Quantification of Fetal DNA by Use of Methylation-Based DNA Discrimination
Quantification of Fetal DNA by Use of Methylation-Based
DNA Discrimination
Anders O.H.Nygren,1Jarrod Dean,1Taylor J.Jensen,1Selena Kruse,1William Kwong,1
Dirk van den Boom,2and Mathias Ehrich2*
BACKGROUND:Detection of circulating cell-free fetal nucleic acids in maternal plasma has been used in non-invasive prenatal diagnostics.Most applications rely on the qualitative detection of fetal nucleic acids to deter-mine the genetic makeup of the fetus.This method leads to an analytic dilemma,because test results from samples that do not contain fetal DNA or are contam-inated with maternal cellular DNA can be misleading. We developed a multiplex approach to analyze regions that are hypermethylated in placenta relative to mater-nal blood to evaluate the fetal portion of circulating cell-free DNA isolated from maternal plasma.
METHODS:The assay used methylation-sensitive re-striction enzymes to eliminate the maternal(unmeth-ylated)fraction of the DNA sample.The undigested fetal DNA fraction was then coamplified in the pres-ence of a synthetic oligonucleotide to permit com-petitive PCR.The amplification products were quantified by single-base extension and MALDI-TOF MS analysis.
RESULTS:Using2independent markers,(sex determin-ing region Y)-box14(SOX14)and T-box3(TBX3),we measured a mean of151copies of fetal DNA/mL plasma and a mean fetal fraction of0.13in samples obtained from pregnant women.We investigated242 DNA samples isolated from plasma from pregnant and nonpregnant women and observed an analytical sensi-tivity and specificity for the assay of99%and100%, respectively.
CONCLUSIONS:By investigating several regions in paral-lel,we reduced the measurement variance and enabled quantification of circulating cell-free DNA.Our results indicate that this multiplex methylation-based reac-tion detects and quantifies the amount of fetal DNA in a sample isolated from maternal plasma.
?2010American Association for Clinical Chemistry Since its discovery,circulating cell-free fetal DNA(ccff DNA)3isolated from maternal plasma(1)has drawn much attention because it provides genetic informa-tion about the fetus with a reduced risk of complica-tions compared with invasive procedures(2).Several applications for noninvasive prenatal diagnosis (NIPD)are available for the detection of fetal sex (3,4),rhesus D blood type(5,6),and paternal-derived mutations(7,8).Despite the achievements made in both qualitative and quantitative analysis of fetal DNA in maternal plasma(9),there are still limitations to this technology.Most applications rely on differences in DNA sequence between the fetal and maternal ge-nomes.In these applications,absence of the investi-gated fetal sequence would correspond to noninher-itance of the paternal allele.A lack of detectable signal, however,could also be due to a low fetal DNA concen-tration in the sample(10).Many factors can contribute to these false-negative results,but all can lead to false interpretation of the fetal DNA component.Therefore, a method that can be used to detect and quantify a universal fetal DNA marker is needed(11,12).
Until recently the search for a universal fetal marker was largely restricted to genetic polymor-phisms located on the autosomal chromosomes(13), but to ensure at least1informative marker a more complex test is necessary that consumes a large portion of the available DNA sample.A potential alternative to sequence-based detection of fetal DNA is to use other factors to discriminate between maternal and fetal DNA(14–16).Recently several independent studies have presented a comprehensive set of genomic regions in which fetal-specific tissue is differentially methyl-ated compared with the corresponding maternal pe-ripheral blood mononuclear cells(PBMCs)(17–21). This epigenetic difference can be explored to establish an assay that targets the fetal portion of the cell-free DNA in plasma.
1Sequenom Center for Molecular Medicine,San Diego,CA;2Sequenom,San Diego,CA.
*Address correspondence to this author at:3595John Hopkins Court,San Diego 92122,CA.Fax858-202-9084;e-mail MEhrich@https://www.360docs.net/doc/083393683.html,.
Received February26,2010;accepted July20,2010.Previously published online at DOI:10.1373/clinchem.2010.146290
3Nonstandard abbreviations:ccff DNA,circulating cell-free fetal DNA;NIPD, noninvasive prenatal diagnostics;PBMC,peripheral blood mononuclear cell; FQA,fetal quantifier assay.
