Mutagenesis vol. 23 no. 1 pp. 57–65, 2008
Advance Access Publication 29 November 2007
doi:10.1093/mutage/gem044
Chromosome 17 and 21 aneuploidy in buccal cells is increased with ageing and in
Alzheimer’s disease
Philip Thomas1,2,* and Michael Fenech1
1
CSIRO Human Nutrition, PO Box 10041, Adelaide BC, Adelaide, South
Australia 5000, Australia and 2Discipline of Physiology, School of Molecular
and Biomedical Sciences, The University of Adelaide, Adelaide, South
Australia 5005, Australia
Alzheimer’s disease (AD) is a premature ageing syndrome
characterized by cognitive impairment arising from
neuropathological changes occurring within specific areas
of the brain. We report a 1.5-fold increase in trisomy
21 (P < 0.001) and a 1.2-fold increase in trisomy 17
(P < 0.001) in buccal cells of Alzheimer’s patients compared to age- and gender-matched controls. Chromosome
17 and chromosome 21 monosomy and trisomy increase
significantly with age (P < 0.001). Down’s syndrome, which
exhibits similar neuropathological features to those observed in AD also showed a strong increase in chromosome
17 monosomy and trisomy compared to matched controls
(P < 0.001). These results suggest that an increased
incidence of aneusomy for both chromosomes 17 and 21
may contribute to the aetiology of AD. We also investigated
aneuploidy rates in hippocampal brain cells, which have
been shown to have a low rate of cell division, which may
be relevant in potential incidence of non-disjunction.
However, aneuploidy rate for chromosomes 17 and 21 in
the nuclei of hippocampus cells of brains from Alzheimer’s
patients and controls were not significantly different. These
results are suggestive that the aneuploidy events investigated which are increased beyond the incidence in normal
ageing may be influenced by genetic factors that may
predispose to AD, but are unlikely to be a primary cause of
AD brain pathology.
Introduction
Alzheimer’s disease (AD) is a complex progressive neurodegenerative disorder of the brain and is the commonest form of
dementia (1). Neurodegenerative changes appear firstly within
the entorhinal cortex and then progress to the hippocampus
eventually disrupting learning and short-term memory (2,3).
The disease is clinically defined by progressive memory loss,
visuospatial and language impairment and various psychiatric
and behavioural changes (1,4–6). The two histopathological
structures present within the brain that positively identify AD
conclusively at post-mortem are the neurofibrillary tangles and
the amyloid-based neuritic plaques (5,6).
Neurofibrillary tangles are composed of the microtubuleassociated protein tau, the gene of which is located on
chromosome 17q21.1 (7). Tau is important as it associates with
tubulin in the formation of microtubules. Microtubules impart
shape and structure to cells and also form the basis of a cellular
transport network allowing the movement of neurotransmitters,
micronutrients and organelles that are essential for normal
cellular function (8). Microtubules also provide points of
attachment for chromosomes during cell division, which if
disrupted may result in an increased incidence of chromosome
malsegregation (9). The neurofibrillary tangles contain structures known as paired helical threads comprised of tau which
are hyperphosphorylated. This hyperphosphorylation leads to
a dissociation between tubulin and tau, resulting in a breakdown
of the brain transport system leading to loss of biological
activity, cell death and potential microtubule dysfunction
leading to chromosome malsegregation (7,8). The second
histopathological feature in the brain of AD patients is the
amyloid-based neuritic plaques. These consist of the 42 amino
acid b amyloid peptide (Ab42), which originates from the
aberrant proteolysis of the amyloid precursor protein (App), the
gene (APP) of which is located on chromosome 21q21 (10).
Down’s syndrome (DS) like AD is a premature ageing
syndrome characterized by various degrees of chromosome 21
trisomy and an elevated incidence of genome instability (11,12).
DS individuals develop dementia and manifest brain changes that
are histopathologically indistinguishable from AD, usually during
the third or fourth decade of life (13,14). For this reason it has been
suggested that both AD and DS share underlying mechanisms
involving susceptibility to chromosome malsegregation events,
particularly those involving chromosome 21. Alteration in gene
dosage and expression for the TAU and APP gene resulting from
aneuploidy may be an important contributor to the aetiology of
AD. It has not yet been determined whether aneuploidy of
chromosome 17 has a higher incidence in AD and DS and whether
gene dosage and altered gene expression of the TAU gene may
play a potential contributory role in AD and DS pathology.
