Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)
Noninvasive Prenatal Testing for Wilson Disease by Use of Circulating Single-Molecule Amplification and Resequencing Technology (cSMART)
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DOI：
10.1373/clinchem.2014.229328
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发表时间：
2015-01-01
影响因子：
9.3
通讯作者：
Wu, Lingqian
中科院分区：
文献类型：
--
作者：
Lv, Weigang;Wei, Xianda;Wu, Lingqian
背景技术背景：通过使用基于PCR的策略进行单基因疾病的无创产前检测（NIPT）需要精确定量母体血浆中循环的突变胎儿等位基因。该研究描述了一种称为循环单分子扩增和重测序技术（cSMART）的新型检测方法的开发和验证，用于计数血浆中的单个等位基因分子。在这里，我们证明了cSMART的适用性NIPT，威尔逊病（WD）作为proof of of concept.METHODS：我们使用桑格和全外显子组测序，以确定家族性ATP 7B（ATP酶，铜++转运，β多肽）基因突变。对于cSMART，用独特的条形码标记单分子并使其环化，通过反向PCR靶向并复制等位基因。通过测序和计数确定唯一的单个等位基因分子，并根据原始母体血浆样本中突变等位基因的百分比确定胎儿基因型。利用桑格和全外显子组测序，我们在每个家系中定位了致病性ATP 7B突变，并证实了先证者对WD的最初诊断。经过验证cSMART与定义的血浆模型模仿胎儿遗传的父亲，母亲，或父母双方的突变等位基因，我们回顾性地表明，在第二次怀孕的胎儿基因型分配的侵入性检测和NIPT是concordance.CONCLUSIONS：我们开发了一个可靠的和准确的NIPT检测，正确诊断胎儿基因型在4妊娠WD的风险。这种新技术有可能作为其他单基因疾病的NIPT的通用策略，因为它只需要父母的致病突变的知识。(C)2014年美国临床化学协会
BACKGROUND: Noninvasive prenatal testing (NIPT) for monogenic diseases by use of PCR-based strategies requires precise quantification of mutant fetal alleles circulating in the maternal plasma. The study describes the development and validation of a novel assay termed circulating single-molecule amplification and resequencing technology (cSMART) for counting single allelic molecules in plasma. Here we demonstrate the suitability of cSMART for NIPT, with Wilson Disease (WD) as proof of concept.METHODS: We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu++ transporting, beta polypeptide) gene mutations. For cSMART, single molecules were tagged with unique barcodes and circularized, and alleles were targeted and replicated by inverse PCR. The unique single allelic molecules were identified by sequencing and counted, and the percentage of mutant alleles in the original maternal plasma sample was used to determine fetal genotypes.RESULTS: Four families with WD pedigrees consented to the study. Using Sanger and whole-exome sequencing, we mapped the pathogenic ATP7B mutations in each pedigree and confirmed the proband's original diagnosis of WD. After validation of cSMART with defined plasma models mimicking fetal inheritance of paternal, maternal, or both parental mutant alleles, we retrospectively showed in second pregnancies that the fetal genotypes assigned by invasive testing and NIPT were concordant.CONCLUSIONS: We developed a reliable and accurate NIPT assay that correctly diagnosed the fetal genotypes in 4 pregnancies at risk for WD. This novel technology has potential as a universal strategy for NIPT of other monogenic disorders, since it requires only knowledge of the parental pathogenic mutations. (C) 2014 American Association for Clinical Chemistry