Identification of Missense Extracellular Matrix Gene Variants in a Large Glaucoma Pedigree and Investigation of the N700S Thrombospondin-1 Variant in Normal and Glaucomatous Trabecular Meshwork Cells.

PURPOSE: Primary open-angle glaucoma (POAG) is a complex heterogeneous disease. While several POAG genes have been identified, a high proportion of estimated heritability remains unexplained. Elevated intraocular pressure (IOP) is a leading POAG risk factor and dysfunctional extracellular matrix (ECM) in the trabecular meshwork (TM) contributes to elevated IOP. In this study, we sought to identify missense variants in ECM genes that correlate with ocular hypertensive POAG. METHODS: Whole-genome sequencing was used to identify genetic variants in five members of a large POAG family (n = 68) with elevated IOP. The remaining family members were screened by Sanger sequencing. Unrelated normal (NTM) and glaucomatous (GTM) cells were sequenced for the identified variants. The ECM protein levels were determined by Western immunoblotting and confocal and electron microscopy investigated ECM ultrastructural organization. RESULTS: Three ECM gene variants were significantly associated with POAG or elevated IOP in a large POAG pedigree. These included rs2228262 (N700S; thrombospondin-1 (THBS1, TSP1)), rs112913396 (D563 G; collagen type VI, alpha 3 (COL6A3)) and rs34759087 (E987K; laminin subunit beta 2 (LAMB2)). Screening of unrelated TM cells (n = 27) showed higher prevalence of the THBS1 variant but not the LAMB2 variant, in GTM cells (39%) than NTM cells (11%). The rare COL6A3 variant was not detected. TSP1 protein was upregulated and COL6A3 was down-regulated in TM cells with N700S subject to mechanical stretch, an in vitro method that mimics elevated IOP. Immunofluorescence showed increased TSP1 immunostaining in cell strains with N700S compared to wild-type TM cells. Ultrastructural studies showed ECM disorganization and altered collagen type VI distribution in GTM versus NTM cells. CONCLUSIONS: Our results suggest that missense variants in ECM genes may not cause catastrophic changes to the TM, but over many years, subtle changes in ECM may accumulate and cause structural disorganization of the outflow resistance leading to elevated IOP in POAG patients.

Analysis of interleukin-20 receptor complexes in trabecular meshwork cells and effects of cytokine signaling in anterior segment perfusion culture.

Purpose: Inflammatory responses may be involved in the glaucomatous process. Our previous studies mapped a T104M mutation in interleukin-20 receptor beta (IL-20RB) in a family with primary open angle glaucoma (POAG). IL-20RB can heterodimerize with IL-20RA to propagate signals from IL-20 family cytokines, IL-19, IL-20, and IL-24 (the type I receptor complex), or it can heterodimerize with IL-22RA to propagate signals from IL-20 and IL-24 (type II receptor complex). In this study, we investigated IL-20 heterodimeric receptor complexes in the trabecular meshwork (TM) compared to dermal fibroblast cell cultures, and examined the phosphorylation of signal transducer and activator of transcription (STAT)-1, -3, and -5 following exposure to IL-20 family cytokines. Additionally, we determined the effects of IL-20 family cytokines on outflow rates in anterior segment perfusion culture, an in vitro model of intraocular pressure (IOP) regulation. Methods: Primary human TM (HTM) cells were grown from dissected TM tissue, and IL-20 receptor expression was investigated with PCR. A Duolink assay was performed to investigate in situ IL-20 receptor protein interactions in HTM or dermal fibroblasts, and Imaris software was used to quantitate the association of the heterodimeric complexes. Phosphorylation of STAT-1, -3, and -5 were evaluated in HTM or dermal fibroblasts using Western immunoblotting after exposure to IL-10, IL-19, IL-20, IL-22, or IL-24. Anterior segment perfusion culture was performed in human cadaver and porcine eyes treated with IL-20, IL-19, or IL-24. Results: All of the IL-20 receptors, IL-20RA, IL-20RB, and IL-22RA1 were expressed in HTM cells. Two isoforms of IL-20RA were expressed: The V1 variant, which is the longest, is the predominant isoform, while the V3 isoform, which lacks exon 3, was also expressed. The Duolink assay demonstrated that the type I (IL-20RA-IL-20RB) and type II (IL-22RA1-IL-20RB) receptors were expressed in HTM cells and dermal fibroblasts. However, in the HTM cells, the type I receptor was present at significantly higher levels, while the type II receptor was preferentially used in the dermal fibroblasts. The HTM cells and the dermal fibroblasts predominantly phosphorylate the Ser727 site in STAT-3. The dermal fibroblasts had higher induction of phosphorylated STAT-1 compared to the HTM cells, while neither cell type had phosphorylated STAT-5 in the cell lysates. The outflow rates in the human anterior segment cultures were increased 2.3-fold by IL-20. However, IL-19 and IL-24 showed differential responses. For IL-19 and IL-24, 50% of the eyes responded with a 1.7- or 1.5-fold increase, respectively, while the other half did not respond. Similarly, perfused porcine anterior segments showed "responders" and "non-responders": IL-20 responders (2.3-fold increase in outflow, n=12) and non-responders (n=11); IL-19 responders (2.1-fold increase, n=7) and non-responders (n=5); and IL-24 responders (1.8-fold increase, n=12) and non-responders (n=5). Conclusions: Type I and type II IL-20 receptor complexes are expressed in human TM cells with predominant expression of the type I receptor (IL-20RA and IL-20RB), which propagates signals from all three IL-20 family cytokines. However, there was a variable response in the outflow rates following perfusion of cytokines in two different species. This may explain why some people are more susceptible to developing elevated IOP in response to inflammation.

