探讨锚定多重PCR联合二代测序法用于产前诊断β-地中海贫血中的可行性研究
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摘要
目的探索与完善β-地中海贫血的无创产前检测技术,进一步探讨孕妇外周血浆游离胎儿DNA对胎儿β-地中海贫血无创性产前诊断的临床应用价值。方法从2015年1月至2017年3月在海南医学院第一附属医院产前诊断中心就诊的孕妇中,选取5例夫妇为不同的β-地中海贫血基因型的家系,经孕妇及其丈夫知情同意后签署知情同意书。在妊娠16w至22w时行羊膜腔穿刺术抽取羊水这一传统的有创产前诊断方法,运用PCR联合RBD技术检测出胎儿β-地贫基因型,将此项检测结果作为产前诊断β-地贫的金标准;采用EDTA K2抗凝采血管分别抽取夫妇双方的前臂外周血,运用锚定多重PCR联合二代测序等一系列技术操作,通过检测孕妇外周血血浆中游离胎儿DNA(circulation free fetal DNA,cff DNA)获得胎儿地贫基因型,将其与金标准作比对,比较二者的一致性,从而推论孕妇外周血血浆cff-DNA对诊断胎儿β地中海贫血的准确性和稳定性。运用锚定多重PCR(AMP)联合二代测序法这一无创性产前检测方法,经过对比计算得出胎儿是否存在与其父亲一致的β地中海贫血基因突变型,将其与金标准对比。结果 1.运用锚定多重PCR(AMP)联合二代测序法经对比计算得出5个家系中,有2个家系的胎儿存在与其父亲一致的β地中海贫血突变型,其余3个家系的胎儿不存在与父亲一致的β地中海贫血突变型。2.存在与其父亲一致的β地中海贫血突变型的2个胎儿,其羊水地贫基因型均遗传了来自其父亲的β地中海贫血基因突变型;而其余3个不存在与其父亲一致的β地中海贫血基因突变型的胎儿,其羊水地贫基因型均没有遗传来自其父亲的β地中海贫血基因突变型。结论目前胎儿地贫基因检测基于有创性手术获得胎儿DNA进行基因诊断,有一定的流产和宫内感染的风险。我们迫切需要找到安全、无创、准确、简便、早期诊断的方法,cff-DNA的发现开启了无创产前诊断的新篇章,且基于二代测序平台的NIPT技术无创产前诊断胎儿唐氏综合征已广泛应用于临床,其准确性可达99%,这为地贫无创产前诊断研究提供了新的思路。
Objective:To explore and improve the noninvasive prenatal testing technology of beta-thalassemia,and to further explores the clinical value of fetal free DNA in maternal peripheral plasma in noninvasive prenatal diagnosis of beta-thalassemia.Methods:Five families diagnosed as carriers of the beta-thalassemia gene mutation and these couples with different beta thalassemia genotypes were recruited.The pregnant woman and her husband signed informed consent.The traditional invasive prenatal diagnosis method of amniocentesis to extract amniotic fluid at 16 weeks to 22 weeks of gestation was performed.PCR combined with RBD technology was used to detect the genotype of fetal beta-thalassemia genotypes,and the results of beta-thalassemia genotype was taken as the gold standard for prenatal diagnosis of beta-thalassemia genotypes.Using EDTA K2 anticoagulant mining respectively take a couple of peripheral blood and the blood using anchor multiple PCR in combination with the next generation sequencing and a series of technical operation,through the detection of pregnant women free fetal DNA in peripheral blood plasma(circulation free fetal DNA,DNA CFF)for testing fetal beta-thalassemia genotypes.With the gold standard at,the consistency of comparison,deduce that the accuracy and stability which peripheral plasma fetal DNA is use for diagnosing beta thalassemia.A noninvasive prenatal testing method,anchored multiple PCR(AMP)combined with second-generation sequencing,was used to determine whether the fetus had a mutated beta-thalassemia gene consistent with its father through comparative calculation and to compare it with the gold standard.Results:1.The results of comparative calculation using anchored multiple PCR(AMP)combined with next generation sequencing method showed that,among the 5 families,2 fetuses in 2 families had the mutation of beta-thalassemia consistent with their fathers,and the other 3 families did not have the mutation of beta-thalassemia consistent with their fathers.2.There are two fetuses with the same mutation of beta-thalassemia as their fathers,whose amniotic thalassemia genotypes all inherit the mutation of beta-thalassemia gene from their fathers;The other three fetuses,which did not have a mutated version of beta thalassemia that was consistent with their father,did not have a mutated version of beta thalassemia that was inherited from their father.Conclusion:Nowadays,the traditional method of gene detection is based on invasive surgery,it has a certain risk of abortion and intrauterine infection.Therefore,it is urgently needed a safe,noninvasive,accurate,simple and early diagnosis method.The discovery of cff-DNA opened a new chapter of noninvasive prenatal diagnosis,and NIPT technology based on the two generation sequencing platform has been widely used in clinical diagnosis of Down syndrome,its accuracy can reach 99%,which provides new ideas on the research of noninvasive prenatal diagnosis.
