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WO2016034231A1 - Procédé de détection d'une contamination dans un test prénatal non-invasif de détermination du sexe fœtal - Google Patents

Procédé de détection d'une contamination dans un test prénatal non-invasif de détermination du sexe fœtal Download PDF

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Publication number
WO2016034231A1
WO2016034231A1 PCT/EP2014/068817 EP2014068817W WO2016034231A1 WO 2016034231 A1 WO2016034231 A1 WO 2016034231A1 EP 2014068817 W EP2014068817 W EP 2014068817W WO 2016034231 A1 WO2016034231 A1 WO 2016034231A1
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dna
pair
fetal
amplicon
sample
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Ceased
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PCT/EP2014/068817
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Inventor
Gabriel MINÁRIK
Barbora IZRAEL
Peter CELEC
Tomáš SZEMES
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Geneton SRO
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Geneton SRO
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection

Definitions

  • This invention relates generally to non-invasive prenatal diagnostics based on the analysis of circulating free fetal DNA in maternal blood.
  • it relates to the detection of contaminating male DNA in prenatal fetal gender test which allows elimination of false-positive results and thus contributes to the reliability of the non-invasive prenatal fetal gender determination.
  • the main application for the circulating fetal DNA in maternal plasma is the use in the screening for the Down syndrome [2]. This possibility becomes available for many pregnant women with the spread of the high throughput sequencing technology. Even more, it is now possible, although still very costly, to sequence the whole fetal genome from DNA isolated from maternal plasma. However, beyond these methods requiring sophisticated equipment, there are several simple PCR based analyses that can be done.
  • RhD genotyping and fetal sex determination are among those that have already advanced into routine clinical and/or commercial use [3].
  • the accuracy of the particular analyses reaches 99% and more.
  • It is of commercial interest as many pregnant women want to know the gender of their baby very early just out of curiosity. In several countries this possibility has, of course, ethical aspects that have to be taken into account.
  • the possibility to detect fetal sex early during the first trimester is also of clinical value. It enables a very precise prognosis for the fetus of risk for X-chromosome linked genetic disorders [4].
  • the non-invasive fetal sex determination is based on a simple PCR targeting the male Y-chromosome sequences [5].
  • a single copy gene is used as a target, typically the SRY gene.
  • multi-copy sequences such as DYS14 are more suitable for qualitative assessments due to higher sensitivity of the PCR.
  • Plasma DNA isolated from non-pregnant women and from pregnant women with a female fetus should not contain any Y-chromosome sequences.
  • a biological exception might be the vanishing twin syndrome [6]. Nevertheless, there is always a small but important risk of technical variability due to contamination during blood sampling and centrifugation, plasma DNA isolation or during the PCR setup.
  • the problem indicated above is solved, at least partially, by the present invention, which provides a method for detection of contaminating (i.e. non-fetal) male DNA in a sample of circulating free DNA isolated from maternal plasma.
  • the method is useful in eliminating false-positive results in non-invasive prenatal fetal gender test.
  • the inventors designed simple, fast and cheap PCR method targeted at Y-chromosome sequences of various lengths and they found that it can be used for reliable detection of external (i.e. contaminating) male DNA in DNA samples from maternal plasma.
  • Particular feature of fetal DNA in maternal blood - its fragmentation - was used as base for the method.
  • the size of the fragments of fetal DNA present in maternal plasma varies but is mostly about 146 bp. Contrary to such an extreme fragmentation, the contaminating male DNA should be less fragmented than the fetal DNA, therefore qPCR (quantitative real-time polymerase chain reaction) assays targeted on the same chromosomal region but producing amplicons with specifically different sizes can be used for differentiation of contaminated and non-contaminated samples.
  • the qPCR targeted at short and long Y-chromosome sequences can distinguish fetal male DNA (that will be positive only for short PCR targets) and contaminating (non-fetal) male DNA (that will be positive for both, short and long PCR fragments).
  • the inventors utilized one known PCR assay (commonly used for prenatal fetal gender determination) and designed 3 novel PCR assays with amplicon sizes reaching 84 bp, 177 bp, 194 bp and 290 bp, all targeted to DYS14 sequence (GenBank:X74029.1). Amplicon size 84 bp falls well below the peak of cell free fetal DNA isolated from maternal plasma, the other were designed to be larger than this, but still bellow the maximum fragment length of cell-free fetal DNA circulating in maternal plasma. All four assays performed well on control male DNA samples were these reached similar qPCR amplification efficiencies.
  • the first aspect of the present invention relates to a method for detecting contaminating non-fetal male DNA in a sample of circulating free DNA isolated from maternal plasma.
  • the method comprising the following steps: a) obtaining a sample of cDNA isolated from maternal plasma, b) on this sample, performing the fist quantitative PCR assay specific for target Y-chromosome sequence with the first pair of oligonucleotide primers providing the first amplicon of the size less then about one nucleosome equivalent (146 bp, the length the DNA from one nucleosome) and determining Ct of this assay, Ctl, c) on the same sample performing the second quantitative PCR assay on the same target Y-chromosome sequence with the second pair of oligonucleotide primers providing the second amplicon of the size longer then about one nucleosome equivalent but shorter than about two nucleosome equivalents (292 bp) and determining Ct of this assay, Ct2, d) determination of Ct difference as difference between Ct2 and Ctl, and e) identification of contaminated sample on the basis of the value of Ct difference, wherein low value (i.e. value
  • target Y-chromosome sequence is DYS14 sequence.
  • the first pair and the second pair of oligonucleotide primers share one common oligonucleotide primer.
  • the first pair of primers consists of the sequences SEQ. ID: NO. 1 and SEQ. ID: NO. 2 and the second pair consists of the sequences SEQ. ID: NO. 1 and SEQ. ID: NO. 3, wherein the first pair of primers provides in PCR the first amplicon of 84 bp and the second pair of primers provides in PCR the second amplicon of 177 bp.