Consensus recommendations for trabecular meshwork cell isolation, characterization and culture.

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma.

Evidence is accumulating to suggest that mutations in the Ankyrin and SOCS Box-containing protein-10 (ASB10) gene are associated with glaucoma. Since its identification in a large Oregon family with primary open-angle glaucoma (POAG), ASB10 variants have been associated with disease in US, German and Pakistani cohorts. ASB10 is a member of the ASB family of proteins, which have a common structure including a unique N-terminus, a variable number of central ankyrin (ANK) repeat domains and a suppressor of cytokine signaling (SOCS) box at the C-terminus. Mutations in ASB10 are distributed throughout the entire length of the gene including the two alternatively spliced variants of exon 1. A homozygous mutation in a Pakistani individual with POAG, which lies in the center of the SOCS box, is associated with a particularly severe form of the disease. Like other SOCS box-containing proteins, ASB10 functions in ubiquitin-mediated degradation pathways. The ANK repeats bind to proteins destined for degradation. The SOCS box recruits ubiquitin ligase proteins to form a complex to transfer ubiquitin to a substrate bound to the ANK repeats. The ubiquitin-tagged protein then enters either the proteasomal degradation pathway or the autophagic-lysosomal pathway. The choice of pathway appears to be dependent on which lysine residues are used to build polyubiquitin chains. However, these reciprocal pathways work in tandem to degrade proteins because inhibition of one pathway increases degradation via the other pathway. In this publication, we will review the literature that supports identification of ASB10 as a glaucoma-associated gene and the current knowledge of the function of the ASB10 protein. In addition, we present new data that indicates ASB10 expression is up-regulated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1α. Finally, we will describe the emerging role of other SOCS box-containing proteins in protein degradation pathways in ocular cells.

The Role of the IL-20 Subfamily in Glaucoma.

Glaucoma is a common disease that leads to loss of peripheral vision and, if left untreated, ultimately to blindness. While the exact cause(s) of glaucoma is still unknown, two leading risk factors are age and elevated intraocular pressure. Several studies suggest a possible link between glaucoma and inflammation in humans and animal models. In particular, our lab recently identified a T104M mutation in IL-20 receptor-B (IL-20RB) in primary open angle glaucoma patients from a large pedigree. Several of the interleukin- (IL-) 20 family of cytokines and receptors are expressed in ocular tissues including the trabecular meshwork, optic nerve head, and retinal ganglion cells. The DBA/2J mouse develops high intraocular pressures with age and has characteristic optic nerve defects that make it a useful glaucoma model. IL-24 expression is significantly upregulated in the retina of these mice, while IL-20RA expression in the optic nerve is downregulated following pressure-induced damage. The identification of a mutation in the IL-20RB gene in a glaucoma pedigree and changes in expression levels of IL-20 family members in the DBA/2J mouse suggest that disruption of normal IL-20 signaling in the eye may contribute to degenerative processes associated with glaucoma.

