As with the 15 controls, samples had been collectedfrom women undergoing biochemical or ultrasoundscreening. The median SOD activity was 28.5 act. U,which was similar to the controls and did not appearto depend on gestation: 32.3 act. U at 15±16 weeks (19samples), 27.5 act. U at 17±18 (30), 27.8 act. U at19±20 (30), 29.3 act. U at 21±22 (10) and 28.5 act. U at23±25 (12). There was no obvious association betweenSOD and maternal age. When the series was dividedinto six age groups the median levels were: 29.8 act. Uamong the 10 samples from women aged 18 oryounger, 28.8 act. U for those aged 19±22 (32), 27.5act. U aged 23±26 (28), 26.0 act. U aged 27±30 (14),31.0 act. U aged 31±34 (11) and 29.5 act. U aged 35±42(6).

Our results con®rm the ®ndings in maternal serum(Ognibene et al. 1999). From a ®gure in theirpublication we have estimated the MoM value foreach sample allowing the two series to be combined.The overall median for all 14 cases was 1.21 MoM,and six (43%) had levels above the 90th centile in thecontrols (1.27 MoM). These are too few results tomake any ®rm judgements about the potential of SODin Down syndrome screening. However, the assaymethod in serum is simple and the data so far aresuf®ciently encouraging to warrant larger studies,ideally including cases from the ®rst trimester ofpregnancy.

There is no obvious explanation for the observedincrease in SOD activity during pregnancies affectedby Down syndrome. One possibility is that it resultsfrom a maternal response to the high SOD levelsfound in the affected fetus (Baeteman et al., 1985).After delivery the mothers of affected infants have anincrease of free-radical processes and a substantial

decrease of SOD activity (Arbuzova, 1998). It ispossible to speculate that during the affected preg-nancy itself an increase in maternal SOD is needed toreduce the oxidant±antioxidant imbalance and pre-serve the pregnancy.

ACKNOWLEDGEMENTS

We thank the Wellcome Trust for supporting thiswork.

Howard Cuckle1 and Svetlana Arbuzova2

1Reproductive Epidemiology, Centre for ReproductionGrowth and Development, 26 Clarendon Road,Leeds LS2 9NZ, U.K.2Interregional Medico-Genetic Center, Hospital No. 1,57 Artem Street, 340000 Donetsk, Ukraine

REFERENCES

Arbuzova SB. 1996. Free radicals in origin and clinical manifestationof Down's syndrome. Cytol Genet 30: 25±34 (Russian).

Arbuzova S. 1998. Why it is necessary to study the role ofmitochondrial genome in trisomy 21 pathogenesis? Down Syn-drome Res Pract 5: 126±130.

Baeteman MA, Mattei MG, Baret A, Gamerre M, Mattei JF. 1985.Immunoreactive SOD-1 in amniotic ¯uid, amniotic cells and®broblasts from a trisomy 21 fetus. Acta Paediatr Scand 74:697±700.

Chevari S, Chaba I, Sekey Y. 1985. The role of SOD-1 in cell'soxidative processes and method of its detection in biologicalliquids. Lab Diagn 11: 678±681.

Ognibene A, Ciuti R, Tozzi P, Messeri G. 1999. Maternal serumsuperoxide dismutase (SOD): a possible marker for screeningDown syndrome affected pregnancies. Prenat Diagn 19:1058±1060.

Fetal DNA in maternal plasma: the prenatal detection of a paternally inheritedfetal aneuploidy

Fetal DNA in maternal plasma and serum has beenimplicated for non-invasive prenatal diagnosis ofpaternally inherited dominant disorder (Lo et al.,1997,1998). Fetal DNA can be detected in maternalplasma as early as 7 weeks' gestation, and the con-centrations of fetal DNA in total maternal plasmarange from 3.4% in early pregnancy to 6.2% in latepregnancy (Lo et al., 1998). Up to now, prenataldetection of fetal aneuploidy from maternal blood hasbeen focused on searching the intact fetal cells, fromwhich nuclei can be used for ¯uorescence in situhybridization (FISH) analysis. However, using fetalDNA in maternal plasma to determine fetal aneu-

ploidy has not yet been demonstrated. Here, we reporta new possibility for detection of paternally inheritedfetal aneuploidy by analysing DNA in maternalplasma.

We studied a pregnant woman with a fetus havingpaternally inherited aneuploidy. The pregnant womanwas a 26-year-old primigravida, referred due toabnormal sonographic ®ndings of fetal holoprosen-cephaly and cyclopia. Collection of parental bloodsamples was immediately made. Amniocentesisperformed later showed fetal distal 3p trisomy(3p23ppter) and 7q36 deletion, 46,XX,der(7)t(3;7)(p23;q36)pat, resulting from a paternal t(3;7) recipro-

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cal translocation. The amniocentesis was performed at23 gestational weeks. The maternal blood sampleswere collected before amniocentesis.

