Novel MIP gene mutation causes autosomal-dominant congenital cataract.

AIM: To identify disease-causative mutations in families with congenital cataract. METHODS: Two Chinese families with autosomal-dominant congenital cataract (ADCC) were recruited and underwent comprehensive eye examinations. Gene panel next-generation sequencing of common pathogenic genes of congenital cataract was performed in the proband of each family. Sanger sequencing was used to valid the candidate gene mutations and sequence the other family members for co-segregation analysis. The effect of sequence changes on protein structure and function was predicted through bioinformatics analysis. Major intrinsic protein (MIP)-wildtype and MIP-G29R plasmids were constructed and microinjected into zebrafish single-cell stage embryos. Zebrafish embryonic lens phenotypes were screened using confocal microscopy. RESULTS: A novel heterozygous mutation (c.85G>A; p.G29R) in the MIP gene was identified in the proband of one family. A known heterozygous mutation (c.97C>T; p.R33C; rs864309693) in MIP was found in the proband of another family. In-silico prediction indicated that the novel mutation might affect the MIP protein function. Zebrafish embryonic lens was uniformly transparent in both wild-type PCS2+MIP and mutant PCS2+MIP. CONCLUSION: Two missense mutations in the MIP gene in Chinese cataract families are identified, and one of which is novel. These findings expand the genetic spectrum of MIP mutations associated with cataracts. The functional studies suggest that the novel MIP mutation might not be a gain-of-function but a loss-of-function mutation.

Mitochondrial TXNRD2 and ME3 Genetic Risk Scores Are Associated with Specific Primary Open-Angle Glaucoma Phenotypes.

PURPOSE: Genetic variants in regions that include the mitochondrial genes thioredoxin reductase 2 (TXNRD2) and malic enzyme 3 (ME3) are associated with primary open-angle glaucoma (POAG) in genome-wide association studies (GWASs). To assess their clinical impact, we investigated whether TXNRD2 and ME3 genetic risk scores (GRSs) are associated with specific glaucoma phenotypes. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 2617 patients with POAG and 2634 control participants from the National Eye Institute Glaucoma Human Genetics Collaboration Hereditable Overall Operational Database (NEIGHBORHOOD) consortium. METHODS: All POAG-associated single nucleotide polymorphisms (SNPs) in the TXNRD2 and ME3 loci were identified using GWAS data (P < 0.05). Of these, 20 TXNRD2 and 24 ME3 SNPs were selected after adjusting for linkage disequilibrium. The correlation between SNP effect size and gene expression levels was investigated using the Gene-Tissue Expression database. Genetic risk scores were constructed for each individual using the unweighted sum of TXNRD2, ME3, and TXNRD2 + ME3 combined risk alleles. Age- and sex-adjusted odds ratios (ORs) for POAG diagnosis were calculated per decile for each GRS. Additionally, the clinical features of patients with POAG in the top 1%, 5%, and 10% of each GRS were compared with those in the bottom 1%, 5%, and 10%, respectively. MAIN OUTCOME MEASURES: Primary open-angle glaucoma OR per GRS decile, maximum treated intraocular pressure (IOP), and prevalence of paracentral visual field loss among patients with POAG with high versus low GRSs. RESULTS: A larger SNP effect size strongly correlated with higher TXNRD2 and lower ME3 expression levels (r = 0.95 and r = -0.97, respectively; P < 0.05 for both). Individuals in decile 10 of the TXNRD2 + ME3 GRS had the highest odds of POAG diagnosis (OR, 1.79 compared with decile 1; 95% confidence interval, 1.39-2.30; P < 0.001). Patients with POAG in the top 1% of the TXNRD2 GRS showed higher mean maximum treated IOP compared with the bottom 1% (19.9 mmHg vs. 15.6 mmHg; adjusted P = 0.03). Patients with POAG in the top 1% of the ME3 and TXNRD2 + ME3 GRS showed a higher prevalence of paracentral field loss than the bottom 1% (72.7% vs. 14.3% for ME3 GRS and 88.9% vs. 33.3% for TXNRD2+ME3 GRS; adjusted P = 0.03 for both). CONCLUSIONS: Patients with POAG with higher TXNRD2 and ME3 GRSs showed higher treated IOP and a greater prevalence of paracentral field loss. Functional studies exploring how these variants impact mitochondrial function in patients with glaucoma are warranted. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found after the references.

Genome-Wide Association Study Identifies Two Common Loci Associated with Pigment Dispersion Syndrome/Pigmentary Glaucoma and Implicates Myopia in its Development.

