MELAS综合征无创性基因突变分析方法研究

Identification of an ideal noninvasive method to detect A3243G gene mutation in MELAS syndrome

目的研究携带A3243G突变的线粒体脑病病-酸酸中毒-卒中样发作（MELAS）综合征患者及其母系亲属的外周血、尿、毛囊、唾液和部分患者的肌肉组织A3243G基因突变率，找到用于检测突变率更敏感的组织，建立更佳的、无创性的检查手段。方法收集25例MELAS患者及其母系亲属33例的外周血、尿、毛囊、唾液和部分患者的肌肉组织提取DNA，利用聚合酶链反应-限制性片段长度多态性（PCR-RFLP）技术检测各组织中A3243G突变，根据酶切产物电泳条带密度值问的比例计算突变型线粒体DNA的含量，比较各组织A3243G突变率的差异。结果25例患者外周血、尿、毛囊、唾液均检测到A3243G突变，尿液中A3243G突变率（62％±9％）明显高于外周血中的突变率（36％±10％）（t=-11．13，P〈0．01）。5例患者进行了肌活检，肌肉突变率44．9％-75．1％，尿液中A3243G突变率和肌肉中A3243G突变率存在正相关，（r=0．900，P=0．037）。33例母系亲属中有28例在至少一种组织中检测到A3243G突变，尿液中A3243G突变率33．0％（5．0％～70．4％）明显高于外周血中的突变率8．0％（0～33．3％）（z=-4．197，P〈0．01）。患者及其母系亲属唾液和毛囊组织中A3243G突变率与外周血相比差别均不大。结论在MELAS患者及其母系亲属中，尿液中A3243G突变率均明显高于外周血，尿液A3243G突变率分析可能是优于外周血线粒体分析的一种无创性方法。

Objective To identify a better non-invasive method to detect the carrier of mitochondrial A3243G mutation, a cause of mitochondrial encephalopathy-laetic acidosis-stroke like episode （MELAS） syndrome. Methods DNA was extracted from the peripheral blood, urine, hair follicle, and saliva of 25 MELAS syndrome patients carrying A3243G mutation and their mothers and other maternal relatives, 33 persons in number, and the muscle tissues from 5 patients obtained by biopsy. A3243G mutation was detected by PCR-RFLP method, and the A3243G mutation ratio was identified by measuring the density of each band and calctdation with the software AlphaEase 5.0. Results A3243G mutations were detected in all tissues of the 25 MELAS patients. The A3243G mutation ratio in urine was 62% ± 9% , significantly higher than that in the blood [ （36%± 10% ）, t = - 11.13, P 〈0. 01 ]. A3243G mutations were detected in at least one tissue of the 28 maternal relatives. The A3243G mutation rates in their urine samples was 33.0% （ 5.0% -70.4% ）, significantly higher than that in their blood samples [ 8.0% （ 0- 33.3% ）, z = -4. 197 ,P 〈0.01 ]. There was no significant difference in A3243G mutation ratio among the samples of hair follicle, saliva, and blood. Conclusion The A3243G mutation ratio in urine is significantly higher than those in blood samples of the patients and their maternal relatives. A noninvasive method, A3243G mutation ratio analysis of urine is superior to that in blood.