Interleukin-20 receptor expression in the trabecular meshwork and its implication in glaucoma.

PURPOSE: To determine whether interleukin-20 receptors (IL-20R) are expressed in trabecular meshwork cells and the effect of a T104M mutation in IL-20R2 on downstream cellular functions. METHODS: Evaluation of signal transducer and activator of transcription (STAT)3 phosphorylation and generic matrix metalloproteinase (MMP) activity in primary open angle glaucoma (POAG) dermal fibroblasts (pHDF) with the T104M IL-20R2 mutation were compared with normal human dermal fibroblasts (HDF). Expression of IL-20R1 and IL-20R2 in human trabecular meshwork (HTM) cells was determined by immunohistochemistry and western immunoblotting. RESULTS: A T104M mutation in IL20-R2 was identified in a large POAG family in which the GLC1C locus was originally mapped. pHDFs harboring this mutation had significantly increased phosphorylated STAT3 (pSTAT3) activity compared with normal HDFs. However, stimulation with either IL-19 or IL-20 for 15 min resulted in significantly decreased levels of pSTAT3 in pHDFs compared with controls. Generic MMP activity was significantly decreased in pHDFs compared with controls after stimulation with IL-20 for 24 h. Both IL-20R1 and IL-20R2 receptors were expressed in HTM cells by western immunoblot and immunofluorescence, and they appeared to be up-regulated in response to cytokine treatment. CONCLUSIONS: A T104M mutation in IL-20R2 significantly impacts the function of this receptor as shown by decreased pSTAT3 levels and generic MMP activity. Reduced MMP activity may affect the ability of glaucoma patients to alter outflow resistance in response to elevated intraocular pressure.

Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells.

PURPOSE: Ankyrin repeat and suppressor of cytokine signaling (SOCS) box containing protein-10 (ASB10) was recently identified as a gene that causes primary open-angle glaucoma. Here, we investigated endogenous ASB10 protein expression in human trabecular meshwork (HTM) cells to provide the first clues to the biologic function of this protein. METHODS: Primary HTM cells were cultured and immunostained with anti-ASB10 and various biomarkers of the ubiquitin-mediated proteasomal and autophagy-lysosomal degradation pathways. Cells were imaged with confocal and high-resolution structured illumination microscopy. Colocalization was quantified using Imaris Bitplane software, which generated a Pearson's correlation coefficient value. Coimmunoprecipitation of ASB10-transfected cells was performed. RESULTS: Immunofluorescence and confocal analysis showed that ASB10 was localized in intracellular structures in HTM cells. Two populations were observed: small, spherical vesicles and larger, less abundant structures. In the ASB10-silenced cells, the number of large structures was significantly decreased. ASB10 partially colocalized with biomarkers of the ubiquitin-mediated proteasomal pathway including ubiquitin and the α4 subunit of the 20S proteasome. However, ASB10 itself was not ubiquitinated. ASB10 also colocalized with numerous biomarkers of specific autophagic structures: aggresomes (histone deacetylase 6 [HDAC6] and heat shock protein 70 [HSP70]), autophagosomes (light chain 3 [LC3] and p62), amphisomes (Rab7), and lysosomes (lysosomal-associated membrane protein 1 [LAMP1]). Pearson coefficients indicated strong colocalization of large ASB10-stained structures with the α4 subunit of the 20S proteasome, K48 and K63-linked ubiquitin antibodies, p62, HSP70, and HDAC6 (Pearson's range, 0.59-0.82). Coimmunoprecipitation assays showed a positive interaction of ASB10 with HSP70 and with the α4 subunit of the 20S proteasome. Super-resolution structured illumination confocal microscopy suggested that the smaller ASB10-stained vesicles aggregated into the larger structures, which resembled aggresome-like induced structures. Treatment of HTM cells with an autophagy activator (MG132) or inhibitors (wortmannin, bafilomycin A1) significantly increased and decreased the number of small ASB10-stained vesicles, respectively. No discernible differences in the colocalization of large ASB10-stained structures with ubiquitin or HDAC6 were observed between dermal fibroblasts derived from a normal individual and a patient with primary open-angle glaucoma carrying a synonymous ASB10 mutation. CONCLUSIONS: Our evidence suggests that ASB10 may play a role in ubiquitin-mediated degradation pathways in TM cells.

