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Biomedical Research

Short communication: Unexpected segregation ofchromosome and Common fragile site expression induced by 5-Azacytidine exposure in human lymphocytes of Down's syndrome patients

Author(s): Ajit K Saxena, A K Srivastava, Gajendra Singh

Vol. 18, No. 1 (2007-01 - 2007-04)

Biomedical Research2007; 18 (1): 31-34

Ajit K Saxena, A K Srivastava٭, Gajendra Singh

Centre of Experimental Medicine and Surgery, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
٭Department of Anatomy, *King George Medical University, Lucknow, India

Key words: Mammalian genome, -Azacytidine, prometaphase, centromere, premature condensation

Accepted November 23, 2007

Abstract

Preferential breakge of chromosomes at specific sites are known as chromosomal fragile sites are distributed all over the mammalian genome. The -Azacytidine (-AZC) induced expression of “common fragile sites” and other chromosomal changes were evaluated in prometaphase/ metaphase chromosomes obtained from peripheral blood lymphocytes from clinically diagnosed Down’s syndrome patients. The present study possibly for the first time reveals significantly (p<0.001) high incidence of common fragile sites expression (12%), centromere breakage (6%) and more then 3% of premature condensation (PMC) of chromosomes in cell poplution when compared with controls, suggesting that high incidencec of common fragile sites expression due to demethylation action of DNA by 5 - AZC could reveal differential gene activity in mentally retarted patients.

Introduction

After the discovery of first fragile site in 1965 it took several years to get recognition as common feature of human/mammalian chromosomes. Chromosomal fragile sites are nonrandom distributed loci are prone to form breaks/ gaps at metaphase stage of cell division under specific culture conditions that either inhibit DNA replication or facilitate repair mechanism termed chromosomal “fragile sites”. These fragile sites are distributed throughout on the human genome except Y- chromosome. Currently “common fragile sites” are also located near or at structural defective regions where DNA is liable to form breaks in cancer cells because of close proximity of “protooncogene”. The genetic instability of “common fragile sites” have possible role in development of genetic damage including involvement of chromosome breakage and gene amplification [1]. -Azacytidine (-AZC) is a potent inhibitor of DNA methylation and S-phase specific cytotoxic agent of cell - cycle shows highly decondesed and extended morphology of chromosomes including fragile sites and other chromosomeal features like exchange and breaks/gape at first mitotic division of cell differentiation [2]. Fragile sites are a manifestation of an altered state of genetic activity in areas associated with transcription because of concordance of CpG islands.The study of “common fragile sites” are very difficult phenomeneon because of presence in low frequency of cell poplution [3,4]. Although the incidence varies in two different sexes i.e. about 1:1500 males and 1:2500 females in population [5]. These “common fragile sites” are sensitive to environment, nutritional or dietry factors and other clastogen to induce centromeric breakage [6,7]. The topographical organization of common fragile sites is highly complex has remained unexplored because of probably lack of unsystematic approach like cloning of fragile sites loci and DNA sequences has not been under taken. The cloning of fragile sites give rise to the concept of dynamic mutation, although less is known about the molecular basis of origin of “common fragile sites” and their biological significance [8].

The study of fragile sites have become of increasing interest over the past two decades because of first genetic curiosity and secondly use as marker for various clinical lesion. In the present study the expression of common fragile site and other cytogenetic features were evaluated in lymphocytes using specific inhibitor (-Azacytidine) of DNA demethylation in Down’s syndrome patients to determine fragility of human chromosomes in Nepalese poplution because of variable complexicity exists in phenotype of families associated with mental retartadion.

Materials and Methods

The peripheral heparinized whole blood (W.B) sample (1.0-1.5 ml) were collected from clinically diagnosed Down’s syndrome patients (n=25) from different age group (4-17 years) in either of male/female sex .Short term lymphocyte cultures were setup under sterile conditions. Human lymphocytes were grown in complete medium (RPMI–1640) with L-glutamine (0.5 mg/ml), FBS (10%), Phytohemagglutinin– M, and antibiotics (100 IU \ml penicillin,100 microgram streptomycin) for 72 hrs at 370C. -Azacytidine (1ugm/ml) was added to the culture medium 8 hours before harvest of culture. Simultaneously three cultures were harvested using Colchicine (0.01 mg/ml) as mitotic (inhibitors), and (0.56%) KCl as hypotonic solution. Chromosome preparations were made according to routine procedure as details were described earlier [7] stained using 5% Giemsa and analyzed using standard protocol [9,10]. High resolution banding technique was performed using (0.001%) trypsin to determine exact location of break points. At least fifty well spread prometaphase or metaphase plates were selected for each data point for chromosomal analysis.

Data were analysed using normal patients act as controls by applying Student t-test to determine significance level (p -value).

