high resolution melt (HRM) analysis and DNA sequencing.TAP studies show that three proteins expressed in HEK-293T cells (NAP1, TANK and TBKBP1) interact with TBK1. Testing cohorts of NTG and normal controls for disease-causing mutations in TANK, identified a total of nine unique variants including three non-synonymous changes, one synonymous changes and five intronic changes. When analyzed alone or as a group, the non-synonymous TBK1 coding sequence changes were not associated with either NTG or primary open angle glaucoma.TAP showed that NAP1, TANK and TBKBP1 interact with TBK1 and are good candidates for contributing to NTG. A mutation screen of TANK detected three non-synonymous variants. Although, it remains possible that one or more of these TANK mutations may have a role in NTG, the data in this report do not provide statistical support for an association between TANK variants and NTG.

Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG) occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN) and TANK binding kinase 1 (TBK1), cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1) gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308) and matched controls (n = 157) using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05). These data suggest that SQSTM1 mutations are not a common cause of NTG.

To evaluate gonioscopic features and relate them to clinical characteristics in eyes with juvenile-onset primary open-angle glaucoma (JOAG). Goniophotographs of unrelated JOAG patients, presenting between 10–40 years of age, were evaluated and compared with 60 healthy subjects in the same age group. Age of onset, family history of glaucoma, highest untreated IOP and visual field defect (mean deviation) were analyzed and correlated with the gonioscopic features among JOAG patients. Of 126 patients included in the study, 44 (34 %) had a normal open angle (group 1), while 82 (66 %) had developmental anomalies (group 2). Developmental anomalies of the angle were classified as: high iris insertion with or without prominent iris processes (n = 42), a featureless angle (n = 30), and those with prominent iris processes alone (n = 10). There was no difference in age of onset (group 1, 30.5 ± 7 years and group 2, 26.3 ± 9.6 years) (p = 0.07) or the untreated IOP at presentation (group 1; 36 ± 12.5 mmHg and group 2, 38.8 ± 12.3 mmHg; p = 0.37) between the groups. However, those with angle anomalies presented with a greater visual field defect (MD −23.5 ± 10.5 vs −14.8 ± 13 dB; p = 0.02) compared to those with normal appearing angle. While two thirds of JOAG patients present with developmental anomalies of the angle, one third have normal appearing angles. High insertion of the iris is the most common form of gonio dysgenesis observed. Those with angle dysgenesis are more likely to present with severe disease.

Next generation sequencing holds great promise for detecting rare variants underlying complex human traits. Due to their extremely low allele frequencies, the normality approximation for a proportion no longer works well. The Fisher's exact method appears to be suitable but it is conservative. We investigate the utility of various variance-stabilising transformations in single marker association analysis on rare variants. Unlike a proportion itself, the variance of the transformed proportions no longer depends on the proportion, making application of such transformations to rare variant association analysis extremely appealing. Simulation studies demonstrate that tests based on such transformations are more powerful than the Fisher's exact test while controlling for type I error rate. Based on theoretical considerations and results from simulation studies, we recommend the test based on the Anscombe transformation over tests with other transformations.

Recently, we discovered chromosome 12q14 duplications in patients with normal tension glaucoma (NTG) (Fingert et al., 2011). Three different but overlapping chromosome 12q14 duplications that all spanned the TBK1, XPOT, and RASSF3 genes were identified. The duplication of TBK1 was judged to be the most likely cause of NTG in these patients because: 1) TBK1 associates with the product of another NTG gene, optineurin (Morton et al., 2008); 2) Duplication of TBK1 leads to increased transcription of TBK1 (Fingert et al., 2011); and 3) TBK1 is specifically expressed in cells affected by glaucoma pathophysiology (retinal ganglion cells and their axons) (Fingert et al., 2011). Finally, the population-based segment of our previous study identified chromosome 12q14 duplications that span TBK1 in 2 (1.3%) of 152 NTG patients from Iowa (Fingert et al., 2011) suggesting that copy number variations of TBK1 may be responsible for a fraction of all NTG cases. Here we report the first replication study to investigate the role of copy number variations (CNVs) of TBK1 in NTG pathogenesis. The study was approved by local Institutional Review Boards and informed consent was obtained from all study participants. We tested NTG patients and ethnically matched normal control subjects from Japan. NTG patients from New York and North Carolina were also studied. Subjects were tested for duplication of TBK1 using a quantitative PCR assay and microarray analysis of SNPs at chromosome 12q14. Patients were examined by fellowship-trained glaucoma specialists and received complete ophthalmic examinations including gonioscopy, standardized computerized Humphrey (Zeiss, San Leonardo, Ca.) SITA visual field testing, and stereoscopic optic nerve examination. Subjects were diagnosed with open-angle glaucoma when optic nerve damage and corresponding visual field defects were detected in at least one eye as previously described (Alward et al., 1998) and NTG was diagnosed when the maximum untreated IOP was ≤21 mmHg in both eyes. The study dataset consisted of 252 NTG patients and 202 controls from Japan, 29 NTG patients from North Carolina, and 28 NTG patients from New York. DNA samples from each subject were tested for duplication of the TBK1 gene using a quantitative PCR assay (TaqMan Copy Number Assay, Applied BioSystems, Carlsbad, CA) as previously described (Fingert et al., 2011). Briefly, a segment of the TBK1 gene was PCR amplified in triplicate for each DNA sample, as was a control amplicon from a different gene on a different chromosome. Experiments were conducted using a 7900HT PCR machine and data was analyzed to detect CNVs using CopyCaller software (Applied BioSystems, Carlsbad, CA) with default settings. Subjects that exhibited a CNV that spanned TBK1 were retested to confirm its presence. One of 252 (0.40%) unrelated Japanese NTG subjects (patient GGJ-414) was found to carry a TBK1 duplication using quantitative PCR. The duplication in patient GGJ-414 was confirmed using microarray analysis (Affymetrix microarrays, Santa, Clara, CA) as previously described (Fingert et al., 2011). No duplications were detected in the Japanese control subjects or in any of the patients from North Carolina or New York. Further analysis of the genetic data (Partek software package, St. Louis, MO) showed that patient GGJ-414 carried a chromosome 12q14 duplication that extended from 64,802,839 to 65,098,981 bps (human genome build hg19). This 300 kbp duplication encompassed the TBK1 gene as well as XPOT and RASSF3 and is similar in extent to the duplication previously reported in Caucasian NTG subject GGA-1159-1 (Fingert et al., 2011). Patient GGJ-414 (subject II-4 in Figure 1) is a Japanese woman who was diagnosed with NTG at age 42 and has a strong family of disease. Representative disc photos from family members are shown in Figure 2. Other members of this family were tested for the presence of the chromosome 12q14 duplication using the quantitative PCR assay described above. Patient GGJ-414 (Figure 1, subject II-4) has a sister with NTG (Figure 1, subject II-3) and two sons that were diagnosed as NTG suspects, based on large cup-to-disc ratios (Figure 1, subjects III-2 and III-3), also carried the TBK1 duplication. The maximum recorded untreated intraocular pressure for patient GGJ-414 was 18 mm Hg OD and 17 mm Hg OS and central corneal thickness was 521 microns OD and 528 microns OS. One family member (subject II-1, Figure 1) that carried the TBK1 duplication had not been diagnosed with glaucoma when he was last examined at 45 years of age. At this examination, subject II-1 had large cup to disc ratios (0.75 OD and 0.70 OS) and had abnormal glaucoma hemifield test OD. Subject II-1 has not been examined in nine years and lacks further information on glaucoma status. Figure 1 Japanese NTG pedigree Figure 2 Disc Photos Over the last 15 years, family-based linkage studies have identified several genes (i.e. myocilin and optineurin) that are capable of causing POAG with little influence from other genes or environmental factors. These single gene or Mendelian forms of glaucoma represent approximately 5% of POAG cases. The vast majority of glaucoma-causing mutations in myocilin and optineurin are missense and nonsense sequence changes. More recently, we reported that duplication of a segment of chromosome 12q14 that encompasses the TBK1 gene is associated with NTG. Our study first detected a chromosome 12q14 duplication in a large African American pedigree with NTG and later identified two different but overlapping chromosome 12q14 duplications in 2 (1.3%) of 152 unrelated Caucasian NTG patients (Fingert et al., 2011). This is the first confirmation that chromosome 12q14 duplications which encompass the TBK1 gene are associated with NTG. Although it remains possible that other neighboring genes in and around the chromosome 12q14 duplication have a role in the pathogenesis of NTG, there is strong data to suggest that it is duplication of TBK1 that leads to this form of NTG (Fingert et al., 2011). The discovery and confirmation that another gene, TBK1, leads to glaucoma potentially represents a significant advance in glaucoma genetics. TBK1 encodes a kinase that influences gene expression in the NF-κB signaling pathway. Two other NTG genes, optineurin (Rezaie et al., 2002) and toll-like receptor 4 (Zareparsi et al., 2005), also participate in NF-κB signaling. Together these data strongly suggest that this biological pathway has an important role in the pathogenesis of NTG in at least a subset of patients, perhaps via its influence on key cellular processes including apoptosis. Furthermore, duplication of TBK1 is associated with 0.40% to 1.3% of NTG in Caucasians, African Americans (Fingert et al., 2011), and Japanese, suggesting that this defect may have a role in NTG pathogenesis in many ethnicities.

