Posttranslational Modifications,Localization, and Protein Interactions of Optineurin,the Product of a Glaucoma Gene

Background

Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG), is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin.

Methodology/Principal Findings

Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin–green fluorescence protein (GFP) fusion protein formed punctate structures termed “foci” in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time.

Conclusions/Significance

The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP–expressing cell lines are also anticipated to facilitate in-depth studies of optineurin. Furthermore, the demonstrations that optineurin is an aggregation-prone protein and that the foci formation is microtubule-dependent bear similarities to features documented in neurodegenerative diseases, supporting a neurodegenerative paradigm for glaucoma.     

Short-Term Reproducibility of Twenty-Four-Hour Intraocular Pressure Curves in Untreated Patients with Primary Open-Angle Glaucoma and Ocular Hypertension

Purpose

To assess the short-term day-to-day reproducibility of 24-hour intraocular pressure (IOP) curves in various respects in untreated primary open-angle glaucoma (POAG) and ocular hypertension (OHT) patients.

Methods

47 subjects with POAG and 34 subjects with OHT underwent IOP measurements every 2 hours in both eyes for consecutive 48 hours by a non-contact tonometer (NCT). IOP values at each time point were recorded. Mean IOP, peak IOP, time difference of peak IOP between two days and IOP fluctuation were also calculated. Intraclass correlation coefficients (ICCs) and Bland-Altman plots were used to evaluate reproducibility.

Results

ICCs of the entire IOP values for a complete 24-hour curve were 0.577 and 0.561 in POAG and OHT patients, respectively. ICCs of IOP values at different time points ranged from 0.384 (10am) to 0.686 (4am) in POAG patients and from 0.347 (6am) to 0.760 (4am) in OHT patients. ICCs of mean IOP, peak IOP and IOP fluctuation were respectively 0.832, 0.704, 0.367 in POAG patients and 0.867, 0.816 0.633 in OHT patients. Only 37.23% and 35.29% of the peak IOP time points appeared within the time difference of 2 hours in POAG and OHT patients, respectively, while 53.19% and 48.53% appeared within 4 hours in POAG and OHT patients, respectively.

Conclusion

A 24-hour IOP curve in a single day is not highly reproducible in short-term and has limited use for evaluating individual IOP condition. Mean IOP and peak IOP for a 24-hour IOP curve are useful parameters in clinical follow-up, while IOP value at a certain time point, IOP fluctuation and peak IOP time point should be interpreted with caution.     

Estimated Rates of Retinal Ganglion Cell Loss in Glaucomatous Eyes with and without Optic Disc Hemorrhages

Purpose

To evaluate whether optic disc hemorrhages are associated with faster rates of estimated retinal ganglion cell (RGC) loss in glaucoma.

Methods

A longitudinal observational cohort study of 222 eyes of 122 patients with glaucoma recruited from the Diagnostic Innovations Glaucoma Study (DIGS) followed for an average of 3.74±0.85 years. All subjects had optical coherence tomography and standard automated perimetry during follow up. Optic disc hemorrhages were detected by masked evaluation of stereophotographs. Rates of change in estimated numbers of RGCs were determined using a previously described method. A random coefficients model was used to investigate the relationship between disc hemorrhages and rates of change in estimated RGC counts over time.

Results

19 eyes of 18 subjects had at least one disc hemorrhage during follow up. At baseline, average estimated RGC counts in eyes with and without disc hemorrhages were 677,994 cells and 682,021 cells, respectively (P = 0.929). Eyes with optic disc hemorrhages during follow-up had significantly faster rates of estimated RGC loss than eyes without disc hemorrhages (22,233 cells/year versus 10,704 cells/year, P = 0.020). The effect of disc hemorrhages on the rates of estimated RGC loss remained significant after adjusting for confounding variables.

Conclusion

Eyes with disc hemorrhages showed faster rates of RGC loss compared to eyes without disc hemorrhages. These results provide further evidence that disc hemorrhages should be considered as an indicator of increased risk for faster neural loss in glaucoma.     

