CAJ | 학술논문

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SMNl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SMN1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SMN1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.
목적 건립일충준학、쾌첩적방법,정량검측운동신경원존활기인(SMN)적고패수,이편분석비순합결실형척수성기위축증(SMA)환인중SMNl기인적잡합성결실.방법 응용등위기인특이PCR(AS-PCR)분별진행SMN1여SMN2기인적특이확증,용령외2개무관기인작내대조,진행변성고효액상색보법(DHPLC)분석,학정기인고패수.결과 (1)개진적쌍중AS-PCR여DHPLC상결합적기술,능구유효분리SMN1화SMN2기인,통과여대조기인적대비,가준학지판단SMN기인적고패수,SMN1화SMN2기인1～4고패지간불존재중첩.(2)38례비순합결실SMA환인중,20례적SMN1기인위1개고패(52.6%),판단위SMN1기인적잡합성결실,기중15례(75.0%,15/20)적SMN2기인위2개고패,5례(25.0%,5/20)SMN2기인위3개고패.(3)30명SMN1기인순합결실형돌변환자적쌍친중,유24명(80.0%)적SMNl기인위1개고패.결론 본연구소건립적방법능구준학、쾌첩지검측SMN기인적고패수.
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 SMN1 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 SMN2genes 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.