Glaucoma is a group of diseases characterized by progressive excavation of the optic disc caused by loss of the retinal ganglion cell axons. It causes peripheral visual field loss and if untreated can lead to blindness; it is the second leading cause of legal blindness in the United States. Primary open-angle glaucoma (POAG), the most common form in Western populations, is insidious in onset and affects 1% to 2% of the population over age 40. When POAG is observed in individuals under the age of 40 it is called juvenile open-angle glaucoma (JOAG). Increased intraocular pressure is a well-documented risk factor, but not a diagnostic criterion, for POAG.1 More recently, reduced central corneal thickness (CCT) has been recognized as an important risk factor for glaucoma.2 Medical and surgical treatments aimed at reducing intraocular pressure may be effective in preventing progressive visual loss in POAG patients, but treatment is often not implemented until significant, unrecoverable vision loss has occurred due to a lack of symptoms in early disease and delayed diagnosis (see Ref. 3 for a review). Heritability estimates range from 0.36 to 0.57 for features of glaucoma, such as intraocular pressure and optic disc diameter, supporting the assertion that POAG has a strong genetic component.4 Linkage analysis studies in large families segregating POAG in Mendelian fashion have identified 14 loci for the disease5–23 (Table 1). Two causative genes have been identified through fine mapping of such linkage regions including myocilin (MYOC) at 1q23-q245,6 and optineurin (OPTN) at 10p13.11 Together, variants in these genes are estimated to account for approximately 5% of POAG in the population at large.11,24 Table 1. Known Glaucoma Loci Identified through Linkage Analysis In this study, we investigated copy number variants (CNVs) as potential glaucoma-causing variation in individuals with POAG ascertained at the University of Iowa. CNVs are operationally defined as genomic insertions or deletions that are larger than 1 kb and not a result of transposable elements. CNVs often escape detection in traditional studies of genetic variation, such as direct sequencing, single-strand conformation polymorphism analysis, and single-nucleotide polymorphism (SNP) association studies. CNVs are now recognized as a major contributor to human genetic variation and have been associated with other genetically complex disorders including autism, schizophrenia, HIV/AIDS susceptibility, and Crohn's disease.25–34 The eye is a highly specialized organ that has shown significant sensitivity to dosage changes of key developmental and regulatory genes, making glaucoma an excellent phenotype for CNV screening. Select copy number mutations have been known to play a role in glaucoma and related disorders of vision for some time, including deletions of LMXB1, FOXC1, and 4q34, among others.35–37 Gross karyotypic abnormalities of 9p23 and trisomies of chromosome 13 have been associated with developmental glaucoma.38–40 Recent fine mapping of 6p25, the FOXC1 locus, has provided evidence of a spectrum of mechanisms by which deletions and duplications of the FOXC1 gene occur.37,41,42 The initial clue that FOXC1 is involved in glaucoma was based on a chromosomal abnormality found in a single patient, demonstrating the value of identifying rare variants that may identify candidate genes and loci for disease causation. One recent study of 27 glaucoma patients and 12 controls analyzed by array comparative genome hybridization (CGH) methods found no CNVs in either patients or controls.43 However, the ability to detect disease-associated CNVs in this sample was limited due to (1) array CGH probe density (2) small sample size, and (3) single algorithm copy number calls. Before the present study, there have been no large-scale studies of copy number variation (CNV) in glaucoma. The development of software to analyze signal intensity data from high-density SNP-based array platforms, coupled with confirmation by quantitative PCR, enabled us to undertake a detailed cataloguing of CNVs in 400 POAG patients and 500 control subjects.

We report identification of a novel genetic locus (GLC1P) for normal tension glaucoma (NTG) on chromosome 12q14 using linkage studies of an African-American pedigree (maximum non-parametric linkage score = 19.7, max LOD score = 2.7). Subsequent comparative genomic hybridization and quantitative polymerase chain reaction (PCR) experiments identified a 780 kbp duplication within the GLC1P locus that is co-inherited with NTG in the pedigree. Real-time PCR studies showed that the genes within this duplication [TBK1 (TANK-binding kinase 1), XPOT, RASSF3 and GNS] are all expressed in the human retina. Cohorts of 478 glaucoma patients (including 152 NTG patients), 100 normal control subjects and 400 age-related macular degeneration patients were subsequently tested for copy number variation in GLC1P. Overlapping duplications were detected in 2 (1.3%) of the 152 NTG subjects, one of which had a strong family history of glaucoma. These duplications defined a 300 kbp critical region of GLC1P that spans two genes (TBK1 and XPOT). Microarray expression experiments and northern blot analysis using RNA obtained from human skin fibroblast cells showed that duplication of chromosome 12q14 results in increased TBK1 and GNS transcription. Finally, immunohistochemistry studies showed that TBK1 is expressed in the ganglion cells, nerve fiber layer and microvasculature of the human retina. Together, these data link the duplication of genes on chromosome 12q14 with familial NTG and suggest that an extra copy of the encompassed TBK1 gene is likely responsible for these cases of glaucoma. However, animal studies will be necessary to rule out a role for the other duplicated or neighboring genes.

