Comprehensive sequencing of the myocilin gene in a selected cohort of severe primary open-angle glaucoma patients.
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ABSTRACT:
SUBMITTER: O'Gorman L
PROVIDER: S-EPMC6395666 | biostudies-literature | 2019 Feb
REPOSITORIES: biostudies-literature
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Comprehensive sequencing of the myocilin gene in a selected cohort of severe primary open-angle glaucoma patients.
O'Gorman Luke L Cree Angela J AJ Ward Daniel D Griffiths Helen L HL Sood Roshan R Denniston Alastair K AK Self Jay E JE Ennis Sarah S Lotery Andrew J AJ Gibson Jane J
Scientific reports 20190228 1
Primary open-angle glaucoma (POAG) is the most common form of glaucoma, prevalent in approximately 1-2% of Caucasians in the UK over the age of 40. It is characterised by an open anterior chamber angle, raised intraocular pressure (IOP) and optic nerve damage leading to loss of sight. The myocilin gene (MYOC) is the most common glaucoma-causing gene, accounting for ~2% of British POAG cases. 358 patients were selected for next generation sequencing (NGS) with the following selection criteria: Ca ...[more]
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Project description:PURPOSE: To investigate the genetic linkage of primary open-angle glaucoma (POAG) in a Chinese family. METHODS: We have screened for myocilin (MYOC) gene mutations in a glaucoma family of five generations. There are fifty-six members of whom 11 were confirmed to have POAG , two with ocular hypertension were considered as POAG suspect, and the remaining 43 were asymptomatic. We also recruited 200 unrelated healthy Chinese subjects as normal control. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing were used to identify mutations in the three exons of MYOC. Presymptomatic diagnoses were made for the family members seeking consultation based on the results of both clinical examination and genetic analysis. RESULTS: Among three allelic variants identified in this pedigree (Pro13Leu [38 C-->T], Arg76Lys [227G-->A], and Gln337Stop [1009C del]), Pro13Leu and Gln337Stop were reported to be novel mutations while Arg76Lys has been previously documented. Our results show that all 11 POAG patients carry the Gln337Stop mutation and that four POAG patients and one POAG suspect (V:2) were found to have the Pro13Leu mutation. In addition, Arg76Lys polymorphism was identified in two patients and a POAG suspect (V:5). CONCLUSIONS: Pro13Leu and Gln337Stop mutations of MYOC are likely responsible for the etiology of POAG in this pedigree, but the causative mechanism needs further research.
| S-EPMC2530518 | biostudies-literature
Project description:BackgroundGlaucoma is the leading cause of irreversible blindness in the world. Recent evidence indicates a role for genetic susceptibility to primary open-angle glaucoma (POAG). The relation between myocilin polymorphisms and POAG susceptibility has been studied in different populations.MethodsA meta-analysis of 32 published genetic association case-control studies, which examined the relation between POAG and the R46X, R76K, Y347Y, T353I, and Q368X polymorphisms of the myocilin gene, was carried out.ResultsIn meta-analysis, significant associations were observed between POAG risk and two myocilin polymorphisms with summarized odds ratio of 4.68 (95%CI, 2.02-10.85) for Q368X and 2.17 (95% CI, 1.32-3.57) for T353I. Both Q368X and T353I were significantly associated with high-tension glaucoma, with summarized odds ratio of 4.26 (1.69, 10.73) and 2.26 (1.37-3.72). In Westerners, significant association was observed for Q368X mutation (odds ratio, 5.17; 95% CI, 2.16-12.40). However, in Asians it was for T353I (odds ratio, 2.17; 95% CI, 1.32-3.57).ConclusionsThere is strong evidence that myocilin polymorphisms are associated with POAG susceptibility, and the prevalence of myocilin mutations might be ethnicity-dependent in Caucasians for Q368X and in Asians for T353I.