Clinical Chemistry56:10
1627–1635(2010)
Molecular Diagnostics and Genetics
1627
We developed a fetal quantitative assay(FQA)for the simultaneous quantification of both differentially methylated regions and chromosome-Y–specific se-quences.The FQA also includes assays to quantify the total amount of circulating cell-free DNA and controls to measure the fraction of nondigested material.This format allows direct determination of total DNA copy numbers(maternal?fetal),fetal DNA copy numbers, and male-specific copy numbers,and consequently the ability to calculate the fraction of fetal DNA present in the plasma sample.We successfully evaluated this assay in DNA model systems,verified its specificity in plasma samples from nonpregnant women,and in the final step tested its feasibility in a large set of plasma samples from pregnant women.
Materials and Methods
MARKER DISCOVERY
We performed marker discovery by coupling methyl-cytosine immunoprecipitation to CpG island microar-rays(Agilent Technologies)as previously described (22).Regions were selected as differentially methylated if the adjusted P value after microarray analysis was P?0.001.Five differentially methylated regions[T-box3 (TBX3)4;solute carrier family38,member10 (MGC15523);(sex determining region Y)-box14 (SOX14);CDC42effector protein(Rho GTPase bind-ing)1(CDC42EP1);and sialophorin(SPN)]were se-lected,and differential methylation was confirmed by using EpiTYPER analysis as previously described(23). Primer sequences are listed in Table1in the Data Sup-plement that accompanies the online version of this article at https://www.360docs.net/doc/083393683.html,/content/vol56/ issue10.A detailed description of the marker selection and EpiTYPER methods is described in online Supple-mental Data Information1.
ASSAY DESIGN
We designed PCR primers and MALDI-TOF MS TypePLEX?extension primers by using Seque-nom?AssayDesigner4.2software.All oligonucleo-tides were obtained from IDT.The FQA contained 4types of assays for the detection of total copy numbers,fetal methylated copy numbers,chromosome-Y sequences,and controls for digestion efficiency.The oligonucleotide sequences can be found in online Sup-plemental Table2.
MODEL SYSTEM
To determine the limits of quantification of the method,we developed a model system to simulate cir-culating cell-free DNA samples isolated from plasma. These samples contained maternal nonmethylated DNA copies isolated from PBMCs,into which we spiked different amounts of either male or female pla-cental DNA.We spiked the samples with amounts ranging from0%to40%relative to the maternal non-methylated DNA copy numbers while we kept the total number of DNA molecules constant.An additional model was developed to be used as a series of standards when we determined the total number of amplifiable genomic copies in a sample.A subset of different DNA samples isolated from the blood of nonpregnant women was tested.Each sample was diluted to contain approximately1000,500,250,125,63,32,16,8,4,2,or 1methylated copies per reaction.
PLASMA SAMPLES
For this study,whole blood samples of10mL were collected in EDTA tubes and shipped on wet ice.The plasma was isolated by centrifugation of the whole blood at2500g for10min.To eliminate potential cel-lular debris,an additional centrifugation step was per-formed at15000g for10min.All plasma samples were processed within6h of the blood draw.Two sets of DNA were isolated from a total of248unique plasma samples.Set1consisted of48plasma samples from nonpregnant women and set2consisted of200plasma samples from pregnant women.This study was con-ducted according to an institutional review board–approved protocol.All study participants gave in-formed consent.
DNA ISOLATION
DNA was extracted from200?L of buffy coat or0.2g of placental tissue by using the QiaAMP Blood Minikit (Qiagen).
Circulating cell-free DNA was extracted from4mL of plasma with the QIAamp Circulating Nucleic Acid Kit(Qiagen)and eluted in a100-?L volume.
