Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing.

PURPOSE: Next-generation sequencing-based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques with regard to test accuracy and reproducibility have not been fully defined. METHODS: We developed a targeted enrichment and next-generation sequencing approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy, and glaucoma. In preparation for providing this genetic eye disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, and reproducibility, as well as the clinical sensitivity, of the test. RESULTS: The GEDi test is highly reproducible and accurate, with sensitivity and specificity of 97.9 and 100%, respectively, for single-nucleotide variant detection. The sensitivity for variant detection was notably better than the 88.3% achieved by whole-exome sequencing using the same metrics, because of better coverage of targeted genes in the GEDi test as compared with a commercially available exome capture set. Prospective testing of 192 patients with inherited retinal degenerations indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%. CONCLUSION: Based on quantified performance metrics, the data suggest that selective targeted enrichment is preferable to whole-exome sequencing for genetic diagnostic testing.

Different WDR36 mutation pattern in Chinese patients with primary open-angle glaucoma.

PURPOSE: To determine the distribution of WD repeat domain 36 (WDR36) sequence variants in Chinese patients with primary open-angle glaucoma (POAG). METHODS: One hundred and thirty-five unrelated POAG patients (82 high tension glaucoma [HTG], 42 normal tension glaucoma [NTG], and 11 juvenile-onset POAG [JOAG] patients) and 77 unrelated controls were recruited. All 23 coding exons and splicing junctions of WDR36 were sequenced using BigDye Terminator v3.1 cycle sequencing kit. Single nucleotide polymorphism (SNP) and haplotype associations were analyzed using PLINK (version 1.04). RESULTS: Nineteen sequence alterations were identified, and eight of them were novel including two novel nonsynonymous SNPs (L240V and I713V). Except the common I264V polymorphism, no other previously reported disease-causing or disease-susceptibility mutations were found. The novel I713V mutation was observed in three (3.7%) patients with HTG. One intronic SNP, IVS5+30C>T (rs10038177), showed significantly higher frequency of minor allele T in HTG patients (16.5%) than in controls (1.3%; Odds ratio [OR]=15.0, p=7.9 x 10(-7), Bonferroni corrected p=1.5 x 10(-5)). Haplotype GTA, which is composed of rs13153937, rs10038177, and rs11241095, was significantly associated with HTG (OR=22.5, p=0.002, Bonferroni corrected p=0.013). Neither the individual SNPs nor haplotypes of WDR36 were associated with NTG or JOAG (Bonferroni corrected p>0.05). CONCLUSIONS: Findings in this study suggest WDR36 to be associated with sporadic HTG but not with NTG or JOAG. Our results also suggest a different mutation pattern of WDR36 in the Chinese population from other ethnic populations.

AC and AG dinucleotide repeats in the PAX6 P1 promoter are associated with high myopia.

PURPOSE: The PAX6 gene, located at the reported myopia locus MYP7 on chromosome 11p13, was postulated to be associated with myopia development. This study investigated the association of PAX6 with high myopia in 379 high myopia patients and 349 controls. METHODS: High myopia patients had refractive errors of -6.00 diopters or greater and axial length longer than 26 mm. Control subjects had refractive errors less than -1.00 diopter and axial length shorter than 24 mm. The P1 promoter, all coding sequences, and adjacent splice-site regions of the PAX6 gene were screened in all study subjects by polymerase chain reaction and direct sequencing. PAX6 P1 promoter-luciferase constructs with variable AC and AG repeat lengths were prepared and transfected into human ARPE-19 cells prior to assaying for their transcriptional activities. RESULTS: No sequence alterations in the coding or splicing regions showed an association with high myopia. Two dinucleotide repeats, (AC)(m) and (AG)(n), in the P1 promoter region were found to be highly polymorphic and significantly associated with high myopia. Higher repeat numbers were observed in high myopia patients for both (AC)(m) (empirical p = 0.013) and (AG)(n) (empirical p = 0.012) dinucleotide polymorphisms, with a 1.327-fold increased risk associated with the (AG)(n) repeat (empirical p = 0.016; 95% confidence interval: 1.059-1.663). Luciferase-reporter analysis showed elevated transcription activity with increasing individual (AC)(m) and (AG)(n) and combined (AC)(m)(AG)(n) repeat lengths. CONCLUSIONS: Our results revealed an association between high myopia and AC and AG dinucleotide repeat lengths in the PAX6 P1 promoter, indicating the involvement of PAX6 in the pathogenesis of high myopia.

Gene expression profiles of human trabecular meshwork cells induced by triamcinolone and dexamethasone.

