目的建立针对MYOC基因RNAi慢病毒载体的优化及筛选方法,为研究后续突变基因的功能奠定基础。方法 Western Blot筛选RNAi有效靶点,Real-time PCR检测小梁细胞MYOC基因Knock Down效率。结果 Western blotting结果显示含有敲减质粒的组别...目的建立针对MYOC基因RNAi慢病毒载体的优化及筛选方法,为研究后续突变基因的功能奠定基础。方法 Western Blot筛选RNAi有效靶点,Real-time PCR检测小梁细胞MYOC基因Knock Down效率。结果 Western blotting结果显示含有敲减质粒的组别其蛋白表达明显减少;Real-time PCR结果显示在荧光显微镜下观察,与对照组相比含有敲减质粒的组别其MYOC基因的表达明显下降,1#、2#、3#具有确切的干扰效果。结论经过Western blotting及Real-time PCR方法检测所构建的4个干扰靶点的敲减效率,证实RNAi慢病毒干扰效果可靠,且两种方法结果一致,可随机选择1#、2#、3#中的任一位点作为后续干扰靶点。展开更多
AIM： To identify the mutations of MYOC, OPTN, CYPIB1 and WDR36 in a large Chinese family affected by juvenile open angle glaucoma （JOAG）. METHODS： Of 114 members of one family were recruited in this study. Blood ...AIM： To identify the mutations of MYOC, OPTN, CYPIB1 and WDR36 in a large Chinese family affected by juvenile open angle glaucoma （JOAG）. METHODS： Of 114 members of one family were recruited in this study. Blood samples from twelve members of this pedigree were collected for further research. As a control, 100 unrelated subjects were recruited from the same hospital. The exon and flanking intron sequences of candidate genes were amplified using the polymerase chain reaction and direct DNA sequencing. RESULTS： The proband （111：10） was a seventy-three years old woman with binocular JOAG at the age of 31. A recurrent heterozygous mutation （c.1099G〉A） of MYOC was identified in the three JOAG patients and another suspect. This transition was located in the first base pair of codon 367 （GGA〉AGA） in exon 3 of MYOC and was predicted to be a missense substitution of glycine to arginine （p.G367R） in myocilin. Mutations in OPTN, CYPIB1 or WDR36 were not detected in this study. The G367R mutation was not present in unaffected family members or in 100 ethnically matched controls. Other variants of the coding regions of candidate genes were not detected in all participants. To date, this family was the largest to have been identified as carrying a certain MYOC mutation in China, further evidence of a founder effect for the G367R MYOC mutant was provided by our data. CONCLUSION： A MYOC c.1099G〉A mutation in an autosomal dominant JOAG family is identified and the characteristic phenotypes among the patients are summarized. Genetic testing could be utilized in high-risk populations and be helpful not only for genetic counseling, but also for early diagnosis and treatment of affected patients or carriers of inherited JOAG.展开更多
AIM: To investigate whether the gene variants in MYOC and ABCA1 are associated with primary angle-closure glaucoma(PACG) and anterior chamber depth(ACD) and axial length(AL) in samples from northern China. METHODS: Th...AIM: To investigate whether the gene variants in MYOC and ABCA1 are associated with primary angle-closure glaucoma(PACG) and anterior chamber depth(ACD) and axial length(AL) in samples from northern China. METHODS: The present case-control association study consisted of 500 PACG patients and 720 unrelated controls. Each participant was genotyped for eleven single nucleotide polymorphisms(SNPs) in MYOC and ABCA1 genes(rs12076134, rs183532, rs235875 and rs235913 in MYOC, rs2422493, rs2487042, rs2472496, rs2472493, rs2487032, rs2472459 and rs2472519 near ABCA1) using an improved multiplex ligation detection reaction(iMLDR) technique. The genetic association analyses were performed by PLINK using a logistic regression model. The association between genotypes and ocular biometric parameters was performed by SPSS using generalized estimation equation. Bonferroni corrections wereimplemented and the statistical power was calculated by the Power and Sample Size Calculation. RESULTS: Two SNPs rs183532 and rs235875 as well as a haplotype TTC in MYOC were nominally associated with PACG despite the significance was lost after Bonferroni correction. No association was observed between ABCA1 and PACG, neither did the association between these variants and ACD as well as AL. CONCLUSION: The present study suggests MYOC and ABCA1 do not play a part in the pathogenesis of PACG as well as the regulation of ocular biometric parameters in a northern Chinese population. Further investigations with large sample size are needed to verify this consequence.展开更多
