Neurobiology of Aging 27 (2006) 710716

Susceptibility to chromosome malsegregation in lymphocytes

of women who had a Down syndrome child in young age
L. Migliore , G. Boni, R. Bernardini, F. Trippi, R. Colognato,
I. Fontana, F. Copped`e, I. Sbrana
Department of Human and Environmental Sciences, University of Pisa, Via S. Giuseppe 22, 56126 Pisa, Italy
Received 14 October 2004; received in revised form 20 January 2005; accepted 30 March 2005
Available online 7 July 2005

Abstract
Recent findings seem to converge towards a unified hypothesis trying to relate Downs syndrome (DS), trisomy 21 and Alzheimers
disease (AD). The majority of DS individuals develop neuropathological characteristics of AD by the age of 40. Previous cytogenetic studies
performed by us showed an increased frequency of aneuploidy in peripheral lymphocytes and fibroblasts of AD patients and a preferential
occurrence of chromosome 21 in malsegregation events. An increased frequency of AD among young mothers of individuals with DS (MDS)
is reported. This study investigates the cytogenetic characteristics and the predisposition to chromosome malsegregation of peripheral blood
lymphocytes in a group of women (n = 35) who had a Down syndrome child in young age (<35 years) and in a control group (n = 30). We
applied the micronucleus assay and the dual-color FISH in order to assess the susceptibility to malsegregation events. The results indicate a
higher frequency of binucleated micronucleated cells in MDS in respect to the control group (16.1 9.1 versus 8.7 5.4). Moreover, our
data reveal that peripheral lymphocytes of MDS are more prone to chromosome non-disjunction with both chromosomes, 13 and 21, equally
involved.
2005 Elsevier Inc. All rights reserved.
Keywords: Down syndrome; Young mothers; Chromosome malsegregation; Micronucleus assay; Alzheimers disease

1. Introduction
Down syndrome (DS) is the most common pathology due
to an autosomic aneuploidy, with a general population frequency up to 1 in 800 of pregnancies, mainly due to trisomy of
chromosome 21 or by overexpression of genes located within
a segment of it, termed the Down locus [33]. Nowadays it has
been estimated that 329 genes are located on the 21q but only
few of them have been characterized. Those, whose function
has been confirmed on the grounds of common involvement
in the same metabolic pathways or in the same biological
systems, are classified into three groups. The first group of
sixteen genes seems to be involved in the production of energy
at a mitochondrial level and in the metabolism of reactive
oxygen species; the second group seems to be involved in

Corresponding author. Tel.: +39 050 836223; fax: +39 050 551290.
E-mail address: l.migliore@geog.unipi.it (L. Migliore).

0197-4580/$ see front matter 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.neurobiolaging.2005.03.025

the genesis of Down syndrome and Alzheimer disease (AD).

Finally, the last group has a role in folate and methyl groups
metabolism [32].
Together with the increase of the mothers age, also the
incorrect recombinations [14] trigger this non-physiological
condition, which originates from a non-disjunction event,
mainly maternal, during gametogenesis for approximately
92% of all cases [2]. After age 35 the risk of having a DS
offspring increases with increasing maternal age; however,
several young women aged less than 35 give birth to DS
children, suggesting a susceptibility to early chromosome
malsegregation [36]. Moreover, an increased risk to develop
AD in women who had a DS child in early age, compared to
controls, has been observed [36,38].
Several epidemiological studies have also shown that
frequency of AD cases in families with DS individuals
and of DS cases in families with AD subjects are significantly increased [15,16,43]. Moreover, both AD and DS

