Cytogenetic studies in children with Down syndrome and acute leukemia

Leukemia Research 29 (2005) 1241–1246

Cytogenetic studies in children with Down syndrome and acute leukemia Ad´an Valladares a , Virginia Palma-Padilla a , Juan Manuel Mej´ıa-Arangur´e b , Roberto Guevara-Y´anez a , Azucena Lerma-Reyes a , Fabio Salamanca-G´omez a,∗ a

Unit of Medical Research in Human Genetics, National Medical Center, IMSS, CMN siglo XXI, Av. Cuauht´emoc 330, Col. Doctores, CP 06725 Mexico City, Mexico b Unit of Medical Research in Epidemiology, National Medical Center, IMSS, Mexico City, Mexico Received 5 May 2004; accepted 11 March 2005 Available online 25 April 2005

Abstract The frequency of chromosomal alterations was compared among four children groups: those with Down syndrome and acute leukemia (DS/AL), those with acute leukemia (AL), those with only Down syndrome (DS) and healthy children (NC). The frequency of acquired chromosome abnormalities was larger in the AL group, followed by the DS/AL. The gaps and isogaps were more frequent in children with only DS. The polymorphisms of the constitutive heterochromatin were larger in the DS/AL group. These findings appear to imply that more genetic changes are necessary to develop AL in the case of healthy children compared to children with DS. © 2005 Published by Elsevier Ltd. Keywords: Down syndrome; Acute leukemia; Chromosomal alterations; Gaps; Polymorphisms; Constitutive heterochromatin; Two mutational events

1. Introduction Children with Down syndrome (DS) have an elevated risk of developing acute leukemia (AL)—10–20 times higher than the general population [1]. According to the Knudson carcinogenesis model, two mutational events are required for the development of cancer [2]. The presence of an additional 21 chromosome has been reported as a sole acquired abnormality in patients with leukemia and normal constitutional karyotype [3–5]. Nevertheless, the carcinogenic role of the genes located in this chromosome is not well known. The increased frequency of fragile sites in chronic lymphocytic leukemia suggests that these could be related to chromosomal alterations [6]. Fragile sites may play a significant role in chromosomal rearrangements involved in oncogene activation or tumor suppressor gene inactivation [7]. Other studies clearly demonstrated the relevance of genome instability in tumorigenesis [8]. Besides, fragile sites have

been associated with Down syndrome [9]. On the other hand, whether polymorphisms of the constitutive heterochromatin affect the development of AL or the presence of Down syndrome has not been established yet. A cytogenetic study in children with DS with AL and its controls was performed: (i) assuming that the 21 trisomy could be the first mutational event in the development of acute leukemia in children with Down syndrome; and (ii) considering that chromosomes with large constitutive heterochromatin regions, such as chromosome number 1, may be at risk of centromeric instability and be predisposed to centromeric fusion with other chromosomes [10]. This cytogenetic study aims to determine the constitutional chromosomal alterations and the polymorphisms of the constitutive heterochromatin associated to leukemia, and consequently to understand the mechanisms involved in the susceptibility to cancer.

2. Materials and methods ∗

Corresponding author at: Apartado Postal 12-951, M´exico, DF 03020, M´exico. Tel.: +52 55 56276945; fax: +52 55 57610952/55885174. E-mail address: [email protected] (F. Salamanca-G´omez). 0145-2126/$ – see front matter © 2005 Published by Elsevier Ltd. doi:10.1016/j.leukres.2005.03.015

Karyotypes were performed in four groups of children: 10 children with DS and AL (DS/AL); 375 children with

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DS; 126 children with AL and 1000 healthy children (NC) as controls. Karyotypes were also carried out in all the parents of children with DS/AL and in the parents of children with balanced translocation or chromosome rearrangements found in the control groups. Seven out of nine public institutions who take care of children with cancer in Mexico City took part in this study. In addition, these institutions look after 95% of all children who develop cancer in Mexico City. All the cases were also diagnosed by cytochemistry analysis of bone marrow aspirates and specific stains were used to differentiate ALL from AML. We received 10 children with DS/AL in a period of 3 years. The control groups belong to specialized institutions that treat children with DS, such as the Siglo XXI Pediatric Hospital. When any child of this institution developed leukemia, he received attention in any of the hospitals that participated in this study. The Siglo XXI Pediatric Hospital is located in Mexico City and receives children with DS from any part of the city. The healthy children belong to second-level hospitals in Mexico City. The peripheral blood samples or bone marrow were obtained from patients receiving no pharmaceutical or radiological treatment and neither having blood transfusions or infections. All of them gave their previous written consent. The peripheral blood samples and bone marrow were cultured for 72 h in RPMI with phytohemagglutinin. The bone marrow samples were also processed without cultivation, corresponding to direct bone marrow (DBM), and with mitogenic stimulus (BMC). The GTG and C bands, and fluorescence in situ hybridization (FISH) to Philadelphia chromosome (Vy-