Changes in the Circadian Rhythm in Patients with Primary Glaucoma

Purpose

The current study was undertaken to investigate whether glaucoma affects the sleep quality and whether there is any difference between patients with primary glaucoma (primary open angle glaucoma, POAG and primary angle-closure glaucoma, PACG) and healthy subjects, using a validated self-rated questionnaire, the Pittsburgh Sleep Quality Index (PSQI).

Methods

The sleep quality of patients with POAG and PACG was tested against normal controls. Subjects were divided into three sub-groups according to age. Differences in the frequency of sleep disturbances (PSQI score >7) were assessed. The differences of sleep quality within the three groups and within the POAG group depending on the patients’ intraocular pressure (IOP) and impairment of visual field (VF) were also studied.

Results

92 POAG patients, 48 PACG patients and 199 controls were included. Sleep quality declined with age in control and POAG group (tendency chi-square, P<0.05). The prevalence of sleep disturbances was higher in POAG and PACG group than in the control group, the differences were statistically significant. The prevalence of sleep disturbances was higher in patients with PACG, compared to POAG patients in the age interval of 61–80. In POAG group, the ratio of patients with sleep disorders increased with augmented impairment of VF, but the differences were not statistically significant (χ²-test, P>0.05). No significant differences were found in POAG group between patients with a highest IOP in daytime and at nighttime (χ²-test, P>0.05).

Conclusions

The prevalence of sleep disorders was higher in patients with POAG and PACG than in controls. PACG patients seemed to have a more serious problem of sleep disorders than POAG patients between 61 to 80 years old. No correlation was found between the prevalence of sleep disorders and impairment of VF or the time when POAG patients showed a highest IOP.     

Intraocular Pressure-Lowering Effects of Commonly Used Fixed-Combination Drugs with Timolol: A Systematic Review and Meta-Analysis

Background

The first goal of medical therapy in glaucoma is to reduce intraocular pressure (IOP), and the fixed-combination medications are needed to achieve sufficiently low target IOP. The aim of this systematic review and meta-analysis is to evaluate IOP-lowering effect of the commonly used fixed-combination drugs containing 0.5% timolol.

Methods

Pertinent publications were identified through systematic searches. Over 85% of the patients had to be diagnosed with primary open-angle glaucoma (POAG) or ocular hypertension (OHT). Forty-one randomized clinical trials were included in the meta-analysis. The main efficacy measures were the absolute and relative values of mean diurnal IOP reduction, and the highest and lowest IOP reductions on the diurnal IOP curve. The pooled 1- to 3-month IOP-lowering effects after a medicine-free washout period was calculated by performing meta-analysis using the random effects model, and relative treatment effects among different fixed combinations were assessed using a mixed-effects meta-regression model.

Results

The relative reductions for mean diurnal IOP were 34.9% for travoprost/timolol, 34.3% for bimatoprost/timolol, 33.9% for latanoprost/timolol, 32.7% for brinzolamide/timolol, 29.9% for dorzolamide/timolol, and 28.1% for brimonidine/timolol. For the highest IOP decrease, relative reductions ranged from 31.3% for dorzolamide/timolol to 35.5% for travoprost/timolol; for the lowest IOP decrease, those varied from 25.9% for dorzolamide/timolol to 33.1% for bimatoprost/timolol. Both latanoprost/timolol and travoprost/timolol were more effective in lowering mean diurnal IOP than brimonidine/timolol (WMD: 5.9 and 7.0) and dorzolamide/timolol (WMD: 3.8 and 3.3).

Conclusions

All six commonly used fixed-combination drugs containing timolol can effectively lower IOP in patients with POAG and OHT, and both latanoprost/timolol and travoprost/timolol might achieve better IOP-lowering effects among the six fixed-combination agents.     

Neuroretinal rim area and body mass index

Purpose

To examine associations between neuroretinal rim area, pressure related factors and anthropometric parameters in a population-based setting.

Methods

The population-based cross-sectional Beijing Eye Study 2006 included 3251 subjects with an age of 45+ years. The participants underwent a detailed ophthalmic examination. Exclusion criteria for our study were high myopia of more than -8 diopters and angle-closure glaucoma.