L. Migliore et al. / Neurobiology of Aging 27 (2006) 710716

individuals share common clinical and molecular features

like some neuropathological hallmarks which appear in DS
subjects in the same encephalic regions of AD patients
[28].
Although the sporadic form of AD represents the major
number of cases, the familial Alzheimers disease is also
well known, counting for about 10% of the investigated
cases. The analysis of the genetic linkage in fact showed
causative mutations present in three loci likely involved in
the onset and progression of the Alzheimers disease: the
gene for the amyloid precursor protein (APP), mapped on
chromosome 21 (21q21.1), and both the presenilin 1 (PS1)
and 2 (PS2) genes. Several literature data suggest that mutations on those genes could bring to a common scenario: an
increased secretion of the A1-42 peptide responsible for
the formation of amyloid nuclei in the neuritic plaques of
AD patients cerebral cortex [18,40]. Furthermore the overexpression of the APP gene product (A1-42 peptide), due
to the presence of three copies of chromosome 21, is the
major candidate for the onset of AD in DS subjects. To
strengthen this hypothesis there is a case of a 78-year-old
Down woman who did not report any aging dementia or neuropathological hallmarks characteristic of AD. Cytogenetic
analysis has revealed a partial 21 trisomy [46,XX,rec(21)dup
q, inv(21) (p12q22.1)] where the APP gene was expressed
only in duplicate [30]. Although relative density and localization in AD and DS are the same, the onset of the accumulation and deposition of A are earlier in DS [37]. In fact
there is a case reporting the A accumulation in a foetus
with Down syndrome already in the first months of gestation
[42].
Therefore, considering this background, the hypothesis of
a gene dosage unbalance involving specifically gene(s) on
chromosome 21, common to both pathologies, arises. This
is also supported by literature data in AD patients, which
report an increased frequency of peripheral aneuploid cells,
in particular trisomic for chromosome 21 [12,23,24].
Considering also the five-fold increase in AD risk in
mothers who had a Down child at a young age (<35 years)
compared to control mothers, it has been proposed a shared
genetic susceptibility to ageing, raising risk both for having
a child with DS and for developing AD [36,38].
However a unified hypothesis trying to relate Downs
syndrome (DS), trisomy 21 and Alzheimers disease (AD)
suggests that trisomy 21 mosaicism at germ cells level or in
brain cells could account of the familial aggregation of AD
and DS [29].
Aim of this study was to analyze, through the micronucleus test, the cytogenetic characteristics of peripheral blood
lymphocytes of a group of women who had a Down child
at young age, in comparison with a control group of mothers who had a healthy child. Moreover, we have evaluated
the presence of any susceptibility to malsegregation events
in a subgroup of mothers, by applying the fluorescence in
situ hybridization (FISH) with molecular probes for chromosomes 13 and 21.

711

2. Materials and methods

2.1. Study population
The study was based on 35 recruited mothers who had
children affected by Down syndrome below 35 years of age.
The DS pathology of the children (primary trisomy) was confirmed by cytogenetic analysis carried out by the Hospitals
where diagnoses were firstly performed. The control group of
30 mothers, who had no miscarriages or children affected by
genetic disorders and at least an healthy child, was recruited
by us among women employed in the Hospital and in the
University of Pisa. A questionnaire was administered by the
investigator for all the subjects of the study, in order to have
the precise situation of the pregress pathological condition of
the individuals and in order to evaluate the adopted exclusion
criteria (X-ray, infections, or any pharmacological therapy
that could interefere with the micronucleus frequency). Blood
for the subsequent analysis was picked up by venipuncture
in heparinized tubes for a total amount of 5 ml and the experimental work was carried out within 45 h after the drawing
procedure.
2.2. Cytokinesis-block micronucleus assay (CBMN)
The CBMN assay was performed according to the procedure previously reported [24]. Briefly, two paired, independent lymphocyte cultures were set up for each subject. Standard medium is composed by RPMI 1640 (Gibco
BRL, Italy) supplemented with 20% foetal bovine serum
(Gibco BRL), 1.5% phytohemagglutinin (Gibco BRL) and
1% penicillinstreptomycin (Gibco BRL). Cytochalasin B
(Sigma, Italy) at the final concentration of 6 g/ml, was added
to each tube 44 h after the starting of the cultures in order to
block the cytokinesis of dividing cells [9]. Lymphocytes culture was harvested after 72 h. Cells were then treated with an
hypotonic solution (0.075 M KCl) to lyse erythrocytes, prefixed in 3:5 methanol:acetic acid, washed once with methanol
and subsequently fixed twice with 7:1 methanol:acetic acid
fixative solution. The cell solution was finally dropped onto
a cold glass slide. Staining procedure was performed by
immerging the air-dried slides in a 4% Giemsa solution.
Two thousands binucleated cells for each individual were
examined, one thousand from each independent culture replicate, following the scoring criteria adopted by the HUman
MicroNucleus Project [5]. We evaluated the binucleated
micronucleated lymphocytes (BML) frequency (BNMN)
as number of binucleated lymphocytes containing one or
more micronuclei (MN) per 1000 binucleated cells. The ratio
of the percentage of binucleated cells to the total cells scored
(BN%) was also evaluated [9].
2.3. Fluorescence in situ hybridization (FISH)
After an incubation of 72 h, the lymphocytes were fixed
with two subsequent fixing procedures in methanol: acetic
acid in a 3:1 ratio.

