Application of Fetal DNA Detection in Maternal Plasma : A Prenatal Diagnosis Unit Experience
Department of Genetics (CGG,MGH,MJTT,ILS,MRdA,FI,JG,CA,CR) and Department of Gynecology and Obstetrics (JDR), Fundación Jiménez Díaz, Madrid, Spain
Correspondence to: Cristina González-González, Fundacion Jimenez Diaz, Genetica, Avda Reyes Catolicos 2, Madrid, 28040, Spain. E-mail: cgonzalezg{at}megalab.es
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Summary |
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Key Words: fetal DNA maternal plasma non-invasive diagnosis QF-PCR real-time PCR cystic fibrosis Huntington disease fetal RhD
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Introduction |
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In the last few years, some characteristics have been described regarding the behavior of this fetal DNA. Quantitative variations have been observed in gestations at risk for preeclampsia or fetal chromosomal anomalies (Lo et al. 1999; Zhong et al. 2001
).
Advances in laboratory technology have helped the development of this non-invasive fetal diagnosis with the use of real-time PCR (Lo et al. 1998b). Because prenatal diagnosis is an established part of routine obstetrical care in most countries, these non-invasive procedures are very interesting for both laboratory personnel and for parents, because of the low risk for the fetus.
Such reports have prompted us to evaluate the analysis of fetal DNA in maternal plasma as a possible alternative tool in routine laboratory prenatal diagnosis. In our assay, we have used maternal plasma to determine fetal sex, to study the behavior of fetal DNA throughout gestation, to detect two Mendelian inherited disorders: cystic fibrosis (CF) (Gonzalez-Gonzalez et al. 2002) and Huntington disease (HD) (Gonzalez-Gonzalez et al. 2003a
,b
), and to determine fetal RhD status. Different methods were used according to assay requirements.
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Materials and Methods |
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DNA Extraction
DNA was extracted from 0.8 to 2 ml of maternal plasma with the QIAamp DNA Blood Mini Kit (QIAGEN; Hilden, Germany). We also extracted DNA from amniotic fluid and CVS to confirm our results.
All procedures were performed avoiding exogenous DNA contamination and in a sterile room with sterile pipettes and separate areas for DNA extraction and post-PCR handling.
PCR
Fetal DNA Detection
Ten µl of the extracted DNA was used as a template. PCR was performed in a volume of 50 µl, with 1.5 mM MgCl2, 10x PCR buffer (Roche; Mannheim, Germany), 0.2 mM of dNTP (Amersham Pharmacia Biotech, Inc.; Piscataway, NJ) and 1 U of Fast Start DNA polymerase (Roche). We used 10 pmol of each specific primer for DYS390 short tandem repeat (STR) located at the Y chromosome, F: 5'-TAT ATT TTA CAC ATT TTT GGG CC-3' and R: 5'-TGA CAG TAA AAT GAA CAC ATT GC-3'. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems; Foster City, CA). Amplification conditions were 55 cycles at 94C, 30 sec; 55C, 30 sec; and 72C, 30 sec. DNA fragments obtained were diluted 10 times and mixed with deoinized formamide and 0.4 µl of GS-400HD ROX standard size (Applied Biosystems). The mix was heated at 95C for 5 min and resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer) using GeneScan analysis software (Applied Biosystems).
Fetal DNA throughout Gestation
Ten µl of the DNA extracted from maternal plasma was used as a template. DNA from pregnant women and their husbands were also amplified by PCR in a previous analysis to determine an informative STR. We selected primers for the HPTR gene (located on the X chromosome) due to the different sizes of mothers' and fathers' alleles. DNA PCR was performed in a volume of 50 µl, with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM of dNTP (Amersham Pharmacia Biotech), and 1 U of Fast Start DNA polymerase (Roche). We used 10 pmol of each specific primer, F: 5'-CTC TCC AGA ATA GTT AGA TGT AGG TAT-3' and R: 5'-ATG CCA CAG ATA ATA CAC ATC CCC-3'. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems). Amplification conditions were 55 cycles at 94C, 30 sec; 55C, 30 sec; and 72C, 30 sec. DNA fragments were resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer; Applied Biosystems).
