Primary Open-angle Glaucoma Families Research Articles

Serine to proline mutation at position 341 of MYOC impairs trabecular meshwork function by causing autophagy deregulation

Glaucoma is a highly heritable disease, and myocilin was the first identified causal and most common pathogenic gene in glaucoma. Serine-to-proline mutation at position 341 of myocilin (MYOCS341P) is associated with severe glaucoma phenotypes in a five-generation primary open-angle glaucoma family. However, the underlying mechanisms are underexplored. Herein, we established the MYOCS341P transgenic mouse model and characterized the glaucoma phenotypes. Further, we systematically explored the functional differences between wild-type and MYOCS341P through immunoprecipitation, mass spectrometry, and RNA-seq analyses. We found that MYOCS341P transgenic mice exhibit glaucoma phenotypes, characterized by reduced aqueous humor outflow, elevated intraocular pressure, decreased trabecular meshwork (TM) cell number, narrowed Schlemm’s canal, retinal ganglion cell loss, and visual impairment. Mechanistically, the secretion of dysfunctional MYOCS341P accumulated in the endoplasmic reticulum (ER), inducing ER stress and dysregulation of autophagy, thereby promoting TM cell death. We describe an effective transgenic model for mechanistic studies and the screening of therapeutic targets. Our data generated from high-throughput analyses help elucidate the mechanism underlying mutant MYOC-related glaucoma.

Novel mutation in EFEMP1 identified from two Chinese POAG families differentially activated endoplasmic reticulum stress markers and induced glaucoma in mouse.

Primary open-angle glaucoma (POAG) is the most common type of glaucoma. Using whole-exome sequencing, we identified two independent families diagnosed as POAG from the China with a novel EFEMP1 variant (Exon3, c.175A>C p.Met59Leu); Three previouslyreported variants c.1160G>A p.R387Q, c.1189T>C p.Y397H, and c.1429C>T p.R477C in EFEPM1 from 55 sporadic POAG individuals were also identified. The variant c.175A>C p.Met59Leu co-segregated with the disease phenotype within the families. Immunoprecipitation and western blot assays showed that all three EFEMP1 mutants (p.Met59Leu, pArg140Trp, pArg345Trp) increased intracellular protein aggregations, and pMet59Leu and pArg140Arg also enhanced their extracellular proteins secretion, compared to WT in HEK293T. The differential regulations to endoplasmic reticulum (ER) stress markers ATF4, GPR78/94, and CHOP, and differential phosphorylation activations to CREB at Ser133, AKT at Ser473, p44/42 at Thr202/Tyr204, and STAT3 at Tyr705, were also detected among the mutants and WT. Finally, we revealed a significant increment of intraocular pressure and obvious reduction of RGC cells at the sixth weekfollowing intravitreal injection of adenovirus 5 (Ad5) expressing in pMet59Leu compared to WT and GFP controls. Together, variant c.175A>C p.Met59Leu in EFEMP1 is pathogenic and different mutants in EFEMP1 triggered distinct signaling pathways, explaining the reason of mutation-dependent disease phenotypes of EFEMP1.

Whole Exome Sequencing Reveals Novel Candidate Genes in Familial Forms of Glaucomatous Neurodegeneration

Glaucoma is the largest cause of irreversible blindness with a multifactorial genetic etiology. This study explores novel genes and gene networks in familial forms of primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG) to identify rare mutations with high penetrance. Thirty-one samples from nine MYOC-negative families (five POAG and four PACG) underwent whole-exome sequencing and analysis. A set of prioritized genes and variations were screened in an independent validation cohort of 1536 samples and the whole-exome data from 20 sporadic patients. The expression profiles of the candidate genes were analyzed in 17 publicly available expression datasets from ocular tissues and single cells. Rare, deleterious SNVs in AQP5, SRFBP1, CDH6 and FOXM1 from POAG families and in ACACB, RGL3 and LAMA2 from PACG families were found exclusively in glaucoma cases. AQP5, SRFBP1 and CDH6 also revealed significant altered expression in glaucoma in expression datasets. Single-cell expression analysis revealed enrichment of identified candidate genes in retinal ganglion cells and corneal epithelial cells in POAG; whereas for PACG families, retinal ganglion cells and Schwalbe's Line showed enriched expression. Through an unbiased exome-wide search followed by validation, we identified novel candidate genes for familial cases of POAG and PACG. The SRFBP1 gene found in a POAG family is located within the GLC1M locus on Chr5q. Pathway analysis of candidate genes revealed enrichment of extracellular matrix organization in both POAG and PACG.