PURPOSE: To identify genetic variants associated with pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) in unrelated patients and to further understand the genetic and potentially causal relationships between PDS and associated risk factors. DESIGN: A 2-stage genome-wide association meta-analysis with replication and subsequent in silico analyses including Mendelian randomization. PARTICIPANTS: A total of 574 cases with PG or PDS and 52 627 controls of European descent. METHODS: Genome-wide association analyses were performed in 4 cohorts and meta-analyzed in 3 stages: (1) a discovery meta-analysis was performed in 3 cohorts, (2) replication was performed in the fourth cohort, and (3) all 4 cohorts were meta-analyzed to increase statistical power. Two-sample Mendelian randomization was used to determine whether refractive error and intraocular pressure exert causal effects over PDS. MAIN OUTCOME MEASURES: The association of genetic variants with PDS and whether myopia exerts causal effects over PDS. RESULTS: Significant association was present at 2 novel loci for PDS/PG. These loci and follow-up analyses implicate the genes gamma secretase activator protein (GSAP) (lead single nucleotide polymorphism [SNP]: rs9641220, P = 6.0×10-10) and glutamate metabotropic receptor 5 (GRM5)/TYR (lead SNP: rs661177, P = 3.9×10-9) as important factors in disease risk. Mendelian randomization showed significant evidence that negative refractive error (myopia) exerts a direct causal effect over PDS (P = 8.86×10-7). CONCLUSIONS: Common SNPs relating to the GSAP and GRM5/TYR genes are associated risk factors for the development of PDS and PG. Although myopia is a known risk factor, this study uses genetic data to demonstrate that myopia is, in part, a cause of PDS and PG.

EFEMP1 rare variants cause familial juvenile-onset open-angle glaucoma.

Juvenile open-angle glaucoma (JOAG) is a severe type of glaucoma with onset before age 40 and dominant inheritance. Using exome sequencing we identified 3 independent families from the Philippines with novel EFEMP1 variants (c.238A>T, p.Asn80Tyr; c.1480T>C, p.Ter494Glnext*29; and c.1429C>T, p.Arg477Cys) co-segregating with disease. Affected variant carriers (N = 34) exhibited severe disease with average age of onset of 16 years and with 76% developing blindness. To investigate functional effects, we transfected COS7 cells with vectors expressing the three novel EFEMP1 variants and showed that all three variants found in JOAG patients caused significant intracellular protein aggregation and retention compared to wild type and also compared to EFEMP1 variants associated with other ocular phenotypes including an early-onset form of macular degeneration, Malattia Leventinese/Doyne's Honeycomb retinal dystrophy. These results suggest that rare EFEMP1 coding variants can cause JOAG through a mechanism involving protein aggregation and retention, and that the extent of intracellular retention correlates with disease phenotype. This is the first report of EFEMP1 variants causing JOAG, expanding the EFEMP1 disease spectrum. Our results suggest that EFEMP1 mutations appear to be a relatively common cause of JOAG in Filipino families, an ethnically diverse population.

Non-Synonymous variants in premelanosome protein (PMEL) cause ocular pigment dispersion and pigmentary glaucoma.

Pigmentary glaucoma (PG) is a common glaucoma subtype that results from release of pigment from the iris, called pigment dispersion syndrome (PDS), and its deposition throughout the anterior chamber of the eye. Although PG has a substantial heritable component, no causative genes have yet been identified. We used whole exome sequencing of two independent pedigrees to identify two premelanosome protein (PMEL) variants associated with heritable PDS/PG. PMEL encodes a key component of the melanosome, the organelle essential for melanin synthesis, storage and transport. Targeted screening of PMEL in three independent cohorts (n = 394) identified seven additional PDS/PG-associated non-synonymous variants. Five of the nine variants exhibited defective processing of the PMEL protein. In addition, analysis of PDS/PG-associated PMEL variants expressed in HeLa cells revealed structural changes to pseudomelanosomes indicating altered amyloid fibril formation in five of the nine variants. Introduction of 11-base pair deletions to the homologous pmela in zebrafish by the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 method caused profound pigmentation defects and enlarged anterior segments in the eye, further supporting PMEL's role in ocular pigmentation and function. Taken together, these data support a model in which missense PMEL variants represent dominant negative mutations that impair the ability of PMEL to form functional amyloid fibrils. While PMEL mutations have previously been shown to cause pigmentation and ocular defects in animals, this research is the first report of mutations in PMEL causing human disease.

