Background
Acute leukemia (AL) is one of the most common malignancies of early childhood. Leukemias in infants (≤12 months) (IL), even being rare, are recurrently studied because they are associated with a high frequency of early death during the first months of life. Despite advances in most other age groups, the prognosis of infants remains poor [
1,
2]. Therefore, understanding the contributing factors that lead to the emergence of early age leukemia (EAL) represents a major opportunity of prevention. Contributing events include chance, exposure to genotoxic substances, and inherited genetic susceptibility.
Epidemiological and molecular studies have already demonstrated that critical molecular lesions, such as the frequently observed
MLL gene rearrangements (
MLL-r) in IL, occur
in utero in early hematopoietic precursors [
3,
4]. Maternal exposures during pregnancy seem to be associated with the onset of EALs [
5‐
7]. Many attempts to identify inherited susceptibility in childhood leukemia (as a whole) have been made [
8] and some studies have already focused on EAL [
9‐
13]. Common allelic variants in
IKZF1 (7p12.2),
ARID5B (10q21.2), and
CEBPE (14q11.2), which are directly related to hematopoietic differentiation and development, have been repeatedly and significantly associated with childhood acute lymphoblastic leukemia (ALL). Of interest, Xu
et al. presented convincing evidence for modifying effects of genetic
ARID5B variants; in particular these data consistently show a trend for increasing allelic odds ratio as age decreased and the risk varied substantially by ethnicity [
14]. We have made similar observations with age-dependent susceptibility and leukemia emergence EAL [
13]. However, the extent to which germline variations contribute to the acquisition of somatic aberrations that define AL subtypes is yet unknown.
Therefore, we genotyped common variants in IKZF1, ARID5B, and CEBPE in a series of children enrolled in the Brazilian Collaborative Study Group of Infant Acute Leukemia (BCSGIAL) in order to evaluate the frequencies of these inherited polymorphisms and determine their associations by (i) age strata (infants versus children aged between 13 and 24 months); (ii) MLL status and/or type of MLL-r; and (iii) ethnic background. From our data we conclude that distinct ARID5B rs10821936 polymorphism represents a novel risk factor to the acquisition of somatic mutation as it increases the risk to acquired MLL-r in EAL.
Results
The call rate for IKZF1, ARID5B rs10821936 and rs10994982, and CEBPE was respectively 247 of 265 (93.2%), 244 of 265 (92.1%), 246 of 265 (92.8%), and 251 of 265 (94.7%) in the investigated cases. The call rate for each SNP was ≥94% in the control groups. Control genotypes for all four SNPs loci were in Hardy-Weinberg equilibrium (P > 0.05).
The demographic characteristics of cases and controls are shown in Additional file
1: Table S1. There were no statistical differences among cases and controls regarding gender, ethnicity or children age range. The
MLL status was established for 149 ALL and 86 AML patients. The analysis of genomic breakpoints by LDI-PCR within the
MLL breakpoint cluster region was performed in a subset of 55
MLL-r with available biological material and successfully determined in 41 cases.
The distribution of allele frequencies among controls and cases within the major acute leukemia subtypes has been evaluated and the results are shown in Additional file
2: Table S2. The risk of developing the pro-B ALL phenotype was increased for patients with the variant allele of
ARID5B rs10821936 (OR 2.54, 95% CI: 1.36-4.70). Increased risks of developing c-ALL (CD10 positive) have been observed for patients with variant alleles of
ARID5B rs10821936 (OR 2.63, 95% CI: 1.41-4.90) and rs10994982 (OR 3.13, 95% CI: 1.24-7.95). Among patients with AML, an increased risk has been observed for those patients with the homozygous variant of
ARID5B rs10821936 (OR 2.39, 95% CI: 1.10-5.17).