Genetic heterogeneity of primary open-angle glaucoma in Pakistan

BackgroundGlaucoma is a neurodegenerative ophthalmic disorder and is considered among the leading causes of irreversible blindness. Primary open-angle glaucoma (POAG) is the most common type of glaucoma that affects after 30 years of life, progressing slowly, and manifests as decreased visual acuity leading to blindness if not treated. POAG is genetically heterogeneous, inherited most commonly in autosomal dominant mode. Several genes have been reported for POAG with myocilin (Myoc) being most common. The present study has been conducted to screen 25 POAG families with 2 or more affected members for their association with Myoc and CYP1B1 (the most common gene in primary congenital glaucoma). MethodsAfter approval from Institutional Ethical Review Committee (ERC), 25 POAG families were enrolled from the southern province (Sindh) of Pakistan. Written informed consent was obtained from all participating individuals and diagnosis was confirmed by consultant ophthalmologists using various instruments and means. Venous blood was obtained from affected individuals and their normal family members for DNA extraction and subsequent analysis. ResultsAll samples were initially screened for the Myoc gene followed by CYP1B1. Screening for Myoc revealed one previously reported variant c.144G>T in POAG-06 whereas screening for CYP1B1 in all 25 families showed a novel variant c.649G>A in POAG-02. The pathogenicity of the novel variant was confirmed using various bioinformatics tools. ConclusionThis is the first report of any POAG family found associated with a novel variant in CYP1B1 from the southern province of Pakistan whereas one family found associated with a reported variant in Myoc. The remaining 23 POAG families did not found to be associated with either Myoc or CYP1B1 indicating genetic heterogeneity of the population in this part of the world.

Genetic Variants Associated With the Onset and Progression of Primary Open-Angle Glaucoma

We sought to investigate the genetic variants associated with the onset and progression of primary open-angle glaucoma (POAG). Case-control genetic association study. Japanese POAG patients (n= 505) and control subjects (n= 246) were genotyped for 22 genetic variants predisposing to POAG that can be classified into those associated with intraocular pressure (IOP) elevation (IOP-related genetic variants) and optic nerve vulnerability independent of IOP (non-IOP-related genetic variants). The total number of risk alleles of the 17 IOP-related and 5 non-IOP-related genetic variants were calculated as the genetic risk score (GRS), and the associations between the GRS and family history of glaucoma as an indicator of POAG onset and age at the diagnosis of glaucoma as an indicator of POAG progression were evaluated. There was a significant association (P= .014; odds ratio 1.26 per GRS) between the non-IOP-related GRS, but not IOP-related GRS, and a family history of glaucoma in POAG. As the non-IOP-related GRS increased, the risk of a family history of glaucoma increased. In contrast, a significant association (P= .0014; β=-0.14) was found between the IOP-related GRS, but not non-IOP-related GRS, and age at the diagnosis of glaucoma. As the IOP-related GRS increased, age at the diagnosis of glaucoma decreased. The results indicate that non-IOP-related (optic nerve vulnerability) rather than IOP-related (IOP elevation) genetic variants may play an important role in the onset of POAG (family history of glaucoma) and that IOP-related rather than non-IOP-related genetic variants may play an important role in its progression (age at the diagnosis of glaucoma).

The screening of pathogenic locus in a primary open angle glaucoma pedigree

Objective To screen the pathogenic locus and gene in a primary open angle glaucoma(POAG), and to provide a basis for molecular genetic study of POAG. Methods A POAG pedigree with 35 members was diagnosed in Sichuan peoples' Hospital from January to August 2005.The disease history and clinical data were collected.Genome-wide scan was performed for the families.Specific software was used to calculate the LOD value, which based on the allele (haploid) typing result with two-point method to definite the positive loci by the largest LOD value. Results The POAG family had 35 members of 4 generations.18 patients were diagnosed as juvenile open angle glaucoma from visual disc shape abnormality and loss of typical visual field.All of the patients in this family suffered various degrees of binocular vision loss and vision loss in childhood, with poorly visual function.The LOD values of 3 short tandom repeat (STR) markers on chromosome 2 were greater than 3.0, they were D2S2369 (LOD value 4.003 3), D2S2332 (LOD value 3.840 2) and D2S337 (LOD value 4.752 0). There was a genetic linkage near the three genetic markers in the family.The primary glaucoma positive locus was a in chromosome p15 to chromosome p16.2, and the genetic distance was about 9 Mb, locating in between the markers D2S2369 and D2S2397. Conclusions GLCIH is a pathogenic locus for this POAG pedigree, which supplies an evidence for elucidating the pathogenesis of POAG. Key words: Primary open angle glaucoma; Locus; Genome-wide scan

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.