LOXL1 Polymorphisms: Genetic Biomarkers that Presage Environmental Determinants of Exfoliation Syndrome.

An agnostic high throughput search of the genome revealed a robust association between LOXL1 genetic polymorphisms and exfoliation syndrome (XFS), a discovery that likely would not have been possible with candidate or family-based gene search strategies. While questions remain regarding how LOXL1 gene variants contribute to XFS pathogenesis, it is clear that the frequencies of disease-related alleles do not track with the varying disease burden throughout the world, prompting a search for environmental risk factors. A geo-medicine approach revealed that disease load seemed to increase as a function of the distance from the equator. The exact reason for this extraequatorial disease distribution pattern remains unclear, but a greater amount of time spent outdoors is a robust risk factor for XFS, suggesting climatic factors such as ocular solar exposure and colder ambient temperature may be involved in disease pathogenesis. Prospective studies have also implicated higher coffee consumption and lower dietary folate intake in association with incident XFS. The discovery of environmental risk factors for XFS suggests that preventive measures may help to reduce ocular morbidity from XFS.

A Role for Clusterin in Exfoliation Syndrome and Exfoliation Glaucoma?

The multifunctional protein clusterin (CLU) is a secreted glycoprotein ubiquitously expressed throughout the body, including in the eye. Its primary function is to act as an extracellular molecular chaperone, preventing the precipitation and aggregation of misfolded extracellular proteins. Clusterin is commonly identified at fluid-tissue interfaces, and has been identified in most body fluids. It is a component of exfoliation material, and CLU mRNA is reduced in eyes with exfoliation syndrome compared with controls. SNPs located in the CLU genomic region have been associated with Alzheimer disease (AD) at the genome-wide level and several CLU SNPs located in an apparent regulatory region have been nominally associated with XFS/XFG in Caucasians with European ancestry and in south Indians. Interestingly, clusterin associates with altered elastic fibers in human photoaged skin and prevents UV-induced elastin aggregation in vitro. In light of the known geographic risk factors for XFS/XFG, which could include UV light, investigations of CLU-geographic interactions could be of interest. Future studies investigating rare CLU variation and other complex interactions including gene-gene interactions in XFS/XFG cases and controls may also be fruitful. Although CLU has been considered as a therapeutic target in AD, cancer and dry eye, a role for clusterin in XFS/XFG needs to be better defined before therapeutic approaches involving CLU can be entertained.

Association between promoter -1607 polymorphism of MMP1 and lumbar disc disease in Southern Chinese.

BACKGROUND: Matrix metalloproteinases (MMPs) are involved in the degradation of the extracellular matrix of the intervertebral disc. A SNP for guanine insertion/deletion (G/D), the -1607 promoter polymorphism, of the MMP1 gene was found significantly affecting promoter activity and corresponding transcription level. Hence it is a good candidate for genetic studies in DDD. METHODS: Southern Chinese volunteers between 18 and 55 years were recruited from the population. DDD in the lumbar spine was defined by MRI using Schneiderman's classification. Genomic DNA was isolated from the leukocytes and genotyping was performed using the Sequenom platform. Association and Hardy-Weinberg equilibrium checking were assessed by Chi-square test and Mann-Whitney U test. RESULTS: Our results showed substantial evidence of association between -1607 promoter polymorphism of MMP1 and DDD in the Southern Chinese subjects. D allelic was significantly associated with DDD (p value = 0.027, odds ratio = 1.41 with 95% CI = 1.04-1.90) while Genotypic association on the presence of D allele was also significantly associated with DDD (p value = 0.046, odds ratio = 1.50 with 95% CI = 1.01-2.24). Further age stratification showed significant genotypic as well as allelic association in the group of over 40 years (genotypic: p value = 0.035, odds ratio = 1.617 with 95% CI = 1.033-2.529; allelic: p value = 0.033, odds ratio = 1.445 with 95% CI = 1.029-2.029). Disc bulge, annular tears and the Schmorl's nodes were not associated with the D allele. CONCLUSION: We demonstrated that individuals with the presence of D allele for the -1607 promoter polymorphism of MMP1 are about 1.5 times more susceptible to develop DDD when compared with those having G allele only. Further association was identified in individuals over 40 years of age. Disc bulge, annular tear as well as Schmorl's nodes were not associated with this polymorphism.

[Polymorphisms of myocilin and optineurin in primary open angle glaucoma patients].