There is much evidence from previous studies to show that
both AD and DS share an increased susceptibility to genomic
instability events. Micronuclei (biomarkers of chromosome
malsegregation and breakage) are elevated in AD lymphocytes
and were shown to be centromere positive indicating whole
chromosome loss (15). Migliore et al. (16) also showed
a significant increase in micronuclei in the lymphocytes of
young mothers of Down’s syndrome individuals (MDS) and
showed they were more prone to non-disjunction events for
chromosome 13 and 21. A similar predisposition to increased
aneuploidy events for chromosome 13 and 21 has been shown
in lymphocytes of AD patients (17,18). Fibroblast cultures
from sporadic and familial AD patient’s that carry mutations
within the presenilin 1, presenilin 2 and the App were found to
*To whom correspondence should be addressed. Tel: þ61 8 303 8897; Fax: þ61 8 303 8899; Email: [email protected]
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57
P. Thomas and M. Fenech
have an 2-fold increase in the number of trisomy 21 cells
compared to cultures from normal individuals (19). These
findings suggest that aneuploidy may have a contributing role
to early changes in disease development.
The aim of this study was to use fluorescent in situ
hybridization (FISH) and fluorescently labelled DNA probes to
determine the incidence of aneuploidy of chromosomes 17 and
21 in the buccal mucosa and hippocampal brain tissue of
Alzheimer’s patients compared to age- and gender-matched
healthy controls. It was also our intention to investigate any
age-related effects on aneuploidy involving these two chromosomes between a young and old cohort, and whether
aneuploidy for chromosome 17 in the buccal mucosa of DS
was significantly different from controls. Buccal cells (BCs)
were selected for this study because they can be collected noninvasively and originate from the neuroectoderm from which
brain tissue is derived (20). BCs may therefore exhibit genetic
defects common to brain tissue acquired during the early stages
of development.
Methods
Recruitment and characteristics of participants
Approval for this study was obtained from CSIRO Human
Nutrition, Adelaide University, Ramsay Health Care and
Southern Cross University Human Experimentation Ethics
Committees. Participants in this study consisted of four distinct
groups: 30 younger controls (age 18–26 years), 26 older
controls (age 64–75 years), 21 DS individuals (age 5–20 years)
and 54 clinically diagnosed Alzheimer’s patients (age 58–93
years). None of the participants recruited to the study were
receiving anti-folate therapy or cancer treatment. Alzheimer’s
patients were recruited at the College Grove Private Hospital,
Walkerville, Adelaide, South Australia, following their initial
diagnosis and prior to commencement of therapy. Diagnosis of
AD was made by experienced clinicians according to the
criteria outlined by the National Institute of Neurological and
Communicative Disorders and Stroke-AD and Related Disorders Association (21). These are recognized standards used in
all clinical trials. Mini Mental State Examination (MMSE)
scores which are a recognized measure of cognitive impairment
were only available for the Alzheimer’s cohorts. Participants
did not receive any remuneration for their participation.
Alzheimer’s patients were separated into two distinct groups.
One group (younger AD group) was age matched to the older
control, while the second group was classified as the older AD
group. Gender ratio differences between the groups were not
significant. MMSE scores between the two AD groups were not
significantly different. The age of the older control group
compared to the younger AD group was not significantly
different but the younger AD group had a significantly lower age
relative to the older AD group (P , 0.0001). The younger and
older controls were ‘normal’ functioning healthy individuals,
who self-volunteered and consented to the study and did not
report a history of cognitive impairment. Demographic characteristics of the cohorts are shown in Table I. Consent from AD
and DS volunteers was provided by either partners or guardians.
BC collection and slide preparation
BCs were collected using a modified version of the method
used by Beliën et al. (22). Prior to BC collection, the mouth
was rinsed thoroughly with water to remove any unwanted
debris. Small headed toothbrushes (Supply SA, Camden Park,
58
Adelaide, Australia, code 85300012) were rotated 20 times in
a circular motion against the inside of the cheek, starting from
a central point and gradually increasing in circumference to
produce an outward spiral effect. Both cheeks were sampled
using separate brushes. The heads of the brushes were
individually placed into separate 30-ml yellow-top containers
(Sarstedt, Australia Pty. Ltd, Technology Park, Adelaide,
Australia, code, 60.9922.918) containing BC buffer [0.01 M
Tris–HCl (Sigma T-3253), 0.1 M ethylenediaminetetraacetic
acid tetra sodium salt (Sigma E5391) and 0.02 M sodium
chloride (Sigma S5886)] at pH 7.0 and agitated to dislodge the
cells. Cells from both right and left cheeks were transferred into
separate TV-10 centrifuge tubes (Sarstedt, code 60.9921.829)
and spun for 10 min at 1500 r.p.m. (MSE Mistral 2000).
Supernatant was removed and replaced with 10 ml of fresh BC
buffer. The BC buffer helps to inactivate endogenous DNAases
and aids in removing bacteria that may complicate scoring.
Cells were spun and washed twice more, with a final volume of
5 ml of BC buffer being added to the cells. The cell suspension
was vortexed and then homogenized for 2 min in a hand
homogenizer (Wheaton Scientific 0.1- to 0.15-mm gauge) to
increase the number of single cells in suspension. Left and right
cell populations were pooled into a 30-ml yellow-top container
and drawn into a syringe with a 21-gauge needle and expelled
to encourage cellular separation. Cells were passed into a TV10 tube through a 100-lm nylon filter (Millipore Australia Pty.