Variants in the ASB10 Gene Are Associated with Primary Open Angle Glaucoma.

BackgroundRecently nonsynonymous coding variants in the ankyrin repeats and suppressor of cytokine signaling box-containing protein 10 (ASB10) gene were found to be associated with primary open angle glaucoma (POAG) in cohorts from Oregon and Germany, but this finding was not confirmed in an independent cohort from Iowa. The aim of the current study was to assess the role of ASB10 gene variants in Pakistani glaucoma patients.MethodsSanger sequencing of the coding exons and splice junctions of the ASB10 gene was performed in 30 probands of multiplex POAG families, 208 sporadic POAG patients and 151 healthy controls from Pakistan. Genotypic associations of individual variants with POAG were analyzed with the Fisher’s exact or Chi-square test.ResultsIn total 24 variants were identified in POAG probands and sporadic patients, including 11 novel variants and 13 known variants. 13 of the variants were nonsynonymous, 6 were synonymous, and 5 were intronic. Three nonsynonymous variants (p.Arg49Cys, p.Arg237Gly, p.Arg453Cys) identified in the probands were not segregating in the respective families. This is not surprising since glaucoma is a multifactorial disease, and multiple factors are likely to be involved in the disease manifestation in these families. However a nonsynonymous variant, p.Arg453Cys (rs3800791), was found in 6 sporadic POAG patients but not in controls, suggesting that it infers increased risk for the disease. In addition, one synonymous variant was found to be associated with sporadic POAG: p.Ala290Ala and the association of the variant with POAG remained significant after correction for multiple testing (uncorrected p-value 0.002, corrected p-value 0.047). The cumulative burden of rare, nonsynonymous variants was significantly higher in sporadic POAG patients compared to control individuals (p-value 0.000006).ConclusionsVariants in ASB10 were found to be significantly associated with sporadic POAG in the Pakistani population. This supports previous findings that sequence variants in the ASB10 gene may act as a risk factor for glaucoma.

Exome Sequencing Identifies a Missense Variant in EFEMP1 Co-Segregating in a Family with Autosomal Dominant Primary Open-Angle Glaucoma.

Primary open-angle glaucoma (POAG) is a clinically important and genetically heterogeneous cause of progressive vision loss as a result of retinal ganglion cell death. Here we have utilized trio-based, whole-exome sequencing to identify the genetic defect underlying an autosomal dominant form of adult-onset POAG segregating in an African-American family. Exome sequencing identified a novel missense variant (c.418C>T, p.Arg140Trp) in exon-5 of the gene coding for epidermal growth factor (EGF) containing fibulin-like extracellular matrix protein 1 (EFEMP1) that co-segregated with disease in the family. Linkage and haplotype analyses with microsatellite markers indicated that the disease interval overlapped a known POAG locus (GLC1H) on chromosome 2p. The p.Arg140Trp substitution was predicted in silico to have damaging effects on protein function and transient expression studies in cultured cells revealed that the Trp140-mutant protein exhibited increased intracellular accumulation compared with wild-type EFEMP1. In situ hybridization of the mouse eye with oligonucleotide probes detected the highest levels of EFEMP1 transcripts in the ciliary body, cornea, inner nuclear layer of the retina, and the optic nerve head. The recent finding that a common variant near EFEMP1 was associated with optic nerve-head morphology supports the possibility that the EFEMP1 variant identified in this POAG family may be pathogenic.