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L. Migliore et al. / Neurobiology of Aging 27 (2006) 710716

Fig. 1. FISH analysis of non-disjunction events. (A) Normal hybridization pattern of a human binucleated lymphocyte with 4 green spots (centromeric region
of chromosomes 13 and 21) and 2 red spots (Down region 21q22.2) in each nucleus. (B) Abnormal FISH pattern: one nucleus shows 5 green + 2 red spots
indicating a malsegregation event of chromosome 13. (C) FISH pattern due to non-disjunction of chromosome 21. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of the article.)

FISH protocol was performed as follows. The slides were

first equilibrated in 2 SSC solution (saline-sodium citrate
buffer, pH 7) at 37 C for 30 min and subsequently dehydrated
in ethanols (70, 80, 95%) before immerging them for 2 min
in 70% formamide (pH 7.0) at 72 C.
A second step of dehydration was performed and the
slides were air-dried. For this study two probes were used:
the first, biotynilated, for the identification of the 13/21
chromosome centromere (Appligene Oncor, Italy), the second one is a cosmidic probe for the q22.2 region of
chromosome 21 (Appligene Oncor, Italy). The denaturation step was performed according to manufacturer protocols. Twenty microliters of probe were laid over each
slides and the hybridization procedure performed in a wet
chamber at 37 C for at least 16 h. Following hybridization the slides were washed in 2 SSC (pH 7) at 72 C
for 5 min and in 4 SSC (pH 7) at room temperature.
Amplification of the biotynilated FISH probe was performed by labelling with FITC-conjugated avidin and by
probing it with anti-avidin biotynilated antibody, diluted in
immunological buffer (IB) (10% of Non Fat Dry Milk in
4 SSC).
For what is instead concerning the digoxigenated probe,
the revelation of the signals was performed with the antidigoxigenin monoclonal antibody and the anti-mouse antibody TRITC conjugated. Amplification was instead performed by labelling with both anti-mouse (IgG, K subclass, produced in mousemouse hybrid, Boehringer, Italy)
and anti-rabbit (IgG, whole molecule, produced in goat,
Boehringer, Italy) TRITC conjugated antibodies. Antibodies were diluted in IB. Every passage was performed for
30 min at 37 C. After the amplification procedure the slides
were washed for 2 min in a 4 SSC/0.5% Tween 20 solution
and subsequently dehydrated in ethanol scale. The signals
associated to these probes are green and red stained, so
the cells were counterstained with DAPI (1 mg/ml) (Fig. 1).
The evaluation of the slides was performed using the Nikon
Eclipse E800 with a magnitude of 600. For each individual
1000 binucleated cells were analyzed in order to verify the
malsegregation events. In this analysis only the binucleated
cells with a complete hybridization pattern of 12 spots (4
green and 2 red for each of the two nuclei) were considered
(Fig. 1).