sis Inc.), were also carried out. The spontaneous gaps and isogaps were identified in chromosomes without bands. The statistical tests were Fhisher’s exact test, Chi-square (χ2 ) and odds ratio with confidence intervals to 95%. This protocol was approved by the research and ethical committees of the National Medical Center.

3. Results 3.1. Subjects The median age of the group of children with DS/AL having leukemia was 11 years, but the median was 5 years (P = 0.039) for children that only presented AL. The gender relation did not show any statistical significance (P = 0.91). Males were 5/10, 203/375, 71/126 and 529/1000 in the groups of DS/AL, DS, AL and NC, respectively. The most frequent type of leukemia in DS/AL and AL groups was ALL (8/10 and 113/126, respectively). There was no significant different of ALL frequency between the DS/AL and AL groups (P = 0.68). 3.2. Karyotypes In the DS/AL group, the 10 cases showed constitutional regular trisomy and 5 out of 10 presented additional chromosome rearrangements (Table 1). In the DS group, 90.9% of children were identified with constitutional regular trisomy, plus unbalanced translocations (2.7%), eight isochro-

Table 1 Karyotypes in the children with Down syndrome and acute leukemia n = 10

Sample

Karyotypes

1

DBM SPB

47,XX,+21[36] 47,XX,+21[79]

2 3

SPB SPB

47,XX,+21[52] 47,XX,+21[70]

4

DBM SPB

47,XY,+21[8] 47,XY,+21[50]/35–57<2n>,XY+21[3]

5

DBM BMC

47,XX,+21[4] 47,XX,+21[8]

6

DBM BMC SPB

47,XY,+21[14]/52,XY,+21[10]/48,XY,+21+mar1[1] 47,XY,+21[10] 47,XY,+21[50]

7

SPB

8

SPB

47,XY,+21[106]/46,XY[4]/48,XY,+21,+ t (9;22) (q34; q11)[3]/ 94,XXYY, +21,+21,[2]/47,XY,+21,+ t (9;22) (q34; q11), −1[3]/ 48,XY, +21,+mar1[1]/55,XY,+21[1] 47,XY,+21[115]/48,XY,+mar (r) [1]/47,XY,+del (21)(q21.2)[10]

9

DBM BMC SPB DBM

47,XX,+21[75]/35–57<2n>,XX,+21[3]/94,XXXX,[1]/43,XX,−7,−8,−21[1] 47,XX,+21[18]/44,XX,−6,−21[1]/47,XX,del(21)(pter → q22:)[3] 47,XX,+21[19] 47,XX,+21[70]/end 47,XX, +21[20]/35–57<2n>,XX,+21[10]/35–57<2n>, XX,+21,+22,+mar1[1]

10

DBM BMC SPB

47,XY,+21[10] 47,XY, +21[100] 47,XY, +21[100]

n: number of cases; mar: marker; end: endorreduplication; DBM: direct bone marrow; BMC: bone marrow culture; SPB: stimuled peripheral blood.

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Table 2 Karyotypes in peripheral blood of children with Down syndrome n = 375

Percentage

Karyotypes

341 11 10 8 2 1 1 1

90.9 2.9 2.7 2.1 0.5 0.3 0.3 0.3

47,XX,+21 or 47,XY,+21 46,XX/47,XX,+21 or 46,XY/47,XY,+21 46,XX,+ der(14;21)(q10; q10) or 46,XY,+der(14;21)(q10;q10) 46,XX,−21,+i (21)(qter → q11::q11 → qter) or 46,XY,−21,+i (21)(qter → q11:: q11 → qter) 46,XX/46,XX−21, + i (21q) (qter → q11::q11 → qter) 46,XY,−13,−14,+ t (13 q; 14 q), +21 46,XY,−11,+ t (11; 21) (11qter → 11p14::21q21 → 21qter) 46,XY, dup (21) (q11; q22)

n: number of cases, dup: duplication. Table 3 Karyotypes in direct bone marrow of children with acute leukemia n = 126

