Serum Th1 and Th17 related cytokines and autoantibodies in patients with Posner-Schlossman syndrome.

Posner-Schlossman syndrome (PSS) shares some clinical features with uveitis and open angle glaucoma. Cytokines and autoantibodies have been associated with uveitis and open angle glaucoma. However, the role of serum cytokines and autoantibodies in the pathogenesis of PSS remains unknown. This study aimed to evaluate the associations of type 1 T helper (Th1) and Th17 related cytokines and autoantibodies with PSS. Peripheral blood serum samples were collected from 81 patients with PSS and 97 gender- and age-matched healthy blood donors. Th1 and Th17 related cytokines, including interleukin-1ß (IL-1ß), IL-12, tumor necrosis factor-α (TNF-α), interferon- γ (IFN-γ), IL-6 and IL-17, and glucose-6-phosphate isomerase (GPI) were determined by double antibody sandwich ELISA. Anti-nuclear antibody (ANA), anti-keratin antibody (AKA) and anti-neutrophil cytoplasmic antibody (ANCA) were detected by indirect immunofluorescence assay. Anti-cardiolipin antibody (ACA)-IgG, ACA-IgM, ACA-IgA, anti-double stranded DNA (anti-dsDNA) and anti-cyclic citrullinated peptide antibody (anti-CCP) were detected by indirect ELISA. Serum levels of IL-1ß, IL-12 and IL-6 in PSS patients were significantly lower than those in controls (P < 0.003), and these associations survived the Bonferroni correction (Pc < 0.018). There was no significant difference in serum levels of TNF-α, IFN-γ and IL-17 between the PSS and control groups (Pc > 0.12). Positive rate of serum anti-dsDNA in PSS patients was significantly higher than that in the control group (P = 0.002, Pc = 0.018), while positive rates of serum ANA, AKA, ANCA, ACA-IgG, ACA-IgM, ACA-IgA, GPI and anti-CCP in the PSS group were not significantly different from those in the control group (Pc > 0.09). These results suggest that anti-dsDNA may contribute to the pathogenesis of PSS, while Th1 and Th17 related cytokines and other autoantibodies may not be major contributors to PSS.

Screening of the LTBP2 gene in 214 Chinese sporadic CYP1B1-negative patients with primary congenital glaucoma.

PURPOSE: To identify deleterious mutations in the latent transforming growth factor-ß-binding protein 2 (LTBP2) gene in sporadic patients with primary congenital glaucoma (PCG) from a Han Chinese population, which had been excluded for mutations in the CYP1B1 gene. METHODS: In this retrospective case-control study, 36 coding exons and adjacent exon-intron boundaries of LTBP2 were amplified with PCR and screened for mutations with Sanger sequencing in DNA samples of 214 sporadic patients with PCG. Sequence variants identified in the patients with PCG were subsequently screened in 100 unaffected control subjects and the unaffected parents of the patients with PCG who had sequence changes in LTBP2. RESULTS: Eight heterozygous single nucleotide polymorphisms (SNPs) in coding regions of LTBP2 were identified in the patients with PCG. Four of these SNPs were missense changes that resulted in the replacement of amino acids (rs2304707, rs116914994, rs45468895, and rs763035721), two of which (rs2304707 and rs116914994) were also present in the control subjects. No significant differences in the frequencies of the missense SNPs were found between the patients with PCG and the controls. The two missense SNPs, rs45468895 and rs763035721, which were each found in one patient also existed in their unaffected parents, suggesting that these two SNPs were not segregated in these families and are unlikely to be a disease-causative variant. In addition, four synonymous SNPs were detected in the patients with PCG (rs61738025, rs862031, rs199805158, and rs12586758). CONCLUSIONS: The results showed that no deleterious mutations were found in coding regions of LTBP2 in patients with PCG, suggesting that it is not a causal gene for PCG in the Han Chinese population.

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.

Association of polymorphisms of tumor necrosis factor and tumor protein p53 with primary open-angle glaucoma.