| S-EPMC3460926 | biostudies-literature
Project description:PurposeMyocilin (MYOC) is a well-established primary open-angle glaucoma (POAG) risk gene, with rare variants known to have high penetrance. The most common clinically relevant risk variant, Gln368Ter, has an allele frequency of 0.1% to 0.3% in populations of European ancestry. Detection of rare MYOC variants has traditionally been conducted using Sanger sequencing. Here we report the use of genotyping arrays and imputation to assess whether rare variants including Gln368Ter can be reliably detected.MethodsA total of 1155 cases with advanced POAG and 1992 unscreened controls genotyped on common variant arrays participated in this study. Accuracy of imputation of Gln368Ter variants was compared with direct sequencing. A genome-wide association study was performed using additive model adjusted for sex and the first six principal components.ResultsWe found that although the arrays we used were designed to tag common variants, we could reliably impute the Gln368Ter variant (rs74315329). When tested in 1155 POAG cases and 1992 controls, rs74315329 was strongly associated with risk (odds ratio = 15.53, P = 1.07 × 10-9). All POAG samples underwent full sequencing of the MYOC gene, and we found a sensitivity of 100%, specificity of 99.91%, positive predictive value of 95.65%, and negative predictive value of 100% between imputation and sequencing. Gln368Ter was also accurately imputed in a further set of 1801 individuals without POAG. Among the total set of 3793 (1992 + 1801) individuals without POAG, six were predicted (probability > 95%) to carry the risk variant.ConclusionsWe demonstrate that some clinically important rare variants can be reliably detected using arrays and imputation. These results have important implications for the detection of clinically relevant incidental findings in ongoing and future studies using arrays.
| S-EPMC4525674 | biostudies-literature
Project description:Mutations in the myocilin gene (MYOC) are associated with primary open-angle glaucoma (POAG) in many different populations. This study represents the first large survey of MYOC mutations in an African American population.We recruited 529 African American subjects with POAG and 270 African American control subjects in this study. A complete eye examination and blood collection was performed in all study subjects. Genomic DNA was extracted. The entire coding sequence of MYOC was amplified and sequenced using the Sanger method. Identified MYOC variants were compared with previously reported MYOC mutations.We identified a total of 29 MYOC variants including six potential MYOC mutations. Two mutations (Thr209Asn and Leu215Gln) are novel and are found only in cases and no controls. We also identified four previously reported MYOC mutations in cases and no controls (Tyr453MetfsX11, Gln368X, Thr377Met, and Ser393Arg). The overall frequency of glaucoma-causing MYOC mutations in our African American population with POAG was 1.4%.We identified two novel probable glaucoma-causing MYOC mutations (Thr209Asn and Leu215Gln). This study indicates that, despite the high prevalence of POAG, MYOC mutations are rare in the African American population.
| S-EPMC3429360 | biostudies-literature
Project description:PurposeTo describe the phenotype of ocular hypertension and primary open-angle glaucoma in a family with individuals compound heterozygote for Gln368STOP and Thr377Met myocilin (MYOC) mutations.MethodsFamily members of the proband underwent comprehensive ocular clinical examination and DNA sequencing for MYOC mutations.ResultsA 34-year-old woman with marked ocular hypertension was found to carry Gln368STOP and Thr377Met MYOC mutations. Three other siblings carried both mutations, while one carried Gln368STOP alone. Three of five siblings had received treatment for ocular hypertension or early glaucoma, with the average age of diagnosis 28 years; one required trabeculectomy at age 27. The mother of the proband was found to be a carrier for Gln368STOP alone, which indicates that her offspring with both Gln368STOP and Thr377Met carry variants on opposing alleles.ConclusionsThis pedigree is the first report with individuals compound heterozygote for the two most common glaucoma-causing MYOC variants. The combination of mutations manifests a more severe phenotype than either alone. Identification of gene changes associated with glaucoma within the family has enabled unaffected members to stratify their risk of future disease and institute closer monitoring and early treatment.