Percentage

Karyotype

98 3 3 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

77.9 2.3 2.3 1.6 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8

46,XX or 46, XY 46,XX,del(22)(pter → q11:) o´ 46, XY, del (22)(pter → q11:) 46,XX[14]/46, XX, del(22)(pter → q11:)[3] 46,XY[15]/46, XY, del (2)(pter → q13:)[3] 46,XY[17]/45, XY,−7[2] 46,XY[13]/45, XY,−19[2] 46,XX[15]/45, XX,−20[1] 46,XX[15]/45,X/45, XX, −8 [1] 46,XY,−7+ mar1[12] 46,XX,−8+ mar1[15] 46,XY,−22+ mar1[10] 46,XX,−22+ mar1[13] 46,XY[17]/69, XXY[3] 46,XY[15]/46,XY,−8,[1]+ mar1/69, XXY[3] 46,XX[16]/92,XXXX[3] 46,XX[22]/35–57,<2n>,XX[23]/end 46,XX[8] 46,XY[19]/46,XX,del (5)(p14 → qter)[2] 46,XX[22]/69,XX,del (22)(pter → q11:)[3] 46,XX[12]/46,XX,-8,−21,t (8q;21q)[2] 46,XX[17]/48,XX,+mar1,+mar2[3] 46,XX[16]/47,XX,−4,−22,+mar1,+ mar2,+mar3[1] 46,XYq+ [20]/48,XYq+,+8,+21[3]/50,XYq+ ,+mar1,+mar2,+mar3,mar4[3] 46,XYq+[16]/45,X[3]/46,XYqs, −13, + ace (13q12 → 13qter)[1] 46,XX[18]/47,XX−7,+7pter → 7p21::7q32 → 7qter,+ 7p21 → 7q32[2]

n: number of cases; del: deletion; mar: marker; ace: acentric fragment.

mosomes (21q) and a duplication that includes the critical region of Down syndrome. Mosaics were also found (Table 2). In the AL group, 77.9% was diagnosed with constitutional normal karyotype and 22.1% with acquired chromosomal alterations (Table 3). The NC presented normal karyotypes without alterations in peripheral blood samples. The parents’ karyotypes were all normal except for a DS/AL child’s father whose karyotype was 46,XY,[20]/47,XY, + mar[1].

3.3. Acquired chromosomal alterations The frequency of acquired chromosomal alterations was higher in the AL group, followed by that of DS/AL, DS and NC (see Table 4). Comparing the number of chromosomal alterations among the groups, significant differences were found (P < 0.0001). The odds ratio (OR) of acquired chromosomal alterations between DS/AL and DS groups was 84.46 (95% CI 44.63–163.42). The OR was 585.62 (95%

Table 4 Frequency of acquired chromosome alteration and gaps, isogaps in the studied group and its control Alteration

DS/AL

DS

AL

NC

Breaks Deletions Rings Markers Hyperdiploids Hypodiploids Euploids Tetraploids Endoreduplications

0.0140 0.0047 0.0010 0.0081 0.0314 0.0163 0 0.0012 0.0233

0.0008 0 0 0 0 0 0 0 0

0.0158 0.0380 0.0015 0.0069 0.1062 0.0143 0.0123 0.0004 0.0138

0.0001 0 0 0.0001 0 0 0 0 0

Total Gaps and isogaps

0.0990 0.0081

0.0008 0.0558

0.2092 0.0164

0.0002 0.0011

P < 0.0001, test of χ2 . DS/AL: Down syndrome children and acute leukaemia; DS: Down syndrome children; AL: acute leukemia children; NC: normal children.

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Table 5 Frequency of constitutive heterochromatin polymorphisms of chromosomes 1, 9, 16,Y and satellites of D and G groups DS/AL (n = 10) Frequency (x/10) Inv 1 1qh− 1qh+ Inv 9 9qh− 9qh+ 16qh− 16qh+ Dsat Gsat Yq− Yq+ Yqsat Total

DS (n = 375) Frequency (x/375)

AL (n = 126) Frequency (x/126)

NC (n = 1000) Frequency (x/1000)

1 (0.10) 2 (0.20) 6 (0.60) 0 1 (0.10) 7 (0.70) 0 3 (0.30) 0 0 0 1 (0.10) 0

1 (0.0026) 6 (0.0160) 7 (0.0186) 18 (0.0480) 3 (0.0080) 25 (0.0666) 1 (0.0026) 6 (0.0160) 24 (0.0640) 38 (0.1013) 9 (0.0240) 28 (0.0746) 2 (0.0053)

