MAD2033S0047-6374(97)00174-710.1016/S0047-6374(97)00174-7Elsevier Science Ireland LtdTable 1Analysis 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)ha (h)E(h)p (h)F (h)00.7691264307.6506140010.201895780.758270.230873692.3494322.649881E-0210.599522.897792E-0211.5911632.318645E-030.92745822.47911E-030.991633441.52161 lE-046.0864445E-021.604646E-046.417519E-0257.988458E-063.195383E-038.303454E-063.310744E-03**63.49495E-071.39798E-043.149962E-071.153606E-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 (P<0.05); **Highly significant (P<0.01); ***Very highly significant (P<0.001).Table 2Analysis 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)ha (h)E (h)p (h)F (h)00.8166865326.6746140010.16537966.15160.183313573.3254121.674462E-026.697851.793447E-027.17380531.130262E-030.45210481.189847E-030.475955545.721952E-052.288781E-025.958439E-052.385065E-02*52.317391E-069.269562E-042.364868E-069.628423E-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 (P<0.05); **Highly significant (P<0.01); ***Very highly significant (P<0.001).Table 3Frequency 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 individualsGroups of individualsNumber of individualsAge/years (±S.D.)Total number of analyzed cellsTotal number of lesionsMinimum expected no. of lesions (Mariani, 1989)Frequency (%) of lesions in bands characterized as fragile sites2q113p145q316p219q12Younger (DS)2117.99 (±6.32)1050105≥54.87.6———Older (DS)1736.39 (±10.15)85081≥44.912.346.27.46.2Down’s syndrome, ageing and fragile sitesMariliade Arruda Cardoso Smith*BiancaBorsattoDepartamento de Morfologia, Disciplina de Genética, UNIFESP Escola Paulista de Medicina, Rua Botucatu, No. 740, 04023 900 CEP São Paulo, Brazil*Corresponding author. Fax: +55 011 5492127/5708378; e-mail: macsmith.morf@epm.brAbstractFragile 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.KeywordsDown’s syndromeAgeingFragile sites1IntroductionDown’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 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.2Material and methods2.1Selection of subjectsThe 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 São Paulo-Escola Paulista de Medicina (UNIFESP-EPM) and Associação de Pais e Amigos dos Excepcionais (APAE-São Paulo). The average age (±S.D.) of younger DS individuals was 17.99 (±6.32) and the average age (±S.D.) of older individuals was 36.39 (±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.2.2Cytogenetic analysisThe 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)3Results and discussionThere 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). Table 1Table 2Table 3 show the frequency of bands considered fragile sites in younger DS individuals group and older DS individuals group. Fragile site 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., 1968Buhler et al., 1970Williams 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, 1969Gagné et al., 1974Miklos and John, 1979Hultén et al., 1987). Fragile site 5q31 was observed only in 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., 1987Hecht, 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.AcknowledgementsThe authors are grateful to Prof. Eduardo Katchburian, Prof. Antonio dos Santos Clemente and Dr Elisa Maria Doceu Moreira Garcez and to Associação de Pais e Amigos dos Excepcionais (APAE-São 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. 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