文摘目的建立针对MYOC基因RNAi慢病毒载体的优化及筛选方法,为研究后续突变基因的功能奠定基础。方法 Western Blot筛选RNAi有效靶点,Real-time PCR检测小梁细胞MYOC基因Knock Down效率。结果 Western blotting结果显示含有敲减质粒的组别其蛋白表达明显减少;Real-time PCR结果显示在荧光显微镜下观察,与对照组相比含有敲减质粒的组别其MYOC基因的表达明显下降,1#、2#、3#具有确切的干扰效果。结论经过Western blotting及Real-time PCR方法检测所构建的4个干扰靶点的敲减效率,证实RNAi慢病毒干扰效果可靠,且两种方法结果一致,可随机选择1#、2#、3#中的任一位点作为后续干扰靶点。
基金Supported by Natural Science Foundation of China(No.81270999No.81570870)Professor Academic Development Fund of Fujian Medical University(No.JS14019)
文摘AIM： To identify the mutations of MYOC, OPTN, CYPIB1 and WDR36 in a large Chinese family affected by juvenile open angle glaucoma （JOAG）. METHODS： Of 114 members of one family were recruited in this study. Blood samples from twelve members of this pedigree were collected for further research. As a control, 100 unrelated subjects were recruited from the same hospital. The exon and flanking intron sequences of candidate genes were amplified using the polymerase chain reaction and direct DNA sequencing. RESULTS： The proband （111：10） was a seventy-three years old woman with binocular JOAG at the age of 31. A recurrent heterozygous mutation （c.1099G〉A） of MYOC was identified in the three JOAG patients and another suspect. This transition was located in the first base pair of codon 367 （GGA〉AGA） in exon 3 of MYOC and was predicted to be a missense substitution of glycine to arginine （p.G367R） in myocilin. Mutations in OPTN, CYPIB1 or WDR36 were not detected in this study. The G367R mutation was not present in unaffected family members or in 100 ethnically matched controls. Other variants of the coding regions of candidate genes were not detected in all participants. To date, this family was the largest to have been identified as carrying a certain MYOC mutation in China, further evidence of a founder effect for the G367R MYOC mutant was provided by our data. CONCLUSION： A MYOC c.1099G〉A mutation in an autosomal dominant JOAG family is identified and the characteristic phenotypes among the patients are summarized. Genetic testing could be utilized in high-risk populations and be helpful not only for genetic counseling, but also for early diagnosis and treatment of affected patients or carriers of inherited JOAG.
基金Supported by the National Natural Science Foundation of China(No.81460093)the Ningxia Nature Science Funding from the Department of Science and Technology of Ningxia Hui Autonomous Region(No.NZ16194)
文摘AIM: To investigate whether the gene variants in MYOC and ABCA1 are associated with primary angle-closure glaucoma(PACG) and anterior chamber depth(ACD) and axial length(AL) in samples from northern China. METHODS: The present case-control association study consisted of 500 PACG patients and 720 unrelated controls. Each participant was genotyped for eleven single nucleotide polymorphisms(SNPs) in MYOC and ABCA1 genes(rs12076134, rs183532, rs235875 and rs235913 in MYOC, rs2422493, rs2487042, rs2472496, rs2472493, rs2487032, rs2472459 and rs2472519 near ABCA1) using an improved multiplex ligation detection reaction(iMLDR) technique. The genetic association analyses were performed by PLINK using a logistic regression model. The association between genotypes and ocular biometric parameters was performed by SPSS using generalized estimation equation. Bonferroni corrections wereimplemented and the statistical power was calculated by the Power and Sample Size Calculation. RESULTS: Two SNPs rs183532 and rs235875 as well as a haplotype TTC in MYOC were nominally associated with PACG despite the significance was lost after Bonferroni correction. No association was observed between ABCA1 and PACG, neither did the association between these variants and ACD as well as AL. CONCLUSION: The present study suggests MYOC and ABCA1 do not play a part in the pathogenesis of PACG as well as the regulation of ocular biometric parameters in a northern Chinese population. Further investigations with large sample size are needed to verify this consequence.