Ltd, North Ryde, NSW, Australia, code MILNYH02500) held
in a swinnex filter (Millipore, code MILSX0002500) to remove
large aggregates of unseparated cells that hinder slide
preparation and cell analysis. Cells were further spun at 1500
r.p.m. for 10 min and the supernatant was removed. Cells were
re-suspended in 1 ml of BC buffer and the cell concentration
determined by a Coulter counter (Beckman Coulter Model
ZB1; settings: attenuation, 1; threshold, 8, aperture, 1/4;
manometer, 0.5). Cell suspensions were prepared containing
80 000 cells/ml after initial readings from a 1:50 dilution (300
ll/15 ml isoton). Dimethyl sulphoxide (50 ll/ml; Sigma 2650)
was added to help clarify cellular boundaries by further
separating the cells. One hundred and twenty microlitres of cell
suspension was added to cytospin cups and spun at 600 r.p.m.
for 5 min in a cytospin (Shandon cytospin 3). Slides containing
two spots of cells were air-dried for 10 min and then fixed in
ethanol:acetic acid (3:1) for 10 min. Slides for FISH were airdried for 10 min prior to being stored at 70°C in a sealed
desiccated slide box.
Brain tissue collection and slide preparation
Frozen non-fixed hippocampal brain tissue from histopathologically confirmed Alzheimer’s patients (positive for the
presence of amyloid plaques and neurofibrillary tangles) was
provided by the Brain Bank of South Australia, Centre of
Neuroscience, Flinders University. Control hippocampal tissue
from histopathological normal individuals was provided by
both the New South Wales Tissue Resource Centre, Department of Pathology, University of Sydney and the Brain
Bank of South Australia. Tissue provided from the hippocampus was taken from slices. Cores were taken using a special
drill bit and battery-powered drill with the slices being kept on
a bed of ice during retrieval. The removed cores were placed in
a small test tube and stored at 70°C until transported.
Two hundred milligrams of brain tissue was weighed
(Sartorius Scales, type 1475) inside a biological safety cabinet
(Gelman Sciences, Ann Arbour, MI, USA, BH series) and
Aneuploidy is increased in Alzheimer’s buccal cells
Table I. Age, gender ratio and MMSE scores in study groups
Age (mean SD)
Age (range)
Gender ratio
(male/female)
MMSE
(mean SD)
MMSE (range)
Young controls,
n 5 30
DS, n 5 21
Old controlsa,
n 5 26
Younger AD,
n 5 23
Older AD,
n 5 31
Alzheimer’s brain
tissue group,
n 5 13
Control brain
tissue group,
n59
22.47 2.177
18–26
15/15
10.43 5.582
5–20
11/10
68.7 2.6
66–75
11/15
70.7 4.1
58–75
8/15
83.13 4.5
78–93
8/23
75.75 7.8
59–88
5/8
74.10 10.95
60–98
6/3
NA
NA
NA
22.52 3.013
22.13 3.6.76
NA
NA
15–27
12–29
NA 5 not available.
a
Age and gender matched to young AD group.
placed on a slide inside a petri dish and moistened with a few
drops of phosphate-buffered saline (PBS; 120 mM sodium
chloride, 2.7 mM pottasium chloride, 10 mM potassium
phosphate monobasic and 10 mM sodium phosphate dibasic,
pH 7.4). Tissue was minced and chopped thoroughly using
a scalpel blade (Blade No 11, Swann-Morton Ltd, Sheffield,
UK) and transferred to TV-10 tube. PBS washings (2 ml) were
taken of the slide within the inclined petri dish and transferred
to the TV-10 tube. Three washings per slide were made. A
glass pipette was used to draw up and release the cell
suspension to encourage the separation of single cells. Care
was taken to avoid the production of aerosols while pipetting.
The tube was balanced and centrifuged at 2500 r.p.m. for 10
min. The supernatant was removed and replaced with 6 ml of
PBS. The cell pellet was drawn up and released several times
using a glass pipette to further encourage the production of
single cells. Cells were drawn up into a 10-ml syringe and
filtered into a TV-10 tube through a 100-lm nylon filter
(Millipore, code MILNYH02500) held in a swinnex filter
(Millipore, code MILSX0002500) to remove large aggregates
of unseparated cells that hinder slide preparation and cell
analysis. One millilitre of Carnoys fix consisting of ethanol:
glacial acetic acid (3:1) was added and the suspension mixed.
Tubes were spun for 2500 r.p.m. for 10 min. Supernatant was
aspirated and fresh fix added to a volume of 6 ml and centrifuged
at 2500 r.p.m. for 10 min. Fixation was repeated and cells were
re-suspended in 4 ml of fix to produce a cloudy suspension. Cells
were dropped onto clean slides, air-dried for 10 min and placed
in a slide box at 20°C with desiccant ready for FISH analysis.