Results

The study included 2917 subjects with a mean age of 59.8±9.8 years (range: 45–89 years). Mean neuroretinal rim area was 1.97±0.38 mm², mean intraocular pressure 15.6±3.0 mmHg, mean diastolic blood pressure 79.0±5.9 mm Hg, mean systolic blood pressure 133.5±11.1 mmHg, and mean body mass index was 25.5±3.7. In univariate analysis, neuroretinal rim area was significantly associated with optic disc size, open-angle glaucoma, refractive error, age and gender. After adjustment for these parameters in a multivariate analysis, a larger neuroretinal rim area was significantly correlated with a higher body mass index (P<0.001), in addition to be associated with a lower intraocular pressure (P = 0.004), lower mean blood pressure (P = 0.02), and higher ocular perfusion pressure.

Conclusions

In a general population, neuroretinal rim as equivalent of the optic nerve fibers is related to a higher body mass index, after adjustment for disc area, refractive error, age, gender, open-angle glaucoma, intraocular pressure, blood pressure and ocular perfusion pressure. Since body mass index is associated with cerebrospinal fluid pressure, the latter may be associated with neuroretinal rim area. It may serve as an indirect hint for an association between cerebrospinal fluid pressure and glaucoma.     

Glaucomatous and Age-Related Changes in Corneal Pulsation Shape. The Ocular Dicrotism

Purpose

To ascertain whether the incidence of ocular dicrotic pulse (ODP) increases with age, it is more pronounced in glaucomatous than in normal eyes and whether it is related to cardiovascular activity.

Methods

261 subjects aged 47 to 78 years were included in the study and classified into four groups: primary open angle glaucoma (POAG), primary angle-closure glaucoma (PACG), glaucoma suspects with glaucomatous optic disc appearance (GODA) and the controls (CG). Additionally, in each group, subjects with ODP were divided into two age subgroups around the median age. A non-contact ultrasonic method was used to measure corneal indentation pulse (CIP) synchronically with the acquisition of electrocardiography (ECG) and blood pulse signals. ODP was assessed from the acquired signals that were numerically processed in a custom written program.

Results

ODP incidence was about 78%, 66%, 66% and 84% for CG, GODA, POAG, and PACG group, respectively. With advancing age, the ODP incidence increased for all subjects (Δ = 12%), the highest being for the PACG and POAG groups (Δ = 30%). GODA group did not show an age-related increase in the incidence of ODP.

Conclusions

The ocular dicrotism, measured with non-contact ultrasonic method, was found to be a common phenomenon in elderly subjects. The increased ODP incidence in PACG and POAG group may correspond to either higher stiffness of glaucoma eyes, biochemical abnormalities in eye tissues, changes in ocular hemodynamics, may reflect the effect of medications or be a combination of all those factors. The results of GODA group suggest different mechanisms governing their ocular pulse that makes them less susceptible to generating ODP and having decreased predisposition to glaucoma.     

Five-Year Incidence of Primary Open-Angle Glaucoma and Rate of Progression in Health Center-Based Korean Population: The Gangnam Eye Study

Objective

To investigate the 5-year incidence and progression rate of primary open-angle glaucoma (POAG) in a health-center-based Korean population.

Methods

The study population comprised 5,021 subjects who participated in standardized health screening (including non-contact tonometry and fundus photography) at the Gangnam Healthcare Center during the period from January 2005 to December 2006 and again from January 2010 to December 2011. Among these subjects, 948 (18.9%) with findings suggestive of glaucoma were subjected to a comprehensive glaucoma evaluation, which included applanation tonometry and standard automated perimetry. Based on the results, the subjects were diagnosed as POAG suspect or definite POAG.