2.4. Statistical analysis

Data obtained from the micronucleus test, expressed as
mean and standard deviation (S.D.) for each individual have
been elaborated through the multifactorial variance analysis
by including as covariance the age and as categorical variable
the condition of being mother of a DS child (MANOVA).
This method let us to evaluate the statistical significance of
the difference in the binucleated micronucleated lymphocytes
(BML) frequency between MDS and controls, taking into
consideration the bias which are known to influence the analysis of CBMN test. MANOVA test has been used to compare
the frequencies of chromosome 21/13 non-disjunction in the
MDS group and in the controls one.

3. Results
In this study we compare the basal cytogenetic chromosome damage in peripheral blood lymphocytes of a group of
35 mothers who had a Down syndrome child (MDS) at an
age below 35 years with a control group of 30 mothers, of
similar age, who had at least one healthy child.
Table 1 reports the demographic characteristics of the
study population. The distribution of the age between the two
groups is comparable in terms of age mean, 48.03 12.15 in
the MDS group and 43.66 10.97 in controls; in the table is
also presented the mean age at which the MDS group had the
DS child (27.63 5.09).
Table 2 shows the results obtained from the CBMN assay.
The binucleated micronucleated lymphocytes (BML) frequency lays within a range of 2.042.0, with a mean
of 16.1 9.1, in MDS group; instead, the control group
shows a frequency range between 1.5 and 21.0 with a
mean of 8.7 5.4. The mean frequency of BML in MDS
is clearly higher if compared to the control group; this eviTable 1
Study population

Controls
MDS

No.

Age (years) S.D.

Age at the birth of

the DS child

30
35

43.66 10.97
48.03 12.15

27.63 5.09

L. Migliore et al. / Neurobiology of Aging 27 (2006) 710716

Table 2
CBMN test results
BMLa
BN%b

Controls (n = 30)

MDS (n = 35)

8.7 5.4
46.5 8.8

16.1 9.1*
50.7 8.6

a Frequency of micronucleated binucleated lymphocytes calculated analyzing 2000 cells.

b Percentage of binucleated lymphocytes.
* MANOVA: P < 0.001 MDS subjects vs. controls.

713

the events of chromosome non-disjunction involving both

chromosome 13 and 21. Since previous studies had shown
that autosome loss as micronuclei in lymphocytes is too low
to provide sufficient statistical power, we decided, according
to Wang et al. [44], not to take into account the chromosome loss parameter. The mean frequency of non-disjunction
for chromosome 13 is 0.88 0.65% in the MDS group and
0.28 0.18% in controls, instead for the chromosome 21 the
values are 0.70 0.53% and 0.11 0.05%. The total nondisjunction events have a mean frequency of 1.57 1.15%
in mothers with a DS child and 0.39 0.21% in mothers
belonging to the control group, with a statistical significance
by MANOVA (P < 0.01) (Table 3). A significant effect of the
age (P < 0.01) has been found for non-disjunction events for
both chromosomes, 13 and 21, in MDS. A similar effect was
observed in controls but statistically significant (P < 0.01)
only for chromosome 21.

4. Discussion
Fig. 2. Graphical representation of the statistical analysis of the influence of
the age on BML frequency. The graph shows the scatter plot analysis of MDS
(*) and controls (
) data. The lines are the results of regression analysis.
Dashed and filled lines are referred to MDS and control group, respectively.

dence is statistically remarkable by the MANOVA analysis

(P < 0.001). Moreover, the analysis pointed out that a significant effect of the age (P < 0.01) on the increase of the
BML is observable in the control group. Any statistical significant influence by the age is instead observable in MDS
group probably due to the presence of a subgroup of mothers
(n = 3) who show, compared to the rest of the individuals, a
remarkable increase in the BML frequency (Fig. 2).
The FISH analysis for the non-disjunction events has been
instead performed in a subgroup of 22 individuals, 12 MDS
and 10 controls. When the 13/21 alphoid probe is hybridised
in human diploid cells, the detection of four signals indicates a kariotipically normal cell, whereas the presence of five
signals suggests trisomy for either chromosome 13 or chromosome 21. To distinguish between the presences of trisomy
(and monosomy) 21 versus trisomy (and monosomy) 13, we
used a differently labelled probe for chromosome 21q22.2
at the same time. For each subject the frequency has been
evaluated by counting at the fluorescence microscope 1000
binucleated lymphocytes (Fig. 1). Table 3 presents the values
obtained from the MDS and the control group by analyzing
Table 3
FISH analysis results (non-disjunction)a
Non-disjunction, ND (%)