0 5 (0.0396) 8 (0.0634) 3 (0.0238) 0 14 (0.1111) 12 (0.0952) 12 (0.0952) 5 (0.0396) 21 (0.1666) 11 (0.0873) 17 (0.1349) 0

1 (0.0010) 33 (0.0330) 39 (0.0390) 7 (0.0070) 7 (0.0070) 52 (0.0520) 12 (0.0120) 15 (0.0150) 25 (0.0250) 53 (0.0530) 15 (0.0150) 47 (0.0470) 0

21 (2.10)

168 (0.4476)

108 (0.8567)

306 (0.3060)

n: number of cases; DS/AL: Down syndrome and acute leukaemia; DS: Down syndrome; AL: acute leukaemia; NC: normal children. P < 0.0001, test of χ2 , x: number cell with polymorphisms.

CI 415.0–815.02) between AL and NC. However, when we compared the group DSA/L and the group AL, the OR was 0.41 (95% CI 0.32–0.53). In a case of DS/AL group, a deletion was observed in the large arm of chromosome 22. This case was associated with the translocation (9;22) (q34;q11) involving oncogenes ABR/BCL demonstrated by the FISH technique. When the frequency of breaks was compared between the groups DS/AL and DS, the OR was 16.57 (95% CI 6.30–44.39). As the AL group was compared to the NC group, the OR was of 107.0 (95% CI 55.9–207.0). The frequency of markers was higher in the group with DS/AL than in the group with DS and the OR was 19.23 (95% CI 5.0–78.0). The OR was 69.61 (95% CI 29.1–169.0) between the group with AL and the NC group. There were deletions only in the groups with DS/AL and with AL. The lower frequency was in the group with SD/LA and the OR was 0.12 (95% CI 0.04–0.34). 3.4. Gaps, isogaps and polymorphisms of the constitutive heterochromatin The gaps and isogaps in the different groups were more frequently found and were statistically significant in the group with only DS (P < 0.0001; Table 4). In the group with DS/AL, some gaps and isogaps were identified in similar frequencies in 1q12, 13q32, 23q11 and 18q23 chromosome bands. They were found in 3p21 in the case of children with DS. For the AL group, the frequency was in decreasing order (6p21 > 1q12 > 10q25). The frequency was 7q22 > 4q31 > 12q13 in NC. The total number of polymorphisms of the constitutive heterochromatin was larger in children with DS/AL than in children with DS and more frequent in children with AL than in NC. The chromosome 1 inversion and 1qh− were only significant when the group with DS/AL was compared to the group with DS, and when the group with DS/AL was compared to NC group. The pericentromeric chromosome

9 inversion was significant in the group with DS compared to NC and 9qh− was only significant between groups with DS/AL and AL. The Dsat polymorphism was only significant between the DS and NC groups. Gsat was significant between AL compared to NC and DS compared to NC; Yq+ was only significant in AL and NC (Table 5).

4. Discussion Mexican children with Down syndrome get leukemia when they are older (11 years) compared to children from other countries. The median age of children with AL was similar to that showed in previous reports [8,11,12]. It is noteworthy that there are biological differences in the development of AL among the groups with DS/AL and with AL. It was expected, for instance, that children with DS developed leukemia at an earlier age [13]. In children with DS/AL as well as children with AL, the alterations found in direct bone marrow (DBM) are different from the identified in bone marrow culture (BMC). The cytogenetic study in DBM is therefore considered the most informative since it avoids the selective effect produced by the mitogens, in this case, phytohemagglutinin [14]. In two children with DS/AL, a deletion in chromosome 21 was identified (Table 1). This fact could lead to the development of leukemia because of the loss of heterozygosity [15,16] or to the progression of the disease due to the loss of the transcription factors AML1 and ERG [14,17,18]. A boy with DS and 47,XY,+21 [98]/47,XY,−7,del14 + t(7;14),+21[1]/46,XY,−7+t(7;14)[1] karyotype developed ALL 3 months after the study and his karyotype was 47,XY,+21[50]/35-57<2n>,XY+21[3]. It is likely that the translocation could be associated as the second event in the development of the leukemia. The father of a child with DS/AL exhibited the 46,XY [20]/47,XY+ mar [1] karyotype. It is not known if the marker

