Mechanisms of Ageing and Development
101 (1998) 167 – 173

Down’s syndrome, ageing and fragile sites
Marilia de Arruda Cardoso Smith *, Bianca Borsatto
Departamento de Morfologia, Disciplina de Genetica, UNIFESP Escola Paulista de Medicina,
´
Rua Botucatu, No. 740, 04023 900 CEP Sao Paulo, Brazil
˜
Received 15 September 1997; accepted 24 November 1997

Abstract
Fragile sites have been interesting for mapping chromosomal regions involved in disease
and ageing. The chromosomal fragile site expression from 38 Down’s syndrome (DS)
individuals aged 0–48 years was investigated in blood peripheral lymphocytes. Fragile sites
were statistically characterized as the minimum expected number of lesions per band based
on a Poisson distribution. The results showed that the fragile site 2q11 was associated with
the DS condition and fragile sites 5q31, 6p21 and 9q12 with ageing in DS subjects. Fragility
in 6p21 has also been associated with Alzheimer’s disease patients. © 1998 Elsevier Science
Ireland Ltd. All rights reserved.
Keywords: Down’s syndrome; Ageing; Fragile sites

1. Introduction
Down’s syndrome (DS) or trisomy of chromosome 21 is the most common
congenital chromosomal aberration in human beings. The majority of individuals
(95%) with trisomy 21 have three free copies of this chromosome and in a small
percentage of patients (5%), one copy is translocated to another acrocentric
chromosome or is present in mosaic form. DS in marked by particular phenotypes
that include mental retardation, characteristic physical features and reduced life
* Corresponding author. Fax: + 55 011 5492127/5708378; e-mail: macsmith.morf@epm.br
0047-6374/98/$19.00 © 1998 Elsevier Science Ireland Ltd. All rights reserved.
PII S 0 0 4 7 - 6 3 7 4 ( 9 7 ) 0 0 1 7 4 - 7

168

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173

expectancy. This syndrome generally originates in a meiotic nondisjunction, with
an estimated frequency of 1:600–1:800 births. In adults with DS, most of the
clinical signs associated with normal ageing occur prematurely. These individuals show an early cognitive decline with age and generally develop the neuropathological changes of Alzheimer’s disease (AD) (Wisniewski et al., 1992).
Fragile sites are points in the chromosomes where they preferentially show lesions such as breaks or gaps, either spontaneously or after exposure to speciﬁc
tissue culture conditions (Sutherland, 1979, 1985). The fragile sites can be used
as cytogenetic markers in association and linkage studies (Sutherland, 1988;
Sutherland and Hecht, 1995). The use of chromosomal fragile sites has been
useful for mapping chromosomal regions of the genome that contain genetic
loci important for the causation of diseases and/or ageing. In our laboratory,
we observed the chromosomal fragile site 6p21 associated with elderly healthy
people and AD patients (Kormann-Bortolotto et al., 1992). Mariani (1989) proposed a characterization of fragile sites according to a minimum expected frequency of lesions (gaps, breaks and rearrangements) per band based on a
Poisson distribution. Those sites in which the probability of chance recurrence
was less than 5% are considered fragile sites. Up to now no approaches similar
to ours have been described which investigated the occurrence of ‘spontaneous’
chromosome lesions in DS subjects. In the present study we investigated the
expression of fragile sites under low folate culture conditions in Down’s syndrome patients in order to verify whether some fragile sites can be correlated
with the clinical condition of the patients or with their ageing process.

2. Material and methods

2.1. Selection of subjects
The sample of individuals with DS was made up of 38 patients, of whom 21
were between 0 and 25 years old and 17 between 29 and 48 years old. The
individuals were selected and supervised within a co-operative program between
the Universidade Federal de Sao Paulo-Escola Paulista de Medicina
˜
(UNIFESP-EPM) and Associacao de Pais e Amigos dos Excepcionais (APAE¸˜
Sao Paulo). The average age (9 S.D.) of younger DS individuals was 17.99
˜
(9 6.32) and the average age (9S.D.) of older individuals was 36.39 (9 10.15).
All the DS patients had free trisomy. The younger group was made up of 11
females and ten males. The older group had one female and 16 males. The DS
patient groups were divided arbitrarily based on evidence that persons with DS
develop neuropathological changes similar to those of AD after the 4th decade
of life (Malamud, 1972). The older group was almost entirely composed of
people in the 4th and 5th decade of life. The younger group was almost entirely composed of people in the 2nd and 3rd decade of life.