Analysis of MYOC gene mutation in a primary open angle glaucoma family from China

Background Primary open angle glaucoma (POAG) is one of the frequent glaucomatous types,and genetic factor participates in pathogenesis and development of the disease.Recently,MYOC mutation was found to be associated with POAG.Objective This study was to describe the clinical and genetic findings in a POAG family from Luoyang,China.Methods This study protocol was approved by Ethic Committee of Affiliated First Hospital of Henan University of Science and Technology.The study adhered to Declaration of Helsinki.A POAG family with 29 members of 5 generations was surveyed and followed-up for 5-year duration.The mode of inheritance was determined by the pedigree analysis.The periphery blood sample was collected form 12 families and 100 health controls for the extraction of genomic DNA under the informed consent.The third exon and its flanking introns of MYOC were amplified,and quantitative real time PCR products were sequenced,and the structure and function of mutated gene were examined by restriction fragment length polymorphism analysis.The predicted effects of the detected variants on the secondary structure of MYOC protein were evaluated using Garnier-Osguthorpe-Robson (GOR) method,and homology analysis of protein was carried out by Blast software provided by National Center for Biotechnology Information (NCBI).Results This POAG family included 29 members of 5 generations,and the clinical data were not clear in 11 family members.Three individuals from 3 generations were determined POAG,another one was ocular hypertension,and 2 were carriers.Pedigree analysis appeared an autosomal dominant inheritance.In 12 subjects included 6 members genetically affected and 6 members with normal phenotype,the heterozygous mutation was found in the third exon of MYOC gene in 6 genetically affected members,which revealed a T→C transition at position 1021 (p.S341P),resulting in a switch of serine (Ser) to proline (Pro).It was a missense mutation abolished a CviKI-1 restriction site that segregated with the affected members.Secondary structure prediction of p.S341P suggested that myocilin protein was misfolded.Analysis of protein homology and switched Ser was conservative amine acid at position 1021 (p.S341P).No similar change was found in the 6 normal families and the normal controls.Conclusions Ser341Pro MYOC mutation is disease-causing factor in the POAG family of Luoyang.The clinical and genetic features of this mutation warrant further investigation.The mutation spectrum of MYOC is expanded to offer a better diagnosis and treatment for POAG patients. Key words: Glaucoma, open angle/genetics; Pedigree; MYOC gene; Mutation; Base sequence; Cytoskeletal protein; DNA mutational analysis; China

Family‐based analysis identified CD 2 as a susceptibility gene for primary open angle glaucoma in Chinese Han population

Primary open angle glaucoma (POAG) is characterized by optic disc cupping and irreversible loss of retinal ganglion cells. Few genes have been detected that influence POAG susceptibility and little is known about its genetic architecture. In this study, we employed exome sequencing on three members from a high frequency POAG family to identify the risk factors of POAG in Chinese population. Text-mining method was applied to identify genes associated with glaucoma in literature, and protein–protein interaction networks were constructed. Furthermore, reverse transcription PCR and Western blot were performed to confirm the differential gene expression. Six genes, baculoviral inhibitors of apoptosis protein repeat containing 6 (BIRC6), CD2, luteinizing hormone/choriogonadotropin receptor (LHCGR), polycystic kidney and hepatic disease gene 1 (PKHD1), phenylalanine hydroxylase (PAH) and fucosyltransferase 7 (FUT7), which might be associated with POAG, were identified. Both the mRNA expression levels and protein expression levels of HSP27 were increased in astrocytes from POAG patients compared with those from normal control, suggesting that mutation in CD2 might pose a risk for POAG in Chinese population. In conclusion, novel rare variants detected by exome sequencing may hold the key to unravelling the remaining contribution of genetics to complex diseases such as POAG.

Interleukin-20 Receptor Expression in the Trabecular Meshwork and Its Implication in Glaucoma

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. 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. 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. 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.

Genetics of primary open angle glaucoma

Glaucoma is a neurodegenerative disease and one of the leading causes of irreversible blindness, affecting over 60 million people worldwide. At the present time, glaucoma is clinically defined, but the exact etiology is unknown. Genetic studies are one approach to identify the molecules and pathways involved in disease pathogenesis. Familial aggregation of primary open-angle glaucoma (POAG) has long been recognized, and the analysis of POAG families with a Mendelian inheritance form of this disease has been employed to identify multiple loci linked to them. Some causative genes, such as myocilin, optineurin and WD repeat domain 36, have been identified. However, most cases of POAG are considered to be a prevalent, multifactorial disorder. Several association studies have been conducted for candidate genes, and genome-wide association studies recently identified new susceptibility loci for POAG, namely, S1 RNA binding domain 1 region on chromosome 2p21, the caveolin 1 and caveolin 2 regions on 7q31, transmembrane and coiled-coil domain 1 region on 1q24, cyclin-dependent kinase inhibitor 2B antisense RNA on 9p21, the SIX1 and SIX6 regions on 14q24 and, possibly, the regulatory region of 8q22. Further analysis of clinical manifestations caused by specific genes and functional analysis of these genes will contribute to the development of new strategies for the diagnosis and treatment of POAG.

