Leukemia Research 29 (2005) 1241–1246

Cytogenetic studies in children with Down syndrome and acute leukemia

Adan Valladaresa, Virginia Palma-Padillaa, Juan Manuel Mejıa-Arangureb,Roberto Guevara-Yaneza, Azucena Lerma-Reyesa, Fabio Salamanca-Gomeza,∗

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 2005Available 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 acquiredchromosome abnormalities was larger in the AL group, followed by the DS/AL. The gaps and isogaps were more frequent in children witho that moreg©

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eywords:Down syndrome; Acute leukemia; Chromosomal alterations; Gaps; Polymorphisms; Constitutive heterochromatin; Two mutational eve

. Introduction

Children with Down syndrome (DS) have an elevated riskf developing acute leukemia (AL)—10–20 times higher than

he general population[1]. According to the Knudson car-inogenesis model, two mutational events are required forhe development of cancer[2]. The presence of an additional1 chromosome has been reported as a sole acquired abnor-ality in patients with leukemia and normal constitutional

aryotype[3–5]. Nevertheless, the carcinogenic role of theenes located in this chromosome is not well known.

The increased frequency of fragile sites in chronic lym-hocytic leukemia suggests that these could be related tohromosomal alterations[6]. Fragile sites may play a signif-cant role in chromosomal rearrangements involved in onco-ene activation or tumor suppressor gene inactivation[7].ther studies clearly demonstrated the relevance of genome

nstability in tumorigenesis[8]. Besides, fragile sites have

∗ Corresponding author at: Apartado Postal 12-951, Mexico, DF 03020,exico. Tel.: +52 55 56276945; fax: +52 55 57610952/55885174.

been associated with Down syndrome[9]. On the other handwhether polymorphisms of the constitutive heterochromaffect the development of AL or the presence of Down sdrome has not been established yet. A cytogenetic stuchildren with DS with AL and its controls was performe(i) assuming that the 21 trisomy could be the first mutatievent in the development of acute leukemia in childrenDown syndrome; and (ii) considering that chromosomeslarge constitutive heterochromatin regions, such as chrsome number 1, may be at risk of centromeric instaband be predisposed to centromeric fusion with other chrosomes[10]. This cytogenetic study aims to determine the cstitutional chromosomal alterations and the polymorphof the constitutive heterochromatin associated to leukeand consequently to understand the mechanisms involvthe susceptibility to cancer.

2. Materials and methods

Karyotypes were performed in four groups of childr

E-mail address:fasalam@prodigy.net.mx (F. Salamanca-Gomez). 10 children with DS and AL (DS/AL); 375 children with

145-2126/$ – see front matter © 2005 Published by Elsevier Ltd.oi:10.1016/j.leukres.2005.03.015

1242 A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246

DS; 126 children with AL and 1000 healthy children (NC) ascontrols. Karyotypes were also carried out in all the parentsof children with DS/AL and in the parents of children withbalanced translocation or chromosome rearrangements foundin the control groups.

Seven out of nine public institutions who take care of chil-dren with cancer in Mexico City took part in this study. Inaddition, these institutions look after 95% of all children whodevelop cancer in Mexico City. All the cases were also di-agnosed by cytochemistry analysis of bone marrow aspiratesand 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 treatchildren with DS, such as the Siglo XXI Pediatric Hospital.When any child of this institution developed leukemia, he re-ceived attention in any of the hospitals that participated in thisstudy. The Siglo XXI Pediatric Hospital is located in Mex-ico City and receives children with DS from any part of thecity. The healthy children belong to second-level hospitals inMexico City.

The peripheral blood samples or bone marrow were ob-tained from patients receiving no pharmaceutical or radio-logical treatment and neither having blood transfusions orinfections. All of them gave their previous written consent.The peripheral blood samples and bone marrow were culturedfor 72 h in RPMI with phytohemagglutinin. The bone mar-r orre-s nics ce ins Vy-

sis Inc.), were also carried out. The spontaneous gaps andisogaps 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 ethicalcommittees of the National Medical Center.