| S-EPMC3538040 | biostudies-literature
Project description:A substantial fraction of glaucoma has a genetic basis. About 5% of primary open angle glaucoma (POAG) is currently attributed to single-gene or Mendelian forms of glaucoma (ie glaucoma caused by mutations in myocilin or optineurin). Mutations in these genes have a high likelihood of leading to glaucoma and are rarely seen in normal subjects. Other cases of POAG have a more complex genetic basis and are caused by the combined effects of many genetic and environmental risk factors, each of which do not act alone to cause glaucoma. These factors are more frequently detected in patients with POAG, but are also commonly observed in normal subjects. Additional genes that may be important in glaucoma pathogenesis have been investigated using quantitative traits approaches. Such studies have begun to identify genes that control the magnitude of important quantitative features of glaucoma that may also be important risk factors for POAG, such as central corneal thickness. Each of these different approaches to study glaucoma genetics is providing new insights into the pathogenesis of POAG.
| S-EPMC3171270 | biostudies-literature
Project description:PURPOSE: To investigate the genotype and phenotype of juvenile-onset open angle glaucoma (JOAG) in a Chinese family (PN pedigree). METHODS: Each family member was comprehensively examined by an experienced ophthalmologist. The clinical characteristics of the family patients with JOAG were documented. Blood samples were obtained from 22 available participants from the PN pedigree. Linkage analysis was performed to identify the possible chromosome loci. The presence of gene mutation was ascertained by polymerase chain reaction amplification and subsequent direct sequencing. RESULTS: The affected members in the PN pedigree are characterized by early age of onset (mean age at diagnosis is 17 years old), severe clinical presentations, high intraocular pressure (mean IOP of 34.18±2.97 mmHg), and poor response to pharmacological treatment (87.5% of the patients required filtering surgery). The region on chromosome 1 between D1S3464 and D1S1619 was identified in this pedigree by linkage analysis. A Pro370Leu myocilin mutation resulting from a heterozygous C?T transition at the 1,109th nucleotide in exon 3 was detected by gene sequencing. The Pro370Leu mutation co-segregated among all affected individuals of PN pedigree. CONCLUSIONS: The GLC1A Pro370Leu mutation is firmly correlated with a severe POAG phenotype. These data provide clues for the severe disease-causing nature of the Pro370Leu allele. Gene screening may be a useful method for pre-symptom diagnosis and a forewarning to detect the at-risk individuals in familial open-angle glaucoma patients, especially in pedigrees of early-onset.
| S-EPMC3110496 | biostudies-literature
Project description:The total RNA from control and POAG optic nerve lamina cribrosa region was isolated and subjected to analysis on U133A 2.0 affymetrix microarray chip. The results from these two analysis was compared to determine whether PAD II RNA level is altered. Purpose of the study. This study was done to determine which RNA transcripts undergo significant changes in the optic nerve between normal and cadaver primary open-angle glaucoma donors. Using donor tissues from NDRI we have performed proteomic analyses (which will be published shortly), we intend a comparison between the proteomic and RNA changes for select proteins in our study. Sample Collection and Preparation of Labeled Copy RNA; The total RNA from optic nerve was obtained using TRIZOL (Invitrogen Inc., Carlsbad, CA) with modification of recommended protocols. The optic nerve from donor eyes were carefully excised and minced into small pieces first using a scissor and then a scalpel. Prior to use tissue was washed with DEPC water and all solutions were prepared in DEPC water. The minced tissue was placed in a glass homogenizer with 1 ml TRIZOL per 100 mg of tissue and homogenized in a glass homogenizer DUALL 20 (Kimble Kontes Glass Co, Vineland, NJ) with 10 strokes cycles each at room temperature and after freezing with liquid nitrogen for 1 min for 40 cycles. This RNA was extracted with