Microenvironmental regulation by fibrillin-1.

Fibrillin-1 is a ubiquitous extracellular matrix molecule that sequesters latent growth factor complexes. A role for fibrillin-1 in specifying tissue microenvironments has not been elucidated, even though the concept that fibrillin-1 provides extracellular control of growth factor signaling is currently appreciated. Mutations in FBN1 are mainly responsible for the Marfan syndrome (MFS), recognized by its pleiotropic clinical features including tall stature and arachnodactyly, aortic dilatation and dissection, and ectopia lentis. Each of the many different mutations in FBN1 known to cause MFS must lead to similar clinical features through common mechanisms, proceeding principally through the activation of TGFß signaling. Here we show that a novel FBN1 mutation in a family with Weill-Marchesani syndrome (WMS) causes thick skin, short stature, and brachydactyly when replicated in mice. WMS mice confirm that this mutation does not cause MFS. The mutation deletes three domains in fibrillin-1, abolishing a binding site utilized by ADAMTSLIKE-2, -3, -6, and papilin. Our results place these ADAMTSLIKE proteins in a molecular pathway involving fibrillin-1 and ADAMTS-10. Investigations of microfibril ultrastructure in WMS humans and mice demonstrate that modulation of the fibrillin microfibril scaffold can influence local tissue microenvironments and link fibrillin-1 function to skin homeostasis and the regulation of dermal collagen production. Hence, pathogenetic mechanisms caused by dysregulated WMS microenvironments diverge from Marfan pathogenetic mechanisms, which lead to broad activation of TGFß signaling in multiple tissues. We conclude that local tissue-specific microenvironments, affected in WMS, are maintained by a fibrillin-1 microfibril scaffold, modulated by ADAMTSLIKE proteins in concert with ADAMTS enzymes.

Variants in ASB10 are associated with open-angle glaucoma.

The molecular events responsible for obstruction of aqueous humor outflow and the loss of retinal ganglion cells in glaucoma, one of the main causes of blindness worldwide, remain poorly understood. We identified a synonymous variant, c.765C>T (Thr255Thr), in ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) in a large family with primary open angle glaucoma (POAG) mapping to the GLC1F locus. This variant affects an exon splice enhancer site and alters mRNA splicing in lymphoblasts of affected family members. Systematic sequence analysis in two POAG patient groups (195 US and 977 German) and their respective controls (85 and 376) lead to the identification of 26 amino acid changes in 70 patients (70 of 1172; 6.0%) compared with 9 in 13 controls (13 of 461; 2.8%; P = 0.008). Molecular modeling suggests that these missense variants change ASB10 net charge or destabilize ankyrin repeats. ASB10 mRNA and protein were found to be strongly expressed in trabecular meshwork, retinal ganglion cells and ciliary body. Silencing of ASB10 transcripts in perfused anterior segment organ culture reduced outflow facility by ∼50% compared with control-infected anterior segments (P = 0.02). In conclusion, genetic and molecular analyses provide evidence for ASB10 as a glaucoma-causing gene.

Primary open angle glaucoma due to T377M MYOC: Population mapping of a Greek founder mutation in Northwestern Greece.