We collected 5 ml of both paternal and maternalperipheral blood in EDTA-containing tubes. Bloodsamples were centrifuged at 3000 g and the plasma wascarefully removed without disturbing the buffy coat.The maternal plasma sample was recentrifuged and thesupernatant was collected for processing. DNA wasextracted from the buffy coat and 600 ml plasmasamples using a DNA Extraction kit (QIAGEN,Hilden, Germany). We used ¯uorescent polymerasechain reaction (PCR) assays and polymorphic smalltandem repeats (STRs) to analyse DNA in maternalplasma. Five pairs of highly polymorphic primerswere separately used to amplify the followingloci: D3S1297 (chromosome 3pter-p25, heterozygosity82%), D3S1560 (chromosome 3pter-p24.2, heterozyg-osity 82%), D3S1263 (chromosome 3p25-p24.2, het-erozygosity 87%), D3S1293 (chromosome 3p25-p24.2,heterozygosity 80%), and D3S1266 (chromosome3p24.2-p22, heterozygosity 73%) (Perkin-Elmer, Nor-walk, USA) (Internet Web of STRs, 1999a, 1999b).Each of the forward primers was labelled at the5k end with one of the following ¯uorescent dyes:6-carboxy¯uorescein (6-FAM), 4, 7, 2k, 4k, 5k, 7k-hexachloro-6-carboxy¯uorescein (HEX), or 4, 7, 2k,7k-tetrachloro-6-carboxy¯uorescein (TET). The PCRcondition was performed according to the protocolrecommended by the manufacturer. We used 60 ng ofparental white blood cell (WBC) DNA according tooptical density measuring and maternal plasma DNAaliquot equivalent to 1/20 of the starting plasma astemplate. Normal controls were performed by ampli-fying maternal plasma and WBC DNA from womencarrying fetuses not affected by chromosomal aneu-ploidies. After initial denaturation at 95uC for 5 min,10 cycles of PCR ampli®cation were doneÐ15 sdenaturation at 94uC, 15 s annealing at 55uC and30 s extension at 72uC. Subsequently, 20 cycles ofPCR ampli®cation were doneÐ15 s denaturation at89uC, 15 s annealing at 55uC, and 30 s extension at72uC. The ®nal extension was at 72uC for 10 min.The DNA fragments were diluted 10 times (HEX-labelled products) or 20 times (6-FAM- or TET-labelled products) and were then mixed together withformamide and GS-500 TAMRA size standard(Applied Biosystems, Foster City, USA). The DNAfragments were resolved on a DNA sequencer (ABI377 model) by Genescan Analysis 2.1 software(Applied Biosystems, Foster City, USA). The ampli-®ed alleles were sized based on the peaks on theelectrophoretograms. The specimen of this caseshowed disomy for paternal 3p by three informativemarkers: D3S1297, D3S1263 and D3S1293 (Table 1,Figure 1). Therefore, results obtained by PCR assayswere consistent with the cytogenetic results.

Our results have shown the application of poly-morphic markers outside the Y chromosome inmaternal plasma for non-invasive prenatal diagnosis.This may be useful in early non-invasive detection ofchromosomal abnormalities in subsequent pregnancies

in the presence of a fully known paternal balancedtranslocation or gonadal mosaicism. Nevertheless,such an application in prenatal diagnosis is by nomeans without limitations. Firstly, due to the over-riding presence of maternal DNA in the maternalplasma, this method can only be applied to a verysmall number of fetal chromosomal abnormalities ofwhich the aneuploidy involves the inheritance of twocopies of the paternal chromosomal material. Sec-ondly, the exact nature of the aneuploidy should beknown before PCR ampli®cation so that the STRmarkers can be selected in some speci®c regions.

Figure 1ÐRepresentative electrophoretograms for paternal WBCs(upper), maternal WBCs (middle), and maternal plasma (lower)

Table 1ÐGenotypic information of parental WBCs andmaternal plasma 5 STR markers speci®c for 3p obtainedby ¯uorescent PCR assays

MarkersPaternalWBCs

MaternalWBCs

Maternalplasma

D3S1297 225,231 227,229 225,231,227,229D3S1560 243,243 241,243 241,243D3S1263 235,237 239,239 235,237,239D3S1293 120,126 124,130 120,124,126,130D3S1266 291,291 293,295 291,293,295

WBCs, white blood cells.Italics indicate allele of paternal origin.

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Copyright # 2000 John Wiley & Sons, Ltd. Prenat Diagn 2000; 20: 353±357.

Finally, the selected STR markers must have aninformative nature with paternal allele sizes differingfrom those of the mother. Judged by the area of thepaternal peaks in the electrophoretogram of thematernal plasma, the proportion of fetal DNA inour sample is around 30%, which is much higher thanthat predicted by Lo et al. (1998). It is likely that whenamplifying STRs from small amounts of DNA,preferential ampli®cation may occur and consequentlythe fetal DNA in maternal plasma would be over-estimated. As for no over-representation of one of thematernal peaks in the electrophoretogram of thematernal plasma, we speculate that the overwhelmingpresence of maternal DNA in the maternal plasmamay obscure the contribution of the maternallyacquired allele from the fetal DNA in the maternalplasma.

ACKNOWLEDGEMENTS

The authors thank Professor C.H. Rodeck and thereferee for their valuable comments and constructive

criticism. This work was supported by a research grant(NSC-89±2314-B-195±011) from the National ScienceCouncil, Taiwan, R.O.C.

Chih-Ping Chen1,2, Schu-Rern Chern2 andWayseen Wang2

1Department of Obstetrics and Gynecology, MackayMemorial Hospital, Taipei, Taiwan, R.O.C.2Department of Medical Research, Mackay MemorialHospital, and National Yang-Ming University, Taipei,Taiwan, R.O.C.

REFERENCES

Internet Web of STRs. 1999a. http://www.gdb.org/.Internet Web of STRs. 1999b. http://lpg.nci.nih.gov/CHLC/.Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL,

Redman CWG, Wainscoat JS. 1997. Presence of fetal DNA inmaternal plasma and serum. Lancet 350: 485±487.

Lo YMD, Tein MSC, Lau TK, Haines CJ, Leung TN, Poon PMK,Wainscoat JS, Johnson PJ, Chang AMZ, Hjelm NM. 1998.Quantitative analysis of fetal DNA in maternal plasma andserum: implications for noninvasive prenatal diagnosis. Am J HumGenet 62: 768±775.

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Copyright # 2000 John Wiley & Sons, Ltd. Prenat Diagn 2000; 20: 353±357.