长片段PCR-DNA测序方法在Rett综合征诊断中的应用  

Application of long range polymerase chain reaction and DNA direct sequencing in diagnosis of Rett syndrome

作　　者：李美蓉[1] 潘虹[1] 包新华[1] 曹广娜[1] 吴希如[1] 

机构地区：[1]北京大学第一医院儿科,100034

出　　处：《中华儿科杂志》2007年第8期579-582,共4页Chinese Journal of Pediatrics

基　　金：国家自然科学基金(30271374)

摘　　要：目的探讨利用长片段 PCR-DNA 测序方法检测 Rett 综合征(RTT)患儿 MECP2基因突变的可行性及临床意义。方法对40例临床诊断的 RTT 患儿用盐析法从外周血提取基因组DNA,采用长片段 PCR 同时扩增 MECP2基因的第3和第4外显子,用1.5%的琼脂糖凝胶鉴定扩增目的片段的大小,进行 DNA 直接测序。结果在40例 RTT 患儿中有33例患儿 MECP2基因存在突变:无义突变16例;错义突变14例;缺失突变3例,其中有一例为314 bp 的大片段基因缺失。突变以p.T158M 最为多见,占21%(7/33),其后依次为 p.R255X,占12%(4/33),p.R168X 和p.R106W 各占9%(3/33),p.R270X 和 p.Y141X 各占6%(2/33),p.R133C、p.D156H、p.F157L、p.P225R、p.Q244X、p.Q262X、p.R294X、p.R306C、P322L、c.1005delG、c.1005-1318del 314 bp 和 c.1127-1179del 53bp 各占3%(1/33)。结论长片段 PCR 方法鉴定了83%(33/40)的 RTT 患儿存在 MECP2基因突变,目前是一种简单、方便、快速、准确的基因诊断方法,能同时发现常见突变和基因大片段的缺失,有助于 RTT 的诊断。Objective Rett syndrome （RTr, MIM 312750） is a progressive neurodevelopmental disorder that affects females almost exclusively, caused by mutations in MECP2 gene on chromosome Xq28, with symptoms such as autism, severe mental deficiency, deceleration of head growth, ataxia, loss of purposeful hand function and characteristic stereotypic hand movements. Over 80% MECP2 mutations located in the exon 3 and exon 4 were confirmed by our work and large-scale studies. RTr is defined based on clinical presentation. It is difficult to diagnose in the early life without definite biochemical abnormality, but genetic test is helpful for this. The aim of this study was to investigate the feasibility and clinical significance of applying long range polymerase chain reaction （PCR） to RTr diagnosis and establish a simple, economic, efficient method of genetic diagnosis. Method Genomic DNA was extracted using standard procedures from the peripheral blood leukocytes of each patient. Long range polymerase chain reaction（PCR） and DNA direct sequencing were employed to analyze the exon 3 and 4 of MECP2 gene simultaneity in 40 patients with RTr. The PCR products were checked by using 1.5% agarose gel. Result In total, 18 different MECP2 mutations were identified in 33 of the 40 diagnosed sporadic female patients with RTr. Missense mutations were 16, followed by 14 nonsense mutations and 3 deletions. The 314 base pairs large deletion was identified. The p. T158M mutation （21%, 7/33） was the most common, followed in order of frequency by p. R255X （ 12%, 4/33 ）, p. R168X and p. R106W （9%, 3/33） respectively, p. R270X and p. Y141X （6%, 2/33） respectively, p. R133C, p. D156H, p. P157L, p. P225R, p. Q244X, p. Q262X, p. R294X, p. R306C, P322L, c. 1005del G, c. 1005-1318del 314 bp and c. 1127-1179del 53 bp （3%, 1/33）, respectively. Conclusion Long range PCR is a simple, economic, quick, precise method of genetic diagnosis and was able to find 83% MECP2 gene mutations in RTr patients in this study. It is helpful

关 键 词：RETT综合征 聚合酶链反应 染色体蛋白质类 非组蛋白 DNA结合蛋白质类 阻遏蛋白质类 突变 

分 类 号：R748[医药卫生—神经病学与精神病学]