GENETIC TESTING AND MOLECULAR BIOMARKERS
Volume 13, Number 1, 2009
ª Mary Ann Liebert, Inc.
Pp. 73–77
DOI: 10.1089=gtmb.2008.0054
Detection of Parental Origin and Cell Stage Errors
of a Double Nondisjunction in a Fetus by QF-PCR
Ali Irfan Guzel,1 Osman Demirhan,1 Ayfer Pazarbasi,1 Fatma Tuncay Ozgunen,2
Sabriye Kocaturk-Sel,1 and Deniz Tastemir1
Aim: To investigate parental origins and cell stage errors of a double nondisjunction in a fetus. Method: For the
determination of the most common chromosome anomalies, quantitative fluorescent polymerase chain reaction
method using short tandem repeat (STR) DNA markers was applied to a fetus with abnormal ultrasonographic
findings. Parental origin and cell stage errors of the trisomies were inferred by comparing the inherited STR alleles.
Conventional cytogenetic technique was also applied for the confirmation of the aneuploidies. Results: A double
nondisjunction including chromosomes 21 and X (48,XXX,þ21) was detected prenatally in the fetus. The origin of
both chromosomes was maternal, and the errors were in meiosis I for 21 and meiosis II for X. Molecular results were
concordant with cytogenetic results. Conclusion: Molecular techniques could be useful for the pre- and postnatal
diagnosis of the common aneuploidies and determining its parental origin. This kind of study will improve
knowledge about the mechanisms of nondisjunction and enable appropriate and rapid genetic counseling.
dem repeat (STR) regions of any chromosome. Parental origin
of the aneuploidy can be inferred by comparing the inherited
alleles and their relative doses with parental DNA samples,
too (Lamb et al., 1996, Adinolfi et al., 1997; Diego-Alvarez et al.,
2006). QF-PCR is increasingly being considered and proposed
as a complementary investigation or even as an alternative
to conventional cytogenetic analysis in prenatal diagnosis
(Grimshaw et al., 2003; Ogilvie, 2003; Leung et al., 2004).
In this study, we report a fetus diagnosed as trisomy 21 and
X using QF-PCR and karyotype analysis. It was also determined by analyzing the transmission of the STR markers
that the abnormality is arisen by maternal nondisjunction in
meiosis I (M I) and in meiosis II (M II).
Introduction
A
lthough nondisjunction is the most common cause of
chromosomal abnormalities, the event of double aneuploidies is observed rarely (ranges from 0.21% to 2.8% among
karyotyped spontaneous abortions) (Ohno et al., 1991; Reddy,
1997; Li et al., 2005). So far, a number of double aneuploidy
cases have been reported (Hassold and Jacobs, 1984; LordaSanchez et al., 1991; Jaruratanasirikul and Jinorose, 1994;
Tsukahara et al., 1994; Park et al., 1995; Chen et al., 2000; Kovaleva and Mutton, 2005), but the mechanism of it has not
been well studied. Epidemiological analysis of sex chromosome and chromosome 21 double aneuploidy showed that a
48,XXY,þ21 karyotype was associated with advanced maternal age in contrast to a 48,XYY,þ21 karyotype (Kovaleva
and Mutton, 2005). Women with pregnancies at increased risk
of chromosome abnormality (usually because of maternal age,
altered serum metabolites, or ultrasound abnormalities of the
fetus) undergo invasive sampling of either amniotic fluid (AF)
or chorionic villi. Cells from these samples are used for full
karyotype analysis or DNA extraction to be used in molecular
studies (NEQAS, 2000). Quantitative fluorescent polymerase
chain reaction (QF-PCR) entered the field of prenatal diagnosis to overcome the need to culture fetal cells, and hence
allows rapid diagnosis of some selected chromosomal
anomalies (Divane et al., 1994; Pertl et al., 1999). QF-PCR is
based on the amplification of highly polymorphic short tan-
Case and Methods
Case
AF from a fetus that has some positive ultrasonographic
and biochemical findings for fetal Down’s syndrome was taken. Ultrasonography at the time of amniocentesis (18 weeks
of gestation) showed nuchal thickening (10.7 mm), a large
ventricular septal defect, pericardial effusion, and absence of
middle flanks of fifth fingers at both hands.
Peripheral blood samples were taken from the mother (35
years old) and father (40 years old) after they were informed
about the study for the ethical reasons.
Departments of 1Medical Biology and Genetics and 2Obstetrics, Faculty of Medicine, Cukurova University, Adana, Turkey.