A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246

chromosome may be associated with the development of the leukemia in the boy. The presence of marker chromosome in the father can be also due to the clastogenic effect of ethanol that induce chromosomal alterations [19]. It is also possible that the father had acquired the marker after the child’s birth. The frequency of acquired chromosomal alterations was higher in the group with AL, followed by the group with DS/AL (Table 4). It has been reported that the number of chromosomal alterations is higher in children with DS regarding to normal controls because of the oxidative damage of DNA, and of the potential defect in repairing the damaged DNA [20,21]; it was expected that more acquired alterations would be found in the group with DS/AL. Our findings in the group with DS/AL can be explained by the greater susceptibility of these children to the development of AL, due to special mechanisms, such as the disomic homozygosity, originated by the presence of the additional 21 chromosome [1,22–24]. Ten cases of the group with AL were randomly selected in order to monitor the effects of the group size. This helped to confirm the differences in the frequencies of acquired chromosomal alterations between the group with DS/AL and that with AL. In relation to acquired chromosomal alterations, deletions and hypodiploids were more frequent in the AL group and they had been related to an unfavorable prognosis [11]. The spontaneous gaps and isogaps related to fragile sites found in this study were more frequent in children with DS, followed by children with AL. This finding can be due to the reactive oxygen species, to the oxidative damage of DNA [25] and to the potential defect that children with DS showed in repairing DNA [20,21]. The fragile sites found in children with AL are also the most common in the normal Mexican population and the frequency varies according to the studied population [26,27]. The spontaneous gaps and isogaps were different among the four children groups, but were not related to the fragile sites where oncongenes are relocated in leukemias and other neoplasias [7,26,28,29]. No fragile site that has been associated with the DS condition or with the child’s age [9] was found in this study. It is possible that constitutive heterochromatin polymorphisms have no clinical repercussion. However, a remarkable 1qh+ polymorphism was identified [30] in a case of Chediak–Higashi syndrome, an autosomic recessive disorder which exhibits susceptibility to cancer. In this study, the constitutive heterochromatin polymorphisms (Table 5) were less frequent in normal children than those in other groups. These data are in line with what has been reported in other works [31]. The high frequency of the 9qh+ polymorphism found in the group with DS/AL is similar to other reports and it has been associated with chromosome 21 non-disjunction. It is known that other polymorphisms are also found in some pathologies that include acute leukemia [30,31]. As a result, it can be suggested that the increased constitutive heterochromatin could be related to the presence of a chromosome unbalances.

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In conclusion, the frequency of acquired chromosomal alterations was higher in the AL group followed by the DS/AL group, despite of the small number of samples from the group with DS/LA. This seems to imply that more genetic changes are necessary to develop leukemia in healthy children than in children with DS. Fragile sites were more common in groups with DS than in groups with DS/AL and AL. The polymorphisms of constitutive heterochromatin were higher in groups with DS/AL and AL. Consequently, they could be predisposed to a chromosome unbalance.

Acknowledgement This study was supported by FOFOI from “Instituto Mexicano del Seguro Social”.

References [1] Ross JA, Spector LG, Robison LL, Olshan AF. Epidemiology of leukemia in children with Down syndrome. Pediatr Blood Cancer 2005;44:8–12. [2] Knudson Jr AG. Mutation and cancer: statistical study of retinoblastoma. Proc Natl Acad Sci USA 1971;68:820–3. [3] Ohsaka A, Hisa T, Watanabe N, Kojima H, Nagasawa T. Tetrasomy 21 as a sole chromosome abnormality in acute myeloid leukaemia. Fluorescence in situ hybridization and spectral karyotyping analyses. Cancer Genet Cytogenet 2002;134:60–4. [4] Yamamoto K, Nagata K, Hamaguchi H. A new case of CD7-positive acute myeloblastic leukemia with trisomy 21 as a sole acquired abnormality. Cancer Genet Cytogenet 2002;133:183–4. [5] Salido M, Sole F, Espinet B, Fernandez C, Zamora L, Woessner S, et al. Pentasomy 21 with two isochromosomes 21 in a case of acute myeloid leukemia without maturation. Cancer Genet Cytogenet 2002;132:71–3. [6] Fundia A, Giere I, Larripa I, Slavusky I. Spontaneous breakage and fragile site expression in chronic lymphocytic leukemia. Cancer Genet Cytogenet 1998;103:144–8. [7] Gumus G, Sunguroglu A, Tukun A, Sayin DB, Bokesoy I. Common fragile sites associated with the breakpoints of chromosomal aberrations in hematologic neoplasms. Cancer Genet Cytogenet 2002;133:168–71. [8] Arlt MF, Casper AM, Glover TW. Common fragile sites. Cytogenet Genome Res 2003;100:92–100. [9] Smith A, Borsotto B. Down syndrome ageing and fragile sites. Mech Ageing Dev 1998;16:167–73. [10] Wan TS, Ma SK, Au WY, Chan LC. Derivative (1;18)(q10;q10): a recurrent and novel unbalanced translocation involving 1q in myeloid disorders. Cancer Genet Cytogenet 2001;128:3538. [11] Reynolds P, Von BJ, Elkin EP. Birth characteristics and leukemia in young children. Am J Epidemiol 2002;155:603–13. [12] Wiemels JL, Smith RN, Malcolm TG, Eden OB, Alexander FE, Greaves MF. Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and risk of molecularly defined subtypes of childhood acute leukemia. Proc Natl Acad Sci USA 2001;98:4004–9. [13] Lilleyman JS. Acute lymphoblastic leukemia. Eur J Cancer 1997;33:85–90. [14] Hagemeiser A, Grosueld G. Molecular cytogenetics of leukemia. In: Goasguen JE, editor. Biologic diagnostic of leukemias. 6ath ed. New York: Saunders Company; 1996. p. 131–44. [15] Cline JM. The molecular basis of leukemia. N Engl J Med 1994;330:328–36.

