Molecular Human Reproduction vol.6 no.9 pp. 855–860
Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21 by
QF–PCR in 662 selected pregnancies at risk
Winfried Schmidt1, Jutta Jenderny1,3, Kurt Hecher2, B.-Joachim Hackelöer2, Susanne Kerber1,
Lothar Kochhan1 and Karsten R.Held1
1Institute
of Immunology, Pathology, Molecular Biology and Human Genetics (IPM), Lademannbogen 61–63,
22339 Hamburg, and 2Allgemeines Krankenhaus Barmbek, Dept. of Prenatal Diagnosis and Therapy, Rübenkamp 148,
22307 Hamburg, Germany
3To
whom correspondence should be addressed at: Institute of Immunology, Pathology, Molecular Biology and Human
Genetics (IPM), Lademannbogen 61–63, 22339 Hamburg, Germany. E-mail: jenderny@ mail.labor-keeser-arndt.de
A quantitative fluorescent–polymerase chain reaction (QF–PCR) test system with different short tandem
repeat (STR) markers of the X chromosome (SBMA, DXS8377 and DXS1283E) together with the amelogenin
locus (AMXY) was developed for the rapid detection of sex chromosome aneuploidies on uncultured amniotic
fluids. The samples (n ⍧ 662) were also tested with STRs specific for chromosomes 13, 18 or 21, with two
STRs used for each chromosome. In uninformative cases, an additional STR marker was applied. The QF–
PCR data were compared with the results of conventional cytogenetics. One dark red stained specimen
showed an artificial PCR pattern, probably due to maternal contamination. Six sex chromosome aberrations
(four 45,X, one 47,XXY, one mosaic 47,XXY/46,XX) were identified as aneuploid by STRs specific for
chromosome X and AMXY. One pregnancy with a mosaic 45,X/46,XX karyotype was not detected by the
assay. In all, 12 cases with a numerical aberration involving either chromosome 18 or 21 or with a triploidy
were correctly diagnosed by QF–PCR. No information was obtained in one fetal sample with a trisomy 18
due to an uncertain result for two of the three applied STRs specific for chromosome 18 and an uninformative
third STR marker. Two samples with an unbalanced Robertsonian translocation could be identified by QF–
PCR as trisomic for chromosomes 13 and 21 respectively. The results show an excellent agreement between
QF–PCR and cytogenetics with regard to sex chromosome and autosomal aneuploidy detection in prenatal
diagnosis.
Key words: amniocytes/prenatal diagnosis/quantitative fluorescent–polymerase chain reaction (QF–PCR)/sex
chromosome aberrations/trisomy 13, 18 and 21
Introduction
Since 1992, fluorescence in-situ hybridization (FISH) on uncultured amniocytes of pregnancies at risk has been used for the
rapid detection of numerical aberrations of chromosomes X,
Y, 13, 18 and 21 (Klinger et al., 1992; Ward et al., 1993;
Bryndorf et al., 1997; Eiben et al., 1998, 1999; Jalal et al.,
1998; Lewin et al., 2000; Thilaganathan et al., 2000). An
alternate approach for the identification of selected aneuploidies
is the use of fetal DNA amplified by quantitative fluorescent–
polymerase chain reaction (QF–PCR) using short tandem
repeats (STRs) (Mansfield, 1993; Pertl et al., 1996; Findlay
et al., 1998; Toth et al., 1998; Verma et al., 1998). Recently,
the first diagnostic application of this method was described
in a systematic study (Verma et al., 1998). This group screened
2167 samples of uncultured amniotic fluid for the prenatal
diagnosis of trisomy 21. Other authors (Pertl et al., 1999)
demonstrated the detection of major autosomal aneuploidies
by QF–PCR, analysing a large number of chorionic villus
samples. So far, the low level of polymorphism of most
X-specific DNA markers has hampered the use of QF–PCR
for the detection of sex chromosome aberrations. Recently, a
first investigation on peripheral blood samples of patients
© European Society of Human Reproduction and Embryology
with sex chromosome aberrations previously diagnosed by
cytogenetic analysis showed that it is possible to detect patients
with a Turner syndrome or an XXY chromosome constitution
using X22, HPRT and P39 as X-linked markers (Cirigliano
et al., 1999). We tested three other STR markers for the
X chromosome for the identification of sex chromosome
aneuploidies in prenatal diagnosis. We focused our study on
662 amniotic fluid samples of pregnancies at risk, thereby
studying the most relevant fetal sex chromosome and autosomal
aneuploidies.