BACKGROUND: Mutations in the MYOC gene have been shown to explain 5% of unrelated primary open angle glaucoma (POAG) in different populations. In particular, the T377M MYOC mutation has arisen at least three separate times in history, in Great Britain, India, and Greece. The purpose of this study is to investigate the distribution of the mutation among different population groups in the northwestern region of Greece. MATERIALS AND METHODS: We explored the distribution of the "Greek" T377M founder mutation in the Epirus region in Northwestern Greece, which could be its origin. Genotyping was performed in POAG cases and controls by PCR amplification of the MYOC gene, followed by digestion with restriction enzyme. Statistical analyses were performed by an exact test, the Kaplan-Meier method and the t-test. RESULTS: In the isolated Chrysovitsa village in the Pindus Mountains, a large POAG family demonstrated the T377M mutation in 20 of 66 family members while no controls from the Epirus region (n = 124) carried this mutation (P < 0.001). Among other POAG cases from Epirus, 2 out of 14 familial cases and 1 out of 80 sporadic cases showed the mutation (P = 0.057). The probability of POAG diagnosis with advancing age among mutation carriers was 23% at age 40, and reached 100% at age 75. POAG patients with the T377M mutation were diagnosed at a mean age of 51 years (SD +/- 13.9), which is younger than the sporadic or familial POAG cases: 63.1 (SD +/- 11) and 66.8 (SD +/- 9.8) years, respectively. CONCLUSIONS: The T377M mutation was found in high proportion in members of the Chrysovitsa family (30.3%), in lower proportion in familial POAG cases (14.2%) and seems rare in sporadic POAG cases (1.2%), while no controls (0%) from the Epirus region carried the mutation. Historical and geographical data may explain the distribution of this mutation within Greece and worldwide.

The path to open-angle glaucoma gene discovery: endophenotypic status of intraocular pressure, cup-to-disc ratio, and central corneal thickness.

PURPOSE. Primary open-angle glaucoma (POAG) is a complex disease with a genetic architecture that can be simplified through the investigation of individual traits underlying disease risk. It has been well studied in twin models, and this study was undertaken to investigate the heritability of some of these key endophenotypes in extended pedigrees. METHODS. These data are derived from a large, multicenter study of extended, Caucasian POAG families from Australia and the United States. The study included 1181 people from 22 extended pedigrees. Variance components modeling was used to determine the heritabilities of maximum intraocular pressure (IOP), maximum vertical cup-to-disc ratio (VCDR), and mean central corneal thickness (CCT). Bivariate quantitative genetic analysis between these eye-related phenotypes and POAG itself was performed to determine whether any of these traits represent true endophenotypes. RESULTS. Heritability estimates for IOP, VCDR, and CCT (0.42, 0.66, and 0.72, respectively) were significant and show strong concordance with data in previous studies. Bivariate analysis revealed that both IOP (RhoG = 0.80; P = 9.6 x 10(-6)) and VCDR (RhoG = 0.76; P = 4.8 x 10(-10)) showed strong evidence of genetic correlation with POAG susceptibility. These two traits also correlated genetically with each other (RhoG = 0.45; P = 0.0012). Alternatively, CCT did not correlate genetically with risk of POAG. CONCLUSIONS. All the proposed POAG-related traits have genetic components. However, the significant genetic correlations observed between IOP, VCDR, and POAG itself suggest that they most likely represent true endophenotypes that could aid in the identification of genes underlying POAG susceptibility. CCT did not correlate genetically with disease and is unlikely to be a useful surrogate endophenotype for POAG.

Association of POAG risk factors and the Thr377Met MYOC mutation in an isolated Greek population.

PURPOSE: To characterize the MYOC genotype correlation with phenotypes in an isolated Greek population with a high incidence of glaucoma. METHODS: Five hundred thirty-one villagers were enrolled in the study. Participants underwent a comprehensive ophthalmic examination. All three exons of myocilin were bidirectionally sequenced. Power calculations and measured genotype analysis was conducted using the genetic variance analysis program, SOLAR version 4.2, to account for the relatedness between individuals. RESULTS: The participants, 376 of whom were linked in a single 11-generation pedigree, ranged in age from 10 to 95 years with a mean age of 49. Sixty-five individuals had POAG, and 27 of those carried the Thr377Met MYOC mutation. Both peak intraocular pressure and vertical cup-to dis- ratio were significantly associated with the MYOC Thr377Met variant (P = 9 x 10(-14) and P = 9 x 10(-8), respectively), whereas central corneal thickness showed no significant association (P < 0.7). CONCLUSIONS: This village had a high frequency of glaucoma, with 12% of the participants aged 10 to 95 years having the disease. In this cohort, the Thr377Met MYOC mutation was significantly associated with both high intraocular pressures and high vertical cup-to-disc ratios. No association was found with central corneal thickness.