  • Another aspect of the present invention relates to the use of the method described above in the non-invasive prenatal fetal gender test based on the detection of Y-chromosome sequence in maternal plasma for decreasing or eliminating a risk of false-positive results.
  • kits for qPCR detection of contaminating non-fetal male DNA in a sample of circulating free DNA isolated from maternal plasma comprises the first pair of oligonucleotide primers specific for Y-chromosome target sequence providing the first amplicon of the size less then about one nucleosome equivalent and the second pair of oligonucleotide primers specific for the same Y-chromosome target sequence providing the second amplicon of the size longer then about one nucleosome equivalent but shorter than about two nucleosome equivalent.
  • primers in the kit are specific for target Y-chromosome sequence which is DYS14 sequence (GenBank: X74029.1).
  • the kit can comprise only three different oligonucletides since it can comprise one common primer, in other words, the first pair and the second pair of primers can share one common oligonucleotide primer.
  • the kit comprises oligonucleotide primers having sequences SEQ. ID: NO. 1 and SEQ. ID: NO. 2 and SEQ. ID: NO. 3.
  • the kit can further comprise at least one fluorescently labelled oligonucleotide probe specific for the detection of both the first and the second amplicon produced in qPCR.
  • Figure 2 The comparison of amplification of DNA sample of pregnant woman bearing male fetus (A) and control male DNA (B) with all four tested assays represented with qPCR curve profiles reactions run in duplicates). Among shorter assays the 84 bp assay is the first from the left and the 177 bp assay the second from the left.
  • Figure 3 Differences between Ct values of 177 bp and 84 bp assays calculated for fetal and control male DNA samples (p ⁇ 0.0001).
  • Figure 4 Group analysis and subsequent ANOVA analysis of contaminated and non- contaminated samples presented as differences between Ct values of 177 bp and 84 bp assays (A - contaminated samples, B - non-contaminated samples of pregnant women bearing male fetuses, C - non-contaminated samples of pregnant women bearing female fetuses).
  • Samples Samples of peripheral blood taken from 60 pregnant women bearing female and male fetuses (in 10th - 20th weeks of gestation) were collected after informed consent was acquired. Blood samples were stored at 4 - 8 0 C and processed within 24 h. The plasma was separated by centrifugation two times at 2200 g for 10 min, then aliquot
  • plasma were pooled in a way that it was possible to prepare 40 different pooled samples represented by 10 samples of female and 30 samples of male fetal gender.
  • the pooled samples were prepared from one to three individual samples. Additionally, whole blood samples of ten males were used as control male samples (non-fetal) in the study.
  • DNA extraction Circulating DNA was extracted from 40 individual or pooled plasma samples with use of DSP Virus Kit (Qiagen, Hilden, Germany) according to the standard vacuum extraction protocol. The isolated circulating DNA was eluted in 100 of MBG water. In control male samples DNA was extracted from 200 of whole blood with use of standard protocol and DNA Blood Mini Kit (Qiagen, Hilden, Germany). DNA sample utilization in the examples
  • Example 1 Of the isolated DNA samples - 40 cDNA and 10 control male samples, 20 of cDNA and 10 of control male samples were used in Example 1 to test the ability of assays in qPCR and in Example 2 for detection of difference in performance of different assays regarding to the length of their amplicons.
  • Example 3 10 of remaining 20 cDNA samples (5:5 of male:female bearing pregnancies) were contaminated with control male DNA, while the other 10 (5:5 of male:female bearing pregnancies) were not contaminated. Samples were blinded, analysed and the statistical analysis of results of qPCR was performed.
  • the concentration of male DNA used for contamination of isolated circulating DNA of pregnant women was determined with use of spectrophotometer and the DNA was diluted to the concentration of 50 pg ⁇ L (-7.5 gEq ⁇ L). Ten
  • Isolated circulating DNA was quantified using real-time PCR with singleplex TaqMan probe assays.
  • Four assays (one previously known [5] and three novel assays) with lengths of amplicons 84 bp, 177 bp, 197 bp and 290 bp targeted at DYS14 sequence were designed with use of online tool Primer3 rhttp://biotools.umassmed.edu/bioapps/primer3 www.cgil .
  • the four assays were based on combination of 4 oligonucleotide primers and one common TaqMan probe with sequences and corresponding amplicon localization and lengths summarized in Table 1 and Figure 1, respectively.
  • PCR amplification was performed according to the study [12] that was published by the inventors earlier. Briefly, 5 ⁇ ⁇ of template DNA was used in 15 ⁇ ⁇ reaction volume containing final concentrations of 1 x QuantiFast Probe PCR Kit (Qiagen, Hilden, Germany), 1 ⁇ /L of each primer and 0.8 ⁇ /L of TaqMan probe. The following PCR program was used: initial denaturation step at 95 0 C for 3 min, followed by 50 cycles each consisting of denaturation step at 95 0 C for 3 s and combined annealing/extension step at 60 0 C for 30 s. QPCR reactions run in the first phase in duplicates and in the second phase in triplicates.
  • the real-time PCR was performed on Eppendorf Mastercycler ep realplex 4S (Eppendorf, Hamburg, Germany) and Roche LightCycler 480 (Roche, Rotnch, Switzerland) instruments with their corresponding softwares.
  • the Ct value of 38 was set as the cuttof for positive signal of amplification.
  • Fig. 3 demonstrates the difference in performance of the two tested assays with 84 bp and 177 bp amplicons between fetal male cDNA containing and control male DNA containing samples (the lowest tested DNA amount in control male DNA samples was 5 genomic equivalents).
  • Ct difference Prominent shift of Ct value (Ct difference) in cDNA samples was detected, while it was not present in control male DNA samples.).
  • the difference measured as difference between Ct values of 177 bp and 84 bp assays was found to be statistically significant (p ⁇ 0.0001).
  • the present invention reveals the possibility to detect external contamination by male DNA in non-invasive prenatal fetal gender test with assays amplifying amplicons with different but specific sizes in qPCR.
  • the inventors designed and verified simple, fast and cheap method, which uses same target sequence for detection of true-positive and false-positive samples.
  • the principle of the method is universally applicable in non-invasive prenatal testing, e.g. for RhD gene.
  • the use of the method according to the invention may lead to minimizing the proportion of inconclusive results, when external contamination could be the problem.

