| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Technical Briefs |
Departments of
1
Obstetrics and Gynecology, and
2
Medical Research, Mackay Memorial Hospital, Taipei, Taiwan
aaddress correspondence to this author at: Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Rd., Taipei, Taiwan; fax 886-2-25433642, e-mail cpc_mmh{at}yahoo.com
The recent demonstration of fetal DNA in maternal plasma and serum at concentrations much higher than those present in the cellular fraction has introduced new possibilities for noninvasive prenatal diagnosis of paternally inherited dominant disorders (1)(2)(3). To date, prenatal detection of fetal aneuploidy in maternal blood has focused on searching intact cells using fluorescence in situ hybridization. The use of fetal DNA in maternal plasma to determine fetal aneuploidy has rarely been described. We previously reported prenatal detection of a paternally inherited fetal aneuploidy from fetal DNA in maternal plasma (4). Here we report the application of such a technique in an additional case involving a mother’s three consecutive pregnancies.
We studied fetal DNA in maternal plasma from a pregnant woman whose
fetuses possibly had paternally inherited aneuploidy. Her husband had a
balanced reciprocal translocation between the long arm of chromosome 10
and the short arm of chromosome 22, 46,XY,t(10;22)(q24.1;p11.2). The
woman’s karyotype was normal. During her first pregnancy, genetic
amniocentesis was performed at 19 weeks of gestation, and the maternal
blood sample was collected at 22 gestational weeks before termination
of the pregnancy. In contrast, during her second and third pregnancies,
the maternal blood samples were collected at 14 and 18 gestational
weeks, respectively, before amniocentesis. The amniocentesis of
the first pregnancy revealed fetal distal 10q trisomy
(10q24.1
qter), 46,XX,der(22)t(10;22)(q24.1;p11.2), resulting from
paternal t(10;22) reciprocal translocation. The amniocentesis of the
second and third pregnancies showed a balanced translocation the same
as the paternal karyotype, 46,XY,t(10;22)(q24.1;p11.2).
We collected 5 mL of both paternal and maternal peripheral blood into EDTA-containing tubes. Blood samples were centrifuged at 3000g, and the plasma was carefully removed without disturbing the buffy coat. The maternal plasma sample was recentrifuged, and the supernatant was collected for processing. DNA was extracted from buffy coat and 600-µL plasma samples using a DNA extraction reagent set (QIAamp® DNA Blood Mini Kit). We used fluorescent PCR assays and polymorphic small tandem repeats (STRs) to analyze DNA in maternal plasma. Five pairs of highly polymorphic primers were used separately to amplify the following loci (www.gdb.org): D10S541 (chromosome 10q22-q23; heterozygosity, 78%), D10S574 (chromosome 10q; heterozygosity, 75%), D10S534 (chromosome 10q23-q25; heterozygosity, 78%), D10S187 (chromosome 10q; heterozygosity, 84%), and D10S186 (chromosome 10q; heterozygosity, 81%). Each of the forward primers was labeled at the 5' end with one of the following fluorescent dyes: 6-carboxyfluorescein (FAM); 4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein (HEX); or 4,7,2',7'-tetrachloro-6-carboxyfluorescein (TET).
The PCR conditions followed the protocol recommended by the
manufacturer. We used 60 ng of parental white blood cell (WBC) DNA
(according to the measured absorbance) and a maternal-plasma DNA
aliquot equivalent to 1/20 of the starting plasma as template. Normal
controls were performed by amplifying maternal plasma and WBC DNA from
women carrying fetuses not affected by chromosomal aneuploidies. After
initial denaturation at 95 °C for 5 min, 10 cycles of PCR
amplification were performed: denaturation for 15 s at 94 °C,
annealing for 15 s at 55 °C, and extension for 30 s at
72 °C. Subsequently, 20 cycles of PCR amplification were performed:
denaturation for 15 s at 89 °C, annealing for 15 s at
55 °C, and extension for 30 s at 72 °C. The final extension
was at 72 °C for 10 min. The DNA fragments were diluted 10-fold
(with HEX-labeled products) or 20-fold (with 6-FAM- or TET-labeled
products) and were then mixed with formamide and GS-500 TAMRA size
standard (Applied Biosystems). The DNA fragments were resolved on a DNA
sequencer (ABI 377 model) by Genescan Analysis software (Ver. 2.1;
Applied Biosystems). The sizes of the amplified alleles were estimated
based on the peaks on the electrophoretograms. The specimen
of maternal plasma in the first pregnancy showed disomy for paternal
10q by two informative markers, D10S534 and
D10S186, whereas the maternal plasma samples in the second
and the third pregnancies showed monosomy for paternal 10q (Table 1
and Fig. 1
). Therefore, results obtained by PCR assays were consistent
with the cytogenetic results.
|
|
Our presentation demonstrates the application of polymorphic markers outside the Y chromosome in maternal plasma for noninvasive detection of possible fetal 10q trisomy in three consecutive pregnancies in the presence of a fully known paternal balanced translocation. This demonstration is similar to our previous report of detection of fetal 3p trisomy resulting from a paternal t(3;7) translocation (4). In our study, the earliest gestational age at which fetal DNA was detected was 14 weeks. This suggests that STR analysis of maternal plasma can be used for early second trimester, noninvasive prenatal diagnosis. However, because of the overriding presence of maternal DNA in the maternal plasma, our method should be applied with caution to fetal aneuploidy involving the inheritance of two copies of the paternal chromosomal material, and well-selected, informative STR markers should be used. Recently, Tang et al. (5) successfully detected fetal-derived paternally inherited X-chromosomal polymorphisms in maternal plasma and opened up new possibilities for noninvasive investigation of sex-linked genetic disorders and fetal DNA abnormalities in female fetuses.
In conclusion, we have shown that fetal aneuploidy can be detected by analyzing STR markers of fetal DNA in maternal plasma. With the development of polymorphic STR markers, we believe that molecular analysis of fetal DNA in maternal plasma can be used for the noninvasive detection of fetal-derived paternally inherited aneuploidy.
References
The following articles in journals at HighWire Press have cited this article:
|
|
 |
|
  K.C. A. Chan, A. B.Y. Hui, N. Wong, T. K. Lau, T. N. Leung, K.-W. Lo, and Y.M. D. Lo Investigation of the Genomic Representation of Plasma DNA in Pregnant Women by Comparative Genomic Hybridization Analysis: A Feasibility Study Clin. Chem., December 1, 2005; 51(12): 2398 - 2401. [Full Text] [PDF]
|
|
|
|
|
|
K.C. A. Chan, J. Zhang, A. B.Y. Hui, N. Wong, T. K. Lau, T. N. Leung, K.-W. Lo, D. W.S. Huang, and Y.M. D. Lo Size Distributions of Maternal and Fetal DNA in Maternal Plasma Clin. Chem., January 1, 2004; 50(1): 88 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Tachdjian, N. Frydman, F. Audibert, P. Ray, V. Kerbrat, P. Ernault, R. Frydman, and J.-M. Costa Clinical applications of fetal sex determination in maternal blood in a preimplantation genetic diagnosis centre Hum. Reprod., August 1, 2002; 17(8): 2183 - 2186. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L.M. Poon, T. N. Leung, T. K. Lau, K. C.K. Chow, and Y.M. D. Lo Differential DNA Methylation between Fetus and Mother as a Strategy for Detecting Fetal DNA in Maternal Plasma Clin. Chem., January 1, 2002; 48(1): 35 - 41. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