Preparation of chromosome 17 DNA probe
Centromeric chromosome 17-specific DNA probe was generated by PCR labelling with centromere-specific primers for the
chromosome (a17A1: 5#-AATTCGTTGGAAACGGGATAA
TTTCAGCTG-3# and a17B2: 5#-CTTCTGAGGATGCTTCTGTCTAGATGGC-3# (Geneworks, Thebarton, Adelaide,
Australia) (23,24). The length of the amplification product is
227 bp (25). Briefly, PCR was carried out with 0.2 lM of each
primer, 60 lM digoxigenin-11-dUTP (Roche, Basel, Switzerland, No: 1093088), 140 lM dTTP and 200 lM dATP, dCTP
and dGTP (Roche, Basel, Switzerland, No: 1969064). The
PCR was started with pre-denaturation at 94°C for 2 min,
followed by 25 cycles of 92°C for 1 min, 61°C for 2 min, 72°C
for 2 min and terminal extension at 72°C for 10 min.
Chromosome 17 centromeric probe was validated by hybridizing to human metaphases to determine exact position and
ensure no cross-hybridizing had occurred.
Hybridization and detection for chromosome 17
Prior to hybridization and detection, the buccal slides were pretreated with pepsin (Sigma P-7012) [300 lg/ml in 0.01 N HCl
(BDH Lab Supplies, Poole, UK)] for 10 min while lymphoblastoid control slides were treated with (5 lg/ml pepsin in
0.01 N HCl) at 37°C for 10 min and then washed twice in PBS
(pH 7.0) for 5 min at room temperature. Afterwards, the slides
were dehydrated in a cold ethanol series (70, 70, 80, 90 and
100%) for 2 min each and air-dried. Hybridizations were
performed according to a modified technique of Pinkel et al.
(25,26). Slides were denatured at 79°C for 5 min in prewarmed 70% formamide (Sigma Aldrich, Castle Hill, NSW,
Australia, No: 295876)/2 standard saline citrate (SSC) (0.15
M sodium chloride and 0.015 M sodium citrate) and then
dehydrated through an ethanol series (70, 70, 80, 90 and 100%,
for 2 min each) at 4°C. Two microlitres of the digoxigenin-11dUTP-labelled PCR product and 1 ll of 2 mg/ml herring sperm
DNA (Sigma D7290) were mixed with 6 ll hybridization
buffer [55% formamide, 10% dextran (Sigma D 6001) in 1
SSC] and 1 ll of water and denatured at 79°C for 5 min. The
probe mixture was then added to the slides (5 ll per cytospin
spot), a coverslip was applied and sealed with rubber cement.
Hybridization was carried out overnight (18–20 h) in
a humidified chamber at 37°C. Post-hybridization washing at
45°C occurred as follows: twice in 2 SSC for 5 min, twice in
50% formamide (2 SSC, pH 7.0) for 5 min and twice in 0.1
SSC for 5 min followed by a 30-min incubation in block buffer
[3% bovine serum albumin (Sigma, St Louis, MO, USA, No: B
4287)/4 SSC] at 37°C. The signal was detected using antidigoxigenin rhodamine (1:300) (Roche Diagnostics, Basel,
Switzerland, No: 1207750) at 37°C for 30 min followed by
three washes in 4 SSC/0.03% Tween 20 (Sigma P1379) at
45°C for 5 min each. The nuclei were counterstained with 4,6diamidino-2-phenylindole (DAPI, Sigma, No: D9542) (0.1 lg/
ml in 4 SSC with 0.06% Tween 20) for 5 min at room
temperature and coverslipped in anti-fade solution [0.2333 g of
1,4-diazabicyclo-(2.2.2)-octane (Sigma D 2522) in 800 ll
purified water, 200 ll 1 M Tris–HCl (Sigma T 3252) and 9 ml
glycerol (Sigma G 5516)]. The coverslips were sealed with nail
varnish and kept at 20°C in dark.
Hybridization and detection for chromosome 21
Chromosome 21 aneuploidy was investigated using a directly
labelled commercially bought fluorescent probe (Vysis-LSI 21
probe labelled with spectrum orange, No: 32-190002). LSI 21
is 200 kb and contains unique DNA sequences complementary to the loci D21S259, D21S341 and D21S342 (21q22.13–
59
P. Thomas and M. Fenech
q22.2). It was the intention of the authors to probe the slides
simultaneously, but due to delays in acquiring a suitably
labelled chromosome 21 probe and specific filter blocks, slides
were probed independently for both chromosomes.
