多次退火环状循环扩增技术在单细胞水平诊断脊髓性肌萎缩症的应用评估

Multiple annealing and looping-based amplification cycles(MALBAC) technology at the single cell level diagnosis of spinal muscular atrophy application evaluation

目的:应用并评价多次退火环状循环扩增(multiple annealing and looping-based amplification cycles,MALBAC)技术在单细胞水平诊断脊髓性肌萎缩症(spinal muscular atrophy,SMA)基因变异的效率。方法:收集SMN1基因7号外显子纯合缺失、正常皮肤成纤维细胞以及废弃胚胎单个卵裂球细胞,分别使用MALBAC和多重链置换扩增(multiple displacement amplification,MDA)方法进行全基因组扩增(WGA)。Sanger测序检测SMN1及SMN2序列,并对3个微卫星位点进行连锁分析。结果:2种扩增技术的总扩增成功率、等位基因脱扣(ADO)率无统计学差异(P>0.05);MALBAC组诊断准确率为91.7%(67/73),低于MDA组的96.1%(73/76)(P<0.05)。结论:针对SMA疾病开展单细胞水平遗传学诊断,传统MDA方法略优于MALBAC全基因组扩增技术。

Objective： To evaluate diagnosis of gene mutation efficiency of spinal muscular atrophy （spinal muscular atrophy, SMA） by using multiple annealing and looping based amplification cycle technologys （MALBAC） at the single cell level. Methods： Genome DNA of single cell of SMA fibroblasts （homozygous deletion of exon 7 of survival motor neuron gene 1, SMN1） and normal fibroblasts, discarded embryos of single blastomere cells respectively using MALBAC and multiple displacement amplification （MDA） to be amplified first. Then, Sanger sequencing was used to detect SMN1 and SMN2 gene sequences. Three microsatellite loci were used for linkage analysis. Results： Between the two whole genome amplification （WGA） methods, there was no statistical difference in the total success rate of amplification or total allele dropout （ADO） rate. The accuracy rate in MALBAC group [91.7%（67/73）] was lower than that in MDA group [96.0%（73/76）]（P〈0.05）. Conclusion： In view of the SMA disease to carry out the single cell level genetic diagnosis, traditional MDA method is slightly better than MALBAC.