BRIEF REPORT |
Analysis of Cell-free Fetal DNA in Plasma and Serum of Pregnant Women
Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow, Russia
Correspondence to: T.V. Zolotukhina, Research Centre for Medical Genetics, Prenatal Diagnosis, Russian Academy of Medical Sciences, Moskvorechje 1, Moscow, 115478, Russia. E-mail.tvz{at}medgen.ru
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
(J Histochem Cytochem 53:297299, 2005)
Key Words: fetal DNA maternal serum and plasma SRY nested PCR
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aim of this investigation was the development of an efficient nested PCR-based method for the detection of Y-chromosome-specific sequence (SRY) gene in fetal DNA extracted from plasma or serum of pregnant women.
![]() |
Patients |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Cytogenetics |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
DNA Extraction from Plasma or Serum Samples |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
PCR Analysis |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The control PCR for a fragment of amylo-1,6-glucosidase gene (AGL) was carried out as standard assay in a total volume of 50 µl. The part of the AGL gene that is located on 1p21 was amplified. Oligonucleotide primers GL3.5 5'-CCG AGC TTA TTC TGT AGA AG-3' and GL3.6 5'-ACA TGC TCC TGA TGA CTT AC-3' were designed to amplify exons 3334 of AGL. The size of the PCR product was 469 bp.
Nucleotide sequences for the SRY gene and AGL gene were obtained from the Gene Data Bank.
PCR products were analyzed in 2% agarose gel containing ethidium bromide.
PCR fragment of amylo-1,6-glucosidase gene was detected in all samples investigated, confirming the presence of DNA in the samples. Cytogenetic analysis revealed that 24 fetuses had normal female karyotype46,XX, and 36 fetuses had normal male karyotype46,XY.
Specificity of PCR of SRY sequence was 87.5%: specific PCR fragment for Y chromosome was amplified in three DNA samples extracted from plasma or serum of 24 women carrying female fetuses. Thus, false-positive results were 5% (3/60 samples investigated). The sensitivity of the method was 94.5%: specific PCR fragment for Y chromosome was not amplified in two DNA samples extracted from plasma or serum of 36 women carrying male fetuses. Thus, false-negative results were 3.3% (2/60 samples investigated). Detection rate of this method (the coincidence of results of cytogenetic analysis with PCR results) was 91.7% (55/60 samples investigated) (Table 1).
|
Some authors have reported successful applications of fetal cells for prenatal diagnosis of chromosome aneuploidy (Bianchi et al. 1997). However, low concentration of fetal cells in the maternal circulation (on average, one fetal cell per 106 maternal cells) (Simpson and Elias 1994
) combined with a low success rate in sorting of these cells (e.g., by monoclonal antibodies) hampers detection and isolation of fetal cells. Thus, attempts have been tested to detect free fetal nucleic acids instead of fetal cells in maternal blood because fetal cells are exposed to apoptosis (Van Wijk et al. 2000
). As maternal cells also are destroyed, the blood of pregnant women contains different DNA fragments (=cell-free DNA) of fetal and maternal origin. The presence of nucleic acids in human plasma has already been described (Mandel and Metais 1948
). Prior to 1997, it had not been demonstrated that Y-chromosome-specific sequences could be detected by PCR in plasma or serum of pregnant women carrying male fetuses (Lo et al. 1997
).
To detect small amounts of fetal DNA, real-time QF-PCR is the method of choice (Zhong et al. 2000). In cases of known X-linked disease, the possibility to screen for Y-chromosome gene presence in maternal blood allows women to avoid invasive procedures (Rijnders et al. 2004
).
Two rounds of PCR were performed using two pairs of primers for Y-chromosome-specific sequence in the present approach (SRY gene region). The efficiency of nested PCR assay used was 91.7%, correlating well with published data (Lo 2000). PCR fragment for Y chromosome was amplified in three DNA samples extracted from plasma or serum of 24 women carrying female fetuses. Two of the three women were primagravida. In these cases it was probable that there was DNA contamination of samples. A third woman had earlier termination of her pregnancy, and in this case SRY-positive result might be due to a probable previous male pregnancy. It may be that the lymphocytes from this previous pregnancy are the source of cell-free DNA. The persistence of fetal DNA in maternal plasma long after delivery has been reported (Invernizzi et al. 2002
). A long presence of entire fetal cells in maternal circulation is well known (Bianchi et al. 1996
), and it is likely that fetal lymphocytes being degraded by plasma nucleases are followed by the appearance of cell-free DNA.
It is likely that the use of multiplex PCR assay with few Y-chromosome-specific gene markers will allow increased sensitivity and specificity of fetal DNA detection in maternal serum and/or plasma.
In summary, the presence of fetal cell-free DNA in the maternal circulation is a good, low-cost approach for the future development of novel strategies that are expected to provide non-invasive techniques for early prenatal diagnosis.
![]() |
Footnotes |
---|
Received for publication May 17, 2004; accepted November 12, 2004
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bianchi DW, Zickwolf GK, Weil GJ, Sylvester S, DeMaria MA (1996) Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 93:705708
Bianchi DW, Williams JM, Sullivan LM, Hanson FW, Klinger KW, Shuber AP (1997) PCR quantitation of fetal cells in maternal blood in normal and aneuploid pregnancies. Am J Hum Genet 61:822829[Medline]
Elias S, Simpson JL (1993) Amniocentesis. In Simpson JL, Elias S, eds. Essentials of Prenatal Diagnosis. New York, Churchill Livingstone, 2744
Invernizzi P, Biondi ML, Battezzati PM, Perego F, Selmi C, Cecchini F, Podda M, et al. (2002) Presence of fetal DNA in maternal plasma decades after pregnancy. Hum Genet 110:587591[CrossRef][Medline]
Jackson L, Wapner RJ (1993) Chorionic villus sampling. In Simpson JL, Elias S, eds. Essentials of Prenatal Diagnosis. New York, Churchill Livingstone, 4561
Lee T, Le Shane ES, Messerlian G, Canick JA, Farina A, Heber W (2002) Down syndrome and cell-free fetal DNA in archived maternal serum. Am J Obstet Gynecol 187:12171221[CrossRef][Medline]
Lo YM (2000) Fetal DNA in maternal plasma: biology and diagnostic applications. Clin Chem 46:19031906
Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS (1997) Presence of fetal DNA in maternal plasma and serum. Lancet 350:485487[CrossRef][Medline]
Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 45:184188
Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM (1998) Quantitative analysis of fetal DNA in maternal plasma and serum: implications for non-invasive prenatal diagnosis. Am J Hum Genet 62:768775[CrossRef][Medline]
Mandel P, Metais P (1948) Les acides nucleiques du plasma sanguin chez l'homme. CR Acad Sci Paris 142:241243
Rijnders RJ, Christiaens GC, Bossers B, van der Smagt JJ, van der Shoot CE, de Haas M (2004) Clinical applications of cell-free fetal DNA from maternal plasma. Obstet Gynecol 103:157164
Simpson JL, Elias S (1994) Isolating fetal cells in maternal circulation for prenatal diagnosis. Prenat Diagn 14:12291242[Medline]
Van Wijk IJ, De Hoon A, Jurhawan R, Tjoa ML, Griffioen S, Mulders MA (2000) Detection of apoptotic fetal cells in plasma of pregnant women. Clin Chem 46:729731
Zhong XY, Holzgreve W, Hahn S (2000) Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. BJOG 107:766769[Medline]