Hybridization and detection of the probe was performed
according to the manufacturer’s instructions following a
pre-treatment. Prior to in situ hybridization, the buccal slides
were pre-treated with pepsin (Sigma) (300 lg/ml in 0.01 N
HCl) for 10 min while lymphoblastoid control slides were
treated with (5 lg/ml pepsin in 0.01 N HCl) at 37°C for 10 min
and then washed twice in PBS (pH 7.0) for 5 min at room
temperature. Afterwards, the slides were dehydrated in a cold
ethanol series (70, 70, 80, 90 and 100%, for 2 min each) and
air-dried. Slides were denatured at 79°C for 5 min in prewarmed 70% formamide/2 SSC and then dehydrated through
an ethanol series (70, 70, 80, 90 and 100%, 2 min each) at 4°C
and then placed in a 37°C oven to reach hybridization
temperature. Ten microlitres of probe mixture per slide (1 ll
LSI 21 probe, 2 ll distilled water and 6 ll LSI/WCP
hybridization buffer) was prepared and denatured in a 79°C
water bath for 5 min. Five microlitres of the probe mixture was
added to each buccal cytospin spot, coverslipped and sealed with
rubber cement before incubating at 37°C overnight (16 h) in
a humidified box.
Coverslips were removed with forceps before slides were
washed in three coplin jars of 50% formamide/2 SSC at 45°C
for 10 min. Slides were further washed in coplin jars of 2
SSC and 2 SSC/0.1% Nonidet P40 (Roche, No: 1754599) at
45°C for 10 min. The nuclei were counterstained with DAPI
(0.05 lg/ml in 4 SSC with 0.06% Tween 20) for 1 min 20 sec
at room temperature and coverslipped in anti-fade solution.
The coverslips were sealed with nail varnish and kept at 20°C
in dark.
Scoring method
A minimum of 1000 BCs or hippocampal brain tissue cells
were scored for the presence of one, two or three signals
(Figures 1 and 2). Only nuclei with at least one FISH signal
were scored. In the cases of nuclei with more than one signal,
only those nuclei in which the signals were of similar size and
intensities and were separated by a distance of more than half
the diameter of one FISH signal were scored (27). A Nikon
E600 fluorescence microscope was used with a triple-band
filter (for DAPI, FITC and rhodamine) allowing simultaneous
visualization of the signals from both the fluorochrome and the
nuclear counterstain.
Statistical analyses
One-way analysis of variance (ANOVA) was used to determine the significance of difference between groups for
aneuploidy incidence, whereas pair wise comparison of
significance was determined using Tukey’s test. Gender ratios
were tested using the chi-square test. ANOVA values and
cross-correlation analysis was calculated using Graphpad
PRISM (Graphpad Inc., San Diego, CA, USA). Significance
was accepted at P , 0.05.
Results
The results for the incidence of aneuploidy (monosomy and
trisomy) for chromosomes 17 and 21 in BCs for all groups are
shown in Figures 1, 3 and 4. The results showing the incidence
of aneuploidy (monosomy and trisomy) for chromosomes 17
and 21 in hippocampal brain tissue from Alzheimer’s and
control groups are shown in Figures 2, 5 and 6. Percentages of
aneuploid cells scored in both BCs and brain tissue are outlined
in Table II.
Normal ageing—aneuploidy in BCs
Monosomy of chromosomes 17 and 21 was increased by
222.2% (P , 0.001) and 91.4% (P , 0.001) and trisomy of
chromosomes 17 and 21 was increased by 89.0% (P , 0.001)
and 97.4% (P , 0.001), respectively, in the old controls
relative to the young controls. The total aneuploidy rate was
13.8% for chromosome 17 and 9.6% for chromosome 21 in old
controls compared to 5.0 and 5.0%, respectively, in young
controls.
DS—aneuploidy in BCs
Monosomy and trisomy of chromosome 17 was increased by
292.7% (P , 0.001) and 79.7% (P , 0.001), respectively, in
DS relative to young controls and were at a level similar to that
observed in old controls. Total aneuploidy rate for chromosome 17 in DS was 16.0% compared to 5.0% in young controls
Fig. 1. FISH results showing non-disjunction events in BCs. (a) Chromosome 17 monosomy, (b) chromosome 17 disomy, (c) chromosome 17 trisomy, (d)
chromosome 21 monosomy, (e) chromosome 21 disomy and (f) chromosome 21 trisomy.
60
Aneuploidy is increased in Alzheimer’s buccal cells
Fig. 2. FISH results showing non-disjunction events in hippocampal brain tissue. (a) Chromosome 17 monosomy, (b) chromosome 17 disomy, (c) chromosome 17
trisomy, (d) chromosome 21 monosomy, (e) chromosome 21 disomy and (f) chromosome 21 trisomy.
Fig. 3. Frequency of (a) chromosome 17 monosomy and (b) chromosome 21 monosomy following FISH in 1000 BCs of young controls (n 5 30), DS (n 5 21),
old controls (n 5 26), younger AD (n 5 23) and older AD (n 5 31). Groups sharing the same letters are not significant. Error bars indicate SEM.
and 13.8% in old controls. Results for chromosome 21
aneuploidy could not be meaningfully compared given that
DS is caused by trisomy of chromosome 21.