Detection of a CF Mutation
We studied a family whose first-born son was diagnosed with CF. A molecular study of the son confirmed two different mutations of CF: G542x and Q890x. Molecular analysis of the parents was performed, and we discovered that the mother was a heterozygous carrier of G542x mutation and the father was a heterozygous carrier of Q890x. From the plasma-extracted DNA, 20 µl and 30 µl were used as a template for PCR. We also used DNA from peripheral whole blood from relatives, and water was used as a negative PCR control. For the Q890x locus, 15 pmol of each primer, F: 5'-GGT GCA TGC TCT TCT AAT G-3' and R: 5'-AAG GCA CAT GCC TCT GTG C-3' was used in a 50-µl reaction along with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM of dNTP (Amersham Pharmacia Biotech), and 1.5 U of Fast Start DNA polymerase (Roche). Cycling conditions were 94C, 5 min; and 48 cycles of 94C, 1 min; 56C, 1 min; and 72C, 1.5 min. The Q890x mutation consists of an A to G substitution that creates an MseI restriction site that is used to detect the mutation. The PCR product was incubated at 65C for 3 hr with 20 U of MseI and 3.5 µl of buffer R (MBE) (MBI Fermentas; Quimigranel, Madrid) and water up to a total volume of 35 µl. Obtained fragments were visualized after 2 hr of electrophoresis in a 3% agarose gel and in a 6% polyacrylamide gel stained with ethidium bromide.
HD Diagnosis
We used the quantitative fluorescent polymerase chain reaction (QF-PCR) technology to diagnose paternally inherited HD in maternal plasma. We studied two pregnant women, both at 13 weeks of gestation and married to HD patients, attending our unit for prenatal diagnosis of HD. The prenatal diagnosis is comprised of the extraction of DNA from CVS at 1013 weeks of gestation to detect the number of expansions of the polymorphic (CAG)n repeats in the Huntington gene that causes the disease (9 to 36 in normal individuals and 37 to 86 in HD patients). PCR was performed in a volume of 50 µl with 10 pmol of each primer (F: 5'-ATG GCG ACC CTG GAA AGC TGA TGA A-3' and R: 5'-GGC GGC TGA GGA AGC TGA GGA-3') along with 1.5 mM MgCl2, 10x PCR buffer (Roche), 0.2 mM dNTP (Amersham Pharmacia Biotech), 1 U of FastStart Taq DNA polymerase (Roche) per reaction. Cycling conditions were 94C, 5 min; 55C, 1 min; 72C, 1 min; 50 cycles of 94C, 45 sec; 58C, 40 sec; and a final extension of 72C, 10 min. The forward primer was labeled at the 5' end with the fluorescent dye 6-FAM (Applied Biosystems). DNA fragments were diluted 10 times and mixed together with deionized formamide and 0.4 µl of GS-400HD ROX standard size (Applied Biosystems). The mix was then heated at 95C for 5 min and resolved on a DNA sequencer (ABI PRISM 310 Genetic Analyzer) using GeneScan analysis software (Applied Biosystems).
Fetal RhD Detection
Ten µl of DNA extracted from maternal plasma was used to perform a real-time multiplex PCR. Primers located in exon 7 of the RhD gene were used (F: 5'-CCT CTC ACT GTT GCC TGC ATT-3'and R: 5'-TTA CAA GCG CGT CCG TGA-3') and a TaqMan probe (5'-TAC GTG AGA AAC GCT CA-3') labeled with 6-FAM (Applied Biosystems). As amplification control, primers that amplify the beta-globin gene were used (F: 5'-GTG CAC CTG ACT TCC TGA GGA GA-3'and R: 5'-GAC CCG TCC AAC CAT AGT TCC-3') and TaqMan probe labeled with VIC (5'-TCT GCC GTT ACT GCC CT-3'). PCR conditions were 50 nM of each probe, 50 nM of each primer for beta-globin, and RhD gene with 25 µl of TaqMan Universal Mastermix (all products from Applied Biosystems) in a final volume of 50 µl with 40 cycles of 94C, 1 min; 62C, 1.30 min; and 72C, 1 min.
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Results |
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Discussion |
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Presently, the main purpose of prenatal diagnosis is the detection of fetal aneuploidies. However, due to the overriding presence of maternal DNA, we were able to detect only fetal sequences of paternal origin in maternal plasma, and most of the aneuploides, such as trisomy 21 (Down Syndrome), are maternally inherited. Previous research from our group has been concerned with the analysis of fetal cells in maternal blood (Rodríguez de Alba et al. 2001). The high degree of maternal-cell contamination makes the identification and analysis of single fetal cells difficult and time consuming, but currently it appears to be the only strategy for the non-invasive detection of fetal aneuploidies.
There are many Mendelian disorders in which affected families require prenatal diagnosis. We consider that fetal DNA detection in maternal plasma could be very useful in those cases. The prevalence of CF is very high, depending on the population, and many studies focus on the need for prenatal/neonatal screening of CF (Richards and Grody 2004). CF is a disorder in which the exocrine glands of the epithelia produce abnormally thick secretions of mucus and elevated sweat electrolytes. It is caused by many different mutations (
1000) and is characterized by progressive respiratory and gastrointestinal problems and serious impairments of the pancreas, intestine, and liver. In couples at risk of having a fetus affected by CF, the analysis of fetal DNA in maternal plasma could be an alternative to an invasive procedure. This technique is performed routinely in the laboratory but does not have high sensitivity. Nasis et al. (2004)
have developed an allele-specific real-time PCR to improve the sensitivity and specificity to detect a fetal CF mutation in maternal plasma.
