MYOC Gene Sequencing Analysis in Primary Open-Angle Glaucoma Patients from the Centre Region of Portugal.

INTRODUCTION: Primary open-angle glaucoma is the most frequent subtype of glaucoma. Relatives of primary open-angle glaucoma patients have an increased risk of developing the disease, suggesting a genetic predisposition to the disease. MYOC was the first primary open-angle glaucoma-causing gene identified. This study aimed to identify sequence variations in the MYOC gene that may be responsible for the phenotype in a group of primary open-angle glaucoma patients from the Centre Region of Portugal. MATERIAL AND METHODS: The three coding exons and the proximal splicing junctions of the MYOC gene were studied using a PCR sequencing approach in a group of 99 primary open-angle glaucoma patients. RESULTS: The sequencing analysis enabled the identification of 20 variants, including four in the promoter region, seven in the introns and nine in exons one and three, of which four were missense variants. DISCUSSION: Initially, all four missense sequence variations identified were considered candidates to glaucoma causing disease mutations. However, after literature review, only variant c.1334C>T (Ala445Val) remained as likely responsible for mild late-onset normal tension glaucoma. CONCLUSION: This is the first study performed in a group of primary open-angle glaucoma patients from the Centre Region of Portugal, contributing to the identification of one genetic variant in the MYOC gene and reinforcing the hypothesis that normal tension glaucoma could be also due to MYOC gene mutations.

Introdução: O glaucoma primário de ângulo-aberto é o subtipo mais frequente de glaucoma. Os familiares de doentes com glaucoma primário de ângulo-aberto têm um risco maior de desenvolverem a doença, o que sugere uma predisposição genética para a doença. MYOC foi o primeiro gene causador de glaucoma primário de ângulo-aberto a ser identificado. Este estudo pretendeu identificar variações de sequência no gene MYOC que possam ser responsáveis pelo fenótipo num grupo de doentes com glaucoma primário de ângulo-aberto da Região Centro de Portugal. Material e Métodos: Os três exões codificantes e as regiões adjacentes do gene MYOC foram estudados utilizando o método de PCR-sequenciação num grupo de 99 doentes com glaucoma primário de ângulo aberto. Resultados: A análise de sequenciação permitiu identificar 20 variantes, incluindo quatro na região promotora, sete nos intrões e nove nos exões um e três, das quais quatro eram variantes missense. Discussão: Inicialmente, todas as quatro variações de sequência missense identificadas foram consideradas candidatas a mutações causadoras de glaucoma. No entanto, após análise da literatura, somente a variante c.1334C>T (Ala445Val) permaneceu como provável responsável pelo glaucoma de pressão normal de início tardio. Conclusão: Este é o primeiro estudo realizado num grupo de doentes com glaucoma primário de ângulo aberto da Região Centro de Portugal, contribuindo para a identificação de uma variante genética no gene MYOC e reforçando a hipótese de que o glaucoma de pressão normal também poderá ser causado por mutações no gene MYOC.

Analysis of MYOC gene mutation in a primary open angle glaucoma family from China / 中华实验眼科杂志

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.

Analysis of Myocilin gene regulatory network using a genetic genomics approach / 中华实验眼科杂志

Background The pathogenesis of primary open angle glaucoma(POAG) and high myopia are very complex.To construct the regulatory network of virulence genes and relevant genes that involved in pathogenicity are helpful for reveal of the pathogenesis.Objective The aim of this study was to investigate myocilin(Myoc),a gene that contributes to POAG and high myopia in eyes of BXD Recombinant Inbred(BXD RI)mice and construct the regulatory network of Myoc.Methods The affymetrix microarray system was used to detect the differential expression of Myoc in the eyes of C57BL/6J(B6),DBA/2J(D2) and BXD RI mice.Expression quantitative trait loci (eQTL) mapping was performed to construct the regulatory network of Myoc gene.Results The average expression level of the Myoc gene in the BXD strains was 10.83,and the gene exhibited expression levels ranging from 8.39 in BXD55 mice tol 1.43 in B6 mice.The eQTL mapping for the Myoc gene showed a significant likelihood ratio statistic (LRS) of 21.78.The QTL was mapped in chromosome 2,and Myoc was located on chromosome 1,indicating that the Myoc gene was a trans-acting QTL.Olfml2a was identified to be a candidate upstream gene of Myoc by analysis of bioinformatics.Genetic regulatory network analysis demonstrated that a series of genes associated with Myoc probably played roles in the pathogenesis and development of POAG and high myopia.Conclusions The genetical genomics approach provides a powerful tool for constructing pathways that contribute to complex traits,such as POAG and high myopia.

Study on the toxicity and transfection efficiency of PEI/MYOC gene complexes in vivo / 眼科

Objective To investigate the toxicity and transfection efficiency of PEI-mediated plasmid DNA encoding myocilin (MYOC) gene transfected into trabecular meshwork.Design Experimental study.Participants SD rats.Methods PEI and plasmid encoding MYOC gene complex was prepared and 20?l PEI/DNA complex was directly injected into anterior chamber of SD rat by microinjection. The eyes were extracted at day 1,3,7,and 14 after injection for the structure analysis of trabecular meshwork by HE staining and transmission electron microscope.Expression of the transferred myocilin gene was monitored by fluorescence microscopy. Main Outcome Measures HE staining,immunohistochemistry and electron microscope.Results No significant toxicity or inflammation was detected under the HE staining and electron microscope.PEI/DNA complex were seen in cytoplasm of trabecular meshwork cells. The fluorescence intensity of myocilin expression in the trabecular meshwork cells was increasing with time and peaked on clays 3 and declined afterward.Conclusion Plasmid DNA mediated by PEI could be efficiently transfected into trabecular meshwork cells by directly injecting into anterior chamber without any toxicity.