通过富集母血浆中胎儿游离DNA无创性产前诊断β地中海贫血

Noninvasive prenatal diagnosis of β-thalassemia by enriching cell-free fetal DNA in materal plasma

目的:评价富集母血浆中胎儿游离DNA的实验平台在母血浆中富集、鉴定胎儿游离DNA的适用范围。方法:(1)筛选与父源性β地中海贫血基因连锁的单核苷酸多态性(SNP)位点:利用Haploview软件对20例β地中海贫血患者(其中CD41-42型11例,IVS-II-654型9例)的β地中海贫血基因(HBB基因)进行SNP与单倍型自动化分析,检索出HBB基因内杂合度较高的SNP位点并检测与CD41-42位点及IVS-II-654位点连锁的SNP位点。(2)选取4例孕妇外周血标本,且夫妇双方为不同的β地中海贫血基因类型,利用快速耐受温度-低变性温度共扩增PCR技术(TT-FAST-COLD-PCR)检测父源性β地中海贫血基因类型为IVS-II-654型的孕妇外周血标本,若未直接检测到IVS-II-654位点,则检测仅与IVS-II-654位点连锁的SNP位点;同样的原理,利用完全耐受温度-低变性温度共扩增技术(TT-FULL-COLD-PCR)检测父源性β地中海贫血基因类型为CD41-42型的孕妇外周血标本;同时利用普通PCR技术对上述标本进行检测。结果:(1)检测出IVS-II654位点与rs7480526位点连锁9例;CD41-42位点与rs10768683位点连锁11例;(2)通过TT-FAST-COLD-PCR技术在孕妇外周血中检测出1例父源性致病突变基因(IVS-II-654位点),另外1例未直接检测到父源性致病突变基因,但检测到与其连锁的SNP位点(rs7480526位点),与羊水细胞培养检测结果一致,检出率为100%(2/2);通过TT-FULL-COLD-PCR技术在孕妇外周血中检测出2例父源性致病突变基因(CD41-42位点),与羊水细胞培养检测结果一致,检出率为100%(2/2);而普通PCR技术均未检测到上述父源性致病突变基因及与父源性等位基因连锁的SNP位点。结论:TT-COLD-PCR技术适用于富集母血浆中胎儿游离DNA突变,可用于无创性产前诊断单基因遗传病。

AIM： To establish a kind of simple and efficient method for cell-free fetal DNA（ cff-DNA） enrichment and to investigate its range of applications and the advantages and disadvantages. METHODS：（ 1） The single nucleotide polymorphisms（ SNPs）,which linked to paternal β-thalassemia mutations,were screened. We analyzed the contact between the SNPs in β- thalassemia gene（ HBB gene） and haploid type by the Haploview software,and then selected these close SNPs which have higher heterozygosity with the HBB gene.（ 2） We selected 4 cases of different β-thalassemia mutations with their husband,and then we used TT-FAST-COLD-PCR to enrich the IVS-II-654 mutations in maternal plasma. If the IVS-II-654 mutation was not detected,we detected the SNP which linked to the IVS-II-654 mutation. Similarly,we used TT-FULL-COLD-PCR to enrich the CD41-42 mutations in the maternal plasma. At the same time,we used the conventional PCR to enrich CD41-42 mutation and IVS-II-654 mutation in the maternal plasma. RESULTS：（ 1） Nine cases of the SNP（ rs7480526） linked to the mutation at IVS-II-654 in HBB gene,and 11 cases of the SNP（ rs10768683） linked to the mutation at CD41-42 in HBB gene were detected.（ 2） We detected 1 case who inherited the paternal β-thalassemia mutation（ IVS-II-654）. We did not directly detect patermal IVS-II-654 mutation in maternal plasma,but detected the SNP linked to the IVS-II-654 mutation in the other case and had 100% detection,and 2 cases inherited the paternal β-thalassemia mutations（ CD41-42） in the maternal plasma by TT-FULL-COLD-PCR and had 100% detection. However,we detected nothing by conventional PCR. CONCLUSION： TT-COLD-PCR is applicable to enrich cell-free fetal DNA in maternal plasma and is a method in the field of noninvasive prenatal diagnosis.