Purpose To determine the role of the recently discovered primary open angle glaucoma (POAG) risk factor mapped to chromosome 7q31 in glaucoma patients from Iowa and to determine the expression pattern of genes in the locus in human eyes. Methods A cohort of 545 POAG patients and 297 control subjects from Iowa were genotyped with a single nucleotide polymorphism (SNP; rs4236601) in the chromosome 7q31 locus using a quantitative polymerase chain reaction (PCR) assay. The expression of genes within the 7q31 locus, caveolin-1 (CAV1) and caveolin-2 (CAV2) in human eyes was investigated with immunohistochemistry. Results The minor allele frequency (MAF) of rs4236601 was 27% in control subjects and 29% in POAG patients. We detected no statistical difference when we compared the allele frequencies of rs4236601 between POAG patients and control subjects (p=0.5). Similarly, we detected no statistical difference in the frequency of the three possible rs4236601 genotypes between patients and controls (p=0.22). Immunohistochemistry showed caveolin expression in human retina, ciliary muscle, trabecular meshwork, and Schlemm’s canal. In our small cohort of donor eyes, the genotype of rs4236601 did not obviously influence labeling intensity or distribution of CAV1 and CAV2 in the retina. Conclusions A genome-wide association study of subjects from Iceland mapped the first common genetic risk factor for POAG to a small region of the genome on chromosome 7q31 that contains the caveolin genes CAV1 and CAV2. We were unable to detect this association in our patients from Iowa, suggesting that this risk factor may not have a strong effect in all populations.

PURPOSE. Discovery and description of heritable optic nerve head (ONH) phenotypes have been haphazard. In this preliminary study, the authors test the hypothesis that inheritable phenotypes can be discovered and quantified computationally by estimating three-dimensional ONH shape parameters from stereo color photographs from the Twins Eye Study in Tasmania and determining how much of the variability in ONH shape is accounted for by genetic influence. METHODS. Three-dimensional ONH shape was estimated by an automated algorithm from stereoscopic optic disc color photographs of a random sample of 172 subjects (344 eyes, 45 pairs of monozygotic [MZ] and 41 dizygotic [DZ] twins). Shape resemblances between eyes were quantified with a distance metric. The heritability of the shape resemblance was determined both through the distribution of the discongruence indices and through structural equation modeling techniques (ACE model). RESULTS. Significantly different discongruence indices were found for MZ (1.0286; 95% CI, 0.9872‐1.0701) and DZ twins (1.4218; 95% CI, 1.2631‐1.5804); larger indices for DZ twins indicated that variability was substantially determined by genetic factors. The standardized variances of the A(dditive genetic), C(ommon environmental), and (nonshared) E(nvironmental) components were 0.80, 2.00 10 15 and 0.20, respectively, for all OD, and 0.79, 3.24 10 14 , and 0.21 for all OS. CONCLUSIONS. This preliminary study shows that quantitative phenotyping of the ONH shape from color images leads to phenotypes that can be measured and are largely under genetic control. The association of these inherited phenotypes with genotypes deserves confirmation and further study. (Invest Ophthalmol Vis Sci. 2010;51:5870‐5877) DOI:10.1167/iovs.105527

The complement system plays a crucial role in the progression of age-related macular degeneration (AMD). In this study, the authors sought to evaluate the pathophysiologic roles of complement components C3a and C5a in the human choroid in AMD.Human RPE/choroid was assayed for the presence of C3a and C5a receptors (C3aR and C5aR) using RT-PCR and immunohistochemistry. Choroidal endothelial cell migration and proliferation were evaluated in the presence of C5a. Organ cultures of human choroid were incubated in C5a or bovine serum albumin (BSA) followed by quantitative immunohistochemistry and quantitative PCR for ICAM-1. AMD patients and controls were genotyped at SNPs in the C5R1 and C3AR1 genes.C5aR, but not C3aR, was detected in human choroid. C5a did not promote endothelial cell migration or proliferation. However, choriocapillaris endothelial cells in organ culture responded to C5a by increasing ICAM-1 mRNA and protein. No significant association of SNP genotypes was detected in AMD patients at the C3AR1 and C5R1 genes.The generation of C5a peptides may lead to activation of choriocapillaris endothelial cells in AMD. Activation of the choroidal endothelium may affect the progression of AMD by recruitment of monocytes, leading to additional sequelae of AMD pathogenesis.

Background Age-related macular degeneration (AMD) is a common disease of the elderly that leads to loss of the central visual field due to atrophic or neovascular events. Evidence from human eyes and animal models suggests an important role for macrophages and endothelial cell activation in the pathogenesis of AMD. We sought to determine whether common ancestral variants in genes encoding the selectin family of proteins are associated with AMD. Methods Expression of E-selectin, L-selectin and P-selectin was examined in choroid and retina by quantitative PCR and immunofluorescence. Samples from patients with AMD (n = 341) and controls (n = 400) were genotyped at a total of 34 SNPs in the SELE, SELL and SELP genes. Allele and genotype frequencies at these SNPs were compared between AMD patients and controls as well as between subtypes of AMD (dry, geographic atrophy, and wet) and controls. Results High expression of all three selectin genes was observed in the choroid as compared to the retina. Some selectin labeling of retinal microglia, drusen cores and the choroidal vasculature was observed. In the genetic screen of AMD versus controls, no positive associations were observed for SELE or SELL. One SNP in SELP (rs3917751) produced p-values < 0.05 (uncorrected for multiple measures). In the subtype analyses, 6 SNPs (one in SELE, two in SELL, and three in SELP) produced p-values < 0.05. However, when adjusted for multiple measures with a Bonferroni correction, only one SNP in SELP (rs3917751) produced a statistically significant p-value (p = 0.0029). Conclusions This genetic screen did not detect any SNPs that were highly associated with AMD affection status overall. However, subtype analysis showed that a single SNP located within an intron of SELP (rs3917751) is statistically associated with dry AMD in our cohort. Future studies with additional cohorts and functional assays will clarify the biological significance of this discovery. Based on our findings, it is unlikely that common ancestral variants in the other selectin genes (SELE and SELL) are risk factors for AMD. Finally, it remains possible that sporadic or rare mutations in SELE, SELL, or SELP have a role in the pathogenesis of AMD.

AbstrA ct Background: Three mitochondrial mutations account for 95% of Leber’s hereditary optic neuropathy (LHON) in the European population: G3640A, G11778A and T14484C. The purpose of the study was to investigate the frequency of these mitochondrial DNA mutations in LHON patients from a South Indian population. Methods: LHON was diagnosed by inheritance pattern, ophthalmologic examination, and by exclusion of non-LHON forms of optic neuropathy. Ninety unrelated LHON patients and 20 at-risk family members (5 with LHON and 15 without LHON) underwent molecular screening for the mitochondrial DNA mutations G3640A, G11778A and T14484C by amplification refractory mutation system (ARMS) polymerase chain reaction (PCR). Positive results were confirmed with bi-directional sequencing. Results: The G11778A mutation was detected in 8 of 90 (8.9%) LHON families. The T14484 mutation was detected in 3 of 90 (3.3%) LHON families. No instances of the G3460A mutation were detected. Other variants were incidentally detected by the DNA sequencing assay. Conclusions: Three mitochondrial mutations (G3640A, G11778A and T14484C) account for the vast majority of LHON cases in Europe. However, these mutations were detected in only 11 (12%) of 90 LHON families from Southern India in our study. These results suggest that a different set of LHON-causing mutations is present in the South Indian population than in the European population. Further study of subjects with LHON from India may lead to the discovery of novel diseasecausing mutations and/or genes.

