Author: Chandel, D
Date published: April 1, 2010
Free trisomy 21 is the most common abnormality in Down syndrome (DS). Since the identification of this first human trisomy, many attempts have been made to elucidate factors that influence the rate of meiotic nondisjunction. These efforts have been remarkably unsuccessful; apart from the well established association between increasing maternal age and trisomy, no etiological factor has been convincingly linked to human nondisjunction, although increased maternal age and altered recombination are considered to be the strong factors [2,3]. The rate of non disjunction varies between different chromosomes and the factors underlying non disjunction appear to be highly chromosome specific. Most trisomies arise from maternal meiosis I .
Down syndrome has always been of special interest to human geneticists, because of its historical perspective and also due to the strong correlation between advanced maternal age, increased meiotic nondisjunction, and the birth of an affected child, which provide an interesting model for the investigation of chromosomal abnormalities .Much of our knowledge about chromosomal non-disjunction in man comes from studies in trisomy 21.
The cytogenetic literature is filled with possible etiologies for nondisjunction. The suggested factors include maternal and paternal age effects, genetic predisposition, viral infection, parental irradiation, environmental chemical insults, DNAhaplotypes and interchromosomal effects [4,8]. Because substantial information gap still exists, it is necessary to carefully examine any and all potential factors suggested as etiological causes of nondisjunction.
Heteromorphism of NORs is well known in humans and has been extensively investigated to study the parental origin of extra chromosome 21 in Down syndrome and to understand their clinical significance [5-7]. Double NOR was first proposed by Jackson- Cook et al.  and further analyzed by Spinner et al.  in Down syndrome cases. Further, Ohno et al.  have suggested that an increased frequency of association among acrocentric chromosomes might predispose themto nondisjunction during cell division.
In the present study, the frequency of acrocentric chromosome associations (ACAs) and dNORs were determined in the controls and DS and ascertained whether the results could be associated with Down syndrome.
MATERIALS AND METHODS
The children with Down syndrome were first examined and verified by a consultant physician. Clinical features and detailed family history was recorded in a proforma. 2-3 ml of intravenous blood was collected in heparinized syringes from the probands and age matched control children. Peripheral blood lymphocyte cultures were set up in duplicate in RPMI 1640 medium (Hi-media, India) and harvested at 72 hours, after which the slides were prepared as per the routine protocol . Foremost, routine GTG-banding was performed on the slides to confirm free trisomy 21 . In these confirmed cases, GTG banded and silver nitrate stained  metaphase preparations were used for analysis of acrocentric chromosome associations. Hundred metaphase plates having all the acrocentric chromosomes were analysed for acrocentric chromosome associations in 20 Down syndrome and 20 age matched control children. The presence of two or more acrocentric chromosomes in close proximity at their satellite end was considered to be an association. The slides were scanned to analyze the number and types of association(s) viz., DD, DG, GG andmultiple (DDD, DDG, GGG, DDGG etc.) in each plate.
Metaphases were studied under oil immersion lens (100X) of Nikon microscope. For each sample 100 metaphases were scored according to ISCN guidelines . The data was analyzed using student's't' test.
Studies on association of acrocentric chromosomes in 20 Down syndrome and 20 age matched control children were carried out in G-banded and silver nitrate stainedmetaphase preparations and examples of some of the observations are shown in figure I. The detailed result is shown in figure 2 and Table 1. A total of 2000 cells were scored for controls and for Down syndrome children. In the control children, 442 associations were observed while 885 associations were observed in down syndrome children. In both the groups, maximum of D-G associations were observed, followed by D-D and theG-G associations. Themultiple associations were the least in both the groups (Fig. 2). The frequency of ACAs in the metaphase spreads varied from individual to individual as shown in Table 1. Increased frequency of ACAwas observed in Down syndrome children as compared to the controls and the values were highly significant (p<0.001).
The principle objective of this study was to understand the implication of the acrocentric chromosome associations and dNORs in the Down syndrome children and to demonstrate whether these were in some way responsible for nondisjunction
The presence of nucleolar organizing regions on the short arms of all human acrocentric chromosomes and their intimate association in nucleoli has led to frequent speculation that NORs may be important in nondisjunction involving acrocentric chromosomes. Polani et al.  for the first time suggested that nucleolar persistencemight interfere with the normal pairing process, which ultimately leads to random assortment of univalents at meiosis I. They further suggested that this could be the basis for thematernal age effect in trisomy 21, as nucleoli of older oocytes might be more resistant to the normal breakdown process than the nucleoli of the younger oocytes.