The distributions of allele frequencies in controls and cases and the risk association between genetic variants and acute leukemia further stratified by skin color and by
MLL status are displayed in Additional file
3: Table S3. In overall cases, white and non-white children presented similar risk associations. The heterozygous genotype in
ARID5B rs10821936 increased the risk for
MLL-r leukemia in both white and non-white (OR 2.06, 95% CI: 1.12-3.79 and OR 2.36, 95% CI: 1.09-5.10, respectively). The mutant genotype in
ARID5B SNP rs10821936 significantly increased the risk for
MLL-germline leukemia in white and non-white children (OR 2.69, 95% CI: 1.28-5.66 and OR 3.69, 95% CI: 1.57-8.68, respectively). The heterozygous/mutant genotype in the other
ARID5B rs10994982 also significantly increased the risk for
MLL-germline leukemia in white and non-white children (OR 2.60, 95% CI: 1.09-6.18 and OR 3.55, 95% CI: 1.57-8.68, respectively).
When comparing the ALL cases by age strata (infants
versus children aged between 13 and 24 months), white children with ALL of both age groups presented with an increased risk for
MLL-germline leukemia associated with the heterozygous/mutant genotypes
IKZF1 (OR 5.57, 95% CI: 1.39-22.24 and OR 2.58, 95% CI: 1.02-6.51, respectively). The heterozygous genotype in
ARID5B rs10821936 increased the risk for
MLL-r ALL in both white and non-white infants (OR 2.19, 95% CI: 1.07-4.49 and OR 3.82, 95% CI: 1.21-12.12, respectively), while for children aged between 13–24 months the mutant genotype significantly increased the risk for ALL in white children, regardless the
MLL status (OR 7.11, 95% CI: 2.07-24.45 for
MLL-germline; OR 7.91, 95% CI: 1.47-42.46 for
MLL-r) (Additional file
3: Table S3).
In AML, the only increased risk association was observed among non-white
MLL-r cases with the
ARID5B rs10821936 mutant genotype (OR 4.82, 95% CI: 1.50-15.50), while the
CEBPE variant allele was negatively associated with
MLL-germline AML (OR 0.22, 95% CI: 0.07-0.72) (Additional file
3: Table S3).
The SNPs risk associations between acute leukemia and
MLL status are also shown after statistical adjustment on age and on skin color (Additional file
4: Table S4). The results corroborate with those obtained after stratification, showing that
IKZF1 and
ARID5B rs10994982 variant alleles play a role in the susceptibility to
MLL-germline leukemia while
ARID5B rs10821936 confers increased risk to both
MLL-germline and
MLL-r leukemia.
Because the variant
ARID5B rs10821936 allele was remarkably associated with an increased risk of
MLL-r acute leukemia, we tested whether this risk allele was associated to a specific
MLL TPG or to any of the frequent
MLL breakpoint regions. The risk association between
ARID5B rs10821936 and
MLL-r acute leukemia according to the TPGs and
MLL breakpoint regions compared with controls is shown in Table
1. The individuals with heterozygous/mutant genotype had a higher risk of developing
MLL-AFF1 positive leukemia (OR 2.79, 95% CI: 1.27-6.11) and even higher odds of
MLL-MLLT3 positive leukemia (OR 7.10, 95% CI: 1.54-32.68). Moreover, this increased risk magnitude was also observed for individuals with
MLL breakpoints non-located in
MLL intron 11 (OR 10.25, 95% CI: 2.24-46.81). A multivariate analysis has been performed to address whether the
MLLT3 TPG and the
MLL breakpoint region (exon 9-intron 10) were variables dependent on each other. The results showed that the susceptibility risk of having the
MLL breakpoint localized outside of
MLL intron 11 [(OR 0.88, 95% CI: 0.34–2.30),
P = 0.79] and the
MLLT3 as the TPG [(OR 1.49, 95% CI: 0.86–2.58),
P = 0.15] is cross-dependent.