Age-related macular degeneration (AMD) is a complex genetic disease, with many loci demonstrating appreciable attributable disease risk. Despite significant progress toward understanding the genetic and environmental etiology of AMD, identification of additional risk factors is necessary to fully appreciate and treat AMD pathology. In this study, we investigated copy number variants (CNVs) as potential AMD risk variants in a cohort of 400 AMD patients and 500 AMD-free controls ascertained at the University of Iowa. We used three publicly available copy number programs to analyze signal intensity data from Affymetrix® GeneChip SNP Microarrays. CNVs were ranked based on prevalence in the disease cohort and absence from the control group; high interest CNVs were subsequently confirmed by qPCR. While we did not observe a single-locus “risk CNV” that could account for a major fraction of AMD, we identified several rare and overlapping CNVs containing or flanking compelling candidate genes such as NPHP1 and EFEMP1. These and other candidate genes highlighted by this study deserve further scrutiny as sources of genetic risk for AMD.

Objective: To identify the disease-causing mutation in a large 3 generation pedigree of X-linked congenital nystagmus. Methods: Twenty-three members of a single pedigree, including 7 affected males, 2 affected females, 5 obligate carriers, and 9 unaffected family members were tested for mutations in the FRMD7 gene using PCR-based DNA sequencing assays and multiplex PCR assays for deletions. Results: A hemizygous deletion of exons 2, 3, and 4 of FRMD7 was detected in all affected males in the family and was absent from 40 control subjects. Conclusions: A range of missense, nonsense, frameshift, and splicing mutations in FRMD7 have been shown to cause X-linked congenital nystagmus. Here we show for the first time that large intragenic deletions of FRMD7 can also cause this form of nystagmus.

Purpose While chronic glucocorticoid (GC) therapy leads to ocular hypertension in about one third of individuals, almost all primary open-angle glaucoma (POAG) patients show this response and are called “steroid responders.” Two differentially spliced isoforms of the glucocorticoid receptor (GR), GRα and GRβ, regulate GC responsiveness in trabecular meshwork (TM) cells. GRβ acts as a dominant negative regulator of GC activity and is expressed at lower levels in glaucomatous TM cells, making them more sensitive to GCs. Several arginine/serine-rich splicing factor (SR) proteins have been implicated in alternative splicing of the GR. We have previously demonstrated that immunophilins FKBP5 and FKBP4 are required for GRα and GRβ translocation into the nucleus, which is essential for their biologic activity. The purpose of the present study was to use single nucleotide polymorphism (SNP) genotyping to determine whether there are any allele frequency differences in GR, FKBP4/5, or SR genes between normal control, POAG, and steroid responder populations. Methods Clinically characterized individuals (400 normal controls, 197 POAG, and 107 steroid responders) were recruited from the U. Iowa Ophthalmology Clinics after IRB approved consent. Genotyping of DNA samples for 48 SNPs in SFRS3, SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 was done at GeneSeek using a mass spectroscopy based system. Results All 48 SNPs displayed high call rates (99%). There were no significant differences in allele frequencies or genotypes in SNPs for SFRS5, SFRS9, FKBP4, FKBP5, and NR3C1 between the 3 groups. Up to three SNPs in SFRS3 had p-values

Glaucoma is one of the leading causes of blindness worldwide. This review discusses the clinical features, genetics, molecular biology, and cell biology of glaucoma. The authors present a typical case of glaucoma, together with the ocular findings.

Exfoliation syndrome (XFS) is a common age-related disorder primarily recognized by the pathologic accumulations of a fibrillar exfoliative material in the anterior chamber of the eye but also associated with several other ocular and systemic abnormalities.1–6 In many patients, accumulation of exfoliative material within the iridocorneal angle is accompanied by elevated intraocular pressure (IOP) and glaucoma. Indeed, XFS is the most commonly identified cause of secondary open-angle glaucoma.7 In parallel to the clinical significance of exfoliative material in the diagnosis of XFS, much of the experimental work on XFS syndrome has focused on studies of exfoliative material. Such studies have shown that exfoliative material consists of an irregular conglomeration of randomly cross-banded fibrils approximately 30 nm in diameter surrounded by an amorphous matrix of glycoconjugates.8 Exfoliative material also contains epitopes for a variety of proteins related to elastic microfibers, including fibrillin-1,9 elastin,10 latent TGF-β proteins,11 lysyl oxidase,4 and others.3,4 These results and other experimental work on XFS have led to a hypothesis that XFS is a disease of elastosis. According to this theory, insults such as increased oxidative stress and elevated levels of TGF-β1 likely trigger increased production of elastic microfibrils that are subsequently prone to aggregate and accumulate.4 After aggregation and accumulation of exfoliative material within the anterior chamber, aqueous humor outflow becomes impeded, ultimately resulting in increased IOP and glaucoma. A breakthrough in understanding XFS has been precipitated by genomewide association studies that have begun to unravel the genetic factors underlying XFS. XFS has long been appreciated to have strong hereditary contributions.12,13 Recently, the lysyl oxidase-like protein 1 gene (LOXL1) has been identified as the first known genetic risk factor contributing to XFS.14 Initially identified from a large genomewide association study among Scandinavian patients with glaucoma14 and subsequently replicated in additional populations,15–24 a strong association exists between XFS and two single nucleotide polymorphism (SNP) genetic markers resulting in nonsynonymous changes (rs1048661, R141L; rs3825942, G153D) in LOXL1. These LOXL1 SNPs are highly associated with XFS, and the high-risk alleles of these SNPs occur within most XFS patients. The high-risk haplotype of LOXL1 alleles has a 99% population attributable risk in Caucasian populations.14 However, the influence of LOXL1 in XFS may not be as straightforward as is seemingly indicated by these impressive statistics. A multifactorial risk for XFS is suggested by the extremely high occurrence of high-risk LOXL1 alleles among the general population. Within the original Scandinavian populations studied, the high-risk haplotype of LOXL1 alleles was also detected at a frequency of approximately 50% in the general population, with approximately 25% of the general population homozygous for the haplotype.14 Follow-up studies have also confirmed similar high-carrier frequencies.15–24 Thus, most people with high-risk LOXL1 alleles likely do not have XFS. This indicates that although LOXL1 is an important risk factor for XFS, additional factors must play a role in the pathogenesis of the condition. Here, we identify the Lyst gene as an additional gene potentially important to XFS. B6-Lystbg-J mice homozygous for the beige-J (bg-J) allele recapitulate multiple ocular features of human XFS. Our initial consideration of B6-Lystbg-J mice as a possible model of XFS was based on a resemblance of iris transillumination defects between these mice and human patients with XFS. In testing the anatomic basis for the Lyst-mediated transillumination defects, we found that the transillumination defects were caused by an unusual sawtooth-like morphology of the iris pigment epithelium and were accompanied by the presence of a material resembling human exfoliative material. Interestingly, the molecular basis of the beige mutation was discovered to be a 3-bp deletion. The mutation is predicted to delete a single isoleucine from the carboxyl terminus of the LYST protein within the WD40 domain, potentially disrupting a protein-protein interaction. This work represents one of the first descriptions of a mouse model potentially relevant to XFS and provides a genetic resource for continued study of this novel molecular pathway contributing to XFS.