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
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  • Engineering & Computer Science (AREA)
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  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne la détection d'un ADN masculin contaminant dans un test prénatal de détermination du sexe fœtal, qui permet d'éliminer les résultats faux-positifs et ainsi contribue à la fiabilité de la détermination prénatale non-invasive du sexe fœtal. En particulier, un aspect de l'invention concerne un procédé simple, rapide et économique de détection d'un ADN masculin non-fœtal contaminant dans un échantillon d'ADN libre circulant isolé du plasma maternel. Une caractéristique particulière de l'ADN fœtal dans le sang maternel – sa fragmentation – a été utilisée en tant que base pour ledit procédé. Un autre aspect de l'invention concerne une trousse pour détection par qPCR d'un ADN masculin non-fœtal contaminant dans un échantillon d'ADN libre circulant isolé du plasma maternel.
PCT/EP2014/068817 2014-09-04 2014-09-04 Procédé de détection d'une contamination dans un test prénatal non-invasif de détermination du sexe fœtal Ceased WO2016034231A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018050844A1 (fr) * 2016-09-16 2018-03-22 Qiagen Gmbh Procédé de détermination d'une dégradation d'acide nucléique dans un échantillon dans lequel au moins deux amplicons chevauchants sont produits et deux sondes sont utilisées dans le procédé
CN110106237A (zh) * 2019-05-16 2019-08-09 阿吉安(福州)基因医学检验实验室有限公司 Dys14多态性检测引物及试剂盒