PURPOSE: Triamcinolone acetonide (TA) and dexamethasone (DEX) are corticosteroids commonly used for ocular inflammation, but both can cause ocular hypertension. In this study, the differential gene expression profile of human trabecular meshwork (TM) cells in response to treatment by TA in comparison with DEX was investigated. METHODS: Total RNA was extracted from cultured human TM cells treated with TA or DEX and used for microarray gene expression analysis. The microarray experiments were repeated three times. Differentially expressed genes were identified by an empiric Bayes approach and confirmed by real-time quantitative PCR. RESULTS: TA (0.1 mg/mL) treatment resulted in 15 genes upregulated and 12 genes downregulated, whereas 1 mg/mL TA resulted in 36 genes upregulated and 21 genes downregulated. These genes were mainly associated with acute-phase response, cell adhesion, cell cycle and growth, growth factor, ion binding, metabolism, proteolysis and transcription factor. Two genes, MYOC and GAS1, were upregulated, and three genes, SENP1, ZNF343, and SOX30, were downregulated by both TA and DEX treatment. Eight differentially expressed genes were located in known primary open-angle glaucoma (POAG) loci, including MYOC, SOAT1, CYP27A1, SPOCK, SEMA6A, EGR1, GAS1, and ATP10A. CONCLUSIONS: Differential gene expression profiles of human TM cells treated by TA and DEX, and a dosage effect by TA, were revealed by microarray technology. TA and DEX treatment shared several differentially expressed genes, suggesting a common mechanism to cause ocular hypertension. Some differentially expressed genes located in the known POAG loci are potential candidates for glaucoma genes.

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.

Fine mapping of new glaucoma locus GLC1M and exclusion of neuregulin 2 as the causative gene.

PURPOSE: We recently identified a novel glaucoma locus on 5q22.1-q32, designated as GLC1M, in a family from the Philippines with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG). No mutations in myocilin (MYOC), optineurin (OPTN), and WD-repeat protein 36 (WDR36) were found. Neuregulin 2 (NRG2) is an excellent potential functional as well as positional candidate at GLC1M. The goal of the present study was to evaluate the role of the NRG2 gene in this JOAG family and unrelated JOAG patients and to refine the critical interval for GLC1M. METHODS: Genomic DNA was obtained from 27 family members. All coding exons and splicing sites of NRG2 were screened for sequence alterations by polymerase chain reaction (PCR) and DNA sequencing. A cohort of 92 unrelated JOAG patients and 92 control subjects were genotyped for the three single nucleotide polymorphisms (SNPs) of NRG2 by PCR and DNA sequencing. Haplotype and segregation analyses were performed in the family. Fisher's exact test was used to compare the frequencies of the NRG2 polymorphisms between affected and unaffected subjects in the family and between unrelated JOAG patients and control subjects. RESULTS: Three SNPs were identified: c.98G>A (S33N), IVS3+13A>G (rs889022), and c.1976A>G (G659G). None of them segregated with the JOAG phenotype in this family. No association was found between NRG2 and JOAG in the case-control study (p>0.12). However, further inspection of the haplotypes in the family localized the NRG2 gene telomeric to the disease locus. The critical interval of GLC1M was therefore refined to a region of 28 Mb between D5S2051 and NRG2. CONCLUSIONS: The linkage interval for GLC1M was refined to a smaller region. The NRG2 gene was excluded as the causative gene for JOAG.

Veronicastrum axillare Alleviates Ethanol-Induced Injury on Gastric Epithelial Cells via Downregulation of the NF-kB Signaling Pathway.

We used human gastric epithelial cells (GES-1) line in an ethanol-induced cell damage model to study the protective effect of Veronicastrum axillare and its modulation to NF-κB signal pathway. The goal was to probe the molecular mechanism of V. axillare decoction in the prevention of gastric ulcer and therefore provide guidance in the clinical application of V. axillare on treating injuries from chronic nephritis, pleural effusion, gastric ulcer, and other ailments. The effects of V. axillare-loaded serums on cell viability were detected by MTT assays. Enzyme-linked immunosorbent assay (ELISA) and Real-Time PCR methods were used to analyze the protein and mRNA expression of TNF-α, NF-κB, IκBα, and IKKß. The results showed that V. axillare-loaded serum partially reversed the damaging effects of ethanol and NF-κB activator (phorbol-12-myristate-13-acetate: PMA) and increased cell viability. The protein and mRNA expressions of TNF-α, NF-κB, IκBα, and IKKß were significantly upregulated by ethanol and PMA while they were downregulated by V. axillare-loaded serum. In summary, V. axillare-loaded serum has significantly protective effect on GES-1 against ethanol-induced injury. The protective effect was likely linked to downregulation of TNF-α based NF-κB signal pathway.