Myocilin variations and familial glaucoma in Taxiarchis, a small Greek village.

PURPOSE: To initiate a prospective study of glaucoma in a Greek village reported over 30 years ago to have several large families with primary open-angle glaucoma (POAG). METHODS: A random group of 126 villagers from Taxiarchis, Greece was examined in the village community center. The detailed evaluation included ophthalmic and general history, measurement of blood pressure, intraocular pressure (IOP), and central corneal thickness (CCT) as well as evaluation of the optic nerve status. RESULTS: The incidence of glaucoma approached 18% in this small isolated village. Myocilin variants were present in almost half of the individuals screened with Arg76Lys and Thr377Met being the most common finding (25% and 17%, respectively). Over half of the individuals with the Thr377Met mutation were diagnosed with glaucoma. Two of these patients were homozygous for the Thr377Met mutation. Three individuals with the Arg76Lys polymorphism had glaucoma; however, two of these individuals also had the Thr377Met mutation. Only two patients with pseudoexfoliation were identified. CONCLUSIONS: The incidence of glaucoma and the Thr377Met MYOC mutation in this population is much higher than that reported for other European populations.

Clinical features associated with an Asp380His Myocilin mutation in a US family with primary open-angle glaucoma.

PURPOSE: To determine the glaucoma phenotype of an American pedigree with the myocilin Asp380His. DESIGN: An observational case series study. METHODS: An observational case series study was used to examine a family in which an Asp380His myocilin mutation was segregating. Thirteen family members were examined and medical records were obtained on the remaining two individuals. Blood samples were collected from all 15 participants following the tenets of the Helsinki declaration under the auspices of the Oregon Health & Sciences University Institutional Review Board and screened for myocilin variants by denaturing high-performance liquid chromatography (dHPLC). Any DNA samples with dHPLC data different from the control sample were sequenced for base pair analysis. RESULTS: An Asp380His myocilin mutation was identified in eight members, seven of whom had primary open-angle glaucoma (POAG). The eighth individual had high intraocular pressures (IOPs). The disease presents in this family with extremely high IOPs requiring trabeculectomies to control the pressure. The age at diagnosis ranged from 30 to 45. CONCLUSIONS: This family with an Asp380His myocilin mutation presents with an intermediate phenotype between juvenile- and adult-onset glaucoma. The Asp380 amino acid residue appears to be important in myocilin function based on the finding that substitution of this amino acid with four different amino acids (His, Ala, Asn, or Gly) all result in a similar presentation of POAG that is intermediate between the more severe clinical presentations observed in individuals with the Pro370Leu or Lys423Glu variant and the milder findings in patients with the Gln368Stop mutation.

Investigation of founder effects for the Thr377Met Myocilin mutation in glaucoma families from differing ethnic backgrounds.

PURPOSE: The aim of this study was to determine if there is a common founder for the Thr377Met myocilin mutation in primary open angle glaucoma (POAG) families with various ethnic backgrounds. METHODS: Genomic DNA of 24 POAG-affected individuals from nine pedigrees with the Thr377Met mutation and 104 unaffected family members was genotyped with six microsatellite markers and four single nucleotide polymorphisms. The families were from Greece, India, Finland, the USA, and Australia. To assess the degree of linkage disequilibrium across MYOC in the general population we also investigated data generated from the HapMap consortium. RESULTS: Three distinct haplotypes associated with the Thr377Met myocilin mutation were identified. The families from the USA and Greece, as well as the three Australian families originating from Greece and the former Yugoslavian Republic of Macedonia had one common haplotype. Interestingly, however, HapMap data suggest that linkage disequilibrium across MYOC was not strong. CONCLUSIONS: The Thr377Met myocilin mutation has arisen at least three separate times. Evidence for genetic founder effects in this prevalent age-related, yet heterogeneous, disease has important implications for future gene identification strategies.

Differential expression profile prioritization of positional candidate glaucoma genes: the GLC1C locus.