FQA ASSAY
Methylation-sensitive restriction enzymes(24)were used before PCR to digest the nonmethylated maternal fraction of circulating cell-free DNA.The remaining methylated fetal fraction could then be quantified by using an established mass spectrometry–based method.Quantification was achieved by parallel am-plification of a synthetic oligonucleotide of known concentration followed by MALDI-TOF MS analysis to
4Human genes:TBX3,T-box3,MGC15523,solute carrier family38,member10; SOX14,(sex determining region Y)-box14;CDC42EP1,CDC42effector protein (Rho GTPase binding)1;SPN,sialophorin;ALB,albumin;APOE,apolipoprotein E;RNASEP,ribonuclease P;SRY,sex determining region Y;UTY,ubiquitously transcribed tetratricopeptide repeat gene,Y-linked;MGC15523,solute carrier family38,member10;LDHA,lactate dehydrogenase A;POP5,processing of precursor5,ribonuclease P/MRP subunit;RASSF1A,Ras association(RalGDS/ AF-6)domain family member1.
1628Clinical Chemistry56:10(2010)
separate and quantify the amplification products(25). The limited number of fetal DNA copies introduced a variance in the quantitative measurement;however, the ability of our assay to multiplex and measure sev-eral regions simultaneously helped to reduce this variance(Fig.1).
FQA REACTION
Methylation-based DNA discrimination was per-formed by using25?L of eluted DNA per reaction. For this study all samples were run in triplicate.All reagents and apparatus were obtained from Seque-nom,unless stated otherwise.Digestion of plasma DNA was performed for60min at41°C by the ad-dition of10?L of a mixture containing3.5?PCR Buffer(Sequenom no.1738),2.22mmol/L MgCl2, 10U Hha I(New England Biolabs),10U Hpa II(New England Biolabs),and10U Exo I(New England Bio-labs).The exonuclease was added to eliminate single-stranded DNA that would escape digestion and lead to overestimation of the fetal fraction.After the restriction was complete the enzymes were inac-tivated and the DNA denatured by heating the mix-ture for10min at98°C.The nondigested DNA was PCR amplified by the addition of15?L of a PCR mixture containing1?PCR Buffer(Sequenom no. 1738),0.125mmol dNTPs,5U Fast Start Polymerase (Roche),0.1?mol/L PCR primers,and competitors.
A list of all PCR primers,competitor oligonucleo-tides,and amounts used can be found in online Sup-plemental Table2.PCR was initiated by a5-min incubation at98°C,followed by45cycles(30s at 95°C,30s at64°C,and30s at72°C),and the PCR was ended with a3-min incubation at72°C.After the PCR,5?L of a mix containing1mAnson unit of protease(Qiagen)was added to degrade peptides present in DNA extracted from plasma samples.Pro-tease treatment was performed for30min at55°C and ended with a inactivation step for5min at95°C. To minimize post-PCR–induced variance,each PCR was analyzed in quadruplicate;20?L of the PCR reaction was split into4parallel reactions of5?L and dephosphorylated for40min at37°C by the addition of2?L of a mixture containing0.5U shrimp alkaline phosphatase and5U RNase I f(New England Biolabs)in0.85?shrimp alkaline phospha-tase buffer(Sequenom no.10055).The RNase was added to degrade the large amounts of carrier RNA that were added in the DNA isolation and would interfere with the MALDI-TOF analysis.The reac-tion was ended with a5-min incubation at85°C to inactivate the enzymes.Single-base extension was performed by addition of2?L single-base extension reaction mix to the dephosphorylated primary PCR reaction mix,and included extend primer(see on-Fig.1.Outline of the FQA procedure.
(1),The FQA consisted of13assays in1multiplex reaction, including amplicons for detection of fetal methylated DNA,to-tal number of amplifiable genomes,number of chromosome-Y copies,and control assays for the digestion reaction.Each amplicon for the detection of a fetal methylated sequence was designed to span at least2recognition sites for the methylation-sensitive restriction enzymes(REs)Hha I or Hpa II and sites for PCR primers(FP,forward PCR primer sequence; RP,reverse PCR primer sequence).In addition,a synthetic oligonucleotide[competitor(C)]identical to the DNA targets apart from1nucleotide was designed(G vs A).This artificial allele was designed to have a higher molecular mass than the DNA allele.After isolation of the DNA(2),the sample was digested by using methylation-sensitive restriction enzymes
(3).After a heat-denaturation step to denature the enzymes,
a PCR mixture was added that contained a known amount of each competitor oligonucleotide,which was coamplified to-gether with the nondigested methylated DNA(4).Finally,after PCR and single-base extension(5),the analytes were sepa-rated by using MALDI-TOF MS(6).For every analyte pair,the amount of competitor oligonucleotide was known,hence the ratio obtained between DNA allele and competitor allele was directly proportional to the amount of amplifiable genomic copies present in the sample.
