原發(fā)性開角型青光眼家系MYOC基因突變的研究
發(fā)布時間：2018-10-18 16:49
【摘要】：研究目的探討小梁網(wǎng)糖皮質(zhì)激素誘導(dǎo)反應(yīng)蛋白(MYOC)基因、細胞色素P450超家族1亞家族(CYP1B1)基因與原發(fā)性開角型青光眼(POAG)家系之間的發(fā)病關(guān)系。研究對象家系G-05:3代POAG家系,共采集到5份血樣,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份血樣中6人為患者,3人可疑,其余人正常。2個家系中的患者均已行手術(shù)治療。研究方法(1)對2個家系20人進行嚴格的眼科檢查;(2)抽取2個家系中8個患者及余下可疑者和正常者的外周血4ml;(3)使用Wizard Genomic DNA Purification試劑盒提取和純化20位家系成員外周靜脈血中的基因組DNA(gDAN);(4)參照已知青光眼致病基因MYOC和CPY1B1的DNA序列,設(shè)計和合成相應(yīng)的PCR(polymerase chain reaction聚合酶鏈反應(yīng))引物5對,各選擇2個家系中的一位患者的gDNA作為模板,應(yīng)用PCR技術(shù)擴增MYOC及CYP1B1基因的編碼外顯子及剪切鏈接區(qū)域;(5)將PCR產(chǎn)物純化并進行正向和反向測序;(6)將測序結(jié)果與正常人MYOC基因、CYP1B1基因序列進行對比分析,若發(fā)現(xiàn)突變,繼續(xù)對家系中其他人做突變位點的篩查。研究結(jié)果2個家系的20人中,家系G-05家系及家系G-04均發(fā)現(xiàn)位于MYOC的突變,均符合常染色體顯性遺傳伴不完全外顯率。G-05中3人(2名患者及1名可疑者)攜帶突變MYOC/R46X(c.C136T),即位于MYOC 1號外顯子上第46位密碼子中第1位堿基的雜合突變CT(c.C136T),是一個無義突變,終止密碼子替換編碼精氨酸的密碼子(UGA→Stop,p.R46X),最終翻譯為一個僅有45個氨基酸的截短蛋白。G-04中6名患者、3名可疑者及1名正常者攜帶突變MYOC/P370L(c.C1109T),即3號外顯子上第370位密碼子中第2位堿基的雜合突變CT(c.C1109T),脯氨酸突變?yōu)榱涟彼?p.Pro370Leu)。兩個家系均未發(fā)現(xiàn)位于CYP1B1的基因突變。研究結(jié)論MYOC基因的突變R46X、P370L分別是G-05、G-04家系的致病突變基因,且Pro370Leu與臨床表型嚴重的POAG緊密相關(guān)�；蚝Y查可作為POAG早期診斷一種有效方法,對POAG的可疑者及患者做出預(yù)警并指導(dǎo)早期干預(yù)治療。
[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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R775
本文編號：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.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R775
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