Objective To identify the gene causing retinitis pigmentosa (RP) in an autosomal dominant pedigree. Methods Family members with RP were studied with linkage analysis using single-nucleotide polymorphism and short tandem repeat polymorphic markers. Candidate genes in the linked region were evaluated with DNA sequencing. Results Nineteen family members had a mild form of RP. Multipoint linkage analysis of single-nucleotide polymorphism genotypes yielded a maximum nonparametric linkage score of 19.97 with markers located on chromosome 14q. LOD scores higher than 3.0 were obtained with 20 short tandem repeat polymorphic markers, and recombinants defined a 21.7-centimorgan locus on chromosome 14q. The retinol dehydrogenase 12 ( RDH12 ) gene lies within this locus and was evaluated as a candidate gene. A frameshift mutation (776delG) was detected in all affected family members and was not detected in 158 control subjects. Conclusions Heterozygous mutations in RDH12 can cause autosomal dominant RP with a late onset and relatively mild severity. This phenotype is dramatically different from the other disease associated with mutation in this gene, autosomal recessive Leber congenital amaurosis. Clinical Relevance The demonstration that mutations in a gene previously associated with recessive Leber congenital amaurosis can also cause dominant RP illustrates the wide phenotypic variability of retinal degeneration genes.

Purpose To test patients from southern India for the presence of mutations that most commonly cause Leber congenital amaurosis (LCA) in northern America. Methods A review of the literature identified 177 unique LCA causing mutations in eight different genes: aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), crumbs homolog 1 (CRB1), cone-rod homeobox (CRX), guanylate cyclase 2D (GUCY2D), nephronophthisis 6 (NPHP6), retinol dehydrogenase 12 (RDH12), retinal pigment epithelium-specific protein 65 kDa (RPE65), and retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1). Allele-specific ligation assay and bidirectional sequencing were used to test 38 unrelated LCA patients from southern India for 104 of these mutations, which contribute to more than 30% of the LCA cases in a northern American population. Results Only one participant was found to harbor one of the 104 mutations in the allele-specific assay (homozygous RPE65 Tyr368His). A mutation that was not part of the assay (homozygous RPE65 Tyr143Asp) was incidentally detected in a second patient when an equivocal signal from one allele on the assay was followed up with automated DNA sequencing. Conclusions Mutations that contribute to 30% of the LCA cases in northern America were detected in only 2.6% of LCA cases in our cohort from southern India. There were no instances of IVS26 c.2991+1655 A>G in NPHP6, the most commonly detected mutation in LCA. These data suggest that LCA in India is caused primarily by a different set of mutations in the same genes associated with disease in northern America, or by mutations in other genes that have not yet been discovered. Therefore, mutation-specific assays developed for European and northern American cohorts may not be suited for testing LCA patients from India or other ethnically distinct populations.

Leber Hereditary Optic Neuropathy (LHON) is a maternally inherited blinding disease caused by missense mutations in the mitochondrial DNA (mtDNA). However, incomplete penetrance and a predominance of male patients presenting with vision loss suggest that modifying factors play an important role in the development of the disease. Evidence from several studies suggests that both nuclear modifier genes and environmental factors may be necessary to trigger the optic neuropathy in individuals harboring an LHON-causing mtDNA mutation. Recently, an optic neuropathy susceptibility locus at Xp21-Xq21 has been reported. In this study, we performed X-chromosomal linkage analysis in a large Brazilian family harboring a homoplasmic G11778A mtDNA mutation on a haplogroup J background. We report the identification of a novel LHON susceptibility locus on chromosome Xq25-27.2, with multipoint non-parametric linkage scores of5.00 (P = 0.005) and a maximum two-point non-parametric linkage score of 10.12, (P = 0.003) for marker DXS984 (Xq27.1). These results suggest genetic heterogeneity for X-linked modifiers of LHON.

Primary objective was to investigate clinical characteristics of nonarteritic anterior ischemic optic neuropathy (NA-AION) in three families; secondarily, to test these families for a previously detected mitochondrial mutation in a pedigree with familial NA-AION. Study comprised three families where more than one member developed NA-AION. All patients with NA-AION had a detailed ophthalmic, medical and family history, and comprehensive ophthalmic evaluation at initial visit and on follow-up. One patient from family 1, one from family 2, 41 non-familial NA-AION patients, 97 control subjects and 1,488 patients with suspected Leber hereditary optic neuropathy (LHON) were tested for the presence of mitochondrial mutation (G4132A) in a previously reported genetic study of family 3. Familial NA-AION was found in seven individuals of family 1, four of family 2 and six of family 3. Symptoms, signs and clinical findings of familial NA-AION were similar to classical NA-AION, with two exceptions: familial NA-AION had an earlier onset (47.3 + 8.6 years versus 60.1 + 13.6 years) and a higher frequency of bilateral disease. The G4132A mitochondrial variant was not detected outside family 3. None of the three major mutations associated with LHON (G3460A, G11778A, T14484C) was found among Familial NA-AION patients. The only difference in clinical features between familial NA-AION and classical NA-AION is that the former has an earlier onset and a higher frequency of bilateral disease. The G4132A mutation is not commonly associated with familial NA-AION, and was not detected in patients with non-familial NA-AION. The role of hereditary factors in familial NA-AION remains largely unknown.

Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of beta-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.

PURPOSE. To search for and validate potential molecular pathogenic mechanisms in the trabecular meshwork (TM) responsible for the elevated intraocular pressure (IOP) associated with glaucoma. METHODS. Gene chip arrays were used to identify differential gene expression in glaucomatous TM tissues. Serum amyloid A (SAA) upregulation was subsequently confirmed with quantitative PCR (QPCR) and ELISA. The effect of SAA on gene expression of cultured human TM cells was tested with gene chip arrays and verified with ELISA, and its effect on IOP was evaluated in the human ocular perfusion organ culture. RESULTS. Microarray analysis showed that the expression of SAA2 was increased in TM tissues from donors with glaucoma. This finding was subsequently confirmed by QPCR. The SAA mRNA levels were increased in glaucoma TM tissues by more than 5-fold (P 0.05) and in cultured TM cells derived from donors with glaucoma by 25-fold (P 0.05) compared with controls. SAA protein levels in the TM of glaucoma patients were also significantly (P 0.05) elevated by 2.9-fold. Treatment of cultured human TM cells with recombinant SAA affected gene expression, including a 22-fold up-regulation of interleukin-8 (P 0.001). SAA increased IOP by approximately 40% (P 0.05) in the human ocular perfusion organ culture without any observable changes in the morphology of the tissues involved in aqueous outflow. CONCLUSIONS. These findings indicate that SAA, which is an acute-phase apolipoprotein that plays important roles in infection, inflammation, and tissue repair, may contribute to the pathogenic changes to the TM in glaucoma. (Invest Ophthalmol Vis Sci. 2008;49:1916‐1923) DOI:10.1167/iovs.07-1104