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Clinical Chemistry56:10(2010)1629
line Supplemental Table2),TypePLEX Extend mix,
0.222?TypePLEX buffer,and0.04U TypePLEX en-
zyme.We performed200cycles of single-base exten-
sion using the following program:94°C for30s,
followed by20cycles of94°C for5s followed by5
repetitions of52°C for5s and72°C for5s.After
desalting the mixtures by the addition of6mg Clean
Resin,we transferred15nL of each TypePLEX ex-
tend mixture to a SpectroCHIP?II-G384and re-
corded mass spectra using a MassARRAY system.
Spectra were acquired by using Sequenom Spectro-
Acquire software.The software parameters were set
to acquire20shots from each of9raster positions.
The resulting mass spectra were summed,and peak
detection and intensity analysis were performed by
using Sequenom Typer4software.
DATA ANALYSIS
For each assay,we calculated the fraction of each
individual DNA analyte and synthetic competitor by
dividing the intensity of the representative allele by
the sum of both allele intensities:fraction(DNA
analyte)?Intensity(DNA signal)/[Intensity(DNA signal)?Intensity(Competitor signal)].For quantification of the total number of molecules present in the reac-
tion,the DNA-specific analyte for each amplicon
was divided by the competitor-specific analyte to
give a ratio.We could then determine the total num-
ber of DNA molecules by multiplying the ratio by the
number of added competitor molecules.The num-
ber of fetal DNA copies in each sample was calcu-
lated by using the mean of the Y-chromosome–spe-
cific markers for male fetuses and the mean of the
methylated fraction for all samples.Because the total
amount of nucleic acid present in a sample is a sum
of maternal and fetal nucleic acids,the fetal contri-
bution can be considered to be a fraction of the
larger,background maternal contribution.There-
fore,the chromosome-Y–derived fetal fraction
[k(chr Y)]of the total nucleic acid present in the sam-
ple is equal to the equation:k(chr Y)?2?R(chr Y)/ R(Total);where R is the ratio between the DNA analyte and the competitor analyte multiplied by the number of competitor oligonucleotides added.All copy num-bers below20,i.e.,those for which the fraction of DNA allele was below0.1,were considered as background.A similar calculation for the fetal fraction was performed by using the methylation-specific markers.In contrast to Y-chromosome–specific markers,these markers were from diploid targets.Thus,the fetal fraction [k(Meth)]can be determined by using the equation: k(Meth)?R(Meth)/R(Total),with the assumption that the marker regions are fully methylated.Results
MARKER DISCOVERY
Using methyl-CpG immunoprecipitation coupled with high-resolution oligonucleotide microarray anal-ysis,we analyzed8sample pairs derived from placental tissue and maternal buffy coat.A subset of these re-gions was confirmed by using Sequenom?EpiTYPER?technology.For the FQA we selected5candidate re-gions,CDC42EP1,MGC15523,SOX14,SPN,and TBX3,which showed increased placental methylation and no methylation in maternal PBMCs(see online Supplemental Fig.1).
ASSAY SETUP
To enable quantification we used a competitive PCR approach.This method is well established and was re-cently used for validation of DNA copy number vari-ants(26).This method allows for simultaneous mea-surement of both high total copy numbers and low fetal copy numbers,because each assay is independently amplified.In this approach we used placental methyl-ation as the discriminating factor to identify fetal-derived DNA,and relied on methylation-sensitive re-striction enzymes to digest the maternal portion of the DNA sample.In our assay format,incomplete diges-tion of maternal DNA would lead to an overestimation of the fetal fraction.Hence,it was important to design an amplicon that minimized incomplete digestion of target DNA.For optimal digestion efficiency we used2 methylation-sensitive restriction enzymes and selected only target regions that contained at least2restriction sites.
