原发性开角型青光眼家系MYOC基因突变的研究
发布时间：2018-10-18 16:49
【摘要】：研究目的探讨小梁网糖皮质激素诱导反应蛋白(MYOC)基因、细胞色素P450超家族1亚家族(CYP1B1)基因与原发性开角型青光眼(POAG)家系之间的发病关系。研究对象家系G-05:3代POAG家系,共采集到5份血样,其中2人患病,2人可疑,其余人正常。家系G-04:5代POAG家系,共11人患病,采集到的15份血样中6人为患者,3人可疑,其余人正常。2个家系中的患者均已行手术治疗。研究方法(1)对2个家系20人进行严格的眼科检查;(2)抽取2个家系中8个患者及余下可疑者和正常者的外周血4ml;(3)使用Wizard Genomic DNA Purification试剂盒提取和纯化20位家系成员外周静脉血中的基因组DNA(gDAN);(4)参照已知青光眼致病基因MYOC和CPY1B1的DNA序列,设计和合成相应的PCR(polymerase chain reaction聚合酶链反应)引物5对,各选择2个家系中的一位患者的gDNA作为模板,应用PCR技术扩增MYOC及CYP1B1基因的编码外显子及剪切链接区域;(5)将PCR产物纯化并进行正向和反向测序;(6)将测序结果与正常人MYOC基因、CYP1B1基因序列进行对比分析,若发现突变,继续对家系中其他人做突变位点的筛查。研究结果2个家系的20人中,家系G-05家系及家系G-04均发现位于MYOC的突变,均符合常染色体显性遗传伴不完全外显率。G-05中3人(2名患者及1名可疑者)携带突变MYOC/R46X(c.C136T),即位于MYOC 1号外显子上第46位密码子中第1位碱基的杂合突变CT(c.C136T),是一个无义突变,终止密码子替换编码精氨酸的密码子(UGA→Stop,p.R46X),最终翻译为一个仅有45个氨基酸的截短蛋白。G-04中6名患者、3名可疑者及1名正常者携带突变MYOC/P370L(c.C1109T),即3号外显子上第370位密码子中第2位碱基的杂合突变CT(c.C1109T),脯氨酸突变为亮氨酸(p.Pro370Leu)。两个家系均未发现位于CYP1B1的基因突变。研究结论MYOC基因的突变R46X、P370L分别是G-05、G-04家系的致病突变基因,且Pro370Leu与临床表型严重的POAG紧密相关。基因筛查可作为POAG早期诊断一种有效方法,对POAG的可疑者及患者做出预警并指导早期干预治疗。
[Abstract]:Objective to investigate the relationship between trabecular meshwork glucocorticoid inducible reactive protein (MYOC) gene, cytochrome P450 superfamily 1 subfamily (CYP1B1) gene and primary open-angle glaucoma (POAG) family. Subjects five blood samples were collected from G-05: 3 POAG families, 2 of them were ill, 2 were suspicious, and the rest were normal. There were 11 patients in G-04: 5 POAG family, 6 of 15 blood samples were patients, 3 were suspicious, and the rest were normal. All the patients in 2 families had been treated surgically. Methods: (1) strict ophthalmologic examination was performed on 20 individuals from 2 families; (2) peripheral blood of 8 patients from 2 families and the remaining suspicious and normal individuals were extracted from 4 ml; (3) peripheral blood was extracted and purified from 20 family members using Wizard Genomic DNA Purification kit. The genomic DNA (gDAN); (4 in venous blood follows the DNA sequence of MYOC and CPY1B1 genes known to cause glaucoma. Five pairs of primers for PCR (polymerase chain reaction were designed and synthesized. The gDNA of one patient from two families was selected as template. PCR technique was used to amplify the encoding exons and splicing link regions of MYOC and CYP1B1 genes; (5) to purify PCR products and carry out forward and reverse sequencing; (6) to compare the sequencing results with those of normal human MYOC gene and CYP1B1 gene. Continue screening for mutation sites in other families. Results the mutation in MYOC was found in both G-05 and G-04 families of 20 individuals from 2 families. In G-05, 3 patients (2 patients and 1 suspicious person) carried mutated MYOC/R46X (c.C136T), a heterozygous CT (c.C136T) at the first base of codon 46 on exon 1 of MYOC. The termination codon replaces the arginine encoded codon (UGA Stop,p.R46X), and is translated into a 45-amino acid truncated protein .G-04 in 6 patients, 3 suspicious individuals and 1 normal person with mutant MYOC/P370L (c.C1109T), that is, the 370th position on exon 3. The heterozygous mutation of the second base in the codon was CT (c.C1109T) and proline mutated to leucine (p.Pro370Leu). No CYP1B1 gene mutation was found in the two families. Conclusion the mutation of MYOC gene R46XP370L is a pathogenic gene of G-05G -04 family, and Pro370Leu is closely related to POAG with severe clinical phenotype. Gene screening can be used as an effective method for early diagnosis of POAG. It can warn suspicious patients and patients of POAG and guide early intervention therapy.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R775
本文编号：2279748
[Abstract]:Objective to investigate the relationship between trabecular meshwork glucocorticoid inducible reactive protein (MYOC) gene, cytochrome P450 superfamily 1 subfamily (CYP1B1) gene and primary open-angle glaucoma (POAG) family. Subjects five blood samples were collected from G-05: 3 POAG families, 2 of them were ill, 2 were suspicious, and the rest were normal. There were 11 patients in G-04: 5 POAG family, 6 of 15 blood samples were patients, 3 were suspicious, and the rest were normal. All the patients in 2 families had been treated surgically. Methods: (1) strict ophthalmologic examination was performed on 20 individuals from 2 families; (2) peripheral blood of 8 patients from 2 families and the remaining suspicious and normal individuals were extracted from 4 ml; (3) peripheral blood was extracted and purified from 20 family members using Wizard Genomic DNA Purification kit. The genomic DNA (gDAN); (4 in venous blood follows the DNA sequence of MYOC and CPY1B1 genes known to cause glaucoma. Five pairs of primers for PCR (polymerase chain reaction were designed and synthesized. The gDNA of one patient from two families was selected as template. PCR technique was used to amplify the encoding exons and splicing link regions of MYOC and CYP1B1 genes; (5) to purify PCR products and carry out forward and reverse sequencing; (6) to compare the sequencing results with those of normal human MYOC gene and CYP1B1 gene. Continue screening for mutation sites in other families. Results the mutation in MYOC was found in both G-05 and G-04 families of 20 individuals from 2 families. In G-05, 3 patients (2 patients and 1 suspicious person) carried mutated MYOC/R46X (c.C136T), a heterozygous CT (c.C136T) at the first base of codon 46 on exon 1 of MYOC. The termination codon replaces the arginine encoded codon (UGA Stop,p.R46X), and is translated into a 45-amino acid truncated protein .G-04 in 6 patients, 3 suspicious individuals and 1 normal person with mutant MYOC/P370L (c.C1109T), that is, the 370th position on exon 3. The heterozygous mutation of the second base in the codon was CT (c.C1109T) and proline mutated to leucine (p.Pro370Leu). No CYP1B1 gene mutation was found in the two families. Conclusion the mutation of MYOC gene R46XP370L is a pathogenic gene of G-05G -04 family, and Pro370Leu is closely related to POAG with severe clinical phenotype. Gene screening can be used as an effective method for early diagnosis of POAG. It can warn suspicious patients and patients of POAG and guide early intervention therapy.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R775
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