Materials and methods
Patients and cytogenetic analysis
The selected indications of prenatal diagnosis included advanced
maternal age (⬎35 years, n ⫽ 341), abnormal fetal ultrasonographic
signs with or without advanced maternal age (n ⫽ 134), positive test
results after maternal blood biochemical screening methods (n ⫽ 75),
a previous fetus or child with a chromosomal aberration (n ⫽ 15) or
other indications (n ⫽ 97). These indications were parents’ anxiety
(n ⫽ 73), pregnancies complicated by a feto–fetale transfusion
syndrome (n ⫽ 18), X-chromosomal defects (n ⫽ 3) and maternal
855
W.Schmidt et al.
Table I. Primers and short tandem repeat markers (STRs) used for sexing and for the detection of sex
chromosome aberrations and selected autosomal trisomies. The markers were used for each chromosome with a
third marker (shown in brackets) being used in uninformative cases
Chromosome
Marker primer sequence (5⬘-3⬘)
Heterozygosity
X chromosome marker
SBMA-F-FAM
SBMA-R
DXS8377-F-JOE
DXS8377-R
(DXS1283E-F-FAM
DXS1283E-R)
FAM – TCC GCG AAG TGA AGA AC
CTT GGG GAG AAC CAT CCT CA
JOE – CAC TTC ATG GCT TAC CAC AG
GAC CTT TGG AAA GCT AGT GT
6-FAM – AGT TTA GGA GAT TAT CAA GCT GG
GTT CCC ATA ATA GAT GTA TCC AG
0.902
Chromosome 13 marker
D13S258-F-JOE
D13S258-R
D13S303-F-FAM
D13S303-R
(D13S256-F-TAMRA
D13S256-R)
JOE – ACC TGC CAA ATT TTA CCA GG
GAC AGA GAG AGG GAA TAA ACC
6-FAM – ACA TCG CTC CTT ACC CCA TC
TGT ACC CAT TAA CCA TCC CCA
6-TAMRA – CCT GGG CAA CAA GAG CAA A
AGC AGA GAG ACA TAA TTG TG
Chromosome 18 marker
D18S1002-F-TAMRA
D18S1002-R
D18S51-F-FAM
D18S51-R
(D18S499-F-JOE
D18S499-R)
6-TAMRA – CAA AGA GTG AAT GCT GTA CAA ACA GC
CAA GAT GTG AGT GTG CTT TTC AGG AG
6-FAM – CAA ACC CGA CTA CCA GCA AC
GAG CCA TGT TCA TGC CAC TG
JOE – CTG CAC AAC ATA GTG AGA CCT G
AGA TTA CCC AGA AAT GAG ATC AGC
0.812
Chromosome 21 marker
D21S11-F
D21S11-R-TAMRA
D21S1411-F-FAM
D21S1411-R
(D21S1413-F-FAM
D21S1413-R)
ATA TGT GAG TCA ATT CCC CAA G
6-TAMRA – TGT ATT AGT CAA TGT TCT CCA G
6-FAM – ATG ATG AAT GCA TAG ATG GAT G
AAT GTG TGT CCT TCC AGG C
6-FAM – TTG CAG GGA AAC CAC AGT T
TCC TTG GAA TAA ATT CCC GG
0.900
Amelogenin locus
HUMAMG-F-Fam
HUMAMG – R
6-FAM – CCC TGG GCT CTG TAA AGA ATA GTG
ATC AGA GCT TAA ACT GGG AAG CTG
0.950
0.886
0.875
0.909
0.750
0.802
0.710
0.933
0.875
F ⫽ forward primer; R ⫽ reverse primer.
virus infections (n ⫽ 3). At least 3–6 ml of amniotic fluids were
used for cell cultivation and subsequent cytogenetic analysis according
to standard techniques. A minimum of 11 metaphases was analysed.