A genome-wide scan maps a novel juvenile-onset primary open-angle glaucoma locus to 15q.

PURPOSE: To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open-angle glaucoma (JOAG). METHODS: A complete ophthalmic examination was conducted, and genomic DNA was obtained from 25 members of a Chinese family, of which eight were confirmed as having JOAG. Myocilin (MYOC), optineurin (OPTN), and WD repeat-domain 36 (WDR36) were screened for sequence alterations, by PCR and direct sequencing. Subsequently, a genome-wide scan was performed (Prism Linkage Mapping Set MD-10; Applied Biosystems, Inc., Foster City, CA). Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on 15q were analyzed. The significance of the lod score was tested with simulation analyses by using FASTLINK. Haplotypes were constructed with Simwalk2. Three candidate genes, NR2E3, SMAD6, and CLN6, located within the critical region, were screened for mutations. RESULTS: MYOC, OPTN, and WDR36 mutations were excluded in all family members. A maximum two-point lod score of 3.31 at theta = 0.0 was obtained for the marker D15S125. Four adjacent markers, rs2030040, rs169169963, D15S153, and D15S131, gave two-point lod scores of 2.41, 2.90, 3.02, and 2.68, respectively, at theta = 0.0. Haplotype analysis and recombination mapping further confined this region to 15q22-q24 within a genetic distance of 16.6 Mb flanked by D15S1036 and rs922693. No mutations were found in the coding exons and splicing junctions of NR2E3, SMAD6, and CLN6. CONCLUSIONS: The results provide evidence for the mapping of a novel locus for JOAG at 15q22-q24. A further search for the disease-causing gene in this new JOAG locus is in progress.

A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q.

PURPOSE: To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG) and to screen the novel glaucoma gene WD repeat domain 36 (WDR36). METHODS: Complete ophthalmic examination and genomic DNA were obtained from 27 family members, in which nine were confirmed JOAG patients. Myocilin (MYOC), optineurin (OPTN), and WDR36 were screened for mutations by polymerase chain reaction and direct sequencing. Genome-wide scanning was carried out using the ABI PRISM Linkage Mapping Set MD-10. Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on chromosome 5q were also analyzed. The significance of LOD scores was tested with simulation analyses using FASTLINK. Haplotypes were constructed using Simwalk2. RESULTS: MYOC or OPTN mutations were excluded in all family members. A maximum LOD score value of 4.82 at theta=0.00 was obtained for the marker D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at theta=0.00. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32 within a region of 36 Mb flanked by D5S2051 and D5S2090. Screening of the novel WDR36 glaucoma-associated gene, which lies centromeric to the disease interval, revealed no mutations within any of the 23 coding exons or splicing junctions. CONCLUSIONS: Our results provided the mapping of a novel locus for JOAG at 5q and excluded coding or splicing junctions mutations within the WDR36 gene.

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.

Gene mapping for primary open angle glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of visual impairment and blindness worldwide. To date, at least 20 genetic loci for POAG have been reported. Only 3 causative genes are identified from these loci: myocilin (MYOC), optineurin (OPTN) and WD repeat domain 36 (WDR36), which together account for less than 10% of POAG. Only a portion of POAG follows Mendelian inheritance, and a considerable fraction results from a large number of variants in several genes, each contributing small effects. Over the past 10 years, there has been vigorous research on mapping the POAG genes. The main technological approaches are functional cloning, family linkage analysis, genome-wide scan, case-control association study, and microarray analysis. Association studies found 16 genes related to POAG, but reports on glaucoma-causing effects of these genes are conflicting. Ten microarray gene expression studies related to POAG have been published. A number of genes potentially related to POAG have been identified, and they provide a good resource to select candidate genes for mutation analysis in association studies. While linkage studies remain a mainstay, the current trend is to use genome-wide association studies to map genes for POAG. This review gives an overview of the efforts in the past decade to identify the POAG genes through linkage studies, genome-wide scans, case-control association studies and microarray studies. In the near future such comprehensive studies are expected to greatly advance our understanding of the genetic basis of POAG and provide information for effective glaucoma therapy.

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.

Association of apolipoprotein E polymorphisms with normal tension glaucoma in a Chinese population.

