应用UP-GC-MS和基因分析诊断甲基丙二酸尿症.pdfVIP

应用 UP-GC-MS 和基因分析诊断甲基丙二酸尿症 摘 要 目的 探讨尿素酶预处理-气相色谱-质谱法（UP-GC-MS）在甲基丙二酸尿症 （MMA）诊断治疗中的应用价值。采用基因测序分析甲基丙二酸尿症患儿具体突 变类型。 方法 收集 157 例遗传代谢病高危患儿尿液，经去尿素﹑加内标(正十七酸) ﹑除蛋白和真空干燥等处理，残余物进行三甲基硅烷化衍生后进样，应用尿素酶 预处理-气相色谱-质谱法分析。通过聚合酶链反应(PCR)对 2 个家系的患儿及其 父母的MMACHC基因外显子进行扩增并测序。 结果 157例高危儿中，8例尿液中可检测到明显的甲基丙二酸、甲基柠檬酸 等（甲基丙二酸尿症尿液标志物）信号。在总离子流图（TIC）上，两种物质的 绝对、相对保留时间分别是 6.78min、0.396min 和 8.52min、0.24min。在质谱 图上，甲基丙二酸的特征离子（m/z，TMS）分别为73﹑147和247。2例确诊MMA 患儿进行了家系（7人）基因分析，均表现为MMACHC基因2号外显子的146_154 位缺失CCTTCCTGG，4号外显子609位G突变为A。 结论 UP-GC-MS 分析尿液成分是确诊甲基丙二酸尿症的重要而可靠方法。中 国患者及其家属发现了MMACHC基因新的遗传突变146_154位缺失CCTTCCTGG。 关键词 甲基丙二酸尿症，尿素酶预处理-气相色谱-质谱法， MMACHC，基因 分析 Ⅰ Diagnosis of methylmalnic aciduria (MMA) using UP-GC-MS and gene analysis Abstract Objective To investigate the value of urease pretreatment-gas chromatography-mass spectrometry (UP-GC-MS) in the diagnosis of methylmalnic aciduria (MMA). To analyze the mutation types of methylmalonic aciduria patients by gene sequencing . Methods 157 urine samples were collected from the populations with high risk of Inherited metabolic disorders. Urea was decomposed with urease, heptadecanoic acid was added as an internal standard, protein was denaturated with ethanol and precipitate was removed by centrifugation, evaporation was performed to dryness, the residue was trimethylsilylly derivatized with BSTFA/TMCS, and then analyzed with GC-MS. The exons of MMACHC gene were amplified from the 2 MMA families with the patients and their parents by PCR，The PCR products were analyzed by sequencing. Results Large quant