Quantitative fluorescence–polymerase chain reaction (QF–
PCR)
Genomic DNA was extracted from 1–2 ml of uncultured amniotic
cells using a QIAamp blood kit (Qiagen, Germany). All samples
were processed including visibly light or dark red stained amniotic
fluids with potential maternal cell contamination. Visibly light or
dark red samples were washed twice with aqua bidest. All DNA
samples were coded and the analysis was undertaken without knowledge of the fetal karyotype. For sex determination the unique
sequence in the first intron of the X/Y homologous gene amelogenin
(AMXY) was amplified (Nagafuchi et al., 1992). For the detection
of sex chromosome aberrations and selected autosomal trisomies two
different STR markers were used per chromosome. In uninformative
cases, a third STR marker was employed. The STR markers were
selected from genome data because of their high heterozgosity rates
and good results under multiplex PCR conditions. The primers used
for the different STR markers and for sexing are shown in Table I.
One of the primers was labelled (5⬘ end) with a fluorescent dye
(FAM, JOE or TAMRA; Biometra Gottlin, Germany) to enable the
visualization and analysis of the PCR products. PCR amplification
was performed in a total volume of 50 µl containing genomic DNA
(15 µl of the extracted DNA), 200 µmol/l dNTPs, 4–20 pmoles of
each primer, PCR buffer (Perkin Elmer Applied Biosystems Inc,
856
Table II. Multiplex quantitative fluorescent–polymerase chain reaction (QF–
PCR) samples
QF–PCRs
STRs
Primer quantity
(pmol)
Expected fragment
size (bp)
I
Amelogenin
D21S11
D18S51
D18S1002
D13S303
DXS8337
D21S1411
D13S258
SBMA
4
20
5
10
5
10
10
10
10
106X;112Y
205–245
279–323
286–318
338–354
203–245
283–313
180–296
142–178
II
III
IV
STR ⫽ short tandem repeat.
USA) and 1.0 IU AmpliTaq Gold (Perkin Elmer Applied Biosystems
Inc). Four separate multiplex PCR assays were designed using the
STR markers (see Table II). After the initial denaturation at 94°C for
5 min, 31 cycles of PCR amplification followed (1 min denaturation
at 94°C, 1 min annealing at 60°C, 1 min extension at 72°C, final
extension for 5 min at 72°C.) and performed in a Gene Amp. PCR
system 9700 cycler (Perkin Elmer Applied Biosystems Inc). The
allelic fragments were resolved on a 4.25% denaturing polyacrylamide
gel using a 377 DNA Sequencer (Perkin Elmer Applied Biosystems
Inc). The Genescan 672 software was employed for the analysis and
Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21
Table III. Quantitative fluorescent–polymerase chain reaction (QF–PCR) versus cytogenetic results on 662 amniotic fluid samples tested for sex chromosome
aberrations and selected autosomal trisomies
Cytogenetic results
No. of cases
analysed
QF–PCR results
X and Y chromosomes
neg (n ⫽ 652)
46,XX, or 46,XY
45,X
Mosaic 45,X/46,XX
47,XXY
Mosaic 47,XXY/46,XX
69,XXX or 69,XXY
46,XY,der(13;14)(q10;q10),⫹13
47,XX, or 47,XY,⫹18
47,XX, or 47,XY,⫹21
46,XX,i(21)(q10)
Other unbalanced chromosome
aberrations
Balanced chromosome aberrations
Total
n
n
n
n
n
n
n
n
n
n
n
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
⫽
633
4
1
1
1
3
1
5
5
1
2
n⫽5
n ⫽ 662
X status (n ⫽ 4)
not identified
XXY (n ⫽ 1)
XXY (n ⫽ 1)
XXX or XXY (n ⫽ 3)
QF–PCR artefact?