Objective:To explore and improve the noninvasive prenatal testing technology of beta-thalassemia,and to further explores the clinical value of fetal free DNA in maternal peripheral plasma in noninvasive prenatal diagnosis of beta-thalassemia.Methods:Five families diagnosed as carriers of the beta-thalassemia gene mutation and these couples with different beta thalassemia genotypes were recruited.The pregnant woman and her husband signed informed consent.The traditional invasive prenatal diagnosis method of amniocentesis to extract amniotic fluid at 16 weeks to 22 weeks of gestation was performed.PCR combined with RBD technology was used to detect the genotype of fetal beta-thalassemia genotypes,and the results of beta-thalassemia genotype was taken as the gold standard for prenatal diagnosis of beta-thalassemia genotypes.Using EDTA K2 anticoagulant mining respectively take a couple of peripheral blood and the blood using anchor multiple PCR in combination with the next generation sequencing and a series of technical operation,through the detection of pregnant women free fetal DNA in peripheral blood plasma(circulation free fetal DNA,DNA CFF)for testing fetal beta-thalassemia genotypes.With the gold standard at,the consistency of comparison,deduce that the accuracy and stability which peripheral plasma fetal DNA is use for diagnosing beta thalassemia.A noninvasive prenatal testing method,anchored multiple PCR(AMP)combined with second-generation sequencing,was used to determine whether the fetus had a mutated beta-thalassemia gene consistent with its father through comparative calculation and to compare it with the gold standard.Results:1.The results of comparative calculation using anchored multiple PCR(AMP)combined with next generation sequencing method showed that,among the 5 families,2 fetuses in 2 families had the mutation of beta-thalassemia consistent with their fathers,and the other 3 families did not have the mutation of beta-thalassemia consistent with their fathers.2.There are two fetuses with the same mutation of beta-thalassemia as their fathers,whose amniotic thalassemia genotypes all inherit the mutation of beta-thalassemia gene from their fathers;The other three fetuses,which did not have a mutated version of beta thalassemia that was consistent with their father,did not have a mutated version of beta thalassemia that was inherited from their father.Conclusion:Nowadays,the traditional method of gene detection is based on invasive surgery,it has a certain risk of abortion and intrauterine infection.Therefore,it is urgently needed a safe,noninvasive,accurate,simple and early diagnosis method.The discovery of cff-DNA opened a new chapter of noninvasive prenatal diagnosis,and NIPT technology based on the two generation sequencing platform has been widely used in clinical diagnosis of Down syndrome,its accuracy can reach 99%,which provides new ideas on the research of noninvasive prenatal diagnosis.
引文
[1]黄烁丹,张惠琴,邹婕,等.广东省梅州地区地中海贫血的分子流行病学调查[J].热带医学杂志,2011,11(7):788-790.
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[8]Lo YM,Tein MS,Lau TK,et al.Quantitative analysis of fetal DNA in maternal plasma and serum:implications for noninvasive prenatal diagnosis[J].Am J Hum Genet,1998,62:768-775.
[9]Chiu RW,Lau TK,Leung TN,et al.Prenatal exclusion of betathalassaemia major by examination of maternal plasma[J].Lancet,2002,360(9338):998-1000.
[10]Chiu RW,Cantor CR,Lo YM.Non-invasive prenatal diagnosis by single molecule counting technologies[J].Trends Genet,2009,25:324-31.
[11]Ding C,Chiu R W,Lau TK,et al.MS analysis of singlenucleotide differences in circulating nucleic acids:Application to noninvasive prenatal diagnosis[J].Proc Natl Acad Sci USA,2004,101:10762-10767.
[12]Li Y,Di Naro E,Vitucci A,et al.Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma[J].JAMA,2005,293:843-849.
[13]陈萍,李慕军,周林英等.荧光PCR方法检测孕妇血浆胎儿DNA的β地中海贫血基因突变[J].广西医科大学学报,2008,25(2).
[14]Weigang Lv,Xianda Wei,Ruolan Guo,et al.Non-invasive prenatal testing for wilson disease by use of circulating singlemolecule amplification and resequencing technology(cSMART)[J].Clinical Chemistry,2015,61:1-10.