Results

The 5-year incidences of POAG suspect and definite POAG were 0.84% (42 subjects) and 0.72% (36 subjects), respectively. The rate of progression from POAG suspect to definite POAG was 4.75% per year. In subjects with a baseline intraocular pressure (IOP) >21 mmHg, the incidence of POAG suspect or definite POAG was significantly higher than in those with a baseline IOP≤21 mmHg (32% vs. 1.05%; P<0.001). A multivariate analysis showed that the progression from POAG suspect to definite POAG was significantly associated with older age (odds ratio [OR], 1.07; 95% confidence interval [CI], 1.03–1.10), higher baseline IOP (OR, 1.10; 95% CI, 1.01–1.24), higher body mass index (BMI) (OR, 1.15; 95% CI, 1.03–1.31), higher education level (OR, 1.57; 95% CI, 1.05–2.17), and higher hematocrit level (OR, 1.22; 95% CI, 1.08–1.43).

Conclusions

In the health-center-based Korean population, the 5-year incidence of POAG was 0.72%, and the rate of progression from POAG suspect to definite POAG was 4.75% per year. This study identified old age, high baseline IOP, high BMI, high level of education, and high hematocrit level as significant risk factors for incident POAG.     

Myocilin Polymorphisms and Primary Open-Angle Glaucoma: A Systematic Review and Meta-Analysis

Background

Glaucoma is the leading cause of irreversible blindness in the world. Recent evidence indicates a role for genetic susceptibility to primary open-angle glaucoma (POAG). The relation between myocilin polymorphisms and POAG susceptibility has been studied in different populations.

Methods

A meta-analysis of 32 published genetic association case-control studies, which examined the relation between POAG and the R46X, R76K, Y347Y, T353I, and Q368X polymorphisms of the myocilin gene, was carried out.

Results

In meta-analysis, significant associations were observed between POAG risk and two myocilin polymorphisms with summarized odds ratio of 4.68 (95%CI, 2.02–10.85) for Q368X and 2.17 (95% CI, 1.32–3.57) for T353I. Both Q368X and T353I were significantly associated with high-tension glaucoma, with summarized odds ratio of 4.26 (1.69, 10.73) and 2.26 (1.37–3.72). In Westerners, significant association was observed for Q368X mutation (odds ratio, 5.17; 95% CI, 2.16–12.40). However, in Asians it was for T353I (odds ratio, 2.17; 95% CI, 1.32–3.57).

Conclusions

There is strong evidence that myocilin polymorphisms are associated with POAG susceptibility, and the prevalence of myocilin mutations might be ethnicity-dependent in Caucasians for Q368X and in Asians for T353I.     

Inactivation of MARCH5 Prevents Mitochondrial Fragmentation and Interferes with Cell Death in a Neuronal Cell Model

Purpose

To study the impact of the mitochondrial ubiquitin ligase MARCH5 on mitochondrial morphology and induction of apoptosis using an in vitro model of neuronal precursor cells exposed to glaucoma-relevant stress conditions.

Methods

RGC5 cells transfected with expression constructs for MARCH5, MARCH5^H43W, Dpr1^K38A or vector control were exposed to either elevated pressure of 30 mmHg, oxidative stress caused by mitochondrial electron transport chain (ETC) inhibition, or hypoxia-reoxygenation conditions. Mitochondrial morphology of RGC5 cells was analyzed following staining of the mitochondrial marker cytochrome c and photoactivatable GFP (PAGFP) diffusion assay. Induction of apoptotic cell death in these cells was determined by analyzing the release of cytochrome c from mitochondria into the cytosol and flow cytometry.

Results

Exposure of RGC5 cells to oxidative stress conditions as well as to elevated pressure resulted in the fragmentation of the mitochondrial network in control cells as well as in cells expressing MARCH5. In cells expressing inactive MARCH5^H43W or inactive Drp^K38A, mitochondrial fragmentation was significantly blocked and mitochondrial morphology was comparable to that of control cells under normal conditions. Exposure of RGC5 cells to elevated pressure or oxidative stress conditions induced apoptotic cell death as assessed by cytochrome c release and DNA staining, while expression of dominant-negative MARCH5^H43W or Drp1^K38A did significantly delay cell death.

Conclusion

Preventing mitochondrial fragmentation through interference with the mitochondrial fission machinery protects neuronal cells from programmed cell death following exposure to stressors physiologically relevant to the pathogenesis of glaucoma.     