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173

169

2.2. Cytogenetic analysis
The cytogenetic analysis was done on the phytohaemagglutinin-stimulated
lymphocytes from these individuals. These cells were cultured for 72 h in TC 199
medium, which is a low folic acid medium, known to enhance the appearance of
folate sensitive fragile sites, with 7% fetal calf serum. The investigation of fragile
sites was carried out in a blind test using at least 50 cells (all containing 47
chromosomes) per individual. All cells were initially analyzed by conventional
staining. Chromosomal lesions (gaps, breaks or rearrangements) were recorded and
the slides were destained with ﬁxative solution (methanol:acetic acid 3:1) and
restained with G-banding technique for analysis of the same metaphases afterwards.
The number of lesions in each band site were computed and only those sites were
considered fragile in which the probability of random recurrence was less than 5%
(Mariani, 1989)

3. Results and discussion
There was a total of 105 lesions in the younger DS individuals group and there
were 81 among older DS individuals group. These differences were not statistically
signiﬁcant according to the Mann–Whitney test (zcalc = 0.75 and zcrit = 1.64). Thus,
ageing in DS did not increase the number of chromosomal lesions. The older DS
sample is almost formed by men, however some ﬁndings from the literature have
not shown differences between sexes related to the incidence of autosomal fragile
sites (Sutherland 1982). A band was considered a fragile site based on a Poisson
distribution that indicated the minimum expected frequency of events (lesions) per
band (Mariani, 1989). Tables 1 –3 show the frequency of bands considered fragile
sites in younger DS individuals group and older DS individuals group. Fragile site
Table 1
Analysis of the expected distribution based on the total number of events found in 21 younger DS
individuals between 0 to 25 years old (105 lesions), number of identiﬁable bands per haploid set (400)
and number of analyzed metaphases (1050), according to Mariani (1989)
h

a (h)

E(h)

p (h)

F (h)

0
1
2
3
4
5
6

0.7691264
0.2018957
2.649881E-02
2.318645E-03
1.52161 lE-04
7.988458E-06
3.49495E-07

307.6506
80.75827
10.59952
0.9274582
6.0864445E-02
3.195383E-03
1.39798E-04

1
0.2308736
2.897792E-02
2.47911E-03
1.604646E-04
8.303454E-06
3.149962E-07

400
92.34943
11.59116
0.9916334
6.417519E-02
3.310744E-03**
1.153606E-04***

Column h, events per band; Column a (h), probability of ﬁnding h events at a given band; Column E
(h), expected number of bands showing h events; Column p (h), probability of ﬁnding h or more events
at a given band; Column F (h), expected number of bands that would show h or more events.
* Signiﬁcant (PB0.05); ** Highly signiﬁcant (PB0.01); *** Very highly signiﬁcant (PB0.001).

170

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173

Table 2
Analysis of the expected distribution based on the total number of events found in 17 older DS
individuals between 29 to 48 years old (81 lesions), number of identiﬁable bands per haploid set (400)
and number of analyzed metaphases (850), according to Mariani (1989)
h

a (h)

E (h)

p (h)

F (h)

0
1
2
3
4
5

0.8166865
0.165379
1.674462E-02
1.130262E-03
5.721952E-05
2.317391E-06

326.6746
66.1516
6.69785
0.4521048
2.288781E-02
9.269562E-04

1
0.1833135
1.793447E-02
1.189847E-03
5.958439E-05
2.364868E-06

400
73.32541
7.173805
0.4759555
2.385065E-02*
9.628423E-04**

Column h, events per band; Column a (h), probability of ﬁnding h events at a given band; Column E
(h), expected number of bands showing h events; Column p (h), probability of ﬁnding h or more events
at a given band; Column F (h), expected number of bands that would show h or more events.
* Signiﬁcant (PB0.05); ** Highly signiﬁcant (PB0.01); *** Very highly signiﬁcant (PB0.001).

3pl4 was common to both groups. The occurrence of this fragility was similar in
younger and older DS groups. Band 3pl4 is widely cited as the most common
fragile site in humans (Simonic and Gericke, 1996). Recently a gene called FHIT
has been reported that is located in this chromosomal region. This gene is either
completely or partially missing in many neoplasias such as colon, breast and lung
cancer which suggests it may be a tumour suppressor gene. Fragile site 6p21 was
observed only in the older DS group. The gene of a microtubule associated tau like
protein (MTBT2) is located in this band (Kidd et al., 1989). Thus, this gene may be
related to chromosomal alterations which involve defects at the level of microtubules and may also be a primary mechanism of the ageing process of DS
syndrome. Fragile site 2q11 was observed both in older DS group and younger DS
group. This chromosomal region has been described as an unstable secondary
constriction on the long arm of this chromosome in patients with different clinical
features (Lejeune et al., 1968). Other cases with this secondary constriction show
that clinical features can vary considerably, with phenotypes that include mental
retardation, congenital heart malformation and growth retardation, as well as some
healthy individuals (Ferrante et al., 1968; Buhler et al., 1970; Williams and Howell,
1976). Whereas this chromosomal alteration might be considered a normal variant,
Anneren and Gutstavson (1981) suggest that cell lines with it might have a
pathological effect if they occur at critical stages in the organogenesis. The fragility
in chromosome 9ql2 was exclusively observed in older DS patients. Fragile site 9ql2
is a chromosomal region formed by a constitutive heterochromatin, with a still
unclear biological function. Previous studies have also shown a possible increase of
chromosome 9 pericentric inversion among DS subjects and it has been suggested
that this inv(9)(qh) predisposes the carrier parent to nondisjunction. Cytogenetic
studies show an interaction between the heterochromatin and the acrocentric
chromosomes during the cell division process that can interfere in disjunction and
chromosome distribution (Natarajan and Ahnstrom, 1969; Gagne et al., 1974;
´
Miklos and John, 1979; Hulten et al., 1987). Fragile site 5q31 was observed only in
´

