XRCC4 rs28360071 intronic variant is associated with increased risk for infant acute lymphoblastic leukemia with KMT2A rearrangements

Abstract

Early age acute leukemia (EAL) shows a high frequency of KMT2A-rearrangements (KMT2A-r). Previous investigations highlighted double-strand breaks arising from maternal exposure to xenobiotics during pregnancy as a risk factor for EAL and KMT2A-r. In this case-control study, we investigated the relationship between EAL and genetic variants of the nonhomologous end-joining (XRCC6 rs5751129, XRCC4 rs6869366 and rs28360071), since they might affect DNA repair capacity, leading to KMT2A-r and leukemogenesis. Samples from 577 individuals (acute lymphoblastic leukemia-ALL, n=164; acute myeloid leukemia-AML, n=113; controls, n=300) were genotyped. No significant association was found for rs5751129 and rs6869366, whereas rs28360071 was associated with an increased risk for ALL with KMT2A-r (IIxID: OR - Odds ratio 2.23, CI 1.17-4.25, p=0.014). Bone marrow samples from ALL patients showed a higher expression of XRCC4 compared to AML patients (p=0.025). Human Splicing Finder 3.1 predicted that the deleted allele of rs28360071 is potentially associated with the activation of a 5’ cryptic splice site in intron 3 of XRCC4. The sequencing of cDNA did not show any differences on the splicing process for the rs28360071 genotypes. Our results suggest that the deleted allele for rs28360071 increases the risk for ALL with KMT2A-r, but not by modifying the XRCC4 expression levels or its structure.

Keywords:
Early age acute leukemia; nonhomologous end-joining; XRCC4; XRCC6; KMT2A

Introduction

Early age acute leukemia (EAL) here defined as children aged up to 24 months old at the diagnosis) is a heterogeneous group of hematopoietic malignancies that includes neonatal and infant leukemia and is the leading cause of death by neoplastic diseases in this age group (Noone et al., 2018). The etiology of EAL might be related to an interplay between individual genetic susceptibility and exposure to genotoxic agents with potential carcinogenic action (Alexander et al., 2001; Pombo-de-Oliveira and Andrade, 2016). Common somatic aberrations found in EAL are rearrangements involving the KMT2A gene (Lysine K-specific Methyltransferase 2A, also named Mixed Lineage Leukemia, MLL) (Felix, 2001). Since the KMT2A protein plays an essential role in the positive regulation of gene expression during human fetal development and hematopoiesis, (KMT2A-r - KMT2A rearranged) promotes both infant acute lymphoblastic leukemia (ALL - Acute lymphoblastic leukemia) and infant acute myeloid leukemia (AML) (Slany, 2009). ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Translocations can occur after the generation of double-strand breaks (DSB - Double strand break) along one or more chromosomes and incorrect chromosomal rejoining. Several studies have highlighted that KMT2A-r generation occurs in-utero (Greaves and Wiemels, 2003). The association between EAL, KMT2A-r and maternal exposure to substances that can cross the placental barrier and cause DSBs in fetal DNA has been reported in epidemiological studies (Hutt and Kalf, 1996; Alexander et al., 2001; Pombo-de-Oliveira and Koifman, 2006). In the absence of proper DNA repair mechanisms, the maintenance of genomic integrity after exposure to these compounds would be affected, thus increasing the risk of KMT2A-r and EAL.

In eukaryotic cells, DSBs are primarily repaired by nonhomologous end-joining (NHEJ), which might occur in two different pathways: canonical NHEJ (c-NHEJ) and alternative NHEJ (a-NHEJ - Alternative non-homologous end-joining) (Deriano and Roth, 2013). C-NHEJ starts with the recognition of the extremities of DSBs by the heterodimer formed by Ku70 and Ku80 proteins, encoded by the genes XRCC6 and XRCC5, respectively. Another central element involved in c-NHEJ is XRCC4 (X-Ray Cross Complementing 4), which encodes a core harbor protein that activates and leads DNA Ligase IV (LIG4) to the damage site to rejoin the strands (Lieber, 2008). However, a-NHEJ occurs in the absence of some c-NHEJ (Canonical non-homologous end-joining) central elements, including Ku70/80 and XRCC4 proteins, and it is considered an error-prone and unfaithful repair mechanism, potentially leading to the formation of deletions and chromosome translocations (Kabotyanski et al., 1998; Simsek and Jasin, 2010).