Diabetes Mellitus as a Risk Factor for Open-Angle Glaucoma: A Systematic Review and Meta-Analysis

Objective

To determine the association between diabetes mellitus (DM) and primary open-angle glaucoma (POAG).

Methods

This is a systematic review and meta-analysis of case-control and cohort studies. The literature search included two databases (PubMed and Embase) and the reference lists of the retrieved studies. Separate meta-analyses for case-control studies and cohort studies were conducted using random-effects models, with results reported as adjusted odds ratios (ORs) and relative risks (RRs), respectively.

Results

Thirteen studies—seven case-control studies and six population-based cohort studies—were included in this meta-analysis. The pooled RR of the association between DM and POAG based on the risk estimates of the six cohort studies was 1.40 (95% CI, 1.25–1.57). The pooled OR of the association between DM and POAG based on the risk estimates of the seven case-control studies was 1.49 (95% CI, 1.17–1.88). There was considerable heterogeneity among the case-control studies that reported an association between DM mellitus and POAG (P<0.001) and no significant heterogeneity among the cohort studies (P = 0.377). After omitting the case-control study that contributed significantly to the heterogeneity, the pooled OR for the association between DM and POAG was 1.35 (95% CI, 1.06–1.74).

Conclusions

Individuals with DM have an increased risk of developing POAG.     

The p53 Codon 72 PRO/PRO Genotype May Be Associated with Initial Central Visual Field Defects in Caucasians with Primary Open Angle 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.     

The evaluation of the oxidative stress parameters in patients with primary angle-closure glaucoma

Objective

To clarify the presence of oxidative stress in patients with primary angle-closure glaucoma (PACG) and to investigate the relationship between oxidative stress and PACG.

Methods

Fifty patients with primary angle-closure glaucoma and fifty healthy controls of matched age and gender were included in the study prospectively. Serum samples were obtained to detect the oxidation degradation products malondialdehyde (MDA), conjugated diene (CD), 4-hydroxynonenal (4-HNE), advanced oxidation protein products (AOPP), protein carbonyl (PC), ischemia-modified albumin (IMA) and 8-hydroxydeoxyguanosin (8-OHdG).

Results

The concentration of MDA and CD in PACG patients was significantly higher than those of the control subjects (P<0.05, P<0.01). The serum 4-HNE concentrations were increased in PACG patients, but the differences with those of the healthy controls were not statistically significant. Compared to normal subjects, there was significant higher in serum AOPP and PC in PACG patients (P<0.01). PACG patients had higher levels of 8-OHdG in serum with respect to the comparative group of normal subjects (P<0.01). When plasma IMA levels in the PACG group were compared with those in the control group, significant increases in IMA were observed in the former (P<0.05).

Conclusions

Our study demonstrated that IMA is a new biomarker available for assessing oxidative stress in PCAG. Oxidative stress is an important risk factor in the development of primary angle-closure glaucoma. Increased levels of oxidative stress products may be associated with primary angle-closure glaucoma.     

Identification of Candidate Serum Proteins for Classifying Well-Differentiated Small Intestinal Neuroendocrine Tumors

Background

Patients with well-differentiated small intestine neuroendocrine tumors (WD-SI-NETs) are most often diagnosed at a metastatic stage of disease, which reduces possibilities for a curative treatment. Thus new approaches for earlier detection and improved monitoring of the disease are required.

Materials and Methods

Suspension bead arrays targeting 124 unique proteins with antibodies from the Human Protein Atlas were used to profile biotinylated serum samples. Discoveries from a cohort of 77 individuals were followed up in a cohort of 132 individuals both including healthy controls as well as patients with untreated primary WD-SI-NETs, lymph node metastases and liver metastases.

Results

A set of 20 antibodies suggested promising proteins for further verification based on technically verified statistical significance. Proceeding, we assessed the classification performance in an independent cohort of patient serum, achieving, classification accuracy of up to 85% with different subsets of antibodies in respective pairwise group comparisons. The protein profiles of nine targets, namely IGFBP2, IGF1, SHKBP1, ETS1, IL1α, STX2, MAML3, EGR3 and XIAP were verified as significant contributors to tumor classification.

