论文部分内容阅读
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 To explore the feasibility and clinical significance of using long PCR-DNA sequencing to detect MECP2 gene mutations in children with Rett syndrome (RTT). Methods Genomic DNA was extracted from peripheral blood of 40 clinically diagnosed RTT children by salting-out method. The 3 and 4 exons of MECP2 gene were amplified by long-segment PCR and identified by 1.5% agarose gel Increase the size of the fragment, direct DNA sequencing. Results Of 40 children with RTT, there were 33 cases of MECP2 gene mutations: nonsense mutation in 16 cases; missense mutation in 14 cases; deletion mutation in 3 cases, of which a case of 314 bp large fragment gene deletion. The mutations were the most common in p.T158M, accounting for 21% (7/33), followed by p.R255X, accounting for 12% (4/33), p.R168X and p.R106W each accounting for 9% (3/33 ), p.R270X and p.Y141X each 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 and c.1127-1179del 53bp each accounted for 3% (1/33). Conclusion The long PCR method identified 83% (33/40) of RTT patients with MECP2 gene mutation, which is a simple, convenient, rapid and accurate method for gene diagnosis, which can find both common mutations and large gene deletion , Contribute to the diagnosis of RTT.