OBJECTIVE: To detect the single nucleotide polymorphisms (SNPs) of the myocilin (MYOC) and optineurin (OPTN) genes, and to investigate their associations with high tension glaucoma (HTG) and normal tension glaucoma (NTG). METHODS: SNPs were detected using polymerase chain reaction (PCR), followed by conformation sensitive gel electrophoresis (CSGE) and fluorescent labeling automated DNA sequencing among 94 unrelated patients with HTG, 48 unrelated patients with NTG, and 77 unrelated control subjects. RESULTS: Fourteen MYOC sequence alterations were identified, five of them: V53A, I304I, T347T, 1-126T > C, and IVS2 + 172C > A, were novel. Among them, V53A was for the first time found in primary open angle glaucoma (POAG) patient. R76K usually occurred with the promoter polymorphism 1-83G > A. No sequence alterations in the MYOC gene showed significant differences among the HTG, NTG and control subjects (all P > 0.05). A total of 12 sequence alterations were identified in the OPTN gene, and three of them: V161M, I407T and L211L, were novel. Among them, I407T and L211L were found only in the HTG patients. The allele and genotype frequencies of T34T in the NTG patients were significantly higher than those of the controls (P = 0.001 and 0.004 respectively). In HTG, only the allele frequency of T34T was 24% (23/96), significantly higher than those of the NTG group (16.5%, 31/188) and the control group (9.1%, 14/154) (both P < 0.05). In addition, IVS8 + 20G > A was found only in the HTG (3.1%, 3/96) and NTG patients (3.7%, 7/188), and had significantly higher frequencies in the HTG and NTG patients when compared with the controls (P = 0.016 and 0.014, and P = 0.027 and 0.026). CONCLUSION: Polymorphisms in the MYOC and OPTN genes are associated with POAG in Chinese people. Moreover, sequence alterations not causing amino acid changes may play a role in the pathogenesis of POAG.

[Digenic association of RHO and RP1 genes with retinitis pigmentosa among Chinese population in Hong Kong].

OBJECTIVE: To identify the mutation patterns of RHO and RP1 genes in the Chinese patients with retinitis pigmentosa (RP) and to explore their potential interactions in the pathogenesis of RP. METHODS: Sequence alterations in the entire coding region and splice sites of RHO and RP1 gene were screened in 151 RP affected probands and 150 unrelated controls who were all Hong Kong Chinese. Additional 46 relatives of 12 RP probands carrying possible mutations in RHO or RP1 were recruited for segregation analysis. Univariate analysis, multivariate analysis and genotype-pedigree disequilibrium test were used to examine the associations of polymorphisms in these two genes with RP. RESULTS: Two mutations in the RHO gene, 5211delC and P347L, were identified each in one proband from the 151 probands, accounting for 1.3% of the RP patients. Two mutations in the RP1 gene, R677X and D984G, were identified each in one proband from the 151 probands, also accounting for 1.3% of the RP patients. In univariate analysis, non-coding sequence variants in the RHO gene, -26G > A, was found to increase the risk of RP, while R872H in the RP1 gene was likely to be a protective factor for RP. Multivariable logistic regression analysis and haplotype analysis confirmed these associations. CONCLUSION: The prevalences of RHO and RP1 mutations among the RP patients in Chinese population are both less than reported in other populations. Besides the disease-causing mutations, non-coding sequence alterations may also be a modifier for RP. The potential interactions between RHO and RP1 suggest a digenic etiology for RP.

[Recent progress in molecular genetics and gene therapy for retinitis pigmentosa].

Retinitis pigmentosa (RP) is a common genetic eye disease affecting about 1 in 3500 people worldwide with pan-ethnic occurrence. So far there is no effective treatment for RP. This paper gives an overview on recent advances in molecular genetics of RP with emphasis on the important gene mutations for diagnosis and prognosis, and a review on gene therapy of RP.

Polymorphisms in the myocilin promoter unrelated to the risk and severity of primary open-angle glaucoma.