Controls (n = 10)
MDS (n = 12)
a
*

Chr 13

Chr 21

0.28 0.18
0.88 0.65*

0.11 0.05
0.70 0.53*

ND total frequency

0.39 0.21
1.571 1.15*

Results obtained by analyzing 1000 binucleated lymphocytes.

P < 0.01 MDS subject vs. controls.

In our study the analysis with the micronucleus test led

us to reveal a statistically significant increase of binucleated
micronucleated lymphocytes frequency in the group of mothers who had a Down child at a young age compared to the
control group. The increased MN frequency is indeed the
clear sign of cytogenetic damage, chromosome breakage or
loss, in those mothers core of this study.
Many previous studies have reported a positive correlation
between the increased frequency of micronuclei in peripheral blood lymphocytes and the age [4,10,22], in particular
in women, where the preferential involvement of X chromosome in non-disjunction events has been demonstrated
[6,7,31,47]. In our study the positive correlation with age is
clearly present when the control group is analyzed, instead
when the MDS group is considered, a positive trend is found
but it does not result statistically significant (Fig. 2). The
results relative to the increased frequency of micronucleated cells can account only and partially for the chromosome loss, since the other possible mechanism leading to
aneuploidy, non-disjunction, is not evaluated with the classical version of the assay (evaluation of binucleated lymphocytes Giemsa-stained). To take into account both possibilities we have performed experiments of dual-color FISH
with two DNA probes, one for the centromeric regions of
chromosome 13 and 21 and one for the Down syndrome
critical region (21q22.2), which allow us to have the quantitative evaluation of both the events, related to both chromosomes. We are aware that the two chromosomes investigated only correspond to about 5.28 percent of the genome,
but we were interested in verifying the possible preferential occurrence of chromosome 21 malsegregation. The
choice of a unique probe specific for chromosome 21 and
13 is due to the fact that these two philogenetically related
chromosomes share large blocks of identical alphoid DNA
sequences.

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L. Migliore et al. / Neurobiology of Aging 27 (2006) 710716

In the binucleated lymphocytes of the MDS, compared

to controls, we have observed a major incidence of spontaneous non-disjunction, without a specific involvement of
any of the two chromosomes. Moreover, a positive influence of the age has emerged on the non-disjunction, both in
MDS and in controls even if with different endpoints. In fact
our data show that for the control group the age influences
only non-disjunction of chromosome 21, instead for MDS
both chromosomes are involved. These data are in complete
agreement with previous results [39], which reveal that the
age interferes with the non-disjunction of chromosome 21,
confirming that the aging process can lead to malsegregation
events [39].
The results we obtained can be related to findings of
Schupf et al. [36,38], who however linked the increased risk
to develop AD to the hypothesis of an early aging of the
young mothers. In other terms, they said that mothers who
had a Down child in young age could be biologically elderly
than their real age, fact which could give rise to an increase
of DS offspring and to the risk to develop AD [36,38]. A
chromosome malsegregation susceptibility could anyway be
linked to premature aging processes: aged women have an
increase of aneuploid lymphocytes [4,10,47], but we think
that a genetic predisposition to chromosome malsegregation
could lead to a mosaicism both at meiotic (germ cells) and
at mitotic level (e.g. peripheral blood lymphocytes as well
as brain cells) and, whenever chromosome 21 is involved, it
can predispose those women to DS pregnancies and to subsequently develop AD, in agreement with the hypothesis that a
chromosome 21 trisomy mosaicism could contribute to sporadic forms of AD [29].
Several factors could potentially be able to determine a
change in the normal chromosome arrangement, some of
these are chromosome specific and could influence the tendency of chromosomes to non-disjunction [45]. Clusters of
genes like the nucleolus organizing regions (NORs), coding for the ribosomal 18S and 28S RNA and mapped on the
short arm of all human acrocentric chromosomes, belong to
this last category. The chromosomes where the NORs are
located (13, 14, 15, 21 and 22) constitute the group where
the trisomies are more frequently found, so these regions
are suggested to be one of the major cause for the susceptibility of chromosomes to non-disjunction. Another factor
surely involved in chromosome segregation, although limited to the meiosis process, is represented by the chromosome
length which is known to be negatively correlated with a nonphysiological segregation; this could be explained more in
details by the minor number of chiasmata that are formed in
the prophase I of the meiosis during recombination [1]. Since
the chiasmata, during the meiotic phase, maintain the homologues together, they are considered a necessary and sufficient
condition for a correct segregation, so a less number of chiasmata reflect an increased risk of non-disjunction [13,14].
In the chromosome segregation an important role should be
attributed also to the microtubular system. Of course a misregulation of the microtubuli assembly could be implicated