PURPOSE. Numerous genetic diseases and environmental stimuli affect optic nerve morphology. The purpose of this study was to identify the principal heritable components of visible optic nerve head structures in a population-based sample of twins. METHODS. Fifteen optic nerve specialists viewed stereoscopic optic nerve head photographs (Stereo Viewer-II; Pentax Corp., Tokyo, Japan) from 50 randomly selected monozygotic or dizygotic twin pairs. Before viewing, each expert was questioned about which optic nerve head traits they believed were inherited. After viewing a standardized teaching set, the experts indicated which twin pairs they thought were monozygotic. Participants were then questioned about how their decisions were reached. A rank-ordered Rasch analysis was used to determine the relative weighting and value applied to specific optic nerve head traits. RESULTS. The proportion of twin pairs for which zygosity was correctly identified ranged from 74% to 90% (median, 82%) across the panel. Experts who correctly identified the zygosity in more than 85% of cases placed most weighting on shape and size of the optic disc and cup, whereas experts with the lowest scores placed greater weighting on the optic nerve head vasculature in reaching their decisions. CONCLUSIONS. In determining the genetic components of the optic nerve head, the results of this study suggest that the shape and size of the optic disc and cup are more heritable and should receive a greater priority for quantification than should vascular features. (Invest Ophthalmol Vis Sci. 2007;48: 2469‐2475) DOI:10.1167/iovs.06-1470

To evaluate a novel automated segmentation algorithm for cup-to-disc segmentation from stereo color photographs of patients with glaucoma for the measurement of glaucoma progression.Stereo color photographs of the optic disc were obtained by using a fixed stereo-base fundus camera in 58 eyes of 58 patients with suspected or open-angle glaucoma. Manual planimetry was performed by three glaucoma faculty members to delineate a reference standard rim and cup segmentation of all stereo pairs and by three glaucoma fellows as well. Pixel feature classification was evaluated on the stereo pairs and corresponding reference standard, by using feature computation based on simulation of photoreceptor color opponency and visual cortex simple and complex cells. An optimal subset of 12 features was used to segment all pixels in all stereo pairs, and the percentage of pixels assigned the correct class and linear cup-to-disc ratio (LCDR) estimates of the glaucoma fellows and the algorithm were compared to the reference standard.The algorithm was able to assign cup, rim, and background correctly to 88% of all pixels. Correlations of the LCDR estimates of glaucoma fellows with those of the reference standard were 0.73 (95% CI, 0.58-0.83), 0.81 (95% CI, 0.70-0.89), and 0.86 (95% CI, 0.78-0.91), respectively, whereas the correlation of the algorithm with the reference standard was 0.93 (95% CI, 0.89-0.96; n = 58).The pixel feature classification algorithm allows objective segmentation of the optic disc from conventional color stereo photographs automatically without human input. The performance of the disc segmentation and LCDR calculation of the algorithm was comparable to that of glaucoma fellows in training and is promising for objective evaluation of optic disc cupping.

To determine the phenotype of an Australian pedigree with the myocilin (MYOC) Gly252Arg mutation, comparing it with other pedigrees carrying the same mutation.All recruited subjects underwent a comprehensive clinical examination, including optic disc assessment, applanation tonometry, and visual field measurement. Mutation analysis was performed through direct sequencing. Haplotype analysis was performed using microsatellite markers around the MYOC gene.Eight Gly252Arg mutation carriers with glaucoma were identified from the same pedigree. Carriers' mean +/- SD age at diagnosis was 46.3 +/- 11.4 years (range, 31-60 years). Highest recorded intraocular pressure ranged from 27 to 42 mm Hg (mean +/- SD, 32.4 +/- 5.6 mm Hg). Cup-disc ratios in the worst eye ranged from 0.6 to 0.9. Six of the 8 individuals had undergone filtration surgery. A common founding haplotype between MY5 and D1S218 was found for Caucasian individuals tested with this mutation. One subject was compound heterozygotic for the MYOC Gly252Arg mutation and a novel MYOC Gly244Val variant.Although a common founder for Gly252Arg across Caucasian subjects was found, the phenotype from this Australian MYOC mutation-carrying pedigree is less severe than previously described. The severity of glaucoma caused by the Gly252Arg mutation may be similar to the Thr377Met MYOC mutation, yet is more severe than the most common Gln368Stop mutation.Since its implication in glaucoma, much work has been performed investigating the clinical features of MYOC-related glaucoma. Given the strong genotype-phenotype correlations with MYOC disease-causing variants, health care professionals armed with such molecular information are able to accurately counsel patients on their likely disease course. Our work suggests that the disease associated with MYOC Gly252Arg is less severe than previously described in other pedigrees with this specific mutation.

Corrigendum: A common variant mapping to CACNA1A is associated with susceptibility to exfoliation syndrome

Purpose To describe a multigenerational family with autosomal dominant inheritance of cavitary optic nerve head (ONH) anomalies and abnormal ONH vasculature. Design Description of a single family with inherited eye disease. Methods A four-generation pedigree was investigated. Examination included visual acuity, slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, and ophthalmoscopy. Visual fields and fundus photography were obtained when possible. Results Seventeen clinically affected individuals and two obligate carriers were identified. Most (64.7%) affected persons had bilateral involvement. Visual acuity in affected eyes ranged from 20/20 to no light perception. Although the appearance of affected nerves varied greatly, most lacked a well-formed central retinal artery and instead had multiple radial cilioretinal arteries. Prominent cupping was seen in most affected nerves. Four individuals for whom information was available were treated for glaucoma, but none had documented elevated IOP. Four eyes of two patients demonstrated progressive ONH cupping at normal IOPs. Nine (56.3%) of the 16 individuals for whom we had data had evidence of serous macular detachments; five of these had bilateral macular disease. Conclusions A large family with autosomal dominant inheritance of cavitary ONH anomalies and abnormal vasculature is presented. Clinical phenotypes varied markedly. Progressive ONH cupping was documented in four eyes of two patients. Genetic linkage analysis of this family has identified the chromosomal location of a gene responsible for ONH development. This may provide insight into the pathogenesis of glaucomatous ONH damage.

Genetic factors have long been implicated in the pathophysiology of primary open-angle glaucoma (POAG). Recently, myocilin, a gene of unknown function, was associated with both juvenile open-angle glaucoma (JOAG) and POAG. Forty-three different myocilin mutations have been reported in open-angle glaucoma patients, and several large studies have suggested that as a group these mutations are associated with 3–4% of POAG in patient populations worldwide. Support for the pathogenicity of the individual myocilin mutations has been obtained from in vitro assays, statistical methods, and conservation of gene sequence arguments. Several of these myocilin mutations were observed in multiple patients allowing the identification of mutation-specific glaucoma phenotypes (maximum intraocular pressure and age at diagnosis). Associations between myocilin and other forms of open-angle glaucoma have been explored. At present there is no evidence to link myocilin mutations and steroid-induced ocular hypertension or normal-tension glaucoma. Clinical vignettes of POAG patients from four generations of a family harboring the TYR437HIS myocilin mutation are presented, highlighting the benefits of elucidating the genetics of glaucoma.