Table 1
The risk associations between
ARID5B
rs10821936 genotype and
MLL
translocation partner genes or
MLL
breakpoint region, Brazil, 2003-2013
ARID5B
| | | | | | | | | | | |
rs10821936 | | | | | | | | | | | |
TT | 200 | 11 | 1.00 | 6 | 1.00 | 2 | 1.00 | 2 | 1.00 | 5 | 1.00 |
TC | 205 | 27 |
3.26 (1.42-7.49)
| 11 | 2.82 (0.89-8.93) | 12 |
8.62 (1.77-41.94)
| 17 |
12.76 (2.66-61.23)
| 12 |
3.45 (1.08-11.05)
|
CC | 68 | 6 | 1.95 (0.62-6.11) | 4 | 2.12 (0.52-8.63) | 3 | 5.18 (0.79-34.02) | 4 |
6.65 (1.08-41.15)
| 1 | 0.85 (0.09-8.25) |
TC + CC | 273 | 33 |
2.79 (1.27-6.11)
| 15 | 2.53 (0.89-7.22) | 15 |
7.10 (1.54-32.68)
| 22 |
10.25 (2.24-46.81)
| 14 | 2.72 (0.88-8.43) |
We further tested the effect of cumulative variant alleles of
IKZF1,
ARID5B and
CEBPE in the risk susceptibility to EAL (Additional file
5: Table S5). Patients harboring 6–8 variant alleles had significant increased risk to develop ALL older than 12 months-old (OR 1.34, 95% CI: 1.09-1.66) or
MLL-germline leukemia (OR 1.33, 95% CI: 1.06-1.67). However, we could not observe a trend for increasing ORs as the number of risk alleles increased.
Discussion
The molecular epidemiological approach in several genetic studies has raised the concept that most, if not all, childhood leukemia cases originate
in utero[
4]. Previous evidences suggested that the causality factors are likely to be multiple and leukemia subtype-specific, combining both genetic susceptibility and environmental exposures [
17]. Moreover, whether and how the inherited gene variants contribute to the acquisition of the
in utero-acquired somatic alterations frequently found in EAL must be explored.
In this case–control study, we genotyped known susceptibility loci (
IKZF1,
ARID5B, and
CEBPE) in a series of children enrolled in the BCSGIAL. We observed an increased magnitude of ALL risk for children with SNPs in
IKZF1 and
ARID5B. This is expected from the previous genome wide association studies (GWAS) that have been performed in childhood ALL (peak incidence 2–5 years-old) [
18,
19]. Our data do not show evidence that
CEBPE rs2239633 confers increased genetic susceptibility to EAL, in agreement with previous data in IL [
12]. In a recent GWAS,
CEBPE SNPs were strongly related to ALL risk in European Americans, with variable effects in non-European populations [
14]. This result could explain the lack of association in our population.
IKZF1 rs11978267 was associated with the increased risk of
MLL-germline ALL in both infants and older children consistent with results found in previous settings of childhood ALL. Different from ours, the only previous study that has also addressed involvement of
IKZF1 polymorphism in AML has found a contribution of rs11978267 to susceptibility in infant AML overall, irrespective of
MLL-r [
12]. However, because of the differences in number of cases and ethnicity among studied populations, it is difficult to draw conclusions from this comparison. Therefore, further studies focusing on AML will be necessary to verify the
IKZF1 susceptibility role in EAL. As this is an extremely rare disease, pooling studies would be of great interest.
ARID5B gene variants have been systematically shown to increase the risk of childhood ALL in various populations [
14,
18‐
23]. Most of these studies showed that this risk was associated to B-cell precursor ALL, and some of them could distinguish B-hyperdiploid ALL from other subtypes [
18,
19,
24]. This association with B-hyperdiploid ALL has not been reproduced in all studies [
25]. Overall, the
ARID5B gene variants were strongly associated with the risk of EAL in this Brazilian series. This gene encodes a member of the AT-rich interaction domain (ARID) family of DNA binding proteins. The encoded protein forms a histone H3K9
me2 demethylase complex together with PHD finger protein 2 to regulate the transcription of target genes involved in adipogenesis and liver development [
26]. An increased risk of
ARID5B variants in AML had not been reported previously. The gene expression level of
ARID5B is up-regulated in two different AML subtypes (acute megakaryoblastic and promyelocytic leukemia) [
27,
28]. Acute megakaryoblastic leukemia is more frequent in EAL AML opposite to promyelocytic leukemia [
29,
30]. Therefore, it is conceivable that
ARID5B contributes to susceptibly to EAL AML, and an ongoing case–control study is currently underway to answer this question [
31].
