运用DHPLC技术分析非纯合缺失型脊髓性肌萎缩症患儿的SMN基因拷贝数  

Quantitative analysis of SMNI and SMN2 genes based on DHPLC： a reliable method for detection of non-homozygous patients with spinal muscular atrophy

作　　者：龙美娟[1] 宋昉[1] 瞿宇晋[1] 孟岩[2] 王红[1] 金煜炜[1] 黄尚志[2] 

机构地区：[1]首都儿科研究所遗传室,北京100020 [2]中国医学科学院基础医学研究所北京协和医学院基础院医学遗传系,100005

出　　处：《中华医学杂志》2008年第18期1259-1263,共5页National Medical Journal of China

基　　金：国家“十五”科技攻关计划基金资助项目（2004BA720A04）;北京市科学技术委员会研发攻关类基金资助项目（D0906005040491）

摘　　要：目的 建立一种准确、快捷的方法，定量检测运动神经元存活基因（SMN）的拷贝数，以便分析非纯合缺失型脊髓性肌萎缩症（SMA）患儿中SMN1基因的杂合性缺失。方法应用等位基因特异PCR（AS-PCR）分别进行SMN1与SMN2基因的特异扩增，用另外2个无关基因作内对照，进行变性高效液相色谱法（DHPLC）分析，确定基因拷贝数。结果（1）改进的双重AS-PCR与DHPLC相结合的技术，能够有效分离SMN1和SMN2基因，通过与对照基因的对比，可准确地判断SMN基因的拷贝数，SMN1和SMN2基因1-4拷贝之间不存在重叠。（2）38例非纯合缺失SMA患儿中，20例的SMN1基因为1个拷贝（52．6％），判断为SMNl基因的杂合性缺失，其中15例（75．0％，15／20）的SMN2基因为2个拷贝，5例（25．0％，5／20）SMN2基因为3个拷贝。（3）30名SMNl基因纯合缺失型突变患者的双亲中，有24名（80．0％）的SMNl基因为1个拷贝。结论本研究所建立的方法能够准确、快捷地检测SMN基因的拷贝数。Objective To develop a rapid and reliable approach for testing the copy number of survival motor neuron （SMN） gene and analyze the compound heterozygous deletions of SMNI gene. Methods Peripheral blood samples were collected from 38 non-homozygous deletion pediatric patients with SMA, 30 homozygous deletion patients with SMA, and 35 un-related healthy persons. SMN1 and SMN2 genes were amplified separately with allele-specific PCR （AS-PCR）. Meanwhile, two irrelevant genes were amplified as internal quality control respectively. The copy numbers of SMN1 and SMN2 were determined by denaturing high-performance liquid chromatography （DHPLC）. Results （1） A protocol combining multiplex allele-specific PCR and DHPLC was developed to separate SMN1 and SMN2 and to determine the copy numbers of them. The copy numbers of SMN1 and SMN2 varied from 1 to 4 and a clear-cut differentiation among the different copy number ranges could be observed for the two genes. （2） One single copy of SMN1 were detected in 20 of the 38 non-homozygous deletion patients with SMA （52. 6% ）. Heterozygous deletions were determined in these 20 patients. Two copies of SMN2 were detected in 15 of the 20 patients with one copy of SMN1 （75.0%, 15/20）. Other 5 of the 20 patients were with 3 copies of SMN2 （25.0% , 5/20）. （3） One single copy of SMN1 was detected in 24 of the 30 （80%） parents of SMA patients with homozygous deletion. Conclusion SMN copy number can be rapidly and reliably determined by the method of multiplex AS-PCR combined with DHPLC.

关 键 词：脊髓性肌萎缩 儿童 基因 聚合酶链反应 运动神经元存活基因 变性高效 液相色谱法 

分 类 号：R686[医药卫生—骨科学]