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Molecular studies
DNA extraction was performed from AF and blood samples by incubating cell pellets with InstaGene Matrix (BioRad, Hercules, CA). QF-PCR amplifications were performed
using Aneufast (Molgentix SL, Barcelona, Spain) trisomy
detection kit. The kit includes fluorescently labeled primers
for the total 28 predefined STR markers for chromosomes 13,
18, 21, X, and Y (6 for each of chromosomes 13 and 18, 7 for
chromosome 21, and 9 for chromosomes X and Y), Deoxyribonucleotide triphosphate (dNTPs), and Hot Start Taq
polymerase in six multiplex PCR mixtures. Reactions were
prepared (10 mL of the PCR mixtures þ 5–10 ng of DNA and
PCR grade water up to 15 mL) and thermal cycled (15 min at
958C continued with 25 cycles of 40 s at 958C, 90 s at 608C and
40 s at 728C, and then final extension at 608C for 30 min) according to the manufacturer’s protocol. QF-PCR products
(1.5 mL from each mix) were collected in 40 mL Hi-Di formamide (Applied Biosystems, Foster City, CA) containing
0.3 mL of GeneScan500 LIZ (Applied Biosystems) size
standard. After denaturation at 958C for 3 min, the mixture
was allowed to be cooled to 48C, and then capillary electrophoresis was carried out on an ABI 310 Genetic Analyzer
(ABI, Foster City, CA) using POP4 polymer. Analysis of the
results and calculation of the peak areas were performed using
GeneMapper 4.0 software (Applied Biosystems). The criteria
and guidelines for the determination of QF-PCR results of
normal and pathological cases were as follows: in normal individuals who are heterozygous for the STR, the same amount
of fluorescence is generated for both alleles. Therefore, the
ratio between the fluorescent peaks is 1:1. In normal individuals who are homozygous (have the same repeat number) for
GUZEL ET AL.
the STR alleles, the quantification is not possible, and the
marker is uninformative, and the ratio is 1. In trisomic cases,
the three copies of a chromosome can be detected as 1:1:1
(trisomic triallelic) or 2:1 (trisomic diallelic) patterns. Assessment of normal or trisomic copy number is done when at least
two informative markers for each chromosome are detected.
Due to the occasional preferential amplification of the smaller
allele, the ratios between fluorescent peaks may vary within
certain limits: 0.8–1.4:1 or 1.6:1 (for alleles differing $ 20 bp)
for normal cases (ratio: 1:1), and # 0.6 $1.8:1 for trisomic
cases (ratio: 1:1:1, 1:2, or 2:1). Ratios are calculated by dividing the area of the smaller allele by the area of the longer
allele. Taq polymerase slippage during PCR amplification of
repeated sequences can produce extra products that are exactly one repeat smaller than the STR allele; these are called
stutter bands. The proportion of stutter bands is characteristic
for each STR marker and usually does not exceed 15% of the
area of the corresponding allele (Aneufast User Manual; Molgentix SL).
Origin of aneuploidies
Parental origin of the aneuploidies was determined by
comparing the STR alleles of fetus, mother, and father. The
meiotic division error, M I or M II, was inferred on the basis of
nonreduction=reduction stage of the chromosome by comparing the proximal (pericentromeric) markers. If parental
heterozygosity was retained in the trisomic offspring, it is
concluded that the error occurred during M I; if parental
heterozygosity was reduced to homozygosity in the trisomic
offspring, it is concluded that the error occurred during M II
or postzygotic mitosis. Mitotic errors were distinguished from
FIG. 1. Parts from electrophoretograms of the QF-PCR products of five microsatellite markers (D21S1437, D21S1446,
D21S1411, D21S1435, and D21S1414) on chromosome 21 for fetus, mother, and father (electrophoretograms are not in scale).
The fetus was trisomic at all loci (2:1 for D21S1437, 1:2 for D21S1446, 1:1:1 for D21S1435, and D21S1414) except D21S1411,
which is noninformative. Smaller peaks in front (from left to right) of some peaks are stutter peaks. The box under each
fluorescent peak includes molecular size (bp) and area of the peak.
PARENTAL ORIGIN OF DOUBLE NONDISJUNCTION
75
FIG. 2. Parts from electrophoretograms of the QF-PCR products of four microsatellite markers (X22, hypoxanthine phosphoribosyltransferase [HPRT], spinal and bulbar muscular atrophy [SBMA], and DXYS218) on chromosome X for fetus, mother,
and father. X/Y homologous gene amelogenin (AMXY) (on chromosomes X and Y) and Sex-determining Region Y (SRY) (only
on chromosome Y) regions were used for the sex determination (electrophoretograms are not in scale). The fetus was trisomic
in chromosome X (XXX) for all of the markers (1:1:1 for X22, 2:1 for HPRT, 2:1 for SBMA, and 1:2 for DXYS218).
