Prenat. Diagn. 18: 669–674 (1998)

PRENATAL DETECTION OF TRISOMY 13 FROMAMNIOTIC FLUID BY QUANTITATIVEFLUORESCENT POLYMERASE CHAIN

REACTION

1*, 2, 1, -1, 1, 1, 2 1

1Molecular Genetics Laboratory, I. Department of Obstetrics and Gynaecology, Semmelweis,University Medical School, Baross u. 27, Budapest, H-1088, Hungary

2Molecular Oncology, Institute of Pathology, Algernon Firth Building, University of Leeds, Leeds LS2 9LN, U.K.

Received 16 May 1997Revised 15 September 1997Accepted 12 October 1997

SUMMARY

Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. However,this requires lengthy laboratory procedures, high costs and is unsuitable for large-scale screening of pregnantwomen. An alternative method, which is rapid, inexpensive and suitable for diagnosing trisomies, even from singlefetal cells, is the fluorescent polymerase chain reaction (PCR) using polymorphic small tandem repeats (STRs). Inthis paper, we present the method of rapid prenatal detection of trisomy 13 from amniotic fluid using fluorescentPCR and two highly polymorphic STRs (D13S258 and D13S631). The results obtained by quantitative fluorescentPCR amplification of fetal DNA were concordant with amniocyte karyotyping results in all cases. Two cases oftrisomy 13 were detected from 212 amniotic fluids and the results obtained from D13S631 and D13S258 amplifi-cation are presented. In the first trisomy 13 case, a triallelic pattern was detected by both markers, and in the secondcase, D13 markers showed a characteristic 2:1 dosage allele ratio, both of which demonstrate trisomy 13 status. Allother heterozygous disomic samples showed an allele intensity ratio of 1:1. ? 1998 John Wiley & Sons, Ltd.

: trisomy 13; Patau syndrome; prenatal diagnosis; fluorescent polymerase chain reaction; D13S258;D13S631

*Correspondence to: T. Toth, I. Department of Obstetricsand Gynaecology, Semmelweis University Medical School,Baross u. 27, Budapest, H-1088, Hungary. E-mail:toma@noi1.sote.hu

Contract grant sponsor: Medical Research Council; Con-tract grant number: MRC G9530631.

Contract grant sponsor: Special Trustees of the United Leeds

INTRODUCTION

Chromosomal abnormalities are the most fre-quent genetic disorders observed in both livebirthsand miscarriages. Trisomies are the largest groupof chromosome abnormalities and directly result in17 per cent of all fetal deaths (Hook, 1992).

Teaching Hospitals.

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Trisomies for all autosomes, with the exceptionof 1 and 19 have been detected in spontaneousabortions (Hassold et al., 1987), but only threeautosomal trisomies (trisomies 21, 18 and 13) arefound with any significant frequency among livebirths.

Even though more than two-thirds of trisomy 21pregnancies result in fetal death, trisomy 21 (Downsyndrome) has the highest birth prevalence(approximately one affected newborn in 700–800births (Hook, 1981)). The next most commontrisomy observed in prenatal diagnosis is trisomy18 (Edward’s syndrome). Even though 290 percent of trisomy 18 pregnancies result in fetal death,

this syndrome has a birth prevalence of one

670 . .

affected newborn in 1000 live births and results insevere mental retardation, multiple malformationand is generally fatal before one year of age.

Trisomy 13 (Patau syndrome) is the third mostcommon trisomy and 98 per cent of affected chil-dren die before birth (Jacobs et al., 1987), the greatmajority as spontaneous abortion. Although thebirth prevalence of trisomy 13 is low, about1:8000–1:12 000, the prenatal exclusion of thischromosomal abnormality is recommended.

As the frequency of chromosomal abnormali-ties, including trisomy 13, increases with maternalage (Hook, 1983), trisomy screening by prenataldiagnosis (by amniocentesis or chorionic villussampling) has become accepted by women over 35years of age (Ferguson-Smith and Yates, 1984).However, despite the extensive care of pregnantwomen over 35 years of age, the birth prevalenceof trisomies remains high, as two-thirds of fetalchromosomal abnormalities occur in pregnantwomen under 35 years of age. Therefore, to signifi-cantly reduce the birth prevalence of trisomies, awide ranging screening programme of pregnantwomen has been suggested.