chloroform, isoamylalcohol and precipitated with sodium citrate/sodium chloride and isopropanol. The final air-dried RNA precipitate was suspended in DEPC water and stored in â??80oC prior to use. RNA was prepared for hybridization according to the GeneChip Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). The hybridization was performed at Gene Expression Array Core Facility at Case Western Reserve University (www.geacf.net). The RNA sample was cleaned using a column from a Qiagen RNeasy kit, (part # 74104 ) in accordance with the manufacturerâ??s instructions. The volume of DEPC water used to elute the RNA from the Qiagen column was dictated by the nominal mass of the loaded RNA sample. Samples were eluted in 100ml of water, precipitated with 0.5 volume of 7.5M Ammonium Acetate and 2.5 volumes of 100% ethanol at -20oC for 1 hr and centrifuged for 10 min at room temperature. The pellet was drained and washed twice in 500µl of 70% ethanol. Supernatant was aspirated. The pellet was dried briefly (less than 1 min) in a speeedvac (Savant) and re-suspended in a small volume (~ 12â??15ml) of DEPC water. A small aliquot (2ml) of the sample was used for quantitation. Cleaned RNA samples were annotated as â??clnRNAâ?? and stored at -20oC. cDNA Synthesis; The protocol recommended by Affymetrix Inc. for cDNA synthesis was used at all times. The reaction was primed by annealing an oligo-dT primer coupled to a T7 RNA polymerase promoter to the RNA sample. Whenever possible, 8 mg of clnRNA were reverse transcribed using Superscript II reverse transcriptase in a 20ml reaction at 42oC for 1 hr. Second strand synthesis was carried out immediately in a total reaction volume of 150ml in the presence of E. coli DNA polymerase I, RNAse H and DNA ligase. The reaction mixture incubated for 2 hrs at 16oC and for a further 5 min in the presence of T4 DNA pol.. The reaction was terminated by the addition of EDTA. The sample was cleaned with a Qiagen cDNA clean-up column according to the manufacturers directions. The elution buffer volume was 14ml. The eluted samples were stored overnight at -20oC. In-Vitro Transcription (IVT) reaction; In a 0.5ml microfuge tube, complementary RNA (cRNA) was generated in an IVT reaction using a BioArray High Yield ENZO kit (Affymetrix). The 40ml reaction contained 10ml of cDNA sample and was incubated at 37oC in the heating block for 4-5hrs. Samples were mixed by flicking, pulse-centrifuged and returned to the incubator once every 40 minutes. Upon completion of the reaction, IVT samples were adjusted to a volume of 100ml with DEPC-treated water and cleaned using Qiagen RNA clean-up columns. Samples were eluted in 45ml of DEPC-treated water. A 2ml-aliquot was used for quantitation. Corrected concentrations of cRNA (minus the input clnRNA) were determined. A standard fragmentation reaction volume was 40ml in a 0.5 ml microfuge tube and used 20mg of overall IVT RNA in 1X fragmentation buffer (40mM Tris acetate, pH 8.1, 100mM KOAc, 30mM MgOAc). The reaction was incubated at 94oC for 35min in a heat block. After the 35 min incubation the fragmented samples were placed on ice. The standard hybridization cocktail volume was 300ml. 150ml of 2X hybridization buffer was added (final concentrations : 100mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween 20) Herring sperm and acetylated BSA were added to a final concentration of 0.1 and 0.5mg/ml respectively. The amount of fragmentation reaction containing 15mg of cRNA was added to the cocktail and the remaining volume was made up with molecular biology grade water. A 15ml aliquot of a 20X mixture of in-vitro transcripts of bacterial genes BioB BioC BioD and cre as external controls (spikes) were added to the cocktail to give final concentrations of 1.5, 5, 25 and 100pM respectively. Control oligonucleotide (5ml) was added to a final concentration of 50pM. Array Hybridization; Sample Hyridization cocktails (300ml) were removed from storage at â??20oC and thawed in a 45oC temperature block. The samples were incubated at 45oC for 5 min, incubated at 45oC for 5 min and then centrifuged at 15,000g for 5 min. This study used commercially available Affymetrix HG U133A 2.0 arrays only. These are in situ synthesized, high