PURPOSE: To evaluate the role of apolipoprotein E (APOE) polymorphisms in primary open angle glaucoma (POAG). METHODS: A cohort of 400 unrelated Chinese POAG patients was examined, including 294 cases of high tension glaucoma (HTG) and 106 with normal tension glaucoma (NTG). Also studied were 300 unrelated Chinese control subjects. The genotypes of the APOE polymorphisms in exon 4 and in the promoter at positions -491, -427, and -219 were determined by polymerase chain reaction and restriction endonuclease analysis. Frequencies of the genotypes were compared between patients and controls by chi test or Fisher exact test. The association of APOE polymorphisms with POAG phenotypes including age at diagnosis, intraocular pressure (IOP) at diagnosis, highest IOP, cup-disc ratio, and visual field score was investigated by the Kruskal-Wallis test. RESULTS: No significant difference was detected in the frequencies of APOE promoter polymorphisms between POAG patients and control subjects (P>0.0125). For the exon 4 polymorphism, when compared with control subjects, the frequency of epsilon 4 carriers was significantly lower in patients with NTG (P=0.008; odds ratio=0.36, 95% confidence interval=0.17, 0.79) but not in HTG (P=0.07). Compared with -219TT, the -219G carriers had a significant higher age at diagnosis (P=0.0046). No significant association was found between other APOE polymorphisms and POAG phenotypes (P>0.07). CONCLUSIONS: Our findings suggest that the APOE epsilon 4 allele confers a protective effect against NTG, whereas the APOE promoter polymorphisms do not contribute to POAG risk. However, the APOE -219G carriers tended to have later-onset POAG.

[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.

SNPs and interaction analyses of myocilin, optineurin, and apolipoprotein E in primary open angle glaucoma patients.

PURPOSE: To evaluate the association of myocilin (MYOC), optineurin (OPTN), and apolipoprotein E (APOE) genes and their interactions in primary open angle glaucoma (POAG). METHODS: A cohort of 400 unrelated POAG patients (294 high tension glaucoma, HTG, and 106 normal tension glaucoma, NTG) and 281 unrelated control subjects were recruited. All coding exons and splicing junctions in MYOC and OPTN were screened for sequence alterations. Common polymorphisms in APOE were genotyped. Single genes were investigated by univariate and haplotype analysis, and gene-gene interactions by logistic regression and stratified analysis. Multiple comparisons were corrected by the Bonferroni method. Bioinformatics analysis was performed to assess the conservation of mutation sites across species and to predict putative motifs and secondary structures in mutated proteins. RESULTS: Disease-causing mutations in MYOC and OPTN were identified in 1.75% and 1% of POAG patients, respectively. Most of these mutations were highly conserved across species, many predicted to create new motifs or change protein secondary structures. No individual MYOC polymorphisms significantly contributed to HTG or NTG. A haplotype containing the minor allele of the MYOC IVS2+35A>G increased NTG risk (p=0.0001). Three OPTN polymorphisms, T34T, IVS5+38T>G, and IVS8-53T>C increased NTG risk (p<0.0008), while IVS5+38T>G increased HTG risk (p=0.0006). One haplotype that contains the minor alleles of 3 OPTN polymorphisms, T34T, IVS5+38T>G, and IVS7+24G>A, increased NTG risk (p=0.0002). APOE epsilon4 carriers had a decreased NTG risk (p=0.007). Possible gene-gene interactions were found between MYOC, OPTN, and APOE. CONCLUSIONS: Disease-causing mutations in MYOC and OPTN accounted for only a small proportion of Chinese POAG patients. Common polymorphisms in MYOC, OPTN, and APOE might interactively contribute to POAG, indicating a polygenic etiology.

[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.

Absence of myocilin and optineurin mutations in a large Philippine family with juvenile onset primary open angle glaucoma.

PURPOSE: To analyze the role of the two primary open angle glaucoma (POAG) genes, myocilin (MYOC) and optineurin (OPTN), in a large Philippine family segregating autosomal dominant juvenile onset open angle glaucoma (JOAG). METHODS: The coding sequences of the MYOC and OPTN genes were screened in 27 family members by polymerase chain reaction and direct sequencing. The specific MYOC promoter polymorphism (MYOC.mtl) was identified by restriction endonuclease assay. All of the ABI MD-10 microsatellite markers on chromosomes 1, 2, 3, 7, 8, and 10, which harbor the six known POAG loci, were analyzed for linkage with POAG. RESULTS: No mutation was identified in this large kindred. Instead, three polymorphisms (-80G->A, -1000G->C, R76K) in MYOC and four polymorphisms (T34T, M98K, R545Q, IVS7+24G->A) in OPTN were found. All markers flanking the six known POAG loci gave LOD scores not more than 1.1. Non-parametric linkage analysis for all these markers resulted in p values more than 0.05. CONCLUSIONS: Both mutation testing and linkage analysis provide strong evidence against MYOC and OPTN being the causative gene in this large family. It indicates that unidentified genes will underlie the occurrence of glaucoma in this family.

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.

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.