Objective To determine the prevalence and associated phenotype of myocilin ( MYOC ) coding sequence variations and a specific promoter polymorphism(MYOC.mt1) in patients with glaucoma and glaucoma suspects. Methods A consecutive, unselected series of 779 patients (652 with open-angle glaucoma and 127 glaucoma suspects) were recruited from a university medical center and clinically characterized. The coding sequences of the MYOC gene and the MYOC.mt1 locus in the promoter region were screened for sequence variations. We determined the prevalence of MYOC coding sequence mutations and the MYOC.mt1 promoter polymorphism. We also compared the clinical features of individuals with and without mutations and the MYOC.mt1 promoter polymorphism. Results Plausible disease-causing sequence variations (DCVs) in the MYOC gene were found in 3.0% of the entire group. Such variations were found in patients with most forms of open-angle glaucoma studied. Patients with primary open-angle glaucoma (POAG) who harbored coding sequence DCVs were clinically similar to patients without them. Patients who harbored the rarer allele of the MYOC.mt1 promoter polymorphism were no different in any measure of disease severity from those who harbored the more common allele. Conclusions MYOC DCVs were found in approximately 3% of patients with glaucoma and glaucoma suspects. The 2 alleles of the MYOC.mt1 promoter polymorphism were equally distributed among patients with POAG and healthy control subjects. Patients with POAG who harbored the rarer allele of the MYOC.mt1 promoter polymorphism were no different from those with the more common variant in any measure of disease severity. Clinical Relevance Testing for the MYOC.mt1 promoter polymorphism appears to be of no value in the evaluation of patients with glaucoma.

Importance: Normal-tension glaucoma (NTG) is a common cause of vision loss. Objective: To investigate the role of TANK binding kinase 1 (TBK1) gene duplications in NTG to gain insights into the causes of glaucoma that occurs at low intraocular pressure (IOP). Design, Setting, and Participants: In this multicenter case-control study, we investigated patients who met the criteria for NTG, including glaucomatous optic neuropathy, visual field defects, and maximum recorded untreated IOP of 21 mm Hg or less, and matched controls. Participants (N?=?755) were recruited from Southampton, United Kingdom (180 patients and 178 controls), Rochester, Minnesota (65 patients and 12 controls), New York, New York (96 patients and 16 controls), and Iowa City, Iowa (208 controls). Main Outcomes and Measures: Detection of TBK1 gene duplications and comparison of the extent of the identified DNA that is duplicated with prior TBK1 copy number variations associated with NTG. Results: A TBK1 gene duplication was detected in 1 of 96 patients (1.0%) from New York and none of the controls. Analysis of duplication borders with comparative genome hybridization demonstrated that this patient has a novel duplication that has not been previously reported. No gene duplications were detected in any of the other cohorts of patients or controls. Conclusions and Relevance: Duplication of the TBK1 gene is a rare cause of NTG. The identification of another case of NTG attributed to TBK1 gene duplication strengthens the case that this mutation causes glaucoma.

SPECIFIC AIMSGlaucoma is a leading cause of blindness in the world, and the discovery of the glaucoma gene myocilin (MYOC) prompted numerous studies of its role in the molecular pathogenesis of gla...

Background: The myocilin gene is the first glaucoma gene to be associated with primary open-angle glaucoma (POAG). The hereditary subset of POAG and the role of the myocilin gene in our population are not clearly defined. Identification of cases of hereditary glaucoma and a better appreciation of the role of the myocilin gene may allow earlier diagnosis of the disease and optimize management of those at risk for glaucoma. Methods: Patients were recruited from university glaucoma practices in the Greater Toronto area from 1996 to 1998. Pedigree analysis and DNA banking were performed for each participant. Mutational analysis of the myocilin gene by means of single-strand conformation polymorphism analysis and direct sequencing was completed for 140 probands with POAG of diverse ethnic background. Results: A total of 103 patients (55.7%) had a family history of glaucoma. Diseasecausing mutations of the myocilin gene were observed in 7 (5.0%) of the 140 probands, which accounted for 6.5% (5/77) of the familial cases. Most mutations were associated with familial disease, which implies a 50% risk of transmission of a high-risk factor for glaucoma. Interpretation: The hereditary subset of POAG is significant, and heritable glaucoma should always be suspected. In spite of the diversity of the ethnic background of our subjects, the observed prevalence of myocilin gene mutations was comparable to that previously reported, and such mutations do not appear to spare any ethnic group.

In 1997, Stone and colleagues discovered that a gene (GLC1A) on the long arm of chromosome 1 caused a large percentage of autosomal dominant juvenile-onset primary open-angle glaucoma (POAG). GLC1A encodes a 57-kDa protein, known as myocilin, which is expressed in the trabecular meshwork, ciliary body, retina, and 17 of 23 other organs throughout the body (Fingert et al. 1998). In the trabecular meshwork, myocilin production can be induced by corticosteroids, leading some to call it trabecular-meshwork inducible glucocorticoid response (TIGR) protein (Polansky et al. 1997).

(1999). Normal range of hearing associated with myocilin Thr377Met. Ophthalmic Genetics: Vol. 20, No. 3, pp. 205-207.

A substantial proportion of cases of glaucoma have a genetic basis. Mutations causing glaucoma have been identified in the chromosome 1 open-angle glaucoma gene (GLC1A), which encodes a 57-kd protein known as myocilin. The normal role of this protein and the mechanism by which mutations cause glaucoma are not known.We screened 716 patients with primary open-angle glaucoma and 596 control subjects for sequence changes in the GLC1A gene.We identified 16 sequence variations that met the criteria for a probable disease-causing mutation because they altered the predicted amino acid sequence and they were found in one or more patients with glaucoma, in less than 1 percent of the control subjects. These 16 mutations were found in 33 patients (4.6 percent). Six of the mutations were found in more than 1 subject (total, 99). Clinical features associated with these six mutations included an age at diagnosis ranging from 8 to 77 years and maximal recorded intraocular pressures ranging from 12 to 77 mm Hg.A variety of mutations in the GLC1A gene are associated with glaucoma. The spectrum of disease can range from juvenile glaucoma to typical late-onset primary open-angle glaucoma.

The GLC1A gene (which encodes the protein myocilin) has been associated with the development of primary open angle glaucoma. Bacterial artificial chromosomes containing the human GLC1Agene and its mouse ortholog were subcloned and sequenced to reveal the genomic structure of the genes. Comparison of the coding sequences of the human and mouse GLC1A genes revealed a high degree of amino acid homology (82%) and the presence of several conserved motifs in the predicted GLC1A proteins. The expression of GLC1A was examined by Northern blot analysis of RNA from adult human tissues.GLC1A expression was observed in 17 of 23 tissues tested, suggesting a wider range of expression than was recognized previously. The comparison of the human and mouse GLC1A genes suggests that the mouse may be a useful model organism in studying the molecular pathophysiology of glaucoma.[The sequence data described in this paper have been submitted to the GenBank data library under accession nos. AF049791–AF049796.]