Current methods, such as cytogenetic analysis,for detecting chromosomal abnormalities oftenrequire lengthy laboratory procedures and havehigh costs as well as significant delay in providinga diagnosis. These tests are therefore not suitablefor large-scale screening of all pregnant women.An alternative method, which is both rapid, inex-pensive and suitable for diagnosing trisomies, isthe fluorescent polymerase chain reaction usingpolymorphic small tandem repeats (STRs). Thequantitative nature of this technique allows theamount of PCR product to be determined andthus, allow peak ratios to be calculated. Althoughseveral papers have reported the application of thisnewly developed method for trisomy detection(Mansfield, 1993; Eggeling et al., 1993), very fewhave investigated its usefulness in prenatal diag-nosis (Pertl et al., 1994; Adinolfi et al., 1995).

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Furthermore, this method has been reported onlyfor detecting chromosomes 21, 18 and X abnor-malities (Pertl et al., 1996). Although trisomy 13detection using fluorescent PCR has been pro-posed, it has yet to be reported (Pertl et al., 1996).

We report a rapid prenatal detection techniquefor trisomy 13 detection in amniotic fluid usingfluorescent PCR and two polymorphic chromo-some 13 specific tetranucleotide repeat markerswhich increase diagnostic reliability and accuracy.

MATERIALS AND METHODS

DNA preparation

Cells from 1 ml amniotic fluid were collectedby centrifugation at 15 000 rpm for 10 min. Thesupernatant was discarded and 100 ìl of resinfrom ReadyAmp Genomic Purification System(Promega, WI, U.S.A.) was added to the pelletedcells. The subsequent extraction procedure wasperformed as per manufacturer’s protocol. 3 ìlfrom this DNA solution were used in each PCRreaction.

Table I—Primer sequences for the indicated STR markers

Marker Sequence of primers

D13S258 (F) 5*-ACC TGC CAA ATT TTA CCA GG-3*D13S258 (R) 5*-GAC AGA GAG AGG GAA TAA ACC-3*D13S631 (F) 5*-GGC AAC AAG AGC AAA ACT CT-3*D13S631 (R) 5*-TAG CCC TCA CCA TGA TTG G-3*

Fluorescent polymerase chain reaction

PCR ampification was carried out in two separ-ate assays. The first with primers for the D13S631marker and the second with primers for D13S258marker (Genomic Database; The Utah markerdevelopment group, 1995). Primer sequences arelisted in Table I. The D13S631 forward primer waslabelled at the 5* end with FAM, while D13S258forward primer was labelled with HEX fluorescentdye. PCRs were performed in a total volume of25 ìl containing 3 ìl of DNA solution preparedby the Promega kit, 0·9#Taq polymerase buffer(Perkin Elmer), 200 ìM dNTPs, 5 pmol of eachprimer, 1·5 mM MgCl2 and 0·6 U AmpliTaq(Perkin Elmer). After denaturation at 95)C for

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5 min, 30 cycles at 95)C for 30 s, 54)C (D13S631)or 58)C (D13S258) for 45 s and 72)C for 60 s wereperformed in a Perkin Elmer 2400 thermal cycler.Final extension was 30 min at 60)C to avoid peaksplitting difficulties caused by the incomplete add-ing of adenine by Taq polymerase to the end ofPCR product. Extension at 60)C for 30 min allowsthe AmpliTaq to successfully complete thisreaction producing a single sharp allele band.

Separation and quantitation of PCR products

2 ìl of PCR product from both reactions weremixed together with 24 ìl formamide and 1 ìlGenescan-500 TAMRA size standard (AppliedBiosystems, U.S.A.). The mixture was denaturedat 95)C for 3 min and placed on ice until analysis.Electrophoretic analysis was performed by usingPOP4 gel (Applied Biosystems, U.S.A.) andemploying the ABI 310 Genetic Analyser (AppliedBiosystems, U.S.A.). The amplification productswere analysed by Genescan Analysis 2.1 software(Applied Biosystems, U.S.A.) and the relative peakareas were automatically calculated by the soft-ware. As the FAM fluorescent dye is visualized

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as blue and HEX as yellow using the filter C,the PCR products from the two reactions cantherefore be quantified within a single tube.

Fig. 1—Electrophoretogram of a trisomy 13 sample showing triallelic pattern for both D13S258and D13S631 STRs

RESULTS

During a 10 week period, 212 pregnant womenunderwent amniocentesis to rule out fetal chromo-somal abnormalities either due to maternal age orfrom previously detected ultrasound abnormali-ties. Informed consent was obtained from allpatients. 10 ml of amniotic fluid were removedwith 9 ml used for cytogenetic analysis whilst 1 mlwas stored for molecular genetic analysis. Samplesfrom each patient underwent karyotyping andtrisomy 13 diagnosis using D13S258 and D13S631STR markers.