Conclusions

We propose new potential protein biomarker candidates for classifying WD-SI-NETs at different stage of disease. Further evaluation of these proteins in larger sample sets and with alternative approaches is needed in order to further improve our understanding of their functional relation to WD-SI-NETs and their eventual use in diagnostics.     

The Relationship between Visual Field Index and Estimated Number of Retinal Ganglion Cells in Glaucoma

Purpose

To evaluate the relationship between visual field index (VFI) and the estimated number of retinal ganglion cells (RGCs) in glaucoma.

Methods

A multicenter study of 1,245 healthy, glaucomatous and suspected glaucomatous eyes of 1,245 subjects recruited from the Diagnostic Innovations in Glaucoma Study (DIGS) and African Descent and Glaucoma Evaluation Study (ADAGES). All eyes underwent standard automated perimetry (SAP) and time-domain optical coherence tomography (TD-OCT). Estimates of RGC count and percentage of RGCs remaining, compared to age-matched healthy eyes, were calculated from TD-OCT using a previously described formula. Smoothing spline curves were fitted to examine the relationship between VFI and the percent remaining RGCs. The first derivative (i.e., slopes) of these curves was used to explore the relationship between changes in these measures.

Results

The relationships between the VFI and both estimated RGC counts and the percent remaining RGCs were nonlinear. A unit number of VFI loss corresponded to substantially greater loss of estimated RGCs and estimated percentage of RGCs remaining in early compared to late disease.

Conclusions

The relationship between VFI and estimated RGC counts is nonlinear and the index substantially underestimates the amount of neural loss early in the disease. Disease severity should be taken into account when interpreting rates of VFI change over time.     

Common variants in CDKN2B-AS1 associated with optic-nerve vulnerability of glaucoma identified by genome-wide association studies in Japanese

Background

To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated.

Methods and Principal Findings

We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10⁻¹⁰). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients.

Conclusions and Significance

In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma.     

Variants in the ASB10 Gene Are Associated with Primary Open Angle Glaucoma

Background

Recently nonsynonymous coding variants in the ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) gene were found to be associated with primary open angle glaucoma (POAG) in cohorts from Oregon and Germany, but this finding was not confirmed in an independent cohort from Iowa. The aim of the current study was to assess the role of ASB10 gene variants in Pakistani glaucoma patients.

Methods

Sanger sequencing of the coding exons and splice junctions of the ASB10 gene was performed in 30 probands of multiplex POAG families, 208 sporadic POAG patients and 151 healthy controls from Pakistan. Genotypic associations of individual variants with POAG were analyzed with the Fisher’s exact or Chi-square test.

Results

In total 24 variants were identified in POAG probands and sporadic patients, including 11 novel variants and 13 known variants. 13 of the variants were nonsynonymous, 6 were synonymous, and 5 were intronic. Three nonsynonymous variants (p.Arg49Cys, p.Arg237Gly, p.Arg453Cys) identified in the probands were not segregating in the respective families. This is not surprising since glaucoma is a multifactorial disease, and multiple factors are likely to be involved in the disease manifestation in these families. However a nonsynonymous variant, p.Arg453Cys (rs3800791), was found in 6 sporadic POAG patients but not in controls, suggesting that it infers increased risk for the disease. In addition, one synonymous variant was found to be associated with sporadic POAG: p.Ala290Ala and the association of the variant with POAG remained significant after correction for multiple testing (uncorrected p-value 0.002, corrected p-value 0.047). The cumulative burden of rare, nonsynonymous variants was significantly higher in sporadic POAG patients compared to control individuals (p-value 0.000006).

Conclusions

Variants in ASB10 were found to be significantly associated with sporadic POAG in the Pakistani population. This supports previous findings that sequence variants in the ASB10 gene may act as a risk factor for glaucoma.     