Genome-Wide Linkage Scan for Primary Open Angle Glaucoma: Influences of Ancestry and Age at Diagnosis

Primary open-angle glaucoma (POAG) is the most common form of glaucoma and one of the leading causes of vision loss worldwide. The genetic etiology of POAG is complex and poorly understood. The purpose of this work is to identify genomic regions of interest linked to POAG. This study is the largest genetic linkage study of POAG performed to date: genomic DNA samples from 786 subjects (538 Caucasian ancestry, 248 African ancestry) were genotyped using either the Illumina GoldenGate Linkage 4 Panel or the Illumina Infinium Human Linkage-12 Panel. A total of 5233 SNPs was analyzed in 134 multiplex POAG families (89 Caucasian ancestry, 45 African ancestry). Parametric and non-parametric linkage analyses were performed on the overall dataset and within race-specific datasets (Caucasian ancestry and African ancestry). Ordered subset analysis was used to stratify the data on the basis of age of glaucoma diagnosis. Novel linkage regions were identified on chromosomes 1 and 20, and two previously described loci—GLC1D on chromosome 8 and GLC1I on chromosome 15—were replicated. These data will prove valuable in the context of interpreting results from genome-wide association studies for POAG.

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.

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.

Variable Clinical Spectrum of the Myocilin Gln368X Mutation in a Dutch Family with Primary Open Angle Glaucoma

Purpose: To describe the clinical phenotype in a family with primary open angle glaucoma harboring a p.Gln368X mutation in MYOC.Materials and Methods: We identified a proband with primary open angle glaucoma and the p.Gln368X MYOC mutation. She and her six siblings were examined clinically, including Heidelberg Retina Tomography II, and venous blood samples were screened for other variants in MYOC, WDR36, OPTN, and CYP1B1.Results: Four individuals showed the p.Gln368X MYOC mutation, no other genetic variations were assessed. Two of these four siblings had glaucomatous optic disc changes with corresponding visual field losses and abnormal Heidelberg Retina Tomography results by the Moorfields regression analysis, one had abnormal results by the Moorfields regression analysis but no visual field loss, and one showed no glaucomatous signs or symptoms at all. These findings did not correlate with the age of the affected individuals.Conclusion: In the primary open angle glaucoma family described here, we documented a wide range in clinical symptoms, demonstrating a highly variable penetrance of the MYOC p.Gln368X mutation.

Distribution ofWDR36DNA Sequence Variants in Patients with Primary Open-Angle Glaucoma

To determine the distribution of WDR36 sequence variants in a cohort of patients with primary open-angle glaucoma (POAG) in the United States. All of the 23 coding exons and flanking introns of the WDR36 gene were sequenced in 118 probands from families with at least two members affected by POAG, 6 probands from juvenile-onset POAG families, and 108 control individuals. Thirty-two WDR36 sequence variants were found in this population of patients with POAG. Nonsynonymous single-nucleotide polymorphisms (SNPs), including those previously described as "disease-causing" and "disease susceptibility," were found in 17% of POAG patients and 4% of control subjects. Although the distribution of WDR36 variants in the pedigrees did not show consistent segregation with the disease, the WDR36 sequence variants were found more frequently in patients with more severe disease. The results of this study suggest that abnormalities in WDR36 alone are not sufficient to cause POAG. The association of WDR36 sequence variants with more severe disease in affected individuals suggests that defects in the WDR36 gene can contribute to POAG and that WDR36 may be a glaucoma modifier gene.