3. Results

3.1. Subjects

The median age of the group of children with DS/ALhaving leukemia was 11 years, but the median was 5 years(P= 0.039) for children that only presented AL. The genderrelation did not show any statistical significance (P= 0.91).Males were 5/10, 203/375, 71/126 and 529/1000 in the groupsof DS/AL, DS, AL and NC, respectively. The most frequenttype of leukemia in DS/AL and AL groups was ALL (8/10and 113/126, respectively). There was no significant differ-ent 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 constitutionalr hro-mo tri-s chro-

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ow samples were also processed without cultivation, cponding to direct bone marrow (DBM), and with mitogetimulus (BMC). The GTG and C bands, and fluorescenitu hybridization (FISH) to Philadelphia chromosome (

able 1aryotypes in the children with Down syndrome and acute leukemia

= 10 Sample Karyotypes

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

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

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

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

DBM 47,XY,+21[14]/52,XY,+21[10]/48,XY,+21+maBMC 47,XY,+21[10]SPB 47,XY,+21[50]

SPB 47,XY,+21[106]/46,XY[4]/48,XY,+21,+ t (9;2248,XY, +21,+mar1[1]/55,XY,+21[1]

SPB 47,XY,+21[115]/48,XY,+mar (r) [1]/47,XY,+de

DBM 47,XX,+21[75]/35–57<2n>,XX,+21[3]/94,XXXBMC 47,XX,+21[18]/44,XX,−6,−21[1]/47,XX,del(21SPB 47,XX,+21[19]DBM 47,XX,+21[70]/end 47,XX, +21[20]/35–57<2n

0 DBM 47,XY,+21[10]BMC 47,XY, +21[100]SPB 47,XY, +21[100]

: number of cases; mar: marker; end: endorreduplication; DBM: direct bone

egular trisomy and 5 out of 10 presented additional cosome rearrangements (Table 1). In the DS group, 90.9%f children were identified with constitutional regularomy, plus unbalanced translocations (2.7%), eight iso

; q11)[3]/ 94,XXYY, +21,+21,[2]/47,XY,+21,+ t (9;22) (q34; q11),−1[3]/

q21.2)[10]

3,XX,−7,−8,−21[1]q22:)[3]

21[10]/35–57<2n>, XX,+21,+22,+mar1[1]

marrow; BMC: bone marrow culture; SPB: stimuled peripheral blood.

A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246 1243

Table 2Karyotypes in peripheral blood of children with Down syndrome

n= 375 Percentage Karyotypes

341 90.9 47,XX,+21 or 47,XY,+2111 2.9 46,XX/47,XX,+21 or 46,XY/47,XY,+2110 2.7 46,XX,+ der(14;21)(q10; q10) or 46,XY,+der(14;21)(q10;q10)8 2.1 46,XX,−21,+i (21)(qter→ q11::q11→ qter) or 46,XY,−21,+i (21)(qter→ q11:: q11→ qter)2 0.5 46,XX/46,XX−21, + i (21q) (qter→ q11::q11→ qter)1 0.3 46,XY,−13,−14,+ t (13 q; 14 q), +211 0.3 46,XY,−11,+ t (11; 21) (11qter→ 11p14::21q21→ 21qter)1 0.3 46,XY, dup (21) (q11; q22)

n: number of cases, dup: duplication.

Table 3Karyotypes in direct bone marrow of children with acute leukemia

n= 126 Percentage Karyotype

98 77.9 46,XX or 46, XY3 2.3 46,XX,del(22)(pter→ q11:)o 46, XY, del (22)(pter→ q11:)3 2.3 46,XX[14]/46, XX, del(22)(pter→ q11:)[3]2 1.6 46,XY[15]/46, XY, del (2)(pter→ q13:)[3]1 0.8 46,XY[17]/45, XY,−7[2]1 0.8 46,XY[13]/45, XY,−19[2]1 0.8 46,XX[15]/45, XX,−20[1]1 0.8 46,XX[15]/45,X/45, XX,−8 [1]1 0.8 46,XY,−7+ mar1[12]1 0.8 46,XX,−8+ mar1[15]1 0.8 46,XY,−22+ mar1[10]1 0.8 46,XX,−22+ mar1[13]1 0.8 46,XY[17]/69, XXY[3]1 0.8 46,XY[15]/46,XY,−8,[1]+ mar1/69, XXY[3]1 0.8 46,XX[16]/92,XXXX[3]1 0.8 46,XX[22]/35–57,<2n>,XX[23]/end 46,XX[8]1 0.8 46,XY[19]/46,XX,del (5)(p14→ qter)[2]1 0.8 46,XX[22]/69,XX,del (22)(pter→ q11:)[3]1 0.8 46,XX[12]/46,XX,-8,−21,t (8q;21q)[2]1 0.8 46,XX[17]/48,XX,+mar1,+mar2[3]1 0.8 46,XX[16]/47,XX,−4,−22,+mar1,+ mar2,+mar3[1]1 0.8 46,XYq+ [20]/48,XYq+,+8,+21[3]/50,XYq+,+mar1,+mar2,+mar3,mar4[3]1 0.8 46,XYq+[16]/45,X[3]/46,XYqs,−13, + ace (13q12→ 13qter)[1]1 0.8 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 re-gion of Down syndrome. Mosaics were also found (Table 2).In the AL group, 77.9% was diagnosed with constitutionalnormal karyotype and 22.1% with acquired chromosomalalterations (Table 3). The NC presented normal karyotypeswithout alterations in peripheral blood samples. The parents’karyotypes were all normal except for a DS/AL child’s fatherwhose karyotype was 46,XY,[20]/47,XY, + mar[1].