(n ⫽ 1)
Chromosome 13
neg (n ⫽ 622)
Chromosome 18
neg (n ⫽ 636)
Chromosome 21
neg (n ⫽ 642)
Tris 13 (n ⫽ 3)
Tris 13 (n ⫽ 1)
Tris 18 (n ⫽ 3)
Tris 21 (n ⫽ 3)
Tris 18 (n ⫽ 4)
Tris 21 (n ⫽ 5)
Tris 21(n⫽1)
Uninformative (n ⫽ 35) Uninformative (n ⫽ 18) Uninformative (n ⫽ 10)
QF–PCR artefact?
QF–PCR artefact?
QF–PCR artefact?
(n ⫽ 1)
(n ⫽ 1)
(n ⫽ 1)
neg ⫽ negative; Tris ⫽ trisomy.
calculation of the amplification products. The study design was
approved by the regional ethics committee.
Results
A total of 662 amniotic fluid samples were analysed by QF–
PCR (Table III). Of them, 586 (88.5%) were clear samples,
whereas 39 (5.9%) were light and 37 (5.6%) dark red stained.
No sample failed and had to be rerun. Males were diagnosed
by amplification of the X and Y chromosomal PCR products
of the amelogenin locus with a normal 1:1 ratio, females by
the presence of one signal of the X chromosome PCR product.
For the rapid diagnosis of trisomy 21 we used up to three
STR markers, which resulted in an informative PCR pattern
in 98.3% (651/662) of all analysed specimens. 97.3% (643/
662) of all samples were heterozygous and thus informative
for at least one STR marker specific for chromosome 18.
Using up to three STR markers specific for chromosome 13,
94.6% (626/662) of all samples were found to be informative
for at least one STR marker.
The criteria and guidelines for the diagnosis of a normal or
pathological QF–PCR result were as follows: for the diagnosis
of a Turner syndrome a monoallelic pattern of all X-specific
STR markers and the absence of the Y specific amelogenin
locus was necessary (Figure 1). Due to the heterozygosity
indices (Table I), the probability of homozygosity for each X
chromosome marker is extremely low. A normal autosomal
QF–PCR product was assumed after at least one STR marker
for each chromosome displayed a clear diallelic pattern with
a normal 1:1 ratio. The analysis of samples from patients with
a trisomy revealed the presence of either three peaks (ratio
1:1:1) or two peaks with a ratio of 2:1 for the chromosome
specific STR marker (Figure 2). In our laboratory we assumed
a tolerance of ⫾20% for the calculation of the peak ratio.
This calculation is necessary due to the PCR-specific worse
preferential amplification of larger PCR fragments. Under
these conditions, no preferential amplification leading to mis-
diagnosis was observed. One amniotic fluid which was dark
red stained showed an abnormal (no 2:1 ratio) diallelic PCR
pattern in two STR profiles and an unclear triallelic (three
peaks which showed no 1:1:1 ratio) PCR pattern in five STR
profiles. This sample was suspected of being contaminated
and was classified as an artificial PCR product probably due
to maternal contamination (Figure 3). The PCR products were
not compared with maternal ones. Chromosomal analysis of
this sample revealed a normal karyotype. Whenever a QF–
PCR product was pathological, the result was validated within
24 h by QF–PCR with a second independent analysis of 1–2
ml uncultured amniotic fluid of the same sample. In all cases,
the pathological results of the QF–PCRs were consistent with
the cytogenetic analysis [45,X (n ⫽ 4); 47,XXY (n ⫽ 1);
69,XXX or 69,XXY (n ⫽ 3), 47,XX, or XY,⫹18 (n ⫽ 4);
47,XX, or XY,⫹21 (n ⫽ 5)]. One sample with a sex chromosome mosaicism (mos 45,X/46,XX; level III mosaicism) was
misdiagnosed as an XX chromosome complement by QF–
PCR. One case diagnosed as XXY status by QF–PCR displayed
sex chromosome mosaicism (47,XXY/46,XX; level III
mosaicism) by conventional chromosome analysis. No
information was obtained in one fetal sample with a trisomy
18 due to an unclear 2:1 ratio for two of three applied
STRs specific for chromosome 18. The third STR marker was
uninformative. Two cases with an unbalanced Robertsonian
translocation [46,XY,der(13;14) (q10;q10),⫹13; 46,XX,i(21)
(q10)] could be identified by QF–PCR as trisomic for chromosomes 13 and 21 respectively. No false positive QF–PCR results
were observed. Seven aberrant chromosome complements were
not detectable by molecular analysis: After karyotyping, two
cases showed an unbalanced karyotype [46,XX, in-situ
hybridzation del(22)(q11.2); mosaic 47,XY,⫹20/46,XY] and five
samples a balanced chromosome aberration (45,XX,der(13;
14)(q10;q10) de novo; 45,XX,der(13;14) (q10;q10)pat;
46,XX,t(3;8)(p34.1;q25)mat; 46,XY,inv(9)(p24q32) pat; 46,XY,
t(1;17) (p34.1;q25)mat.