with an increased risk of aneuploidy in the dividing cells. A

basic defect in a mechanism controlling microtubular polymerization and/or alteration in the microtubular structures,
responsible for the formation of trisomic cells during mitosis and meiosis, has been evoked by Ford [11]. Moreover,
also in AD the microtubular system alterations, in particular
that of the microtubular associated proteins (MAPs), assume
a relevant role in the onset and progression of the pathology [25]. Together with these findings there are data reporting malfunctions of the mitotic fuse with consequent nonphysiologic chromosome segregation [21], fact that could
explain the high frequency of aneuploid cells, in particular for
those carrying trisomy for chromosome 21, in AD patients
[12,23,24].
Other susceptibility factors are likely involved in the chromosome non-disjunction event allowing the correlation of the
risk to have a Down child in young age with the development of AD in elderly: among those ones with the major
impact are the polymorphisms of the ApoE gene and of
presenilin genes [34]. The presence of the ApoE4 allelic
variant is significantly higher in young mothers with a meiosis II error [3,8] leading to several possible hypotheses on
the mechanisms through which the 4 allele could increase
the risk for DS: the ApoE4 mediated increase in oxidative damage could lead to accelerated aging of the ovary
or it could be a possible specific binding of the ApoE4
isoform to microtubule-associated proteins with a consequent interference in microtubule function during meiosis
[8]. Presenilins seem to positively interfere with the mitotic
process: their colocalization with kinetochores on the nucleoplasmic surface of the inner nuclear membrane suggests
that they may play a role in chromosome organization and
segregation [20]. It has also been observed that a particular polymorphism, in intron 8 on PS1 gene [27] in a
group of mothers which had a Down child in young age,
could be associated to non-disjunction events. The same
polymorphism has also been associated to late onset AD
[46].
More recently interesting studies are focussing on the
levels of folic acid, homocysteine and other relevant factors associated with folate metabolism, as risk factors for
a Down syndrome child. Some of them have observed an
increased frequency of polymorphisms of methylenetetrahydrofolate reductase (MTHFR, C677T) and methionine synthase genes (MTRR, A66G) in mothers with a DS child.
Those studies are based on the evidence that abnormal folate
and methyl metabolism can lead to DNA hypomethylation
and abnormal chromosomal segregation [17,19,26,35,41].
Indeed spontaneous frequency of micronucleated lymphocytes has been found increased in MTRR 66GG individuals
[48].
Thus, in concomitance with the maternal ageing, there
must be cellularmolecular events and other biochemical
pathways, likely genetically determined that could promote
maternal meiotic non-disjunction and, at the same time, as
we demonstrated, also mitotic non-disjunction.

L. Migliore et al. / Neurobiology of Aging 27 (2006) 710716

Acknowledgements
The authors wish to thank to Prof. G. Siciliano, Dr. S.
Bargagna and Dr. P. Simi, for collecting biological samples,
and Dr. S. Molinu for her excellent technical assistance.

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