Familial Primary Open-angle Glaucoma Research Articles

Variants in the PRPF8 Gene are Associated with Glaucoma

Glaucoma is the cause of irreversible blindness worldwide. Mutations in six genes have been associated with juvenile- and adult-onset familial primary open angle glaucoma (POAG) prior to this report but they explain only a small proportion of the genetic load. The aim of the study is to identify the novel genetic cause of the POAG in the families with adult-onset glaucoma. Whole exome sequencing (WES) was performed on DNA of two affected individuals, and predicted pathogenic variants were evaluated for segregation in four affected and three unaffected Dutch family members by Sanger sequencing. We identified a pathogenic variant (p.Val956Gly) in the PRPF8 gene, which segregates with the disease in Dutch family. Targeted Sanger sequencing of PRPF8 in a panel of 40 POAG families (18 Pakistani and 22 Dutch) revealed two additional nonsynonymous variants (p.Pro13Leu and p.Met25Thr), which segregate with the disease in two other Pakistani families. Both variants were then analyzed in a case-control cohort consisting of Pakistani 320 POAG cases and 250 matched controls. The p.Pro13Leu and p.Met25Thr variants were identified in 14 and 20 cases, respectively, while they were not detected in controls (p values 0.0004 and 0.0001, respectively). Previously, PRPF8 mutations have been associated with autosomal dominant retinitis pigmentosa (RP). The PRPF8 variants associated with POAG are located at the N-terminus, while all RP-associated mutations cluster at the C-terminus, dictating a clear genotype-phenotype correlation.

A Histologic Categorization of Aqueous Outflow Routes in Familial Open-Angle Glaucoma and Associations With Mutations in the MYOC Gene in Japanese Patients.

This study evaluated specific relationships between pathogenic mechanisms and genetic polymorphisms in primary open-angle glaucoma (POAG). We analyzed the morphologies of trabeculectomy specimens obtained from patients with familial POAG. We used light microscopy and transmission electron microscopy to examine specimens obtained from 17 eyes of 14 patients with familial POAG. We also conducted exome analyses of two families and used targeted Sanger sequencing to analyze samples obtained from the remaining patients. The POAG cases examined in this study were divided into two groups based on morphologic characteristics. Group A eyes (7 eyes from 5 patients) had an abnormally thick trabecular meshwork (TM), whereas group B eyes (10 eyes from 9 patients) had a TM of normal thickness. The characteristics of the outflow routes in group A eyes were remarkable and included apoptotic TM cells, abnormally thickened TM basement membranes, fused TM beams, and occluded Schlemm's canals. All group A patients harbored mutations (F369L, P370L, T377M, and T448P) in the myocilin (MYOC) gene that were not found in group B patients. Although age matching of morphologic changes in the outflow routes was impossible due to the small sample size, this study suggests that abnormal TM cells may cause sequential damage in abnormally thickened TM basement membranes, TM cell apoptosis, TM beam fusion, and the occlusion of Schlemm's canals. The four detected MYOC mutations appeared to be associated with morphologic changes in the TM and the underlying pathogenesis of a subtype of familial POAG.

Enhanced optineurin E50K–TBK1 interaction evokes protein insolubility and initiates familial primary open-angle glaucoma

Glaucoma is the leading cause for blindness affecting 60 million people worldwide. The optineurin (OPTN) E50K mutation was first identified in familial primary open-angle glaucoma (POAG), the onset of which is not associated with intraocular pressure (IOP) elevation, and is classified as normal-tension glaucoma (NTG). Optineurin (OPTN) is a multifunctional protein and its mutations are associated with neurodegenerative diseases such as POAG and amyotrophic lateral sclerosis (ALS). We have previously described an E50K mutation-carrying transgenic (E50K-tg) mouse that exhibited glaucomatous phenotypes of decreased retinal ganglion cells (RGCs) and surrounding cell death at normal IOP. Further phenotypic analysis of these mice revealed persistent reactive gliosis and E50K mutant protein deposits in the outer plexiform layer (OPL). Over-expression of E50K in HEK293 cells indicated accumulation of insoluble OPTN in the endoplasmic reticulum (ER). This phenomenon was consistent with the results seen in neurons derived from induced pluripotent stem cells (iPSCs) from E50K mutation-carrying NTG patients. The E50K mutant strongly interacted with TANK-binding kinase 1 (TBK1), which prohibited the proper oligomerization and solubility of OPTN, both of which are important for OPTN intracellular transition. Treatment with a TBK1 inhibitor, BX795, abrogated the aberrant insolubility of the E50K mutant. Here, we delineated the intracellular dynamics of the endogenous E50K mutant protein for the first time and demonstrated how this mutation causes OPTN insolubility, in association with TBK1, to evoke POAG.