Because this approach required a double-stranded DNA template,if a portion of the maternal-derived DNA was single stranded,these sequences would es-cape digestion and lead to overestimation of the fetal fraction.Therefore,to protect against overestimating the fetal DNA component,we performed a simulta-neous exonuclease/digestion reaction to remove any single-stranded DNA present in the sample.The effi-ciency of the restriction reaction was monitored by the introduction of2restriction controls into the multi-plex.These assays targeted2regions known to be un-methylated in both maternal blood and placental tis-sue.The FQA was designed to contain4different assay types including:(a)assays for quantification of the to-tal DNA(maternal and fetal),(b)assays for quantifica-tion of chromosome-Y copy numbers,(c)assays for quantification of fetal methylated DNA,and(d)con-trol assays to estimate the digestion efficiency.The re-sulting multiplex contained13assays,3for total DNA quantification[albumin(ALB),apolipoprotein E (APOE),and ribonuclease P(RNASEP)],3for quanti-fication of chromosome Y[2sex determining region Y
1630Clinical Chemistry56:10(2010)
(SRY)and ubiquitously transcribed tetratricopeptide repeat gene,Y-linked(UTY)],5for quantification of differentially methylated fetal DNA[CDC42EP1;sol-ute carrier family38,member10(MGC15523);SOX14; SPN;and TBX3),and2assays as restriction controls [lactate dehydrogenase A(LDHA)and processing of precursor5,ribonuclease P/MRP subunit(POP5)]. MODEL SYSTEM AND VERIFICATION OF DYNAMIC RANGE
To evaluate the performance and dynamic range of our multiplexed assay we designed a model system.Here we used DNA derived from maternal PBMCs to repre-sent the maternal fraction and DNA isolated from pla-centa tissue to represent the fetal component.We ob-tained sample pairs with matching material from8 women carrying a male fetus.First we evaluated the ability to quantify varying amounts of total DNA. Quantification that uses competitive PCR and MALDI-TOF MS relies on the relative evaluation of a target signal against a reference signal.Various pub-lished reports have indicated that the total amount of circulating cell-free DNA is around2000copies/mL of plasma and the fraction of fetal DNA is between3% and20%(27–29).Consequently we chose3000copies of single-stranded competitor DNA for the estimation of total DNA and300copies of competitor DNA for the estimation of fetal DNA.This system could quantify copy numbers between450and7500copies of total DNA per reaction and45–750copies of fetal copies(see online Supplemental Fig.2).On the basis of these re-sults we introduced additional quality criteria:Each sample must contain a minimum of at least450but no more than7500amplifiable copies per reaction and have digestion efficiency above99%to be analyzed. Samples containing copy numbers outside of these ranges can be reanalyzed by adjusting the amount of input DNA.We tested the ability to quantify a fraction of methylated DNA in the presence of unmethylated DNA.We used2000copies of the DNA that repre-sented maternal DNA and added0%,2.5%,5%,10%, 20%,and40%of the DNA that represented fetal DNA. Each individual dilution series was measured with a mean r2of0.99(least squares method)(Fig.2A).We observed a high correlation between each of the5 methylation markers(mean??0.98,Pearson correla-tion)(see online Supplemental Fig.3).The quantifica-tion of Y chromosomal markers is a well-accepted stan-dard for measuring fetal DNA(30).Therefore we used 8sample pairs that had male placental DNA to com-pare the copy numbers obtained by using either the Y chromosomal markers or the methylation-based markers.In Fig.2B,the model system showed correla-tion between the methylation-based quantification and the established method for quantification by using chromosome-Y–specific sequences(??0.98,P?0.001,Pearson correlation).
(A),For copy number quantification,a model system was created that contained a constant number
nonmethylated DNA with varying amounts of spiked-in male placental methylated DNA.
approximately400,200,100,50,25,or0methylated placental copies per reaction.Each measurement was
the mean DNA/competitor ratio obtained from the methylation sensitive assays.Each symbol represents1sample observed;Exp.,experimental.(B),Correlation between methylation markers and chromosome Y.The copy numbers DNA spiked into maternal nonmethylated DNA in varying amounts was calculated by using the ratios obtained methylation assays and the Y-chromosome markers compared to the total copy-number assays.