Number of
individuals

21
17

Groups of
individuals

Younger (DS)
Older (DS)

Total number
of analyzed
cells

1050
850

Age/years
( 9S.D.)

17.99 ( 9 6.32)
36.39 ( 910.15)
105
81

Total number
of lesions

]5
]4

Minimum expected no. of
lesions (Mariani, 1989)

4.8
4.9

2q11

7.6
12.34

3p14

—
6.2

5q31

—
7.4

6p21

Frequency (%) of lesions in bands
characterized as fragile sites

—
6.2

9q12

Table 3
Frequency of lesions in bands characterized as fragile sites, minimum expected number of lesions according to Poisson distribution (Mariani, 1989) and
total number of lesions in lymphocytes from younger DS individuals and older DS individuals

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173
171

172

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173

older DS individuals. In this region, for instance the gene for a lymphokine,
interleukin-13, is located which is expressed in activated human T-lymphocytes.
Minty et al. (1993) suggested that interleukin-13 may be critical in regulating
inﬂammatory and immune responses. It is well known that DS individuals exhibit
an increased frequency of infections and malignancies, especially those involving
the head, neck and respiratory tracts. Finally, fragile site 2q11 has been observed in
both DS groups whereas has not been observed in young and/or elderly control
groups previously analyzed in our laboratory (Kormann-Bortolotto et al., 1992).
Thus, we believe that this fragile site is associated with DS condition. Fragile site
6p21 was observed in the older DS syndrome group, in the elderly control group
and in AD analyzed previously in our laboratory (Kormann-Bortolotto et al.,
1992). Thus, we believe that fragile site 6p21 is associated with the elderly
condition.
Some studies have suggested that the fragile site could be a cytogenetic representation of gene expression (Yunis et al., 1987; Hecht, 1988). Austin et al. (1992) have
shown a clustering of spontaneous aberrations at fragile sites that may indicate that
these regions are physiologically active during the S-phase of the cell cycle. Fragile
sites FRAXA, FRAXE, FRAXF and FRA16A are formed by the (CGG)n trinucleotide repeat unit expansion with subsequent methylation of a adjacent CpG island
that can introduce a recognition site for de novo imprinting (Nancarrow et al.,
1994). Therefore fragile sites could represent adjacent gene expression. Finally, our
ﬁndings have shown fragile site 2q11 associated with DS condition and fragile sites
5q31, 6p21 and 9q12 associated with older DS individuals. These fragile sites could
indicate chromosomal regions or genes to be further investigated in DS ageing or
in this syndrome.

Acknowledgments
The authors are grateful to Prof. Eduardo Katchburian, Prof. Antonio dos
Santos Clemente and Dr Elisa Maria Doceu Moreira Garcez and to Associacao de
¸˜
Pais e Amigos dos Excepcionais (APAE-Sao Paulo) for allowing this co-operative
˜
program. We would also like to thank Elenice Rosana Salas for technical assistance
and Dr Tulio Mariani who provided the statistical computer program. This work
was supported by Sandoz Foundation of Gerontological Research, Fundacao de
¸˜
Amparo a Pesquisa do Estado de Sao Paulo, Brazil (FAPESP), Coordenacao de
`
˜
¸˜
Aperfeicoamento de Nıvel Superior, Brazil (CAPES) and Conselho Nacional de
¸
´
Desenvolvimento Cientıfco e Tecnologico, Brazil (CNPq).
´
´

References
Anneren, G., Gustavson, K., 1981. A fragile secondary constriction on chromosome 2 in ﬁve patients
with different clinical features. Hereditas 95, 63 – 67.