The aim of the present study was to conduct the first investigation focusing on the relationship between XRCC6 rs5751129, XRCC4 rs6869366, and XRCC4 rs28360071 polymorphisms and the risk for developing EAL with KMT2A-r. The rs5751129 is a single nucleotide polymorphism (SNP) within the promoter region of XRCC6, which was previously proved to modulate the risk of other cancer types, probably by lowering the mRNA expression levels (Jia et al., 2015). The XRCC4 rs6869366 is also a SNP located at the promoter region, whereas rs28360071 represents a 30 bp deletion/insertion within intron 3 of XRCC4. The implications of these later variants remain to be clarified. However, both are associated with increased risk for several cancer types, including acute childhood leukemia (Wu et al., 2010; Shao et al., 2013; Jin et al., 2019; Gupta et al., 2020).

Material and Methods

Subjects

In total, 277 samples (164 ALL and 113 AML cases) from Brazilian infants and toddlers (ages <24 months at the diagnosis of acute leukemia) were included in this study. These samples were obtained from the Brazilian Collaborative Study Group of Infant Acute Leukemia studies (2000-2013) (Pieters, 2009). DNA samples from the umbilical cord blood of healthy subjects (n=300) were genotyped as controls. EAL encompasses a group of leukemias that affect neonates (≤31 days), infants (≤12 months), and toddlers (up to 24 months). Our EAL cohort was composed of infant-ALL, which is defined when leukemia diagnosis occurred in children ≤12 months-old, based on its distinct clinical characteristics, such as pro-B immunophetotype (CD10^neg) as well as high frequency of KMT2A-r. On the other hand, the time-frame for AML inclusion was extended to ≤ 24 months of age at diagnosis, given that KMT2A-r are more evenly distributed between infants and toddlers with this leukemia subtype (Pieters, 2009; Creutzig et al., 2012; Emerenciano et al., 2013). In the present study, the exclusion criteria for cases were T-cell precursor ALL, non-hematological or hematological malignancy syndromes with genetic alterations, such as Noonan Syndrome and Down Syndrome, and Myelodysplasic Syndrome. EAL patients with insufficient or low DNA quality samples and without parental consent were excluded from the study.

The diagnosis of acute leukemia subtypes was applied throughout bone marrow morphology, immunophenotyping, and molecular-cytogenetic alterations, according to World Health Organization criteria (Arber et al., 2016).

The race/ethnicity was defined according to the Instituto Brasileiro de Geografia e Estatística (IBGE), which gathers subjects according to skin color self-definition. The subjects were categorized into whites or non-whites. The group of non-whites comprised self-categorized brown or African-descent subjects.

The present study was approved by the Ethics and Scientific Committee of the Instituto Nacional de Câncer (CEP-CONEP # 626.268; April 24^th, 2014).

KMT2A status analysis

Bone marrow RNA extraction, cDNA synthesis, and quantitative polymerase chain reaction (qPCR) were performed as described elsewhere (Lopes et al., 2015). Briefly, RNA was extracted from bone marrow samples using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer’s instructions. Following DNase treatment (Invitrogen, CA, USA), cDNA was synthesized using SuperScript III Reverse Transcriptase (Invitrogen, CA, USA) with 3 μg of purified RNA as a template. KMT2A status was evaluated by reverse transcription-PCR assays for the detection of the main chimeric transcripts (fusion between KMT2A and AFF1, MLLT3, MLLT1, MLLT10, or MLLT4), and/or by fluorescence in situ hybridization (Vysis LSI MLL Dual Color, Break Apart Rearrangement Probe, Abbott Laboratories, Abbot Park, IL, USA) as previously described (Emerenciano et al., 2006; Burmeister et al., 2015).

DNA extraction and genotyping

DNA extraction was performed with the QIAamp DNA Mini kit (Qiagen Inc., Germantown, MD, USA) according to the manufacturer’s protocols. Genotyping was performed by PCR, followed by restriction fragment length polymorphism (RFLP) for XRCC6 rs5751129 (informally cited as -991 T>C) and XRCC4 rs6869366 (informally cited as -1394 T>G) according to a previous report (Tsai et al., 2007). Subsequently, the alleles of insertion (139 bp) and deletion (109 bp) of XRCC4 rs28360071 (informally cited as intron 3 DIP) were detected by 2% agarose gel electrophoresis.