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013129727A1 (fr) * 2012-03-02 2013-09-06 Cheil General Hospital & Women's Healthcare Center Procédé d'analyse permettant de déterminer le sexe d'un fœtus et appareil associé

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013129727A1 (fr) * 2012-03-02 2013-09-06 Cheil General Hospital & Women's Healthcare Center Procédé d'analyse permettant de déterminer le sexe d'un fœtus et appareil associé

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
B. ZIMMERMANN: "Optimized Real-Time Quantitative PCR Measurement of Male Fetal DNA in Maternal Plasma", CLINICAL CHEMISTRY, vol. 51, no. 9, 14 July 2005 (2005-07-14), pages 1598 - 1604, XP055167796, ISSN: 0009-9147, DOI: 10.1373/clinchem.2005.051235 *
KOIDE KEIKO ET AL: "Fragmentation of cell-free fetal DNA in plasma and urine of pregnant women", PRENATAL DIAGNOSIS, CHICHESTER, SUSSEX, GB, vol. 25, no. 7, 1 July 2005 (2005-07-01), pages 604 - 607, XP002609705, ISSN: 0197-3851, [retrieved on 20050720], DOI: 10.1002/PD.1213 *
NAGOYA J MED ET AL: "ORIGINAL PAPER FRAGMENT SIZE ANALYSIS OF FREE FETAL DNA IN MATERNAL PLASMA USING Y-STR LOCI AND SRY GENE AMPLIFICATION", SCI, 1 July 2011 (2011-07-01), XP055167922, Retrieved from the Internet <URL:http://www.med.nagoya-u.ac.jp/medlib/nagoya_j_med_sci/7334/v73n34p129_135.pdf> [retrieved on 20150206] *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018050844A1 (fr) * 2016-09-16 2018-03-22 Qiagen Gmbh Procédé de détermination d'une dégradation d'acide nucléique dans un échantillon dans lequel au moins deux amplicons chevauchants sont produits et deux sondes sont utilisées dans le procédé
EP3512962A1 (fr) * 2016-09-16 2019-07-24 QIAGEN GmbH Procédé de détermination d'une dégradation d'acide nucléique dans un échantillon dans lequel au moins deux amplicons chevauchants sont produits et deux sondes sont utilisées dans le procédé
US11149304B2 (en) 2016-09-16 2021-10-19 Qiagen Gmbh Method for determining nucleic acid degradation in a sample in which at least two overlapping amplicons are produced and two probes are used in the method
US11767553B2 (en) 2016-09-16 2023-09-26 Qiagen, Gmbh Kit for determining nucleic acid degradation
CN110106237A (zh) * 2019-05-16 2019-08-09 阿吉安(福州)基因医学检验实验室有限公司 Dys14多态性检测引物及试剂盒
CN110106237B (zh) * 2019-05-16 2023-06-02 阿吉安(福州)基因医学检验实验室有限公司 Dys14多态性检测引物及试剂盒

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