Subsequently, a large number of investigations were carried out by enlarging upon this initial hypothesis, and two of the first few reports were by Ohno et al.  and Ferguson-Smith and Handmaker . They suggested that the satellite associations and fusion of nucleoli are different manifestations of the same phenomenon and if "sticky" material from the large fused nucleoli persists through cell division, nondisjunction may result.
The reports dealing with the activity of the NORs in trisomic patients are diverse; Hansson andMikkelsen  and Murthy  found a higher association frequency. These reports lend support to the idea that acrocentric chromosome associations play a significant role in nondisjunctional events leading to Down syndrome. However, other scientists did not find such an increase [20,21].
Mirre et al.  proposed that NOR related nondisjunction is derived from an error in chromosome pairing or separation due to physical proximity of acrocentric chromosomes in nucleoli. Schmickel et al.  suggested that recombination errors involving the short arms of non-homologous acrocentric chromo-somes could give rise to non-disjunction. Contrary to this, Green et al.  indicated that the role of NORs in the mechanism of non-disjunction remains unclear.
The results are not consistent probably because of varying criteria of scoring such associations. Study of Sigmund et al.  revealed that the frequencies of ACAs in first, second and third mitosis in human lymphocyte cultures were not the same. They observed a marked decrease of the association frequency with increasing number of cell cycles. Yasseen and Aunuiz  evaluated the contribution of satellite association phenomenon on spermatogenic impairment in infertilemales and observed significant increase in the satellite association.
In the present study, the acrocentric chromosome associations were observed to be significantly higher in DS patients than in controls. The results are in agreement with the previous reports suggesting relationship between ACAs and a nondisjunctional event. The results presented here indicate that the association of two acrocentric chromosomes is very frequent. As theoretically expected, the DG type association is relatively more common as compared to DD and GG type. It is apparent that the frequency of specific type of association depends largely on the number of chromosomes available to take part in the phenomenon. This is very well demonstrated in the present study.
Zankl andNagl  studied trisomic and normal cells in the same individual having trisomy 21 mosaicism and found significantly increased association of chromosome 21 in the trisomic cells. The finding of significant frequency ofGG association in the trisomy 21 cases of the present study may be due to the fact that these individuals possess an extra G group chromosome.
Nazmy et al.  evaluated the role of nucleolus organizer region heteromorphism as an etiological factor for parental nondisjunction in Down syndrome and found a significant difference in the size of the dNOR variants. Jackson-Cook et al.  in their study concluded that dNORs were important in the etiology of trisomy 21, and suggested that the presence of this variant might increase the risk of having a child with DS by as much as 20 fold. Further, Underwood and Giri  indicated that the NOR might be a diagnostic discriminator of malignancy. Jones et al.  have implicated dNOR in non-disjunction of sex chromosome as well. However, these initial reports were not supported by many subsequent studies. Hassold et al.  studied parents of spontaneously aborted trisomic fetuses and chromosomally normal fetuses, and did not observe dNOR in both, the cases and controls. The relationship of dNOR was again questioned by Spinner et al. . Similar conclusions were also made by Schwartz et al.  and Serra and Bova  that a dNOR carrier is not at risk of having a DS offspring.
In the present study too, not a single dNOR was observed in 20 DS children and 20 age matched control children. ACA and the dNORs were scored in the same metaphase spreads. Thus, our observations are contrary to the annotations made by Jackson-Cook et al.  and similar to those of Spinner et al.  and Serra and Bova  that no association exists between the presence of dNOR and non-disjunction. Schmickel et al.  have suggested that the dNORs most likely arise from unequal homologous recombination within the ribosomal genes, and there is no proven relationship between dNORs and any disease or phenotype.
From the observations made in the present study, it could be concluded that an increased acrocentric chromosome associations and not the dNORs can be related to the nondisjunctional event.
 Connor, J.M. and Ferguson-Smith, M.A.: Essential Medical Genetics. Oxford,Blackwell, (1991).
 Abruzzo, M.A. and Hassold, T.J.: Environ. Mol. Mutagen., 25: 38-47 (1995).
 Thomas, S.N., Ennis, S., Sharp, A.J., Durkie, M., Hassold, T.J., Collins, A.R. and Jacobs, P.A.: Hum. Mol.Gen. 11(3): 243-250 (2001).