Fetal Quantifier Assay
Clinical Chemistry56:10(2010)1631
COMPARISON BETWEEN PLASMA DNA FROM NONPREGNANT WOMEN AND GENOMIC DNA OBTAINED FROM PBMCs
To investigate the analytical sensitivity and specificity of this method as well as the identification of sporadic methylation in clinical samples,we analyzed48plasma samples obtained from nonpregnant women.Three samples were excluded because they did not contain any DNA,and each of the remaining45samples passed the previously defined criteria for analysis.We mea-sured a mean of1134(range453–3526)genomic cop-ies/mL https://www.360docs.net/doc/083393683.html,pared with genomic DNA ob-tained from maternal PBMCs,3assays,CDC42EP1, MGC15523,and SPN,had to be excluded from addi-tional analysis because they showed aberrant methyl-ation in the majority of the samples.These results in-dicate that paired maternal PBMCs and placental tissue is not an optimal system of identification of fetal-specific methylation markers.For the remaining2 markers,SOX14and TBX3,we measured copy num-bers per milliliter of plasma that were below the quan-titative range[mean(SD)7(5.5)copy numbers/mL; range0–26copy numbers/mL].Although quantifica-tion within this range is associated with a larger mea-surement variation,the data showed that SOX14and TBX3are not methylated in maternal plasma.
DNA SAMPLES FROM200PREGNANT WOMEN
To estimate the biological variance of differentially methylated regions,we used a set of plasma samples isolated from200pregnant women.Three samples were omitted from analysis because they did not con-tain any DNA;most likely the DNA was lost during the extraction procedure.An advantage with the FQA is that samples containing no DNA can easily be identi-fied owing to the sole amplification of the competitor oligonucleotides.All of the remaining197samples passed QC with regard to total copy numbers within the quantitative range and a digestion efficiency of at least99%.We observed a mean of1245amplifiable copies/mL plasma(range487to4926)and measured a mean of151copies of fetal-derived DNA per mL plasma and a mean fetal fraction of0.132for all sam-ples(Fig.3A).We observed an increasing amount of fetal copy numbers with longer gestation,with means of102,114,and163copies/mL for samples obtained from8-,9-,and10-week pregnancies,respectively.No significant difference was observed between male and female pregnancies(nonpaired t-test:P value?0.05) (Fig.3B).We investigated the correlation between the fetal DNA fraction obtained from either methylation or chromosome Y markers in the samples obtained from male pregnancies(Fig.3C).In this large sample cohort we confirmed the correlation between the methylation-based quantification and the established method for quantification by using chromosome-Y–specific se-quences(??0.85,P?0.001,Pearson correlation).Using the sample set consisting of197samples from pregnant women and45samples from nonpregnant women,we achieved analytical sensitivity and specificity of99%and 100%(Fig.3D).Together these data strongly demon-strate that the presented FQA assay can serve as a sex-and polymorphism-independent method for the quantifica-tion of ccff DNA in a plasma samples from pregnant women.
Discussion
In this study we present the first multiplex methylation-based approach for sex-and polymorphism-independent quantification of ccff DNA isolated from maternal plasma.For a similar method that uses differential methylation of Ras association(RalGDS/AF-6)do-main family member1(RASSF1A),as well as markers for SRY and ALB,3real-time PCR reactions are neces-sary(15).Compared to DNA derived from the cellular compartment of a blood draw,cell-free DNA from plasma is several orders of magnitude less abundant.In particular,the fetal fraction,which constitutes only around15%of all circulating cell-free DNA,is limited. This situation poses2main challenges for a fetal DNA quantification assay.Ideally,the majority of fetal DNA is maintained for the downstream analytical assay and not used for the quantification process itself.Also,be-cause of the low copy numbers that have to be detected, it is desirable to have redundant measurements,which will increase the confidence in the results.Our multi-plexed assay format addresses both of these challenges. All necessary measurements and the digestion controls are run in a single reaction,and because mass spec-trometry allows for high multiplexing,each of these measurements can be performed in duplicate.Our re-sults show that methylation-based quantification of ccff DNA is a feasible method that can overcome the sex-based limitations of the Y chromosomal markers. However,in this study we present only an analysis of the technical feasibility of a multiplex assay for fetal DNA quantification.Therefore the results should be interpreted with caution because a number of ques-tions must be addressed before this assay concept can be implemented for routine clinical use.In particular, the biological stability of the methylation markers has to be further investigated.A perfect methylation marker has to fulfill specific requirements.First,to minimize maternal influence it should exhibit a large difference in methylation between maternal and fetal DNA,in which the maternal-derived DNA is not meth-ylated and the fetal DNA is fully methylated.Second, the methylation difference must be stable,and there-fore not change between individuals across popula-tions or during gestation.Although a vast amount of
1632Clinical Chemistry56:10(2010)
knowledge is available for genetic markers,for epige-netic markers such as DNA methylation,it is still un-clear if these assays can provide the necessary stability. In this study we found2markers that allowed quanti-fication of fetal DNA in197DNA samples isolated from https://www.360docs.net/doc/083393683.html,ff DNA isolated from plasma samples from pregnant women is still a scarce resource.Results of published studies indicate that the mean number of DNA molecules found in1mL of plasma is around 2000,with a fetal component of around15%
(A),Comparison between pregnant and nonpregnant women.