PURPOSE: To investigate the proximal 2.5 kb promoter in the myocilin (MYOC) gene for mutations in Chinese patients with primary open-angle glaucoma (POAG). PATIENTS AND METHODS: We screened for sequence alterations in the MYOC promoter in 88 unrelated Chinese patients with POAG and 94 unrelated individuals without glaucoma, aged 50 years or above, as control subjects. In addition, the specific MYOC.mt1 polymorphism was determined in a total of 212 POAG patients and 221 control subjects. The relationships between POAG phenotype and the identified polymorphisms were studied by univariate analysis, multivariable logistic regression analysis, and haplotype analysis. RESULTS: All polymorphisms identified in this study followed Hardy-Weinberg equilibrium (P > 0.12) both in POAG patients and controls. Both univariate and multivariable logistic regression analyses showed no polymorphism that was significantly associated with the risk of POAG, P > 0.08 and P > 0.044 respectively. Haplotype analysis further indicated no association of MYOC promoter polymorphisms with the susceptibility for POAG (P > 0.1). On the other hand, there was no difference of POAG phenotypes among different genotypes of MYOC.mt1 (P > 0.31). CONCLUSIONS: In this study on the Chinese population, polymorphisms in the MYOC promoter are not related to the risk of POAG. There is no association between the MYOC.mt1 promoter polymorphism with the severity of POAG.

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.

[Single nucleotide polymorphisms of the myocilin gene in primary open-angle glaucoma patients].

OBJECTIVE: To detect single nucleotide polymorphisms (SNPs) of the myocilin (MYOC) gene and to investigate their associations with primary open-angle glaucoma (POAG). METHODS: One hundred and fifty-seven sporadic patients with POAG and 155 unrelated control subjects without POAG were recruited from staff and visitors to the Prince of Wales Hospital between 1998 and 2000. All study subjects are ethnic Chinese living in Hong Kong. The two populations were matched in frequencies of gender and age. The SNPs of the MYOC gene in POAG patients and control subjects were screened and identified by high throughout conformation sensitive gel electrophoresis and fluorescent labeling automated sequencing. The genotype frequencies of each SNP in the two groups were compared by the Chi2 test or Fisher's exact 2-tailed test. RESULTS: A total of seventeen SNPs were identified from 2172 bp long of the MYOC gene, including all 3 exons and adjacent non-coding regions. The identified SNPs were 1-83G --> A, G12R, P16L, A17S, R46X, R76K, R91X, T123T, D208E, L215P, 730+35A --> G, A260A, I288I, E300K, T353I, Y471C and 1515+73G --> C, respectively. Of these, R91X, E300K and Y471C were found only in POAG patients. A significant difference between POAG patients and control subjects was found in the genotype frequencies of 1515+73G --> C. The frequency of the heterozygote (CG) was 0.6% in POAG patients, significantly less than the 4.5% in control subjects (Fisher's exact 2-tailed test, P=0.036, OR=0.136, 95%CI=0.022-0.828). No significant difference was found between the two populations in genotype frequencies of all other SNPs. CONCLUSION: The polymorphisms of the MYOC gene may be related to POAG.

[Antibacterial effect of niaoluqing oral liquid on clinical drug-resistant strains and different serotype strains of Ureaplasma urealyticum in vitro].

OBJECTIVES: To study the antibacterial effect of Niaoluqing Oral Liquid (NOL) on clinical drug-resistant strains and 14 serotype strains Ureaplasma Urealyticum (UU). METHODS: Sixty-three clinical strains of UU were detected to determine their serology and antibiotic susceptibilities by the metabolic inhibition test (MIT). Mininum inhibitory concentration (MIC) was used to evaluate the sensitivity of NOL to different serotypes of UU. The sensitivity of NOL, erythromycin and tetracycline to 63 clinical strains of UU was also studied. RESULTS: In 63 clinical strains of UU, the range of MIC to NOL was from 0.48 mg/ml to 15.63 mg/ml, MIC50 < or = 1.95 mg/ml, MIC90 < or = 3.91 mg/ml. Among them, 31 strains were resistant to tetracycline and 31 were resistant to erythromycin. No obvious correlation between the sensitivity of NOL to UU clinical strains and that of erythromycin and tetracycline to UU clinical strains (P > 0.05). Clinical strains of UU in this experiment contains all of its serotypes, also having a higher sensitivity to NOL (MIC < or = 3.91 mg/ml) except serology 1, 2, 3 and 11 (MIC > or = 7.81 mg/ml). CONCLUSIONS: NOL exerts a strong in vitro antibacterial effect on erythromycin-resistant and tetracycline-resistant clinical strains of UU. All kinds of serotype strains had a higher sensitivity to NOL, too. Chinese medicinal herbs are of momentous significance in the treatment of UU infection.

[Case control study on the risk factors of primary open angle glaucoma in China].

