Service de Génétique et Biologie Moléculaires, HUPC Hôpital Cochin, Paris, France.
Service de Cytogénétique, HUPC Hôpital Cochin, Paris, France.
Clin Chem Lab Med. 2018 Apr 25;56(5):728-738. doi: 10.1515/cclm-2017-0689.
To limit risks of miscarriages associated with invasive procedures of current prenatal diagnosis practice, we aim to develop a personalized medicine-based protocol for non-invasive prenatal diagnosis (NIPD) of monogenic disorders relying on the detection of paternally inherited mutations in maternal blood using droplet digital PCR (ddPCR).
This study included four couples at risk of transmitting paternal neurofibromatosis type 1 (NF1) mutations and four couples at risk of transmitting compound heterozygous CFTR mutations. NIPD was performed between 8 and 15 weeks of gestation, in parallel to conventional invasive diagnosis. We designed specific hydrolysis probes to detect the paternal mutation and to assess the presence of cell-free fetal DNA by ddPCR. Analytical performances of each assay were determined from paternal sample, an then fetal genotype was inferred from maternal plasma sample.
Presence or absence of the paternal mutant allele was correctly determined in all the studied plasma DNA samples.
We report an NIPD protocol suitable for implementation in an experienced laboratory of molecular genetics. Our proof-of-principle results point out a high accuracy for early detection of paternal NF1 and CFTR mutations in cell-free DNA, and open new perspectives for extending the technology to NIPD of many other monogenic diseases.
为了降低当前产前诊断实践中侵入性操作相关的流产风险，我们旨在开发一种基于个体化医学的非侵入性产前诊断（NIPD）方案，该方案依赖于使用液滴数字 PCR（ddPCR）在母体血液中检测父系遗传的突变，以诊断单基因疾病。
本研究纳入了四对有携带 NF1 突变风险的夫妇和四对有携带复合杂合 CFTR 突变风险的夫妇。NIPD 在妊娠 8 至 15 周时与常规的侵入性诊断同时进行。我们设计了特定的水解探针来检测父系突变，并通过 ddPCR 评估游离胎儿 DNA 的存在。从父系样本中确定了每个检测的分析性能，然后从母体血浆样本中推断出胎儿基因型。
在所研究的所有血浆 DNA 样本中，均正确确定了父系突变等位基因的存在或缺失。
我们报告了一种适用于分子遗传学经验丰富的实验室实施的 NIPD 方案。我们的原理验证结果表明，在游离 DNA 中早期检测父系 NF1 和 CFTR 突变具有很高的准确性，并为将该技术扩展到许多其他单基因疾病的 NIPD 开辟了新的前景。