3.3. Acquired chromosomal alterations

The frequency of acquired chromosomal alterations washigher in the AL group, followed by that of DS/AL, DS andNC (seeTable 4). Comparing the number of chromosomalalterations among the groups, significant differences werefound (P< 0.0001). The odds ratio (OR) of acquired chro-mosomal alterations between DS/AL and DS groups was84.46 (95% CI 44.63–163.42). The OR was 585.62 (95%

Table 4Frequency of acquired chromosome alteration and gaps, isogaps in the stud-ied group and its control

Alteration DS/AL DS AL NC

Breaks 0.0140 0.0008 0.0158 0.0001Deletions 0.0047 0 0.0380 0Rings 0.0010 0 0.0015 0Markers 0.0081 0 0.0069 0.0001Hyperdiploids 0.0314 0 0.1062 0Hypodiploids 0.0163 0 0.0143 0Euploids 0 0 0.0123 0Tetraploids 0.0012 0 0.0004 0Endoreduplications 0.0233 0 0.0138 0

Total 0.0990 0.0008 0.2092 0.0002Gaps and isogaps 0.0081 0.0558 0.0164 0.0011

P< 0.0001, test ofχ2. DS/AL: Down syndrome children and acuteleukaemia; DS: Down syndrome children; AL: acute leukemia children;NC: normal children.

1244 A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246

Table 5Frequency of constitutive heterochromatin polymorphisms of chromosomes 1, 9, 16,Y and satellites of D and G groups

DS/AL (n= 10) DS (n= 375) AL (n= 126) NC (n= 1000)Frequency (x/10) Frequency (x/375) Frequency (x/126) Frequency (x/1000)

Inv 1 1 (0.10) 1 (0.0026) 0 1 (0.0010)1qh− 2 (0.20) 6 (0.0160) 5 (0.0396) 33 (0.0330)1qh+ 6 (0.60) 7 (0.0186) 8 (0.0634) 39 (0.0390)Inv 9 0 18 (0.0480) 3 (0.0238) 7 (0.0070)9qh− 1 (0.10) 3 (0.0080) 0 7 (0.0070)9qh+ 7 (0.70) 25 (0.0666) 14 (0.1111) 52 (0.0520)16qh− 0 1 (0.0026) 12 (0.0952) 12 (0.0120)16qh+ 3 (0.30) 6 (0.0160) 12 (0.0952) 15 (0.0150)Dsat 0 24 (0.0640) 5 (0.0396) 25 (0.0250)Gsat 0 38 (0.1013) 21 (0.1666) 53 (0.0530)Yq− 0 9 (0.0240) 11 (0.0873) 15 (0.0150)Yq+ 1 (0.10) 28 (0.0746) 17 (0.1349) 47 (0.0470)Yqsat 0 2 (0.0053) 0 0

Total 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 ofχ2, x:number cell with polymorphisms.

CI 415.0–815.02) between AL and NC. However, when wecompared the group DSA/L and the group AL, the OR was0.41 (95% CI 0.32–0.53).

In a case of DS/AL group, a deletion was observed inthe large arm of chromosome 22. This case was associatedwith the translocation (9;22) (q34;q11) involving oncogenesABR/BCLdemonstrated by the FISH technique.

When the frequency of breaks was compared betweenthe groups DS/AL and DS, the OR was 16.57 (95% CI6.30–44.39). As the AL group was compared to the NC group,the OR was of 107.0 (95% CI 55.9–207.0). The frequency ofmarkers was higher in the group with DS/AL than in the groupwith DS and the OR was 19.23 (95% CI 5.0–78.0). The ORwas 69.61 (95% CI 29.1–169.0) between the group with ALand the NC group. There were deletions only in the groupswith DS/AL and with AL. The lower frequency was in thegroup with SD/LA and the OR was 0.12 (95% CI 0.04–0.34).