density oligonucleotide arrays which can interrogate 22,777 gene entities. The part number is 900444 more detailed information may be found at www.affymetrix.com website. Meanwhile, the affymetrix chip arrays were taken from their storage at 4oC and allowed to come to room temperature. The chamber was filled with 1X hybridization buffer (final concentrations : 100mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween 20). The chip was then preconditioned by incubation in the hybridization oven at 45oC for 10 min. The chip was removed and the chamber drained. Sample cocktails (200ml) were then introduced into the chamber of the chips. The ports were sealed with tough-spots stickers and placed in the hybridization oven and incubated at 45oC with rotation overnight. Next day (16hr later), the incubation was terminated and the chips removed from the oven. Samples were retrieved from the chips and stored again in a freezer at -20oC. The chips were filled with buffer A and placed in a module of Fluidics 400 wash station. The chips were subjected to the Affymetrix â??EuKws4 ver 2â?? programmed series of stringent post hybridization washes, staining and post-staining washes in accordance with Affy protocols. Upon completion of the wash series (approximately 2 hrs), the chips were removed from the Fluidics station. The chips were inspected. Chips were scanned using an Agilent Gene Array scanner 3000 driven by Affymetrix GCOS software. Generation of Expression Values; Microarray Suite, version 5 (Affymetrix), was used to generate *.cel files, and a computer program (Probe Profiler, ver. 1.3.11; Corimbia Inc., Berkeley, CA) developed specifically for the GeneChip system (Affymetrix) was used to convert intensity data into quantitative estimates of gene expression for each probe set. The software identifies informative probe pairs, and down-weights the signal contribution of probe pairs that are subject to differential cross-hybridization effects or that consistently produce no signal. The software also detects and corrects for saturation artifacts, outliers, and chip defects. A probability statistic was generated for each probe set. The probability is associated with the null hypothesis that the expression level of the probe set is equal to zero (background). Genes not significantly expressed above background in any of the samples (P > 0.05) were
2008-06-12 | E-GEOD-2387 | biostudies-arrayexpress
Project description:Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disease. Conjunctival telangiectasias and retinal vascular malformations are known ocular manifestations. We report here the first case of open angle glaucoma in a patient with HHT caused by a nonsense mutation, C471X in the ACVRL1 gene.
| S-EPMC4574785 | biostudies-literature
Project description:Glaucoma is a major cause of irreversible blindness worldwide. There is evidence showing that a subset of the disease is genetically determined. In this study, we screened for mutations in chromosome 1q-linked open-angle glaucoma (GLC1A) in a Chinese family with primary open-angle glaucoma (POAG).A total of 23 members from five generations of a family were enrolled and underwent thorough ophthalmologic examinations. In addition, 200 unrelated healthy Chinese controls were also recruited as normal control. GLC1A gene was amplified by polymerase chain reaction, and DNA sequencing was performed to screen for mutations.Six members were diagnosed as POAG, with severe clinical manifestations, and history of high intraocular pressures. The mean age of disease onset was 26.3 years. However, the others were asymptomatic. In six affected and three asymptomatic members, gene sequencing revealed a mutation c.C1456T in exon 3 of myocilin gene (MYOC). Furthermore, we also identified a novel mutation c.G322A in beta-1,4-galactosyltransferase 3 (B4GALT3) gene in all six affected and three asymptomatic members, which was not reported previously in POAG patients. The two newly identified variants were absent in other family members as well as controls.The mutations c.1456C < T (p.L486F) in MYOC and c.322G < A (p.V108I) in B4GALT3 are likely responsible for the pathogenesis of POAG in this family.
| S-EPMC5146788 | biostudies-literature