Guissart C, Dubucs C, Raynal C, Girardet A, Tran Mau Them F, Debant V, Rouzier C, Boureau-Wirth A, Haquet E, Puechberty J, Bieth E, Dupin Deguine D, Khau Van Kien P, Brechard M P, Pritchard V, Koenig M, Claustres M, Vincent M C
Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France; Equipe Accueil EA7402, Université Montpellier, Montpellier, France.
Laboratoire de Génétique Moléculaire, IURC, CHRU de Montpellier, France.
J Cyst Fibros. 2017 Mar;16(2):198-206. doi: 10.1016/j.jcf.2016.12.011. Epub 2016 Dec 28.
Analysis of cell-free foetal DNA (cff-DNA) in maternal plasma is very promising for early diagnosis of monogenic diseases; in particular, cystic fibrosis (CF). However, NIPD of single-gene disorders has been limited by the availability of suitable technical platforms and the need to set up patient or disease-specific custom-made approaches.
To make research applications more readily accessible to the clinic, we offer a simple assay combining two independent methods to determine the presence or absence of paternally inherited foetal allele p.Phe508del (the most frequent mutation in CF patients worldwide). The first method detects the presence or absence of a p.Phe508del allele by Mutant Enrichment with 3'-Modified Oligonucleotide PCR coupled to Fragment Length Analysis (MEMO-PCR-FLA). The second method detects the p.Phe508del allele with classical Multiplex Fluorescent PCR including five intragenic and extragenic STR markers of the CFTR locus and a specific SRY sequence.
We collected 24 plasma samples from 23 women carrying foetuses at risk for CF and tested each sample using both methods. Our new procedures were successfully applied to 10 couples where fathers carried the p.Phe508del mutation and mothers were carrying a different mutation in the CFTR gene. These simple tests provided clear positive or negative results from the maternal plasma of the pregnant women. We confirmed the presence of cff-DNA in the studied samples by the identification of a tri-allelic DNA profile using a miniSTR kit. All results were correlated with chorionic villus sampling or amniocentesis analyses.
This NIPD approach, easily set up in any clinical laboratory where prenatal diagnosis is routinely performed, offers many advantages over current methods: it is simple, rapid, and cost-effective. It opens up the possibility for testing a large number of couples with offspring at risk for CF.
分析母血中的游离胎儿DNA（cff-DNA）对单基因疾病的早期诊断，尤其是囊性纤维化（CF），具有很大的前景。然而，单基因疾病的无创性产前诊断（NIPD）受到合适技术平台的可用性以及建立患者或疾病特异性定制方法的需求的限制。
为了使临床更容易获得研究应用，我们提供了一种简单的检测方法，结合两种独立方法来确定父系遗传的胎儿等位基因p.Phe508del（全球CF患者中最常见的突变）的存在与否。第一种方法通过3'-修饰寡核苷酸PCR结合片段长度分析的突变富集法（MEMO-PCR-FLA）检测p.Phe508del等位基因的存在与否。第二种方法通过经典的多重荧光PCR检测p.Phe508del等位基因，该PCR包括CFTR基因座的五个基因内和基因外短串联重复序列（STR）标记以及一个特定的SRY序列。
我们从23名怀有CF风险胎儿的女性中收集了24份血浆样本，并使用两种方法对每个样本进行了检测。我们的新程序成功应用于10对夫妇，其中父亲携带p.Phe508del突变，母亲携带CFTR基因的不同突变。这些简单的检测从孕妇的母血中提供了明确的阳性或阴性结果。我们通过使用微型STR试剂盒鉴定三等位基因DNA图谱，证实了研究样本中存在cff-DNA。所有结果均与绒毛取样或羊膜穿刺术分析相关。
这种NIPD方法在任何常规进行产前诊断的临床实验室中都易于建立，与现有方法相比具有许多优势：它简单、快速且具有成本效益。它为检测大量有CF风险后代的夫妇开辟了可能性。