The
ARID5B rs10994982 has only significantly increased the risk in
MLL germline children, in agreement with observations in childhood [
18,
19] and IL [
12]. We observed a major and wider spectrum of risk increase for
ARID5B rs10821936. This is consistent with previously mentioned studies, as this specific SNP has been strongly associated with risk across several populations and leukemia subgroups. In our study, the rs10821936 increased the risk for both
MLL wild-type and
MLL-r ALL and
MLL-r AML patients. One of the most significant findings from this study is that
ARID5B rs10821936 not only differed between EAL and control groups but also distinguished
MLL-MLLT3 positive leukemias from other
MLL-r. Interestingly, a strong association could be observed both by analyzing the TPG (
MLLT3) and the breakpoint location of
MLL (mainly intron 9), and the multivariate model confirmed that these parameters were dependent on each other. Recently, the
MLL recombinome analysis pointed out different tendencies concerning the breakpoints localization when it was analyzed breakpoint distributions together with TPGs [
32]. For that study, the
MLL breakpoint cluster region was subdivided into 3 sub regions (A, exon 9 - intron 9; B, exon 10 - intron 10; C, exon 11 - intron 12). The observed ‘mean breakpoint frequencies’ for these 3 regions in South America (dataset includes our Brazilian samples) was A = 31.9%, B = 21.7%, and C = 43.5%. However, when separating by
MLLT3 TPG and restricted to the infants subgroup, the MBPF was A = 41.8%, B = 13.3%, and C = 42.9%, while in pediatric and adults these ‘mean breakpoint frequencies’ were: 35.7%, 18.8%, 43.8% and 34.2%, 7.59%, 57.0%, respectively. Therefore, recombination affecting
MLLT3 displayed a tendency for
MLL intron 9 breaks in IL. Together, all these data are concordant with our finding that increased risk susceptibility in infants is associated with
MLL-MLLT3 rearrangement. Although future studies will be necessary to confirm this finding and to understand the specific role of this SNP in the pathogenesis, the availability of such rare epidemiological set of cases prompted us to suggest an association between inherited gene variants and specific somatic aberrations in the pathogenesis of
MLL-r EAL.
There are limitations in this present analysis. First, the small number of cases after some subsets stratification raises concern with regards to statistical power. However, given the rarity of this disease, one should consider that the consistency of the associations observed, and the concordance with previously published data indicate good validity and sensitivity of our study. Second, we had missing genotyping calls in some cases and controls that precluded us to have all samples screened uniformly. However, an acceptable call rate has been achieved in either cases or controls and the frequencies obtained did not present any deviation.
We can also mention some study strengths. As replication of GWAS is highly desirable, this is an important contribution of the present Brazilian work, especially because the studies have been so far concentrated to European and American populations. For example, validation sequencing of this ARID5B genomic region has been requested in order to reveal the exact nature of the differences previously observed. Moreover, this report focus on EAL and particularly those harboring MLL-r, and in this context, this study is innovative.
Acknowledgements
We are grateful to the children and their parents for participating in the study. This investigation was supported by the Brazilian National Research Council (CNPq) and Instituto Nacional de Câncer (INCA). The project was partially funded by grants from INCT-Controle do Cancer (CNPq #573806/2008-0, FAPERJ#E026/170.026/2008 and FAPERJ#E-26/110. 509/2010; FAPERJ#E-26/110.823/2012). MSPO has been supported by CNPq research scholarships (#309091/2007) and FAPERJ (#E026/101.562/2010). ME has been supported by Brazilian Ministry of Health through the Institutional Development Program Scholarship.