PURPOSE: To evaluate the variants of 10 genes for association with primary open-angle glaucoma (POAG) in a Chinese population. METHODS: A total of 405 unrelated patients with POAG (255 high-tension glaucoma [HTG], 100 normal-tension glaucoma [NTG], and 50 juvenile-onset open-angle glaucoma [JOAG]) and 201 control subjects were recruited. Seventeen variants in 10 genes with reported association with POAG were genotyped for analysis of allele and haplotype frequencies between cases and control subjects. These genes included CDH1 (cadherin 1, type 1, E-cadherin), CDKN1A (cyclin-dependent kinase inhibitor 1A), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), GSTM1 (glutathione S-transferase mu 1), GSTT1 (glutathione S-transferase theta 1), MTHFR (5,10-methylenetetrahydrofolate reductase), NOS3 (nitric oxide synthase 3), OPA1 (optic atrophy 1), TNF (tumor necrosis factor), and TP53 (tumor protein p53). RESULTS: One SNP (-308G>A; rs1800629) in TNF demonstrated a significant association with HTG (P = 0.012). The allele G frequency was higher in HTG patients than in control subjects (94.6% vs. 90.3%; OR = 1.89). One haplotype consisting of rs1799724 and rs1800629 was significantly associated with HTG (P = 0.015, corrected P = 0.045). One SNP (R72P; rs1042522) in TP53 was significantly associated with NTG (P = 0.018). The allele G frequency was higher in NTG patients than in control subjects (56.1% vs. 45.8%; OR = 1.52). The significance of these associations survived the Bonferroni correction (corrected P < 0.024). Other gene variants were not significantly associated with HTG (P > 0.063) or NTG (P > 0.13). None of the studied variants was significantly associated with JOAG (P > 0.17). CONCLUSIONS: The findings suggest that variants in TNF and TP53 are risk factors for POAG, whereas variants in other studied genes are not major risk factors for POAG, at least in the Chinese population.

Lack of association of polymorphisms in homocysteine metabolism genes with pseudoexfoliation syndrome and glaucoma.

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

The cytotoxic and stress responses of human trabecular meshwork cells treated with triamcinolone acetonide.

PURPOSE: To evaluate the cytotoxic effect of triamcinolone acetonide (TA) on cultured human trabecular meshwork (TM) cells. METHODS: TA (0.1 mg/ml, 1 mg/ml) or the vehicle (benzyl alcohol, 0.0025%, 0.025%) was added to human TM cell cultures on day 0 and collected subsequently on day 1, 3, or 5. The amount of cell proliferations with or without TA treatment was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) assay. All samples were read in triplicate (n=4 in all cases). By using real-time quantitative polymerase chain reaction (PCR), gene expression levels of c-fos, c-jun, caspase-3, c-myc, and p53 were determined after TA treatments at 0 min, 10 min, 20 min, 30 min, 50 min, 80 min, 2 h, 12 h, 24 h, and 48 h. Unpaired t-test was used to test the drug and concentration effects of TA, ANOVA was used to test the time effects of TA, and the Bonferroni test was used to correct multiple comparisons. Apoptosis of TM cells as a result of TA treatment were assessed by the terminal uridyl nick end labeling (TUNEL) assay. RESULTS: Both concentrations of TA caused a significant reduction in the number of human TM cells as early as day 1 and across five days of the treatment period. Significantly increased expressions of c-jun, c-fos, c-myc, p53, and caspase 3 were observed at different time points after both 0.1 mg/ml and 1 mg/ml TA treatment. Significantly increased apoptotic cells were observed after TA treatment for three days. CONCLUSIONS: Our results showed that TA was cytotoxic to human TM cells in culture and the presence of TA caused apoptotic cell death. It gave evidence that the underlying mechanism of TA caused ocular hypertension and may be associated with necrosis and apoptosis of the TM cells.

Novel myocilin mutation in a Chinese family with juvenile-onset open-angle glaucoma.