OBJECTIVE: The purpose of this study is to comprehensively explore the risk factors of primary open angle glaucoma (POAG) in China. METHODS: Two groups of data based on distinct resources were analyzed to explore the risk factors of POAG. One group of data was based on hospital records between 1995 and 2000 which composed of 107 patients with POAG and 149 controls without POAG. The other group of data was based on 40 patients with POAG and 120 matched controls without POAG. The former was designed by non-matched case control study, the latter was done by 1:3 matched case control study. The relationships between POAG and the factors such as age, sex, family history, diabetes, hypertension, intraocular pressure (IOP), cardiovascular diseases, smoking, drinking and mutation of TIGR gene were studied by logistic regression analysis. RESULTS: The simple factor analysis showed that the risk of POAG was related to age, family history, hypertension, IOP, cardiovascular diseases, smoking, drinking and the mutation of TIGR gene (T353I). However, logistic regression analysis confirmed that POAG mainly related to IOP, family history, hypertension, smoking, alcohol intake and the mutation of TIGR gene. CONCLUSION: The most important risk factor of POAG was IOP. Family history, hypertension, smoking and the mutation of TIGR gene were also important risk factors of POAG. However, alcohol intake was a protective factor for POAG.

TIGR/MYOC gene sequence alterations in individuals with and without primary open-angle glaucoma.

PURPOSE: To discover sequence alterations in the TIGR/MYOC gene associated with primary open-angle glaucoma (POAG) in Chinese subjects. METHODS: Two hundred one unrelated Chinese patients with POAG and 291 unrelated individuals without glaucoma, aged 50 years or more, were screened for sequence alterations in the TIGR/MYOC gene by polymerase chain reaction, conformation sensitive gel electrophoresis, and DNA sequencing. Up to 111 more control subjects were screened for some of the alterations. RESULTS: Fourteen sequence variants that lead to amino acid changes were identified. Seven were novel: Pro16Leu, Ala17Ser, Leu95Pro, Leu215Pro, Glu300Lys, Glu414Lys, and Tyr471Cys. Of these, Glu300Lys and Tyr471Cys were found only in POAG. Arg46Stop was found in 4 patients with POAG (2.0%) and 9 of 402 control subjects (2.2%); one control subject was homozygous. IOP showed a trend (P = 0.11) toward a decrease of 1.5 mm Hg among the control subjects, with Arg46Stop compared with matched control subjects without Arg46Stop. Gly12Arg occurred four times as frequently in control subjects as in patients, but the difference was not statistically significant. CONCLUSIONS: Gly12Arg might be negatively associated with POAG, suggesting a protective effect. Three patients with POAG had a sequence change not found in control subjects, for a frequency of possible disease-causing TIGR/MYOC mutations of 1.5% (95% confidence interval [CI] = 0.3%-4.3%). Arg46Stop occurred with similar frequency in patients with POAG and control subjects, suggesting that the reduced amount of TIGR/MYOC predicted to result from this truncation does not dramatically increase or decrease risk of glaucoma.

[Study on single nucleotide polymorphism of TIGR gene in primary open-angle glaucoma patients].

OBJECTIVE: To detect the single nucleotide polymorphism (SNP) of trabecular meshwork-induced glucocorticoid response protein (TIGR) gene and to investigate the association between SNP and primary open-angle glaucoma (POAG) in Chinese Han population. METHODS: High throughput conformation sensitive gel electrophoresis (HTCSGE) and fluorescent labeling automated sequencing method were used to screen and identify the SNPs of TIGR in 82 unrelated patients with POAG. Restriction endonuclease analysis was used to detect the SNPs of TIGR in 150 sex-and age-matched unrelated control subjects without POAG. The frequencies of genotypes and alleles of each SNP between the two groups were compared by chi-square test. RESULTS: A total of six SNPs were identified in TIGR gene with a length of 2172 bp, including all coding regions and part of promoter, introns and 3'UTR region: 1-83(G --> A), G12R, R46X, R76K, IVS2 + 35(A --> G), and T353I. Significant differences were found in frequencies of the genotype TC and the allele T of T353I between POAG patients and control subjects. The frequency of heterozygote TC was 12.20% in POAG patients, significantly higher than that in control subjects (3.33%, kappa(2) = 6.885, P = 0.009, OR = 4.028, OR95% CI = 1.327 approximately 12.223). The frequency of the allele T was 6.10% in POAG patients, significantly higher than that in control subjects (1.67%, kappa(2) = 6.655, P = 0.010). No significant difference was found between the two groups in frequencies of genotypes and alleles of the other SNPs. CONCLUSION: The T353I polymorphism of TIGR gene is associated with POAG in Chinese Han population.

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.