AD—aneuploidy in BCs
Both chromosome 17 monosomy and trisomy were increased
in the younger AD group by 19.4% (P , 0.05) and 16.1%
(P , 0.001), respectively, relative to the old control group, but
there were no differences between the younger and older AD
groups. The total chromosome 17 aneuploidy rate in younger
and older AD groups was 15.7 and 16.4% compared to 13.8%
in the old control group. Chromosome 21 trisomy was
increased by 183.3% (P , 0.001) in the AD groups relative
to the old controls, but there was no difference in chromosome
21 monosomy. MMSE scores were not significantly correlated
with chromosome 17 or chromosome 21 aneuploidy.
AD—aneuploidy in brain hippocampus
Chromosome 17 or chromosome 21 aneuploidy did not differ
significantly in hippocampus tissue of Alzheimer’s cases
and controls. Chromosome 17 and 21 aneuploidy rate in
hippocampus was 18–18.2% and 11.8–12.8% compared to
13.8–16.4% and 9.6–11.6% in BCs of old controls and AD
patients, respectively, suggesting a slightly higher aneuploidy
rate in brain tissue relative to buccal mucosa.
Discussion
The aim of the study was to test the hypotheses that aneuploidy
of chromosomes 17 and 21 in BCs (i) is a risk factor for
neurodegenerative diseases such as AD and DS and (ii)
increases with ageing.
We report a significant 1.2-fold increase in aneuploidy for
chromosome 17 and a 1.5-fold increase in aneuploidy 21 within
the buccal mucosa of AD patients compared to age- and gendermatched controls (Figures 1, 3 and 4). We also report a 1.8-fold
increase in the incidence of chromosome 17 in DS BCs
compared to a young control group (P , 0.001). The DS values
were not significantly different from the old control, even though
the old control group is 50 years senior to the DS group. DS
aneuploidy rates are beyond those expected for normal ageing
61
P. Thomas and M. Fenech
Fig. 4. Frequency of (a) chromosome 17 trisomy and (b) chromosome 21 trisomy following FISH in 1000 BCs of young controls (n 5 30), DS (n 5 21), old
controls (n 5 26), younger AD (n 5 23) and older AD (n 5 31). Groups sharing the same letters are not significant. Error bars indicate SEM.
Fig. 5. Frequency of (a) chromosome 17 monosomy and (b) chromosome 21 monosomy from 1000 hippocampal brain tissue cells of Alzheimer’s patients (n 5 13)
and control hippocampal brain tissue (n 5 9). Groups sharing the same letters are not significant. Error bars indicate SE of the mean.
Fig. 6. Frequency of (a) chromosome 17 trisomy and (b) chromosome 21 trisomy from 1000 hippocampal brain tissue cells of Alzheimer’s patients (n 5 13) and
control hippocampal brain tissue (n 5 9). Groups sharing the same letters are not significant. Error bars indicate SEM (n 5 9).
confirming its status as a premature ageing syndrome. Although
a significant increase in aneuploidy rate for chromosome 17
(P , 0.001) and chromosome 21 (P , 0.001) occurred in BCs
with normal ageing, our data show that the incidence of
chromosome malsegregation in both the AD and DS groups are
significantly increased and beyond the levels expected for
healthy subjects in corresponding age groups. This confirms the
findings from earlier studies that have shown elevated rates of
aneuploidy in AD patients for a number of different chromosomes, such as 13, 18 and 21 indicating higher rates of genomic
instability in this premature ageing syndrome (18,19,28). We
found that the total aneuploidy rate for chromosome 21 was
62
92.1% in DS compared to 5.0% in young controls and 9.6% in
old controls. Most DS individuals are cytogenetically determined as having full trisomy 21. Incomplete hybridization
of the LSI 21 probe to BCs may be responsible for a lower rate of
trisomy 21 with cells appearing disomic and therefore being
scored as false negatives. However, It has been shown that
DS individuals tend to exhibit increased disomy 21 with ageing,
resulting in various frequencies of mosaicism for aneuploidy of
chromosome 21, which are reflected in our findings (29,30).
Given the strong association of trisomy 21 with premature
ageing in DS, it is plausible that elevated trisomy 21 observed in
AD may contribute to accelerated ageing in this condition.