Objectives: To refine the dominant optic atrophy locus, OPA1 , on chromosome 3q and to characterize the phenotype of a 6-generation family pedigree affected with this disease. Methods: Fifty-six family members had a complete eye examination. Clinical records of an additional 3 patients were reviewed. Goldmann perimetry and a 21-chip subtest of the Farnsworth-Munsell 100-Hue test were performed on selected patients. Affected patients, unaffected siblings, and potentially informative spouses were genotyped with short tandem repeat polymorphisms located on chromosome 3. The genotypic data were subjected to linkage analysis. Results: Thirty-four family members were found to be clinically affected. Most experienced vision loss (20/40 or poorer) in the first decade of life. Most (9 of the 16 eyes) progressed to 20/800 or poorer visual acuity by age 60 years, while 2 patients maintained visual acuities of 20/40 at that age. Affected patients had a 2- to 10-fold increase in the error score of a 21-chip subtest of the Farnsworth-Munsell 100-Hue test compared with age-matched unaffected family members. The optic nerve examination revealed temporal pallor and excavation in all affected individuals. Linkage analysis revealed significant lod scores with 9 markers. The highest lod score, 10.1 (u=0), was obtained with marker D3S2305. Analysis of recombinants narrowed the disease interval to approximately 3.8 centimorgans, flanked by D3S3669 (centromeric) and D3S1305 (telomeric). Conclusions: Most patients affected with dominant optic atrophy in this family progressed to legal blindness by middle age. Color vision testing is a sensitive method for detection of affected patients. The dominant optic atrophy locus, OPA1 , has been refined by the identification of new flanking markers: D3S3669 (centromeric) and D3S1305 (telomeric).

Autosomal dominant neovascular inflammatory vitreoretinopathy (ADNIV) is an autoimmune condition of the eye that sequentially mimics uveitis, retinitis pigmentosa, and proliferative diabetic retinopathy as it progresses to complete blindness. We identified two different missense mutations in the CAPN5 gene in three ADNIV kindreds. CAPN5 encodes calpain-5, a calcium-activated cysteine protease that is expressed in retinal photoreceptor cells. Both mutations cause mislocalization from the cell membrane to the cytosol, and structural modeling reveals that both mutations lie within a calcium-sensitive domain near the active site. CAPN5 is only the second member of the large calpain gene family to cause a human Mendelian disorder, and this is the first report of a specific molecular cause for autoimmune eye disease. Further investigation of these mutations is likely to provide insight into the pathophysiologic mechanisms of common diseases ranging from autoimmune disorders to diabetic retinopathy., Author Summary We care for several families with an inherited form of autoimmune inflammation inside the eye. The patients also develop bleeding, scar tissue, and eventually blindness. Using advanced gene analysis methods, we discovered the cause of this disease is gene mutations in the CAPN5 gene. This gene makes a protein, calpain-5, which belongs to a family of calcium-activated enzymes that slice other proteins inside cells. Calpain-5 is expressed in the retina, and the disease mutations alter its location inside the cell. Future studies to understand how this protein causes inflammation and bleeding inside the eye will help us develop treatments for this condition and more common eye diseases with inflammation and bleeding.

The iris plays an essential role in regulating the amount of light passing to the retina and is also important in many human diseases. Several diseases change iris pigmentation, including forms of oculocutaneous albinism, Hermansky-Pudlak syndrome, Chediak-Higashi syndrome, Horner's syndrome, Waardenburg syndrome, and Fuchs' heterochromic iridocyclitis. In addition, other ocular diseases, such as pigment dispersion syndrome and exfoliation syndrome, involve disease-related morphologic changes to the pigmented tissues of the iris. Each of these diseases involves strong hereditary links, but much remains unknown concerning the underlying genetic pathways. In this study, we focused on three of these conditions: albinism, pigment dispersion syndrome, and exfoliation syndrome. In oculocutaneous albinism (OCA), there is reduced or absent pigmentation of the skin, hair, and eyes. Decreased melanin in the eyes can give rise to several ocular abnormalities, including foveal hypoplasia and decreased visual acuity; retinal ganglion cell axon misrouting; and strabismus, nystagmus, iris translucency, color vision impairment, and photophobia.1 The hereditary basis of OCA is complex. There are at least 4 genes that contribute to classic forms of OCA and at least another 12 associated with syndromic forms. The best understood form of OCA, and the most common in many populations, is OCA1.2,3 OCA1 is caused by mutations in the tyrosinase (TYR) gene, which encodes the rate-limiting enzyme necessary for melanin synthesis. Most people with OCA1 are believed to be compound heterozygotes, although in 15% of OCA1 cases, the second mutation remains unidentified.4 Interestingly, TYR appears to also influence many traits beyond pigmentation. For example, tyrosinase mutation is capable of rescuing a mouse model of pigment dispersion,5 but acts to worsen disease in mouse models of developmental glaucoma.6 Clearly, much remains unknown concerning TYR and its influences on ocular disease. In pigment dispersion syndrome, liberated pigment from the iris pigment epithelium becomes aberrantly deposited throughout the anterior chamber. As pigment accumulates in the iridocorneal angle, aqueous humor outflow resistance and intraocular pressure can become elevated.7,8 Although pigment dispersion syndrome has strong hereditary links,9,10 the genetic risk factors remain to be identified. DBA/2J mice develop a form of pigmentary glaucoma involving a pigment-dispersing iris disease, elevated intraocular pressure, and optic nerve damage.11,12 Mutations in two genes encoding melanosomal proteins, Tyrp1 and Gpnmb, are responsible for initiation of the DBA/2J disease process.13 To date, genetic studies of TYRP1 and GPNMB in human pigment dispersion patients have not detected mutations,13,14 suggesting that other genes in a pathway linked to TYRP1 and GPNMB may be the next most logical candidates worthy of consideration. In exfoliation syndrome, a primary diagnostic feature is the presence of fibrillar exfoliative material throughout the anterior chamber of the eye.15 The disease often also involves the dispersion of iris pigment and morphologic changes to the structure of the iris pigment epithelium.16,17 As with pigment dispersion syndrome, accumulations of material within the iridocorneal angle can obstruct aqueous humor outflow, resulting in elevated intraocular pressure and glaucoma. Recently, genetic variations in the LOXL1 gene have been linked with exfoliation syndrome.18 Because the same LOXL1 alleles associated with exfoliation syndrome also occur in the general population at a very high frequency, additional risk factors are presumed to exist. B6-Lystbg-J mice exhibit multiple ocular features resembling exfoliation syndrome, including the presence of an exfoliative-like material, pigment dispersion, and iris transillumination defects caused by an apparent loss of cell–cell adhesions within the iris pigment epithelium.17 Accordingly, LYST and other genes within the LYST genetic pathway are candidates that are likely to contribute to exfoliation syndrome in humans. We report global gene expression patterns of the iris in four strains of mice with identical genetic backgrounds: wild-type C57BL/6J mice with normal irides, albino mice with Tyr mutation, pigment dispersion–prone mice with Tyrp1 and Gpnmb mutations, and exfoliative-like mice with Lyst mutation. In each comparison between these strains, transcriptional changes are presented for select genes of functionally annotated clusters and are also presented according to the magnitude of the ratio of change.