showing the fetal copy numbers based on the SOX14and TBX3methylation markers in DNA samples isolated
vs nonpregnant women.The boxes represent samples isolated from pregnant women at?8weeks of gestation women at?8weeks of gestation(n?23),and nonpregnant women(n?45).The upper and lower whiskers
and95th percentiles.The upper,middle,and lower bars represent the25th,50th,and75th percentiles.(B),Comparison
male and female pregnancies.Box plot of the fetal copy numbers of male vs female DNA samples obtained
isolated from197pregnant women.The upper and lower whiskers represent the5th and95th percentiles.
and lower bars represent the25th,50th,and75th percentiles.No significant difference was observed between
and female samples(n?89)for the methylation markers(P value?0.05,t-test).Meth,methylation-specific chromosome-Y–specific assays.(C),Comparison between methylation and chromosome-Y markers.Paired
the calculated fetal fractions obtained by using the mean of the methylation markers versus the mean
Y-chromosome markers for the male samples.The given values indicated minimal difference between the measurements,thus validating the accuracy and stability of the method.(??0.86,P?0.001,Pearson correlation).
the use of circulating cell-free DNA for discriminating between the pregnant and nonpregnant women.(Sensitivity specificity?100%.)AUC,area under the ROC curve.
Fetal Quantifier Assay
Clinical Chemistry56:10(2010)1633
(27,29,31).Both of these values are subject to a large spread across individuals,and therefore it is desirable to minimize the DNA consumption of an assay that controls for the presence of fetal DNA.New methods for DNA extraction from plasma allow for higher input volumes of up to4mL,and consequently maximize the amount of DNA available for the analytical reaction.
In conclusion,the field of noninvasive prenatal di-agnostic using ccff DNA is still in its infancy.Some assays,like those for the detection of the RhD gene,are currently finding their way into clinical practice.In the future we will also see more quantitative applications such as aneuploidy detection through other assays or instrumentation.All of these new tests rely on a control reaction able to quantify the presence of ccff DNA in the plasma sample(32).
Author Contributions:All authors confirmed they have contributed to the intellectual content of this paper and have met the following3re-quirements:(a)significant contributions to the conception and design, acquisition of data,or analysis and interpretation of data;(b)drafting or revising the article for intellectual content;and(c)final approval of the published article.
Authors’Disclosures of Potential Conflicts of Interest:Upon manuscript submission,all authors completed the Disclosures of Poten-tial Conflict of Interest form.Potential conflicts of interest: Employment or Leadership:A.O.H.Nygren,Sequenom Center for Molecular Medicine;J.Dean,Sequenom Center for Molecular Medi-cine;T.J.Jensen,Sequenom Center for Molecular Medicine;S.T. Kruse,Sequenom Center for Molecular Medicine;W.Kwong,Se-quenom Center for Molecular Medicine;D.van den Boom,Seque-nom;M.Ehrich,Sequenom.
Consultant or Advisory Role:None declared.
Stock Ownership:A.O.H.Nygren,Sequenom;D.van den Boom, Sequenom;and M.Ehrich,Sequenom.
Honoraria:None declared.
Research Funding:None declared.
Expert Testimony:None declared.
Role of Sponsor:The funding organizations played no role in the design of study,choice of enrolled patients,review and interpretation of data,or preparation or approval of manuscript.
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