Aneuploidy is increased in Alzheimer’s buccal cells
Table II. Percentages of monosomy and trisomic cells for chromosomes 17 and 21 in at least 1000 BCs of young controls (n 5 30), DS (n 5 21), old controls
(n 5 26), younger AD (n 5 23) and older AD (n 5 31) and hippocampal brain tissue cells of Alzheimer’s patients (n 5 13) and control hippocampal brain tissue
(n 5 9)
Chromosome 17
Monosomy
Trisomy
Total aneuploidy
Chromosome 21
Monosomy
Trisomy
Total aneuploidy
Young controls
DS
Old controls
Younger AD
Older AD
Alzheimer’s brain tissue
Control brain tissue
3.1
1.9
5.0
12.4
3.6
16.0
10.1
3.7
13.8
12.1
4.3
16.4
11.3
4.4
15.7
14.6
3.6
18.2
14.3
3.7
18.0
3.1
1.9
5.0
2.9
89.2
92.1
5.9
3.7
9.6
5.5
5.4
10.9
5.8
5.8
11.6
8.9
3.9
12.8
7.9
3.9
11.8
Increased aneuploidy for chromosomes 17 and 21, which
could lead to altered gene dosage and expression for tau and
App, may be an important contributing factor leading to the
early stages of the development of AD, if critical regions of the
brain are enriched in aneuploid cells during early development
due to mosaicism. The APP gene present on chromosome 21
may be over-expressed in the small population of cells that are
trisomic and could result in aberrant proteolysis and the
accumulation of the Ab42 which is the core component of the
senile plaques found in the brains of both DS and AD patients
(31–34). This amyloidogenic pathway has traditionally held the
view that the neurotoxic effects of these peptides contribute to
the aetiology of the disease. Recently, this view has been
challenged suggesting that APP itself may play an important
neuronal role leading to cognitive impairment (35–37). The
precise role of these peptides and proteins and their interaction
and potential relationship to the development of AD will need
to be conclusively determined through future studies. App
processing is also altered in terms of the ratio of (Ab42)
compared to the normal 40 amino acid b amyloid peptide,
resulting in an increase of the more neurotoxic and amyloidogenic Ab42. Similarly, an individual who is mosaic for trisomy
17 cells could potentially over-express the microtubuleassociated protein tau which may lead to an increased
aggregation of the protein; this could in turn result in increased
paired helical filaments known to make up the neurofibrillary
tangles that are the other cerebral hallmark of the disease.
Abnormal accumulation or deficiency of tau could lead to
a breakdown of the microtubule system causing chromosomal
malsegregation as a result of microtubule disruption. Similarly,
a breakdown in the neuronal transport system due to
microtubule dysfunction may result in an apparent micronutrient deficiency affecting certain susceptible areas of the
brain known to be affected in AD.
However, it is possible that genes other than APP contained
on chromosome 21 and in particular in the DS critical region
such as SOD1 (copper/zinc superoxide) may influence
pathologies such as oxidative stress and the neurodegeneration
associated with both AD and DS (38–40). Knockout mouse
models for DS which exclude APP and SOD1 genes have
shown that within the brain there are increased levels of
oxidative stress, mitochondrial dysfunction and hyperphosphorylation of tau (41). This would suggest that overexpression of other genes within this region contribute to
pathologies common to both conditions. Recently, cultured
trophoblasts from trisomy 21 placentas were analysed for
genome-wide expression and compared to trophoblasts from
normal first trimester pregnancies. About 750 genes were
significantly over-expressed in the trisomic placenta, with
a 4.5-fold gene abundance being found to map to chromosome
21 compared to that expected from the control microarray. It
was also found that genes implicated in AD were also overexpressed, such as a 2-fold increase in APP and a 3-fold
increase in LOX as well as over-expression of the APP-binding
protein. Other relevant genes that were identified were
associated with ubiquitination and proteosomal degradation
(42). This may be relevant as an alternative epigenetic
mechanism involving post-translational modification of proteins common to both AD and DS. It has recently been shown
that neuroblastoma cells treated with Ab showed an increase in
the DYRK1A transcript, a gene found within the DS critical
region, resulting in elevated rates of tau hyperphosphorylation
(43). This is significant as it provides a mechanism that links
chromosome 21 aneuploidy and possible gene over-expression
with histopathological cerebral hallmarks that are common to
both AD and DS. Alterations in the DYRK1A and presenilin
genes could lead to defects in anterograde fast axonal transport
leading to neurodegeneration affecting brain function (44),
while deficits in cellular transport leading to oxidative stress
may indirectly influence spindle formation and chromosome
segregation in all somatic and dividing cells.
It is plausible that trisomy 21 mosaicism in brain tissue,
particularly within the hippocampus, may have originated from
chromosome malsegregation events early in foetal development
and may be a possible explanation for some sporadic forms of AD
(19,45). Low level in utero mosaicism may be the result of nondisjunction caused by folate deficiency, which has been shown to
increase the rate of chromosome 17 and 21 aneuploidy in
lymphocytes (46). Recent studies have shown an increased
predisposition to chromosome malsegregation in the young
mothers of DS individuals (MDS) (47). Schumpf et al. (47)
showed a 5-fold increase in the risk of AD within this cohort of
young mothers and suggested that these individuals are biologically prematurely aged increasing their susceptibility to
aneuploidy events. Chromosome 21 aneuploidy would predispose
these individuals to DS pregnancies and potentially contribute to
an increased risk for developing AD later in life (47).