Absence of the OPTN mutation in a patient with ALS and familial primary open angle glaucoma

The optineurin (OPTN) gene, known to be implicated in primary open-angle glaucoma (POAG), is the more recent genetic factor linked to ALS. We report the case of a 75 year-old man who developed ALS and whose medical history was dominated by a familial POAG. The absence of OPTN gene mutation in a patient who suffered from two conditions linked to mutations of this gene does not support involvement of OPTN in ALS.

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

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. 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. 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. 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.

Enhanced binding of TBK1 by an optineurin mutant that causes a familial form of primary open angle glaucoma

TANK-binding kinase 1 (TBK1) was identified as a binding partner for Optineurin (OPTN) in two-hybrid screens, an interaction confirmed by overexpression/immunoprecipitation experiments in HEK293 cells and by coimmunoprecipitation of endogenous OPTN and TBK1 from cell extracts. A TBK1 binding site was located between residues 1-127 of OPTN, residues 78-121 displaying striking homology to the TBK1-binding domain of TANK. The OPTN-binding domain was localised to residues 601-729 of TBK1, while TBK1[1-688] which cannot bind to TANK, did not interact with OPTN. The OPTN[E50K] mutant associated with Primary Open Angle Glaucoma (POAG) displayed strikingly enhanced binding to TBK1, suggesting that this interaction may contribute to familial POAG caused by this mutation.

Disease Severity of Familial Glaucoma Compared With Sporadic Glaucoma

To determine whether there is a difference in disease severity between familial and sporadic primary open-angle glaucoma (POAG). A cross-sectional study design compared the distribution of Glaucoma Inheritance Study in Tasmania (GIST) severity scores of patients with genealogically confirmed familial POAG and those with sporadic POAG. The GIST severity scores provide a combined weighting of glaucoma severity based on findings from visual field defects and optic disc analysis, with and without intraocular pressure. A Poisson regression analysis, t test, and chi(2) tests were performed. One thousand twelve (59.5%) of 1700 subjects had familial glaucoma. The mean +/- SD age at examination was greater in the sporadic POAG group compared with the familial group (72.6 +/- 10.3 years vs 70.6 +/- 12.6 years; P = .001). The family group was significantly younger at diagnosis than the sporadic group (mean +/- SD, 61.4 +/- 13.0 years vs 64.0 +/- 12.6 years; P<.001). The GIST severity scores were significantly skewed toward greater disease severity in the familial group compared with the sporadic group (P<.001). Identifying individuals at risk of severe POAG will be more successful if screening programs are developed with appropriate weighting toward those with a positive family history of the disease.

Functional analysis of the glaucoma-causing TIGR/myocilin protein: Integrity of amino-terminal coiled-coil regions and olfactomedin homology domain is essential for extracellular adhesion and secretion

TIGR/MYOC mutations account for 2–4% of the primary open-angle glaucoma (POAG) patients. More than 90% of the known mutations are located within its carboxy-terminus olfactomedin-homology (Olf) domain (amino acids (aa) 245–504). In vitro and in vivo studies showed that several Olf domain mutations prevented myocilin secretion. To investigate if intracellular sequestration was a characteristic feature shared by a majority of the mutations, we analyzed the secretion status of 36 myocilin variants. These encompassed 26 glaucoma-causing mutations and 10 non-disease associated or undefined polymorphisms. As several variants were found to be secreted, we tested for their adhesion to the extracellular matrix (ECM) and/or cell surface. Myocilin variants were generated by site-directed mutagenesis of a vector encoding the human MYOC cDNA. COS-7 or immortalized human trabecular meshwork cells were transfected with wild-type or mutated MYOC constructs. Myocilin levels were estimated by immunoprecipitation and/or immunoblotting. All variants showed identical behaviors in both cell lines; the truncated R46X polypeptide being the only variant which could not be detected in our assays. Of the 35 variants monitored, 20 remained sequestered intracellularly. All of them encoded disease-causing polypeptides carrying Olf domain mutations. Of the 15 variants secreted into the culture medium, six (6) were POAG mutants (of which three (3) located within the Olf domain) while the remaining nine (9) were non-disease causing or undefined polymorphisms. Three (3) of the six (6) secreted mutations caused familial POAG; these were the R126W, T377M and A427T mutants. Both, the T377M and A427T mutants located within the Olf domain. When cells were cultured at 30 °C, a process known to facilitate protein folding, 11 of the 20 sequestered mutants were released in the extracellular medium. Out of the 15 secreted variants tested for their adhesion to the ECM and/or cell surface, only the R82C and L95P polypeptides displayed loss of their adhesive properties. Deletion experiments revealed that the coiled-coil (aa 78–105) and leucine zipper (aa 114–183) motifs were essential for adhesion. These experiments demonstrate that intracellular sequestration might be the primary mechanism contributing to myocilin-related POAG as it was associated with more than 80% of the disease-causing mutants tested in our study. A second mechanism may involve abnormal interaction(s) between myocilin and ECM and/or cell surface proteins. Our data further revealed the importance of the olfactomedin-homology domain for myocilin secretion and the significant role of the N-terminal region for its extracellular interactions.