 Hassold, T.J. The origin of aneuploidy in humans. In : Aneuploidy Etiology andMechanisms (Dellarco,V.L., Voytek, P.E. and Hollaender,A. eds), Plenum Press, NewYork, 36: 103-116 (1985).
 Mikkelsen,M., Poulsen, H., Grinsted, J. and Lange,A.: Ann.Hum.Genet., 44: 17-28 (1980).
 Thuline, H.C. and Puschel, S.M.: Advances in Biomedicine and Behavioral Sciences, Acad. Guild Pub., USA(1982).
 Perez-Castillo,A.,Martin Lucas,M.A. andAbrisqueta, J.A.:Hum.Genet., 72: 80-82 (1986).
 Hassold, T.J. and Jacobs, P.A.: Ann. Rev. Genet., 18: 69-97(1985).
 JacksonCook,C.K., Flannery,D.B., Corey, L.A.,Nance, W.E. and Brown, J.A.:Am. J. Hum. Genet., 37: 1049- 1061 (1985).
 Spinner, N.B., Eunpu,D.L., Schimiickel, R.D,, Zackai, E.H.,McEldrew, D., Bunin, G.R.,McDermid, H. and Emanuel,B.S.:Am. J.Hum.Genet., 44: 631-638(1989).
 Ohno, S., Trujillo, J.M., Kaplan,W.D. and Kinosita, R.:Lancet, 11: 123-125 (1961).
Hungerford, D.A.: Stain Technol., 40: 333-338 (1965).
 Sun, N.C., Chu, E.H.Y. andChang, C.C.: Caryologia, 27:315-324 (1974).
 Goodpasture, C. and Bloom, S.E.: Chromosoma, 53: 37-50(1975).
 Mitelman, F.: An International System for Human Cytogenetic Nomenclature. S. Karger, Basel (1995).
Polani, P.E.,Briggs, J.H., Ford,C.E., Clarke,C.M.,Berg, J.M.: Lancet, 721-724 (1960).
 Ferguson-Smith,M.A. andHandmaker, S.D.: Lancet, 1: 638-640 (1961).
 Hansson, A. and Mikkelsen, M.: Cytogenet. Cell Genet., 20: 194-203 (1978).
Murthy, S.K.: Ind. J. Exp.Biol., 25: 363-367 (1987).
Zankl,H.andNagl,H.:Hum.Genet.,55: 115-117 (1980).
Krishnamurthy,D.S. andAmbani, L.M.: Indian J.Med. Res., 73: 53-60 (1981).
Mirre, C.,Hurtung,M. and Stahl,A.: Proc. Natl.Acad. Sci. (USA), 77: 6017-6021(1980).
 Schmickel, R.D., Gonzalez, I.L., Erickson, J.M.: Nucleolus organizing genes on chromosome 21: recombination and non disjunction. In: Annals of the New York Academy of Sciences (Smith, G. ed.),Ann. N.Y. Acad. Sci., 121-131 (1985).
Green, J.E., Rosenbaum,K.N.,Rapoport, S.I.,Schapiro, M.B. andWhite, B.J.:Clin.Genet., 35: 243-250 (1989).
 Sigmund, J., Schwarzacher,H.G. andMikel saar,A.V.: Hum.Genet., 50: 81-91 (1979).
Yasseen,A.A.,Aunuiz,A.F.: SaudiMed. J., 23(4): 427- 431 (2002).
Nazmy, N.A., Kotb, S.M.,Mokhtar,M.M. and Ismail, S.R.: EastMediterr Health J., 5(2): 299-306 (1999).
Underwood, J.C.E. andGiri,D.D.: J. Pathol., 155: 95-96 (1988).
 Jones, C.,Ahmed, I., Cummings,M.R. and Rosenthal, I.M.:Am. J.Med. Genet., 30: 725-732 (1988).
 Hassold, T.J., Jacobs, P.A., Pettay, D.: Hum. Genet., 76:381-384 (1987).
 Schwartz S, Roulston D, Cohen MM.:Am. J. Hum. Genet., 44:627-630 (1989).
 Serra,A. and Bova,R.:Am. J.Med.Genet., 7: 169-174 (1990).
Division of Human Genetics, Department of Zoology, Gujarat University,Ahmedabad - 380 009,
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Received: December 4, 2009; Accepted: January 12, 2010