M. de Arruda Cardoso Smith, B. Borsatto / Mech. Ageing De6el. 101 (1998) 167–173

173

Austin, M.J.F., Collins, J.M., Corey, L.A., Nance, W.E., Neale, M.C., Schieken, R.M., Brown, J.A.,
1992. Aphidicolin-inducible common fragile sites expression: results from a population survey of
twins. Am. J. Hum. Genet. 50, 76–83.
Buhler, E.M., Luchsinger, U., Buhler, U.K., Mehes, K., Stalder, G.R., 1970. Noncondensation of one
´
segment of a chromosome no 2 in a male with otherwise normal karyotype (and severe hypospadias).
Humangenetik 9, 97–104.
Ferrante, E., Bruni, L., Grimaldi, S., 1968. Rottura paracentromerica del braccio lungo di un
chromosome 2 in una bambina di 9 mesi affetta da cistationuria. ritardo psicomotorio e cataratta..
Riv. Clin. Ped. 81, 612–619.
Gagne, R., Luciani, J.M., Devictoc-Vuillet, M., Stahl, A., 1974. C9 heterochromatin during the ﬁrst
meiotic prophase in human fetal oocyte. Exp. Cell. Res. 85, 111 – 116.
Hecht, F., 1988. Fragile sites, cancer, chromosome break points and oncogenes all cluster in light
G-bands. Cancer Genet. Cytogenet. 33, 1 – 10.
Hulten, M., Saadallah, N., Batanian, J., 1987. Meiotic chromosome pairing in the human male:
´
Experience from surface spread synaptonemal complexes. Chromosomes Today 9, 218 – 229.
Kidd, K.K., Bowcock, A.M., Schmidtke, J., et al., 1989. Report of the DNA committee and catalogs of
cloned and mapped genes and DNA polymorphisms. Cytogenet. Cell Genet. 51, 622 – 947.
Kormann-Bortolotto, M.H., Smith, M. de A.C., Toniolo Neto, J., 1992. Fragile sites, Alzheimer’s
disease and ageing. Mech. Ageing Dev. 65, 9 – 15.
Lejeune, J., Dutrillaux, B., Lafourcade, J., Berger, R., Abonyl, D., Rethore, M.O., 1968. Endoreduplication selective du bras long du chromosome 2 chez une femme et sa ﬁlle. Acad. Sci. Paris 266, 24 – 26.
Malamud, D.N., 1972. Ageing and the Brain, Gaitz, C.M., Plenum, New York.
Mariani, T., 1989. Fragile sites and statistics. Hum. Genet. 81, 319 – 322.
Miklos, G.L.G., John, B., 1979. Heterochromatin and satellite DNA in man: properties and prospects.
Am. J. Hum. Genet. 31, 264–280.
Minty, A., Chalon, P., Derocq, J.M., Dumont, X., et al., 1993. Interleukin-13 is a new human
lymphokine regulating inﬂammatory and immune responses. Nature 362, 248 – 250.
Nancarrow, J.K., Kremer, E., Holman, K., Eyre, H., Dogget, N.A., Le Paslier, D., Callen, D.F.,
Sutherland, G.R., Richards, R.I., 1994. Implications of FRAl6A structure for the mechanism of
chromosomal fragile site genesis. Science 264, 1938 – 1941.
Natarajan, A.T., Ahnstrom, G., 1969. Heterocromatin and chromosome aberrations. Chromosoma 28,
48–61.
Simonic, I., Gericke, G.S., 1996. The enigma of common fragile sites. Hum. Genet. 97, 524 – 531.
Sutherland, G.R., 1979. Heritable fragile sites on human chromosomes I Factors affecting expression in
lymphocyte culture. Am. J. Hum. Genet. 31, 125 – 135.
Sutherland, G.R., 1982. Heritable fragile sites on human chromosomes VIII, Preliminary population
cytogenetic data on the folic-acid-sensitive fragile sites. Am. J. Hum. Genet. 34, 452 – 458.
Sutherland, G.R., 1985. Heritable fragile sites on human chromosomes XII Population Cytogenetics.
Annu. Hum. Genet. 49, 153–161.
Sutherland, G.R., 1988. The role of nucleotides in human fragile site expression. Mutat. Res. 200,
207–213.
Sutherland, G.R., Hecht, F., 1995. Fragile Sites on Human Chromosomes, Oxford University Press,
New York.
Williams, A.J., Howell, R.T., 1976. A fragile secondary constriction on chromosome 2 in a severely
mentally retarded patient. J. Ment. Deﬁc. Res. 21, 227 – 230.
Wisniewski, K.E., Hill, A.L., Wisniewski, H.M., 1992. Down’s Syndrome-Advances in Medical Care,
Willey-Liss, New York.
Yunis, J.J., Soreng, A.L., Bowe, A.E., 1987. Fragile sites are targets of diverse mutagens and
carcinogens. Oncogene 1, 248–250.

.