M II by evaluating medial and distal markers. If the trisomic
individual was reduced to homozygosity at all informative
loci, including at least one each in proximal, medial, and distal
portions of the chromosome, a postzygotic origin was inferred. If the trisomic individual was not reduced to homozygosity at one or more loci, the error was assigned to M II
(Lamb et al., 1996; Nicolaidis and Petersen, 1998; Robinson
et al., 1999; Diego-Alvarez et al., 2006).
mosomes 13, 18, and Y of the fetus, mother, and father (data
not shown).
Both of the extra chromosomes in the fetus were of maternal origin. As it is shown in Figure 3, D21S1414 and
D21S1435 are proximal markers, and maternal heterozygosity
was retained on the chromosome 21 of the fetus for these
markers (Fig. 3). This is the expected pattern for nondisjunction occurring in the first meiotic division.
Cytogenetic analysis
Standard cytogenetic procedures were performed for the
analysis of metaphase chromosomes from the AF and peripheral blood samples.
Results
At the end of analysis of 17 STR markers specific for five
chromosomes (13, 18, 21, X, and Y), aneuploidies were detected for chromosomes 21 and X at the fetus (48,XXX,þ21).
Based on visual inspection of comparative intensities of the
peak areas of the chromosomes 21 and X of the fetus, mother,
and father, it was obvious that the two of three alleles of
the fetus were of maternal origin (Figs. 1 and 2). Maternal
alleles were present as twice the dosage of paternal allele
in the diallelic form for the markers D21S1437, D21S1446,
D21S1411, and DXY218 markers that are homozygous at the
mother. Other markers (D21S1414, D21S1435, and X22)
were in the triallelic form of which two are maternal (Figs. 1
and 2). There were no numerical abnormalities for the chro-
FIG. 3.
Positions of the markers on chromosomes 21 and X.
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GUZEL ET AL.
FIG. 4. Fetal karyotype from AF, 48,XXX,þ21. Trisomic chromosomes are indicated by arrows.
For the chromosome X, only one of the maternal alleles
was present at twice the dosage of paternal allele for spinal
and bulbar muscular atrophy (SBMA) (a proximal marker;
Fig. 3), DXY218, and hypoxanthine phosphoribosyltransferase (HPRT) markers (Fig. 2). But, both of the maternal alleles
were present at the fetus for the X22 that is a distal marker
(Fig. 3). This pattern may have resulted from a recombination
event at this site. This is the expected pattern for nondisjunction occurring in the second meiotic division.
The karyotype showed trisomy 21 and X in all 20 cells examined (Fig. 4). The parents decided to terminate the pregnancy, which was performed at 19 weeks of the gestation. The
karyotypes of both parents were normal (data not shown).
Discussion
Double trisomies are rarely observed, presumably because
double nondisjunctions are rare events, and associated with
inevitable lethality in most cases. Multiple aneuploidies (most
often aneuploidy of the sex chromosomes combined with
trisomy 13, 18, or 21) have been described in different studies
(Hassold and Jacobs, 1984; Jaruratanasirikul and Jinorose,
1994; Tsukahara et al., 1994; Park et al., 1995; Kovaleva and
Mutton, 2005). In an epidemiological study of double trisomies concerning sex chromosomes and chromosome 21 by
Kovaleva and Mutton (2005), 121 cases were reported of
which 16 were 48,XXX,þ21 cases. Molecular evaluation of
double aneuploidy involving a sex chromosome and an autosome is rare. Lorda-Sanchez et al. (1991) reported a live-born
case of 48,XXY,þ21 in which the additional chromosome 21
was derived from a maternal M II nondisjunction, and the
additional X chromosome was the result of a paternal M I
nondisjunction. Other investigators (Park et al., 1995) reported
the prenatal identification of a 48,XXX,þ21 case due to a
double maternal M II nondisjunction. Chen et al. (2000) reported a 48,XXX,þ18 fetus with both additional chromosomes
derived from a maternal M II nondisjunction. Diego-Alvarez
et al. (2006) reported seven double trisomy cases among 321
miscarriages of which 4 were of maternal origin. He concluded that a common maternal age–related mechanism
could be implicated in both single and double trisomy cases,
and meiotic errors could cause similar chromosome-specific
patterns for missegregation. Although few cases of multiple
aneuploidies have been investigated, the literatures and our
current findings suggest that the parental origin of double
nondisjunctions is more commonly maternal. The presence of
a general cellular defect, such as impaired spindle function or
improper signaling of sister chromatid segregation, might
account for this type of event (Carpenter, 1994; Koshland,
1994).
Additional studies of examples of double aneuploidy are
needed to determine the nature of the errors in such cases.
Evaluation of exceptional instances of segregation failure may
be useful in improving our understanding of the general
mechanisms of nondisjunction.
Disclosure Statement
No competing financial interests exist.
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Address reprint requests to:
Ali Irfan Guzel, Ph.D.
Department of Medical Biology and Genetics
Faculty of Medicine
Cukurova University
01330 Adana
Turkey
E-mail: [email protected]