OBJECTIVE: To search for the genetic cause of juvenile-onset open-angle glaucoma (JOAG) in a Chinese family. METHODS: In a 3-generation glaucoma family affected with JOAG or ocular hypertension, we screened myocilin (MYOC) and optineurin (OPTN) for mutations and investigated apolipoprotein E (APOE) polymorphisms in 6 family members, 2 of them patients with JOAG, 2 patients with ocular hypertension, and 2 patients who were asymptomatic. Normal controls included 200 unrelated Chinese subjects. The COS-7 cell line was transfected with expression vectors encoding wild-type or mutated MYOC complementary DNA. Cellular and secreted MYOC proteins were characterized by Western blotting. RESULTS: One missense MYOC mutation, 734G>A: Cys245Tyr, was identified. It occurred in all 4 family members afflicted with JOAG or ocular hypertension but not in asymptomatic family members. No OPTN variations were observed. APOE polymorphism frequencies were similar to those for controls. The Cys245Tyr MYOC mutation cosegregated with the disorder within the family. It was absent in the 200 control subjects. The Cys245Tyr mutant MYOC protein formed homomultimeric complexes that migrated at molecular weights larger than their wild-type counterparts. These mutant complexes remained sequestered intracellularly in COS-7 cells. CONCLUSIONS: The Cys245Tyr MYOC mutation was the genetic cause of JOAG in this Chinese family. This mutation may alter covalent bonds that formed between MYOC cysteines. Clinical Relevance Genetic tests of MYOC mutations may be beneficial to predict new cases of the disease in families with JOAG.

Molecular diagnostics of genetic eye diseases.

Eye diseases can be simple or complex, and mostly of heterogeneous molecular genetics. Some eye diseases are caused by mutations in a single gene, but some diseases, such as primary open angle glaucoma, can be due to sequence variations in multiple genes. In some diseases, both genetic and epigenetic mechanisms are involved, as was recently revealed in the mechanism of retinoblastoma. Disease causative mutations and phenotypes may vary by ethnicity and geography. To date, more than a hundred candidate genes for eye diseases are known, although less than 20 have definite disease-causing mutations. The three common genetic eye diseases, primary open angle glaucoma, age-related macular degeneration, and retinitis pigmentosa, all have known gene mutations, but these account for only a portion of the patients. While the search for eye disease genes and mutations still goes on, known mutations have been utilized for diagnosis. Genetic markers for pre-symptomatic and pre-natal diagnosis are available for specific diseases such as primary open angle glaucoma and retinoblastoma. This paper reviews the molecular basis of common genetic eye diseases and the available genetic markers for clinical diagnosis. Difficulties and challenges in molecular investigation of some eye diseases are discussed. Establishment of ethnic-specific disease databases that contain both clinical and genetic information for identification of genetic markers with diagnostic, prognostic, or pharmacological value is strongly advocated.

Genetic and environmental risk factors for primary open-angle glaucoma.

BACKGROUND: Primary open-angle glaucoma (POAG) is characterized by optic nerve damage and consists of a group of genetically heterogeneous disorders. This study was to investigate the associations of genetic and environmental factors with POAG in a hospital-based Chinese population. METHODS: Thirty-two adult onset POAG patients and 96 age-sex matched control subjects were studied by multivariable logistic regression analysis for the relationships between POAG and its risk factors including family history, diabetes, hypertension, cardiovascular diseases, cigarette smoking, alcohol consumption and polymorphisms of the myocilin and the optineurin genes. RESULTS: Univariate analysis showed that POAG was related to family history, cardiovascular disease, alcohol consumption and a myocilin sequence alteration (T353I) (P < 0.04). Multivariable logistic regression analysis confirmed that POAG was significantly associated with family history (OR = 20.2), hypertension (OR = 3.58), cigarette smoking (OR = 10.8), alcohol consumption (OR = 0.028) and T353I (OR = 6.03, all P < 0.05). CONCLUSIONS: Family history, hypertension, cigarette smoking and T353I in the myocilin gene are risk factors for POAG. Alcohol consumption, however, has a protective effect.

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.