Cytogenetic results indicated two trisomy 13samples from the 212 samples. In each case,molecular genetic analyses were also performed onthe amniotic fluid samples by amplifying STRmarkers on chromosomes 13. One sample (Fig. 1)showed a characteristic triallelic pattern for bothchromosome 13 specific STR markers, while

another sample (Fig. 2) showed a diallelic pattern

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672 . .

with a dosage ratio of 2:1 for both D13S258 andD13S631.

The number of homozygous and heterozygoussamples for the examined markers and relativepeak areas of heterozygous disomic samples areshown in Table II. Out of the 212 samples, onlytwo were homozygous and therefore uninforma-tive for both markers. The electrophoretogram inFig. 3 demonstrates a sample heterozygous forboth investigated STR markers. In all informativecases, PCR results were consistent with cytogenetic

Fig. 2—Electrophoretogram of a further trisomy 13 sample demonstrating a diallelic patternwith a dosage ratio of 2:1 for both D13S258 and D13S631

Table II—Results of disomic samples tested by STR markers D13S258 and D13S631. The mean is calculated fromratios obtained by dividing the larger by the smaller allele area. Of the 212 samples tested, two samples were trisomicfor STR markers

Marker Homozygous HeterozygousTotaltested Heterozygosity

Relative peak areas

Mean SD Range

D13S258 14 196 210 0·93 1·06 0·054 1·00–1·18D13S631 25 185 210 0·88 1·09 0·062 1·00–1·24Both 2 208 210 0·99

results.

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DISCUSSION

The pre- and postnatal detection of chromo-somal abnormalities have been almost exclusivelyperformed by cytogenetic analysis, which requireslive cells and complex conditions, as well as time-intensive laboratory culture to obtain sufficientcells for analysis. In addition, cytogenetic detectionof trisomies cannot be performed reliably fromstored tissues or from single cells.

Fluorescent in situ hybridization (FISH) allows

identification of specific chromosomes when the

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Fig. 3—Electrophoretogram of a disomic sample demonstrating a diallelic pattern with adosage ratio of 1:1 for both D13S258 and D13S631

cells are in interphase or metaphase. AlthoughFISH can be used for detection of trisomies fromamniotic fluid, stored tissues and single cells,several technical difficulties have been reportedand the evaluation of trisomic results using FISHremains to be resolved (Kuo et al., 1991). Fordetection of subtle chromosomal abnormalities(Chu et al., 1994), such as balanced and unbal-anced (Rao et al., 1995) translocations, FISH maybe more suitable than fluorescent PCR, but theadvantage of this PCR technique is that it requiresless time and less fetal cells for diagnosis. Theseadvantages may make fluorescent PCR suitable forlarge-scale prenatal screening.

Fluorescent PCR has now been applied to rapidprenatal detection of trisomies 21, 18 and 13. Theresults of trisomies 21 and 18 detection arereported elsewhere whilst the trisomy 13 results aredescribed in this paper for the first time. Thesepreliminary experiments demonstrate that fluor-escent PCR amplification of STRs can reliably beused for the detection of trisomy 13. Similar obser-vations with chromosome 21 and 18 specificmarkers have already proved the usefulness ofthis technique in the prenatal diagnosis of these

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trisomies. Further STR markers on other chromo-somes are likely to be utilized in a similar mannerallowing detection of other chromosomal abnor-malities by molecular genetic methods rather thancytogenetic analysis.

We believe that using two highly polymorphicSTR markers on each chromosome would allow ahigh diagnostic accuracy in the majority of cases.In non-informative cases, further polymorphicmarker(s) could be amplified.

This technique allows detection of trisomieswithin four hours in urgent cases which allowsamniocentesis and possible termination to beundertaken the same day. This is particularlyimportant when the pregnancy is relativelyadvanced, as delay may lead to gestation exceedingthe legal limit for termination. For comparison,the quickest trisomy 13 detection previouslyobtained was by karyotyping amniotic fetal cellsand took 72 h (Roberts et al., 1993).

As fluorescent PCR can also be performed fromsingle cells (Findlay et al., 1995; Findlay andQuirke, 1996) this technique appears to be suitablefor prenatal diagnosis of trisomies from isolatedfetal cells from maternal blood (Cheung et al.,

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674 . .

1996) or in pre-implantation diagnosis from blast-omeres obtained by embryo biopsy. Further inves-tigations, which are in progress, will determine thereliability of this technique in these fields.

We would like to thank the Medical ResearchCouncil (grant MRC G9530631) and the SpecialTrustees of the United Leeds Teaching Hospitalsfor financial support.

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