OBJECTIVES: To develop and apply a model for prioritization of candidate glaucoma genes. METHODS: This Affymetrix GeneChip (Affymetrix, Santa Clara, Calif) study of gene expression in primary culture human trabecular meshwork cells uses a positional differential expression profile model for prioritization of candidate genes within the GLC1C genetic inclusion interval. RESULTS: Sixteen genes were expressed under all conditions within the GLC1C interval. TMEM22 was the only gene within the interval with differential expression in the same direction under both conditions tested. Two genes, ATP1B3 and COPB2, are of interest in the context of a protein-misfolding model for candidate selection. SLC25A36, PCCB, and FNDC6 are of lesser interest because of moderate expression and changes in expression. Transcription factor ZBTB38 emerges as an interesting candidate gene because of the overall expression level, differential expression, and function. CONCLUSIONS: Only 1 gene in the GLC1C interval fits our model for differential expression under multiple glaucoma risk conditions. The use of multiple prioritization models resulted in filtering 7 candidate genes of higher interest out of the 41 known genes in the region. CLINICAL RELEVANCE: This study identified a small subset of genes that are most likely to harbor mutations that cause glaucoma linked to GLC1C.

The role of the WDR36 gene on chromosome 5q22.1 in a large family with primary open-angle glaucoma mapped to this region.

OBJECTIVE: To determine whether mutations in the WD40-repeat 36 (WDR36) gene are responsible for primary open-angle glaucoma (POAG) that maps to the GLC1G locus in a family with 16 affected family members. METHODS: Ninety-two family members underwent clinical evaluation for POAG on the basis of intraocular pressures, cupping of discs, and visual fields after informed consent was obtained. All 23 exons of WDR36 were sequenced in DNA from 5 affected and 2 unaffected family members. RESULTS: Sixteen family members showed evidence of POAG. A number of sequence variations were identified in family members; most of the variations were previously described single-nucleotide polymorphisms also present in the general population. The 3 new sequence changes were all intronic; 2 were found in only 1 of the family members undergoing screening. CONCLUSIONS: Several polymorphisms, including known single-nucleotide polymorphisms, were identified; however, none of these were consistent with disease-causing mutations. A mutation in a noncoding region of WDR36 may be responsible for POAG in this family, or another gene in this region may be the actual cause of glaucoma in this family. CLINICAL RELEVANCE: The finding that the WDR36 gene is probably not the responsible gene in this family further documents the genetic heterogeneity of POAG.

A large GLC1C Greek family with a myocilin T377M mutation: inheritance and phenotypic variability.

PURPOSE: POAG is a complex disease; therefore, families in which a glaucoma gene has been mapped may carry additional POAG genes. The goal of this study was to determine whether mutations in the myocilin (MYOC) gene on chromosome 1 are present in two POAG families, which have previously been mapped to the GLC1C locus on chromosome 3. METHODS: The three exons of MYOC were screened by denaturing (d)HPLC. Samples with heteroduplex peaks were sequenced. Clinical findings were compared with genotype status in all available family members over the age of 20 years. RESULTS: A T377M coding sequence change in MYOC was identified in family members of the Greek GLC1C family but not in the Oregon GLC1C family. Individuals carrying both the MYOC T377M variant and the GLC1C haplotype were more severely affected at an earlier age than individuals with just one of the POAG genes, suggesting that these two genes interact or that both contribute to the POAG phenotype in a cumulative way.

Introductory ophthalmic genetics.

Rapid progress is occurring in molecular cell biology and genetics in the understanding of basic cellular mechanisms and the potential for genetic therapy. As new methods of genetic prognosis and treatment become available, and diseases are redefined in genetic terms, it is essential that clinicians know more about genetic therapy. This article provides a basic outline of gene therapy.

The genetic loci of open-angle glaucoma.

As Posner stated in 1949, the bottom line "to the patient and to his family is..., whether his disease will follow a mild course or will lead to blindness". The final goal of genetic research is the identification of the causal genes in the patient, to aid the ophthalmologist in predicting the outcome, in determining diligent treatment is required, and ultimately, in providing the tools for preventing blindness.