Serum Antibodies against CD28– A New Potential Marker of Dismal Prognosis in Melanoma Patients

Background

Autoantibodies against CD28 have been found in patients with autoimmune and atopic diseases. These antibodies may act as superagonists and activate T cells but may also be antagonistic or induce immunosuppressive effects by activating regulatory T cells. Autoimmunity in melanoma patients has been discussed controversially.

Objective

We investigated 230 melanoma patients for the occurrence of CD28 antibodies and the effect of the latter on overall and progress-free survival.

Methods

We constructed an ELISA assay to measure CD28 serum antibodies. 230 patients with melanoma and a control-group of 625 patients consistent of 212 patients with virus hepatitis b or c, 149 patients with allergies, 78 patients with psoriasis, 46 patients with plasmocytoma and 140 healthy blood donors were investigated for the occurrence of CD28 antibodies.

Results

CD28 abs occur at a higher percentage in patients with melanoma and in patients with viral hepatitis than in other groups investigated (p<0.001). Occurrence of CD28 abs is significantly higher in patients receiving interferons independent from the underlying disease (p<0.001). In vitro CD28 serum antibodies have an inhibitory effect on the CD28 receptor as they lead to reduced stimulation of Jurkat cells. Presence of CD28 was correlated with a higher risk of dying from melanoma (p = 0.043), but not with a significantly shortened overall survival or progression-free survival.

Conclusion

Interferon therapy appears to induce the production of CD28 abs. In light of reports that these CD28 abs induce immunosuppressive Tregs and – as our data show – that they are inhibitors of CD28 receptor mediated stimulation, the continuation of therapies with interferons in melanoma patients developing CD28 antibodies should be critically reconsidered, since our data indicate a worse outcome of patients with CD28 abs.     

Neutralizing and IgG Antibodies against Simian Virus 40 in Healthy Pregnant Women in Italy

Objective

Polyomavirus simian virus 40 (SV40) sequences have been detected in various human specimens and SV40 antibodies have been found in human sera from both healthy individuals and cancer patients. This study analyzed serum samples from healthy pregnant women as well as cord blood samples to determine the prevalence of SV40 antibodies in pregnancy.

Methods

Serum samples were collected at the time of delivery from two groups of pregnant women as well as cord bloods from one group. The women were born between 1967 and 1993. Samples were assayed by two different serological methods, one group by neutralization of viral infectivity and the other by indirect ELISA employing specific SV40 mimotopes as antigens. Viral DNA assays by real-time polymerase chain reaction were carried out on blood samples.

Results

Neutralization and ELISA tests indicated that the pregnant women were SV40 antibody-positive with overall prevalences of 10.6% (13/123) and 12.7% (14/110), respectively. SV40 neutralizing antibodies were detected in a low number of cord blood samples. Antibody titers were generally low. No viral DNA was detected in either maternal or cord bloods.

Conclusions

SV40-specific serum antibodies were detected in pregnant women at the time of delivery and in cord bloods. There was no evidence of transplacental transmission of SV40. These data indicate that SV40 is circulating at a low prevalence in the northern Italian population long after the use of contaminated vaccines.     

Mapping Cortical Thickness of the Patients with Unilateral End-Stage Open Angle Glaucoma on Planar Cerebral Cortex Maps

Purpose

To estimate and compare cerebral cortex thickness in patients with unilateral end-stage glaucoma with that of age-matched individuals with unaffected vision.

Methods

14 patients with unilateral end-stage primary open angle glaucoma (POAG) and 12 age-matched control individuals with no problems with vision were selected for the study based on detailed ophthalmic examination. For each participant 3D high-resolution structural brain T1-weighted magnetization prepared MR images were acquired on a 3.0 T scanner. Brain cortex thickness was estimated using the FreeSurfer image analysis environment. After warping of subjects'' cortical surfaces to FreeSurfer common space, differences between POAG and control groups were inferred at the group analysis level with the General Linear Model.

Results

The analysis performed revealed local thinning in the visual cortex areas in the POAG group. Statistically significant differences form 600 mm² clusters located in the Brodmann area BA19 in the left and right hemisphere.

Conclusion

Unilateral vision loss due to end-stage neuropathy from POAG is associated with significant thinning of cortical areas employed in vision.