3.4. Gaps, isogaps and polymorphisms of theconstitutive heterochromatin

The gaps and isogaps in the different groups were morefrequently found and were statistically significant in thegroup with only DS (P< 0.0001; Table 4). In the groupwith DS/AL, some gaps and isogaps were identified in sim-ilar frequencies in 1q12, 13q32, 23q11 and 18q23 chromo-s chil-d de-c was7

tiveh inc Lto redt asc ome

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

4. Discussion

Mexican children with Down syndrome get leukemiawhen they are older (11 years) compared to children fromother countries. The median age of children with AL wassimilar to that showed in previous reports[8,11,12]. It isnoteworthy that there are biological differences in the devel-opment of AL among the groups with DS/AL and with AL. Itwas expected, for instance, that children with DS developedleukemia at an earlier age[13].

In children with DS/AL as well as children with AL, thealterations found in direct bone marrow (DBM) are differ-ent from the identified in bone marrow culture (BMC). Thecytogenetic study in DBM is therefore considered the mostinformative since it avoids the selective effect produced bythe mitogens, in this case, phytohemagglutinin[14].

In two children with DS/AL, a deletion in chromo-some 21 was identified (Table 1). This fact could leadt s ofh is-e L1a 1[k andh 3].I thes

Y[ er

ome bands. They were found in 3p21 in the case ofren with DS. For the AL group, the frequency was inreasing order (6p21 > 1q12 > 10q25). The frequencyq22 > 4q31 > 12q13 in NC.

The total number of polymorphisms of the constitueterochromatin was larger in children with DS/AL thanhildren with DS and more frequent in children with Ahan in NC. The chromosome 1 inversion and 1qh− werenly significant when the group with DS/AL was compa

o the group with DS, and when the group with DS/AL wompared to NC group. The pericentromeric chromos

o the development of leukemia because of the loseterozygosity[15,16] or to the progression of the dase due to the loss of the transcription factors AMnd ERG [14,17,18]. A boy with DS and 47,XY,+2

98]/47,XY,−7,del14 + t(7;14),+21[1]/46,XY,−7+t(7;14)[1]aryotype developed ALL 3 months after the studyis karyotype was 47,XY,+21[50]/35-57<2n>,XY+21[

t is likely that the translocation could be associated asecond event in the development of the leukemia.

The father of a child with DS/AL exhibited the 46,X20]/47,XY+ mar[1] karyotype. It is not known if the mark

A. Valladares et al. / Leukemia Research 29 (2005) 1241–1246 1245

chromosome may be associated with the development of theleukemia in the boy. The presence of marker chromosome inthe father can be also due to the clastogenic effect of ethanolthat induce chromosomal alterations[19]. It is also possiblethat the father had acquired the marker after the child’s birth.

The frequency of acquired chromosomal alterations washigher in the group with AL, followed by the group withDS/AL (Table 4). It has been reported that the number ofchromosomal alterations is higher in children with DS re-garding to normal controls because of the oxidative damageof DNA, and of the potential defect in repairing the damagedDNA [20,21]; it was expected that more acquired alterationswould be found in the group with DS/AL. Our findings inthe group with DS/AL can be explained by the greater sus-ceptibility of these children to the development of AL, dueto 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 randomlyselected in order to monitor the effects of the group size.This helped to confirm the differences in the frequencies ofacquired chromosomal alterations between the group withDS/AL and that with AL.

In relation to acquired chromosomal alterations, deletionsand hypodiploids were more frequent in the AL group andthey had been related to an unfavorable prognosis[11].

The spontaneous gaps and isogaps related to fragile sitesf S,f tot DNA[ wedi

them fre-qT ng thef itesw r neop tedwi

or-p ark-a fC isor-d thecl ups.T otherw smf ndi ction.I omep ,i chro-m e un-b

In conclusion, the frequency of acquired chromosomal al-terations was higher in the AL group followed by the DS/ALgroup, despite of the small number of samples from the groupwith DS/LA. This seems to imply that more genetic changesare necessary to develop leukemia in healthy children than inchildren with DS. Fragile sites were more common in groupswith DS than in groups with DS/AL and AL. The polymor-phisms of constitutive heterochromatin were higher in groupswith DS/AL and AL. Consequently, they could be predis-posed to a chromosome unbalance.

Acknowledgement

This study was supported by FOFOI from “Instituto Mex-icano del Seguro Social”.

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