Identification of a new GLC1A mutation in a sporadic, primary open-angle glaucoma in Japan

Glaucoma is a major cause of blindness characterized by progressive degeneration of the optic nerve and elevated intraocular pressure. Recent studies have revealed a genetic basis for a substantial proportion of cases of familial primary open-angle glaucoma (POAG) and the gene causing the abnormality has been identified. Sequence variations that meet the criteria for a probable disease-causing mutation have been found in the American and European populations. In this study, we examined 58 cases of sporadic glaucoma from Japan to clarify the relationship between the mutations of the GLC1A gene and sporadic glaucoma in Japan. We have examined 33 POAG, 17 primary closed-angle glaucomas, 6 normal-tension glaucomas and 2 steroid-induced glaucomas for mutation of the GLC1A gene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct DNA sequencing studies. We identified a previously unreported GGT right curved arrow GAT transition at codon 451 in exon 3, resulting in a glycine to asparagine substitution in one POAG patient. No other mutations of the GLC1A gene were found in other types of glaucoma. These findings further emphasize the importance of GLC1A mutation in the development of POAG.

Detection of a New TIGR Gene Mutation in a Japanese Family With Primary Open Angle Glaucoma

Purpose: To describe a new mutation of the trabecular meshwork-inducible glucocorticoid response protein (TIGR) gene in a Japanese patient with familial primary open angle glaucoma (POAG). Methods: Standard ocular examinations were performed on the 44-year-old patient, his sister, and mother. DNA sequencing was used to identify the mutation. We also developed a DNA diagnostic method for detecting this missense mutation by polymerase chain reaction-induced mutation restriction analysis (PCR-IMRA). Results: The patient, father, and sister had been diagnosed as having POAG. The patient and his sister had a Thr448Pro mutation (C→A transition at the nucleotide number 1419) in exon 3. This mutation has not been reported before. Conclusions: Gene analysis is promising for an early diagnosis among the family members of familial POAG patients and will contribute to early therapy before an occurrence of irreversible visual impairment.

Association study of the 5' flanking regions of endothelial-nitric oxide synthase and endothelin-1 genes in familial primary open-angle glaucoma.

1. Endothelium-derived substances are important regulators of the microcirculation. Endothelium-derived nitric oxide (NO), which is catalysed by nitric oxide synthase (NOS), is a potent modulator of vascular tone in the human ophthalmic artery, which is normally in a state of constant vasodilation due to the actions of NO. Endothelin-1 (ET-1) produces vasoconstriction of the anterior optic nerve vasculature and may be associated with glaucomatous optic neuropathy. The aetiology of primary open-angle glaucoma (POAG) remains largely unknown. Thus, alterations in the regulatory sequences of the genes coding for endothelium-derived NOS (eNOS) and ET-1 may have important effects in the development of POAG and were looked for in the present study. 2. In 56 patients with familial POAG and in 100 control subjects with no family history of hypertension or POAG, we examined the 5' flanking sequences of the eNOS and ET-1 genes, which contain many positive and negative regulatory regions affecting gene transcription, using polymerase chain reaction-based single strand conformation polymorphism analysis, to search for alterations. No variant in the promoter region of the ET-1 gene was observed in familial POAG or controls. Using three primer sets spanning 706 b.p. of the eNOS gene, we observed alterations in 11 of 56 (20%) familial POAG members and in seven of 100 (7%) controls. Sequence analysis demonstrated a C/T substitution at the 5' sequence position nucleotide (nt) -690 from the transcription start site, which lies between the cAMP regulatory element (nt -726 to -732) and an activator protein-1 binding domain (nt -655 to -661). 3. In summary, genotypic and allelic frequency analysis found no association between alterations in the promoter region of the ET-1 gene and familial POAG. A variant in the promoter region of the eNOS gene was seen in a significant percentage of familial POAG patients. Future expression studies will determine whether this polymorphism results in altered eNOS gene expression.