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W.Schmidt et al.
Figure 1. Electropherogram of the amplification products from a sample with a Turner syndrome (45,X). The x axis shows the calculated length
of the polymerase chain reaction (PCR) products. The y axis shows fluorescent intensities in arbitrary units. Amplification of the marker AMXY
(amelogenin) resulted in one peak corresponding to the X chromosome. Only single peaks of fluorescent activity were observed after QF–PCR
with the X-specific STR markers DXS8377 (third line, bold) and SBMA (fourth line, bold) respectively. Amplification of all autosomal STR
markers used in this set resulted in a normal heterozygous PCR pattern. The DNA sample was heterozygous for D21S11 and D18S51 (first line),
D18S1002 and D13S303 (second line), D21S1411 (third line) and D13S258 (fourth line) respectively. The alleles of each short tandem repeat
(STR) marker were in a normal 1:1 dosage ratio.
Figure 2. Electropherogram of the amplification products from a sample with a trisomy 21. The x axis shows the calculated length of the
polymerase chain reaction (PCR) products. The y axis shows fluorescent intensities in arbitrary units. Amplification of the marker AMXY
(amelogenin) resulted in one peak corresponding to the X chromosome. Three peaks of fluorescent activity were observed after QF–PCR with
the chromosome 21 specific short tandem repeat (STR) marker D21S11 (first line) and two peaks with a 2:1 ratio were observed with the STR
marker D21S1411 (third line) respectively. Amplification of the other autosomal and sex chromosome STR markers used in this set resulted in a
normal heterozygous PCR pattern. The STR marker D18S1002 revealed an uninformative PCR result.
Discussion
The study presents the results of the first large clinical application
of QF–PCRs for the rapid detection of sex chromosome and
autosomal aneuploidies for chromosomes X and Y, 13, 18 and 21
on 662 uncultured clear and visibly contamined amniotic fluids as
an adjunctive test to conventional cytogenetic analysis. Only one
sample showed an artificial PCR pattern after applying all selected
chromosome specific STR markers. In all other specimens, the high
858
level of heterozygositiy of the X-specific STR markers SBMA,
DXS8377 and DXS1283E in combination with the X/Y homologous gene amelogenin locus resulted in an informative PCR result.
In normal female samples there was no single case of homozygosity
for all applied X-linked markers. Turner syndrome (45,X, n ⫽ 4)
was correctly identified by QF–PCR, showing only single SBMA,
DXS8377 and/or DXS1283E peaks of fluorescence activity. In
two cases, an XXY status could also be diagnosed by the assay.
Detection of aneuploidy in chromosomes X, Y, 13, 18 and 21
Figure 3. Electropherogram of the amplification products from a dark red stained sample with an artificial polymerase chain reaction (PCR)
pattern probably due to maternal contamination. The x axis shows the calculated length of the PCR products. The y axis shows fluorescent
intensities in arbitrary units. Amplification of the marker AMXY resulted in two peaks (1:1 ratio; X:Y) corresponding with the X and Y
chromosome. Amplification of the other short tandem repeat (STR) markers resulted in an abnormal (no 2:1 ratio) diallelic PCR pattern (two
STR profiles) and in an unclear triallelic (three peaks which showed no 1:1:1 ratio) PCR pattern (five STR profiles).