1246

A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246

[16] S´anchez GI. Consequences of chromosomal abnormalities in tumor development. Ann Rev Gene 1997;31:429–53. [17] Kempski HM, Chessells JM, Reeves BR. Deletions of chromosome 21 restricted to the leukemic cell of children with Down syndrome and leukemia. Leukemia 1997;11:1973–7. [18] Look TA. Oncogenic transcription factors in the human acute leukemias. Science 1997;278:1059–64. [19] Madrigal BE, Pi˜na A, Vidal P, Rubio D. Presencia de alteraciones citogen´eticas en pacientes con alcoholismo. Rev Patol Clin 1990;37:33–6. [20] Ankathil R, Kusumakumary P, Nair MK. Increased levels of mutagen-induced chromosome breakage in Down syndrome children with malignancy. Cancer Genet Cytogenet 1997;99:126–8. [21] Jovanovic SV, Clements D, MacLeod K. Biomarkers of oxidative stress are significantly elevated in Down syndrome. Free Radic Biol Med 1998;25:1044–8. [22] Feingold E, Lamb NE, Sherman SL. Methods for genetic linkage analysis using trisomies. Am J Hum Genet 1995;56:475–83. [23] Niikawa N, Deng HX, Abe K, Harada N, Okada T, Tsuchiya H, et al. Possible mapping of the gene for transient myeloproliferative syndrome at 21q11.2. Hum Genet 1991;87:561–6. [24] Shen JJ, Williams BJ, Zipursky A, Doyle J, Sherman SL, Jacobs PA, et al. Cytogenetic and molecular studies of Down syn-

[25]

[26]

[27] [28]

[29]

[30]

[31]

drome individuals with leukemia. Am J Hum Genet 1995;56:915– 25. Carratelli M, Porcaro L, Ruscica M, De Simone E, Bertelli AA, Corsi MM. Reactive oxygen metabolites and prooxidant status in children with Down’s s´ındrome. Int J Clin Pharmacol Res 2001;21: 79–84. Sutherland GR. Hereditable fragile sites on human chromosomes. XII. Population cytogenetics. Ann Hum Genet 1985;49(Part 2):153–61. Simonic I, Gericke SG. The enigma of common fragile sites. Hum Genet 1996;97:524–31. Coquelle A, Pipiras E, Toledo P, Buttin G, Debatisse M. Expression of fragile sites triggers intrachromosomal mammalian gene amplification and sets boundaries to early amplicans. Cell 1997;89:215–25. Debatisse M, Coquelle A, Toledo P, Buttin G. Gene amplification mechanisms: the role of fragile sites. Recent Results Cancer Res 1998;154:216–26. Salamanca F, Salazar M, Amezcua M. Chromosome one polymorphism in a girl with the Chediak–Higashi syndrome. Acta Cytol 1978;2:402–6. Tsezou A, Kitsiou T, Kosmidis H, Paidousi K, Katsouyanni K, Sinaniotis C. Constitutive heterochromatin polymorphisms in children with acute lymphoblastic leukemia. Pediatr Hematol Oncol 1993;10:7–11.