Adult-Onset Primary Open Angle Glaucoma Does Not Localize to Chromosome 2cen-q13 in North American Families

Glaucoma is one of the leading causes of irreversible blindness in the world and is characterized by elevated intraocular pressure, optic nerve atrophy, and progressive visual field loss. Primary open angle glaucoma (POAG) is the most common subtype of glaucoma in the United States. Recently, Stoilova and coworkers [Genomics 1996;36:142–150] identified a locus for POAG on chromosome 2 (2cen-q13) in families primarily located in the United Kingdom. We examined families with POAG identified within the US for linkage to the 2cen-q13 locus. A total of 18 families with POAG were used in the analysis. Of 77 family members, 46 were classified as affected and 31 were either glaucoma suspects or considered normal. Eight highly polymorphic and informative markers flanking and distributed throughout the region were used. Parametric lod score analysis was performed using both a dominant and recessive low penetrance or ‘affecteds-only’ model. Multipoint affected sibpair exclusion mapping was also performed. Lod score (both models) and sibpair analysis excluded linkage of the POAG phenotype to the 2cen-q13 region in these families. These data suggest that the chromosome 2cen-q13 locus does not explain a substantial amount of genetic variation in familial POAG.

PCR-SSCP analysis of the glucocorticoid-responsive element of the atrial natriuretic peptide gene in familial primary open-angle glaucoma.

1. Familial primary open-angle glaucoma (POAG) is a heterogeneous disease of unknown aetiology and the elucidation of the underlying genetic mechanisms contributing to phenotypic expression will be essential if earlier diagnosis of at-risk individuals and more specific medical treatment can be achieved. In a significant percentage of patients with POAG, intraocular pressure increases in response to topical ocular glucocorticoids. 2. Atrial natriuretic peptide (ANP) assists in the regulation of intraocular pressure levels and binding of the glucocorticoid receptor dimer to the glucocorticoid-responsive element in intron 2 of the ANP gene has been shown to increase ANP mRNA levels in vitro. We amplified and examined this sequence in the ANP gene by PCR-SSCP analysis in 100 patients with familial POAG and in 60 normal control subjects. No base alterations in the amplified product were found. 3. Thus, the present study found no evidence for an alteration in the sequence of the glucocorticoid-responsive element of the ANP gene that could alter ANP gene transcription in patients with familial POAG. The mechanism responsible for the increase in intraocular pressure levels in response to glucocorticoids is most likely independent of the glucocorticoid-responsive element in the ANP gene.

The Atrial Natriuretic Peptide Gene in Patients with Familial Primary Open-Angle Glaucoma

Family history is a major risk factor in the development of primary open-angle glaucoma. The atrial natriuretic peptide system has been implicated in the underlying pathophysiology of the disease. This study looked for any alterations in the ANP gene and 5′ proximal promoter regions of the ANP gene, in 53 patients from familial primary open-angle glaucoma families. The ANP gene was amplified by a long-PCR technique from peripheral blood DNA. Gross insertions or deletions in the gene were sought and allelic frequencies at two restriction fragment length polymorphism (RFLP) sites within the gene (ScaI,HpaII) were compared with allelic frequencies obtained from 60 normal controls with no known family history of glaucoma or ocular hypertension. PCR-based single strand conformation polymorphism analysis was then used to search for possible point mutations in the 5′ proximal promoter region of the ANP gene, which is known to contain regulatory elements which modify gene transcription. No gross alterations in the ANP gene or differences in allelic frequencies at the RFLP sites within the gene were observed. PCR-SSCP analysis of the 5′ proximal promoter region of the gene revealed mutations in 10 patients in the −595 to −384bp region (19% of patients). Mutations in the 5′ proximal promoter region of the ANP gene may contribute to altered ANP transcription in at least a proportion of patients with familial glaucoma.

2138 Exclusion of chromosome 1q as the susceptibility locus in familial primary open angle glaucoma

2138 Exclusion of chromosome 1q as the susceptibility locus in familial primary open angle glaucoma

Exclusion of chromosome 1q as the susceptibility locus in familial primary open angle glaucoma

Exclusion of chromosome 1q as the susceptibility locus in familial primary open angle glaucoma