The Brazilian Collaborative Study Group of Infant Acute Leukemia listed as co-authors and Brazilian Institutions:
Appendix
Co-authors: Institutions
Isis Quesado Magalhães, José Carlos Cordoba: Hospital da Criança de Brasília, DF (n, 24)
Theresa Christina Lafayette, Virginia Maria Cóser: Hospital Universitário de Santa Maria (n,24)
Maria D Dorea, Flavia N.Serafim Araujo, Lilian Maria Burlachini: Sociedade de Oncologia da Bahia-Salvador, BA (n,21)
Andrea Gadelha Nobrega, Eloisa C. E. Fialho, Flavia Cristina F. Pimenta, Glaceanne Torres da Luz Mamede: Hospital Napoleão Laureano- João Pessoa, PB (n,19)
Terezinha de Jesus Marques Salles: Hospital Universitário Oswaldo Cruz -CEON, Universidade de Pernambuco (n,17)
Jane Dobbin, Alexandre Apa: Hospital do Câncer –INCA, Rio de Janeiro, RJ (n,17)
Silvia Brandalise, Vitória R. Pinheiro: Centro Infantil Dr. Domingos Boldrini, Campinas, SP (n,17)
Adriana Martins de Souza, Soraya Rouxinol: Instituto Pediatria Puericultura Martagão Gesteira -Rio de Janeiro, RJ (n,16)
Eny G. Carvalho, Ana M. Marinho da Silva, Jozina M. de Andrade Agareno: Hospital Martagão Gesteira-Salvador, BA (n,14)
Mara A. D. Pianovski, Tiago Hessel Tormen: Hospital de Clinicas - Curitiba, PR (n,14)
Patricia Carneiro de Brito, Loretta S.C. Oliveira: Hospital Araujo Jorge - Goiânia, GO (n,14)
Imarui Costa, Denise Bousfield: Hospital Infantil Joana de Gusmão -Florianópolis, SC (n,11)
Fernando de Almeida Wernerck: Hospital dos Servidores do Estado- Rio de Janeiro, RJ (n,10)
Teresa Cristina Cardoso: Hospital Manoel Novais Sta Casa de Misericórdia-Itabuna, BA (n,10)
Marcelo Santos de Souza, Rosania Baseggio: Hospital Regional Rosa Pedrossian - Campo Grande, MS (n,9)
Renato Melaragno, Gustavo Neves: Hospital Santa Marcelina-São Paulo, SP (n,9)
Alejandro M. Arancibia: Hospital Estadual de Bauru, Hospital Amaral Carvalho, Jaú - SP (n,8)
Lilian Maria Cristofani: Instituto da Criança Professor Pedro de Alcantara-São Paulo, SP (n,8)
Wellington Mendes, Cecília M. Lima da Costa: Hospital A.C. Camargo-São Paulo, SP (n,7)
Gilberto Ramos, Joaquim C.Aguirre Neto: Hospital das Clinicas-Belo Horizonte, MG (n,7)
Maria Lucia Marinho Lee: Instituto de Oncologia Pediátrica - São Paulo, SP (n,7)
Nilma Pimentel de Brito: Hospital Aristidez Maltez-Salvador, BA (n,6)
Renata S. Carvalho Gurgel: Hospital Universitário Alcides Carneiro-Campina Grande, PB (n,5)
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
ME wrote the manuscript. ME and TCB performed genotyping assays. BAL contributed significantly with controls’ data collection and DNA samples preparation. CBB and CA performed cytogenetic-molecular studies to characterize MLL rearrangements. AF performed infant leukaemia cases diagnosis and collected their clinical-demographic data. CM has performed molecular analysis to determine the MLL breakpoints. RM provided the conditions to MLL breakpoint analysis and contributed with revision of the manuscript. ME and MSPO contributed to the conception of the study, writings and critical analysis of the data. All co-authors of the Brazilian Collaborative Study Group of Infant Acute Leukaemia contributed with clinical and demographical data. All authors read and approved the final manuscript.