多次退火环状循环扩增和多重置换扩增技术在β地中海贫血基因变异诊断效率中的差异分析

Comparison of multiple annealing and looping-based amplification cycles(MALBAC) and multiple displacement amplification(MDA) in variation diagnosis of β-thalassemia disease at single-cell level

目的:探讨最适的β地中海贫血疾病HBB基因单细胞全基因组扩增方法。方法:60份β地中海贫血成纤维细胞(HBB基因变异位点CD17和IVSⅡ654)和48份废弃胚胎单个卵裂球进行多次退火环状循环扩增法(MALBAC)和多重置换扩增法(MDA)扩增及高通量测序,比较位点检测率、等位基因脱扣(ADO)率及扩增均一度等。结果:β地中海贫血疾病HBB基因MALBAC技术位点检测率(100%)高于MDA技术(96.3%);CD17和IVSⅡ654的ADO率MALBAC技术为9.09%和0.00%,MDA技术为23.08%和19.23%;对编码人β-珠蛋白的HBB基因附近60个SNP位点检测显示MALBAC技术ADO率为12.04%,MDA技术为21.25%;MALBAC技术拷贝数变异检测变异系数为0.13,MDA技术为0.15。结论:β地中海贫血单细胞诊断MALBAC法优于M D A法。

Objective： To detect the anplication efficacy on β-thalassemia of multiple annealing and looping- based amplification cycles （MALBAC）. Methods： Totally 60 fibroblasts samples with variation of CD 17 and IVS 11 654 were amplified at single-cell level by MALBAC and multiple displacement amplification （MDA）, respectively. Totally 48 single-blastomere derived from discarded embryos, were amplified by MALBAC and MDA. Re-sequencing with high depth was performed to detect difference of genome coverage, uniformity, allele drop out （ADO） rates between two methods. Results： The success rate ofHBB gene amplification with fibroblasts samples was 100,00% in MALBAC and 96.29% in MDA. ADO rate for CD17 allelic was 9.09% in MALBAC and 23.08% in MDA. ADO rate for IVS I1654 allelic was 0.00% in MALBAC and 19.23% in MDA. ADO rate for 60 SNP markers close to the HBB gene was 12.04% in MALBAC and 21.25% in MDA. The coefficient of variation （CV） of copy number variation （CNV） at single-cell level detection in MALBAC and MDA was 0.13 and 0.15, respectively. Conclusion： MALBAC has a higher level of uniformity, specificity and lower ADO rate in pre-implantation genetic diagonosis （PGD） for [3-thalassemia at single-cell level than that of MDA. Combined with haplotype analysis with SNP markers, MALBAC is superior to MDA in PGD of β-thalassemia disease.