[15]Thessalia Papasavva,Wilfred FJ van IJcken,Christel EM Kockx,et al.Next generation sequencing of SNPs for non-invasive prenatal diagnosis:challenges and feasibility as illustrated by an application toβ-thalassaemia[J].European Journal of Human Genetics,2013,21:1403-1410.
[16]JJ Chen,JAMA Tan,KH Chua,et al.Non-invasive prenatal diagnosis using fetal DNA in maternal plasma:a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms[J].BMJ Open,2015,5:e007648.
[17]Luisella Saba,Maddalena Masala,Valentina Capponi,et al.Noninvasive prenatal diagnosis of beta-thalassemia by semiconductor sequencing:a feasibility study in the sardinian population[J].European Journal of Human Genetics,2017,25,600-607.
[18]Giulia Breveglieri,Anna Travan,et al.Postnatal and non-invasive prenatal detection ofβ-thalassemia mutations based on Taqman genotyping assays[J].PLoS One,2017,12(2):e0172756.
[2]张新华,周英杰,李平萍,等.广西南宁市农村育龄人群地中海贫血筛查及基因型和血液学参数分析[J].中华流行病学杂志,2006,27(9):769-772.
[3]徐湘民,张新华,陈荔丽.地中海贫血预防控制操作指南[M].第1版.北京:人民军医出版社.2011.27-111.
[4]赵芳,何小洪,程静,等.广州地区1571例胎儿地中海贫血产前基因型与血液学特征分析[J].实用医学杂志,2016,32(21):3562-3565.
[5]黄林环,方群,曾瑞萍,等.地中海贫血胎儿产前基因型诊断结果与血象特点的分析[J].中华儿科杂志,2006,10:760-763.
[6]Lo YM,Corbetta N,Chamberlain PF,et al.Presence of fetal DNAin maternal plasma and serum[J].Lancet,1997,350:485-487.
[7]Lun FM,Chiu RW,Allen Chan KC,et al.Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma[J].Clin Chem,2008,54:1664-1672.
[8]Lo YM,Tein MS,Lau TK,et al.Quantitative analysis of fetal DNA in maternal plasma and serum:implications for noninvasive prenatal diagnosis[J].Am J Hum Genet,1998,62:768-775.
[9]Chiu RW,Lau TK,Leung TN,et al.Prenatal exclusion of betathalassaemia major by examination of maternal plasma[J].Lancet,2002,360(9338):998-1000.
[10]Chiu RW,Cantor CR,Lo YM.Non-invasive prenatal diagnosis by single molecule counting technologies[J].Trends Genet,2009,25:324-31.
[11]Ding C,Chiu R W,Lau TK,et al.MS analysis of singlenucleotide differences in circulating nucleic acids:Application to noninvasive prenatal diagnosis[J].Proc Natl Acad Sci USA,2004,101:10762-10767.
[12]Li Y,Di Naro E,Vitucci A,et al.Detection of paternally inherited fetal point mutations for beta-thalassemia using size-fractionated cell-free DNA in maternal plasma[J].JAMA,2005,293:843-849.
[13]陈萍,李慕军,周林英等.荧光PCR方法检测孕妇血浆胎儿DNA的β地中海贫血基因突变[J].广西医科大学学报,2008,25(2).
[14]Weigang Lv,Xianda Wei,Ruolan Guo,et al.Non-invasive prenatal testing for wilson disease by use of circulating singlemolecule amplification and resequencing technology(cSMART)[J].Clinical Chemistry,2015,61:1-10.
[15]Thessalia Papasavva,Wilfred FJ van IJcken,Christel EM Kockx,et al.Next generation sequencing of SNPs for non-invasive prenatal diagnosis:challenges and feasibility as illustrated by an application toβ-thalassaemia[J].European Journal of Human Genetics,2013,21:1403-1410.
[16]JJ Chen,JAMA Tan,KH Chua,et al.Non-invasive prenatal diagnosis using fetal DNA in maternal plasma:a preliminary study for identification of paternally-inherited alleles using single nucleotide polymorphisms[J].BMJ Open,2015,5:e007648.
[17]Luisella Saba,Maddalena Masala,Valentina Capponi,et al.Noninvasive prenatal diagnosis of beta-thalassemia by semiconductor sequencing:a feasibility study in the sardinian population[J].European Journal of Human Genetics,2017,25,600-607.
[18]Giulia Breveglieri,Anna Travan,et al.Postnatal and non-invasive prenatal detection ofβ-thalassemia mutations based on Taqman genotyping assays[J].PLoS One,2017,12(2):e0172756.