Results

In the present cytogenetic study of “common fragile sites” in Down’s syndrome patients and other chromosomal features were observed in -Azacytidine induced lymphocytes culture condition in prometaphase or metaphase stage of cell -division as details are well documented in tabulated form mention below.

Table 1: Showing various type of cytogenetic features including common fragile sites in Down’s syndrome patients induced by -Azacytidine in human lymphocytes.

S.No Type of chromosomal
aberration
% frequency
(aberrate/cell)
1. Fagile sites expression
(3p14, Xq21.3)
12.0
2. Centeromeric breakage 6. 0
3. P M C٭ 3.0

Significance value p<0.001,
PMC= Premature condensation of chromosomes

The frequency of “common fragile sites” (3p14,Xq21.3) was 12% noticed first time while cenetromeric breakage showing 6%, while premature condensation of chromosomes (PMC) were 3 % observed in lymphocytes of Down’s syndrome patients in Nepalese poplution. Stastical analysis shows highly significant (p<0.001) differences.

Discussion

The present stydy of “common fragile sites” has an important implication on assessment of potential risk in developing mentally retarted babies because these fragile sites exhibits to display a number of characteristic in DNA instability being preferential sites for chromosomal breaks/gaps in individuals due to having extra copy of chromosome i.e trisomy -21 (Down’s syndrome). The frequency of such chromosomal aberration per cell was 12% as observed first time in patients of Down’s syndrome with typical phenotypes having low I.Q. Findings of the present study reveals higher incidence of “common” fragile sites (3p14.3 , Xq21.3) expression what is reported in literature which varies from 5% in cell poplution [6] suggesting probably due to habuitual food / nutritutional habit although differential sensitivity of fragile sites (6q 21) induced by -AZC as observed in Alzheimers disease (11). Food habits includes insufficient consumption of folic acid deficiency which enhances common fragile site expression in cell pollution [7]. At molecular basis, fragile sites are dynamic mutations of naturally occurring CGG trinucleotide repetitive sequences and their appearance in cell population depends on the degree of mutation and genetics variation. Although, phenotypic expression varies from individual to individual depending on the involvement of CGG trinucleotide repeats i.e. from permutations range (54-200 repeats) have no phenotypic effect, but when size of repeat increases(>200 repeats) which correspond to full mutation range shows abnormal phenotypic expression like moderate to severe mental retardation, facial anomalies, macroordchidism [12].

-Azacytidine, known to be associated with DNA demethylation process, hence methylation process in the DNA has been regulated to control mechanism of gene activity connected with genetic imprinting. This disorder is methylation induced abnormal transcription process, results in an expansion of trinucleotide (CGG repeat) gene failure to translate gene product (FMRP), a key protein required for synaptic maturation and function of neuranal cell [13,14]. The changes of fragile site expression usually lack of tissue specificity which indicate the importance of one or more fundamental mechanism operating to produce the changes associated with clinical phenotype [15].

However additional emphasis is required for early detection of such disorder in poplution with clinical significant suggesting an increased risk of developing such neurological disorder (Down syndrome) in specific community because of association with genetics instability, amplification and over expression of amplified genes play an important role in clinical lesion. 5–Azacytidine induced cells showed delay in entering mitosis, because of increased significant abnormal divisions with increased chromosomal aberrations such as centromeric breakage as observed in the present study suggesting probably due to “fragility” of chromosome exist at/near to centromeric region [16]. The present study has important implications on the assessment of potential risk in nutritional deficient diet in Nepalese population. The mode of transmission i.e., inheritances of “common fragile sites” (carriers) have not been known. Therefore, it can be concluded from the present study that such type of genetic disorder with specific chromosome break point which arises under 5 - AZC induced culture condition in Down’s syndrome patient’s population in Nepalese population which shows little higher frequency perhaps due to different culture condition [17]. Earlier study has also shown higher incidence of fragile site expression in mentally retarted patients in folic acid deficient culture condition suggesting that folic acid is an important componenet required during nutritional supplements for Nepalese population [7]. However, further study is required wheather this study may contribute both the evolutionary diversity through increased crossing over, formation of unstable repeats sequences and variable methylation process after -AZC exposure in lymphocytes of Down’s syndrome patients. These changes might have altered the expression of multigene disorder such as Down’s syndrome resulting in the production of complex phenotypes in families of mental retardation. This study is further continuing to collect large number of sample to explore the mechanism of miltigenic disorder in human population.

Acknowledgement

AKS is thankfully acknowledged to BPKIHS Research grant letter no.acad.729/059/060 for financial assistance. Author is also grateful to Mr.Sushil Kamat for technical support.

References

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Correspondence:
A K Saxena
Centre of Experimental Medicine and Surgery
Institue of Medical Sciences
Banaras Hindu University
Varansi 221005, India
e-mail: draksaxena1 ( at ) rediffmail.com

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