We used QF-PCR for the study of HD as a more sensitive method to detect fetal DNA than conventional acrylamide. HD is a dominantly inherited disease with a variable age of onset and causes motor abnormalities, gradual loss of cognition, and ultimately, death. A parental study was always performed to determine the size of the alleles for the CAG repeats. In both studies, we correctly diagnosed the fetal status at an early gestational age. An important aim of our study was to perform a fetal diagnostic study early in pregnancy, when CVS or amniocentesis might be done.
More experience in this field is needed to confirm the results obtained in our study. At present, our group is working with more patients. It is likely that the analysis of fetal DNA by QF-PCR could be very useful as a pretest suitable for monitoring paternally inherited expanded alleles in HD. The low quantity of fetal DNA in the maternal circulation and interference from an excessive amount of maternal DNA make it necessary to use very sensitive methods such as QF-PCR to analyze fetal mutations. This method would also permit the diagnosis of other paternally inherited fetal disorders, but different approaches are needed for each specific situation. Additional advantages of this technique are its feasibility and the rapidity of carrying it out.
To improve the sensitivity of fetal DNA detection, we decided to introduce the technology of real-time PCR. Many authors have described a sensitivity and specificity close to 100% for fetal DNA detection using this technique (Lo et al. 1998b). We considered fetal RhD status detection to be a useful tool in cases of sensitized pregnancies of RhD-negative women with partners heterozygous for the RhD gene. These pregnancies continue to present a considerable obstetric problem of hemolytic disease of the newborn, requiring transfusions in utero and having a mortality risk. Our study demonstrates promising results with 90% correct diagnosis. To determine the sensitivity and usefulness of this technique, an assay with a larger number of samples is being performed in our laboratory. It is also important to consider the RhD polymorphism prevalent in many ethnic populations (deletions, pseudogenes) to avoid incorrect results (Wagner and Flegel 2000
).
Our experience with the analysis of fetal DNA in maternal plasma demonstrates the reliability of these approaches. As a prenatal diagnosis unit, we focus on the clinical application of fetal DNA detection, mainly in the prenatal diagnosis of Mendelian disorders. These procedures could be used as an alternative method prior to CVS or amniocentesis, for those parents discouraged from having an invasive procedure.
Understanding the technical parameters affecting the reliability of the detection of fetal DNA in maternal plasma is very important for its use as a routine prenatal diagnosis procedure. We have observed that after phlebotomy of the mother, it is necessary to obtain the plasma as soon as possible and then aliquot and freeze it until time of DNA extraction. We collected maternal blood and processed it for no longer than 24 hr. A few samples were processed after this time (48 and 72 h), and using these samples we were able to detect maternal DNA but not fetal DNA. Previous studies indicate that fetal DNA in maternal plasma is stable even 24 hr after collection (Angert et al. 2003). The time a sample spends in the tube before processing could not only affect the amount of total DNA by releasing DNA due to apoptosis of maternal cells, but also affect the stability of free-cell fetal DNA. The overriding maternal DNA interferes with fetal DNA amplification, and the degradation of fetal DNA is obviously inconvenient for PCR; therefore, it is important to consider the processing time as an important factor. These data need to be studied at length to establish protocols for the transmission of samples from distant places.
Another limitation of the use of fetal DNA PCR for diagnosis is the possibility of false-positive results. These are attributed mainly to PCR product carry-over, but we believe that sample handling is also an important factor. It is essential to avoid any risk of exogenous DNA contamination; this mandates the use of sterile pipettes and filter tips, and separate sterile areas for DNA extraction and PCR handling. Real-time PCR offers, to date, the highest level of safety and represents the most secure amplification procedure because of its closed-tube systems and the multiplex PCR design that includes an internal PCR amplification control.
Johnson et al. (2004) have evaluated a standard protocol to determine whether fetal DNA detection could be reproducible in multiple laboratories. They reported that their PCR procedure is reliable but suggested that an optimized protocol for DNA extraction would be useful to improve the results. We conclude that fetal detection from maternal plasma is a promising technique but that more experience in this field is needed. Further studies are essential to avoid the interference of maternal DNA to obtain a non-invasive diagnosis with results as precise as those obtained by conventional procedures. Poon et al. (2002)
have focused on the biology of epigenetic phenomena, reasoning that using DNA methylation differences between the mother and fetus, it may be possible to overcome this limitation. It is highly likely that advances in technology will allow the analysis of fetal DNA in maternal plasma to soon be routine in prenatal diagnosis.
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Acknowledgments |
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Footnotes |
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Received for publication May 17, 2004; accepted September 23, 2004
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Literature Cited |
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