Identification of a novel adult-onset primary open-angle glaucoma (POAG) gene on 5q22.1

Glaucoma is a leading cause of blindness in virtually every country. Development of an accurate diagnostic test for presymptomatic detection of individuals at risk is an urgent requisition for this condition. Herein, we report mapping of a new adult-onset primary open-angle glaucoma (POAG) locus on 5q22.1 (GLC1G) and identification of its defective gene. Mutation screening of seven candidate genes from the GLC1G critical region (approximately 2 Mb between D5S1466 and D5S2051) identified only one significant alteration in the WDR36 (WD40-repeat 36) gene. This mutation (i.e. D658G) was segregated in all affected members of our first GLC1G-linked family but it was absent in 476 normal control chromosomes. Further screening of WDR36 in a total of 130 POAG families revealed 24 DNA variations. Overall, four mutations (N355S, A449T, R529Q and D658G) were identified in 17 (5.02-6.92%) unrelated POAG subjects, 11 with high-pressure and six with low-pressure glaucoma. These mutations were absent in a minimum of 200 normal control chromosomes and, further they were conserved between WDR36 orthologues in mouse, rat, dog, chimp and human. WDR36 is a novel gene with 23 exons, which encodes for 951 amino acids and a protein with multiple G-beta WD40 repeats. By northern blotting, two distinct mRNA transcripts of 5.9 and 2.5 kb were observed in human heart, placenta, liver, skeletal muscle, kidney and pancreas. WDR36 gene expression in lens, iris, sclera, ciliary muscles, ciliary body, trabecular meshwork, retina and optic nerve were established by RT-PCR. In mouse, two transcripts of 3.5 and 2.9 kb showed analogous expression patterns to human. mRNA expressions were detected in 7-, 11-, 15- and 17-day-old developing mouse embryos. In summary, WDR36 is a novel causative gene for adult-onset POAG at the GLC1G locus. Specific ocular expressions and observed mutations are consistent with WDR36 role in etiology of both high- and low-pressure glaucoma.

Novel trabecular meshwork inducible glucocorticoid response mutation in an eight-generation juvenile-onset primary open-angle glaucoma pedigree

Objective This study aimed to update a large kindred with juvenile-onset primary open-angle glaucoma (POAG) first described in 1940 and to identify the underlying genetic cause of the disease. Design Molecular genetic study of a single kindred, including clinical examination, retrospective review of clinical and family history records, linkage analysis, and mutation screening. Participants The retrospective review included 957 members of a single large family. The linkage study included 40 members of 1 branch of the family in which juvenile-onset POAG is segregating in an autosomal-dominant pattern. Mutation screening included 15 at-risk family members with juvenile-onset POAG, probands of 40 families with adult-onset POAG, probands of 11 additional unrelated juvenile-onset POAG families, and 43 unrelated normal control subjects. Intervention Clinical and family history records were obtained, ophthalmologic examinations were performed, and blood samples were drawn for use in genotyping. Main outcome measures Allele sizes of microsatellite repeat genetic markers from the vicinity of the GLC1A glaucoma gene on chromosome 1q were assigned based on size fractionation of DNA fragments generated by polymerase chain reaction (PCR). Linkage was established by the method of lod scores. Mutations were identified by determination of the DNA sequence of PCR products amplified from the trabecular meshwork inducible glucocorticoid response (TIGR) gene. Glaucoma status for purposes of linkage and mutation analysis was based on a combination of ophthalmologic examination, clinical records, family history, and previously published information. For some individuals reported in the pedigree, but not included in the genotyping studies, less information was available as presented in the text and tables. Results Autosomal-dominant POAG was confirmed or reported for 78 members of an 8-generation family. Linkage analysis showed significant evidence for linkage of juvenile-onset POAG in one branch of the family to D1S452 (maximum lod score of 6.42 at a recombination fraction of 0.00) and other markers in the vicinity of the GLC1A gene on chromosome 1q. Screening of the TIGR gene identified a mutation that results in substitution of asparagine for isoleucine at codon 477 near the carboxyterminal end of the protein. Conclusions The authors’ findings strongly suggest that the juvenile-onset POAG locus in this family is the GLC1A locus and that the underlying cause of the disease is the lle477Asn TIGR mutation that cosegregates with juvenile-onset POAG in one branch of this large family. Lack of samples from deceased individuals prevented the authors from determining whether reported adult-onset cases in this family could also be attributed to the lle477Asn TIGR mutation. Absence of the lle477Asn TIGR mutation from other juvenile- and adult-onset POAG families implies that this TIGR mutation is not a common cause of glaucoma.