In one of these samples chromosome analysis revealed a sex
chromosome mosaicism (47,XXY/46,XX), whereas the QF–PCR
test was not able to detect the normal cell population. QF–PCR
results leading to false negative results were found in only one
case with mosaicism of the X chromosome (mosaic 45,X/46,XX).
Although the QF–PCR products showed clear heterozygous STR
profiles for DXS8377 and DXS1283 respectively, cytogenetic
analysis yielded a sex chromosome mosaicism with the presence
of a second population of cells with an X karyotype. False positive
QF–PCR results were not observed. Within the current study, a total
of 29 chromosomal abnormalities was diagnosed by cytogenetic
analysis. Five of them showed a balanced chromosomal
complement, mainly due to a familial Robertsonian or reciprocal
translocation or inversion. Of the 24 unbalanced chromosome
complements with a significant risk of phenotypic abnormalities,
20 (83%) were identified by QF–PCR.
FISH on uncultured amniotic cells is an alternative rapid method
to QF–PCR. However, our results showed that the QF–PCR based
diagnostic method has many advantages over FISH. As shown by
several groups contamination by maternal blood was shown to
pose a problem for aneuploidy detection by FISH. Two groups
(Ward et al., 1993; Jalal et al., 1998) reported promising FISH
data (only 2.3%, e.g. 0.3% of their samples failed), whereas Bryndorf et al. (1997) were not able to reproduce their results and Eiben
et al. (1998, 1999) excluded all specimens appearing bloody or
brownish. However, in 98.7% of all our samples (75/76) suspected
of being contaminated, we did not find an abnormal STR pattern.
Therefore, the potential risk of misdiagnosis in blood stained
amniotic fluids seems to be very low by QF–PCR, thereby increasing the detection rate of chromosome aberrations, which is most
important for the assay efficiency and consequently for the clinical
utility. Moreover, in those of our cases where a contamination
could be suspected, STR profiles of the fetal and the corresponding
maternal blood sample could provide valid and accurate informa-
tion about potential maternal contamination so that a correct interpretation is possible. Furthermore, 1–2 ml of uncultured amniotic
fluid is sufficient for the QF–PCR analysis of aberrations of the
sex chromosomes and all three autosomal chromosomes. FISH
with a maximum probe set of five different chromosomes tests
(using commercially available DNA probes) usually requires
2–5 ml of amniotic fluids (Eiben et al., 1998; Jalal et al., 1998). In
our laboratory, the QF–PCR technique with one automatic DNA
scanner and one technician allows the investigation of 18 amniotic
fluid samples within 8 h. This is not possible with the FISH
procedure: After the harvesting procedure and standard hybridization for a minimum of 6 h (Jalal et al., 1998), each FISH analysis
requires a time intensive individual microscopic procedure (0.5 h
per case).
In summary the study demonstrates the feasibility of accurate
diagnosis of Turner syndrome, other common fetal sex chromosome and autosomal aneuploidies by QF–PCR within 1 day
which is not possible by standard cytogenetic methods. Altogether
the QF–PCR method is rapid and reliable, and we believe that
it will be more efficient than alternative methods.
Acknowledgements
The authors thank C.Böttcher, K.Gey, Y.Grossmann, N.Reuter, and
C.Schomburg for technical assistance. Finally, we thank the many
patients and all gynaecologists who participated in these studies.
W.Schmidt developed the molecular technique and carried out most of
the practical work. J.Jenderny initiated the study and summarized the
data. We received most of the pathological cases from B.J.Hackelöer
and K.Hecher. L.Kochhan acted as a substitute for W.Schmidt. J.Jenderny
and S.Kerber handled the cytogenetic analysis and K.R.Held supervised
the project overall.
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Received on February 8, 2000; accepted on June 14, 2000
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