The risk of aneuploidy due to chromosome malsegregation
is likely to be increased in cerebral tissues undergoing postmaturation neurogenesis such as the dentate gyrus of the
hippocampus involved in short-term memory and learning and
the glial cells (48). Both populations of cells have been shown
to continue cell division in the adult brain and so could be
potential candidates to accumulate aneuploid cells throughout
an individual’s lifetime (49). Our data shows that mosaicism
for chromosomes 17 and 21 occurred within the hippocampus
of histopathologically confirmed Alzheimer’s patients. However,
the aneuploidy rate for these two chromosomes was not
63
P. Thomas and M. Fenech
significantly different from control hippocampal tissue (Figures
2, 5 and 6). High rates of monosomy for both chromosomes 17
and 21 were observed in both AD and control hippocampal cells.
During FISH analysis, the morphology of the nucleus appeared
normal and not condensed and thus excludes the latter as an
explanation that may have lead to close proximity of parental
chromosomes resulting in a single FISH signal. However, it
is possible that incomplete hybridization of the probe may
have lead to an elevated percentage rate of false positives for
monosomy being recorded. It has been shown previously in
FISH studies involving lymphocytes that monosomy levels are
2.5-fold higher than trisomic cells and our results in BCs are
comparable with these earlier findings (46). Only further cerebral
aneuploidy studies will either confirm or refute these preliminary
findings. These results suggest that elevated chromosome 17 and
21 aneuploidy is unlikely to be a causative factor of AD on its
own, unless higher rates of aneuploidy of these chromosomes
were present much earlier in the life of AD cases and rates of
mitosis in neural tissue was reduced relative to controls. An
alternative possibility is that abnormal expression of tau and App
occurs to a greater extent in chromosome 17 and 21 aneuploid
cells in AD relative to controls. Studies investigating the
relationship between tau and App expression and chromosome
17 and 21 aneuploidy are needed to address this question.
Polymorphisms of presenilins and ApoE genes that are
known risk factors for both AD and DS are likely to play
a pivotal role in chromosome malsegregation. Mutated
presenilin genes that contribute to early onset familial AD have
been shown to produce proteins localized within the nuclear
membrane, centrosomes and kinetochores indicating a role in
chromosome segregation and organization (50). An association
has also been shown between polymorphisms within intron 8 of
the presenilin 1 gene and errors within maternal meiosis II that
led to trisomy 21 (51). This same polymorphism has also been
identified as conferring an increased risk for late onset AD
(19,52,53). It has been shown that certain alleles of ApoE
influence the age of onset of dementia in both AD and DS. The
ApoE4 allele has been shown to influence the development of
sporadic AD and DS. DS individuals with two copies of this
allele develop more severe forms of AD neuropathology
compared to DS individuals lacking the e4 allele (18,54,55).
The ApoE4 allele has also been shown to be significantly more
common among young mothers of DS children, suggesting that
this allele may predispose these individuals to chromosome 21
non-disjunction and a potential influential role for this allele on
chromosome malsegregation (56).
Micronutrient deficiency may also have an influence upon
chromosome malsegregation in both AD and DS. It has been
shown that AD patients tend to be deficient in vitamins such as
folate and B12 (57,58). Folate and B12 play an important role in
DNA metabolism and the maintenance of methylation patterns
that could modify gene expression (59,60). There is evidence to
show that abnormal folate metabolism, possibly as a result of
polymorphisms within genes of the folate/methionine pathway,
may result in a state of DNA hypomethylation in the centromeric
regions of chromosomes leading to abnormal chromosome
segregation (61–63). In addition folate deficiency has been show
to increase aneuploidy of chromosomes 17 and 21 (46).
In conclusion, the results of this study show that (i)
aneuploidy of chromosomes 17 and 21 increases with ageing
in BCs, (ii) AD patients exhibit an abnormally high rate of
chromosome 17 and 21 aneuploidy in BCs compared to
controls, (iii) DS is associated with increased aneuploidy of
64
chromosome 17 in BCs and (iv) despite these differences in
BCs, aneuploidy of chromosomes 17 and 21 in hippocampus
tissue did not differ between AD cases and controls. These
results suggest that aneuploidy of chromosomes 17 and 21 may
be caused by genetic factors that predispose to AD but are
unlikely to be a primary cause of brain pathology in AD.
Funding
CSIRO, Division of Human Nutrition, Adelaide, South
Australia.
Acknowledgements
The authors would like to thank Associate Professor Michael Roberts for his
valuable contribution and comments during the compilation of the manuscript.
The authors gratefully acknowledge Jane Hecker and Jeffrey Faunt for
clinically diagnosing the Alzheimer patients and for providing the authors with
MMSE scores. Lastly, the authors greatly appreciate the efforts made by all the
individuals who consented to participate in this study.
Conflict of interest statement: None declared.
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Received on July 23, 2007; revised on October 2, 2007;
accepted on October 2, 2007
65