Gene expression

We evaluated XRCC4 mRNA expression in 42 samples (25 ALL and 17 AML) by qPCR using the following primer sequences: 5’- TGGACTGGGACAGTTTCTGA-3’ (forward) and 5’- CTGCTCCTGACAACAATGCT- 3’ (reverse), as previously described (Shao et al., 2014). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was used for the normalization of XRCC4 transcript using the 5’-CAACAGCCTCAAGATCATCAGCAA-3’ (forward) and 5’-AGTGATGGCATGGACTGTGGTCAT-3’ (reverse) primers. The experiments were performed in triplicates, and Ct medians were used to determine the relative expression by the 2^-ΔCt method (Livak and Schmittgen, 2001).

Sequencing

For evaluating if the splicing process is different among the three genotypes for rs28360071, a pair of primers on exon 3 (Forward: 5’-GGAGCAGGACCAGCTGATGTAT-3’) and exon 4 (Reverse: 5’-TTTCTGCAATGGTGTCCAAGC-3’) of XRCC4 cDNA was designed. After that, cDNA samples from five random patients (two carrying II, one carrying ID (Insertion/Deletion. Heterozygous genotype of rs28360071) and two carrying DD (Deletion/Deletion. Homozygous genotype of deletion allele of rs28360071) genotypes) were amplified by PCR. The amplified fragment resulting from the splicing process was expected as a 175 bp fragment (Figure S3b). PCR fragments were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter Company, MA, USA), and sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific Inc.) on a 3500 Genetic Analyser (Thermo Fisher Scientific Inc.). Experiments were performed in triplicates. Representative chromatograms are shown in Figure S3.

Statistical and in silico analysis

The chi-square test was used for the comparison of sociodemographic frequencies between cases and controls, such as child age, sex, ethnicity/skin color, and KMT2A status. Expected genotypes frequencies of the genetic polymorphisms were calculated by Hardy-Weinberg (HW) with GenePop Web version 4.5.1 (http://genepop.curtin.edu.au/). The Odds Ratios (ORs) with 95% confidence intervals (CI) were calculated using chi-square and the Fisher's exact tests. Disease risk was calculated for dominant (wild-type homozygote vs. polymorphic heterozygote + polymorphic homozygote), recessive (wild-type homozygote + heterozygote vs. polymorphic homozygote), codominant (wild-type homozygote vs. heterozygote or wild-type homozygote vs. polymorphic homozygote), and additive (wild-type homozygote vs. heterozygote + two times polymorphic homozygote) models. The Statistical Product and Services Solutions statistical package 22.0 (SPSS Inc., Chicago, IL, USA) was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs) and p values while unconditional logistic regression was used to estimate adjusted odds ratios (aORs). For all analyses, the p values < 0.05 were considered significant. Bonferroni correction for testing three SNPs was applied to the adjusted p value. The Bonferroni-corrected statistical significance level set at p values < 0.017 (0.05/3) indicated statistically significant results. The qPCR statistical analysis was performed with GraphPad Prism 5 (GraphPad Software, Inc., California, USA). The comparison among genotypes in relation to XRCC4 expression was performed using the Kruskal-Wallis tests. The Mann-Whitney test was used for analyses related to KMT2A status.

The online bioinformatics tool Human Splicing Finder version 3.1 software http://www.umd.be/HSF3/index.html (HSF, 2019) was applied to predict the potential functional impact of the indel polymorphism at XRCC4 splicing sites. This software is an international reference that combines twelve different algorithms to identify and predict the effect of a variant on splicing motifs, including the acceptor and donor splice sites, the branch point, and auxiliary sequences known to either enhance or repress splicing.

Results

XRCC4 deleted allele rs28360071 increases the risk for ALL in patients with KMT2A-r

The frequency distribution of demography characteristics, childhood leukemia subtypes, and KMT2A status are described in Table 1. The children were ≤ 2 years old at the diagnosis of acute leukemia, with a mean age of 13 ± 5.6 months at the time of diagnosis (ALL= 12 ± 7.0 months and AML = 13 ± 5.7 months). The distribution of cases and controls by sex was similar, but regarding to ethnicity, there was a higher number of whites in both groups (p= 0.010). All genotype distribution of polymorphisms was in Hardy-Weinberg equilibrium (^{Table S1}).

Table 1 -
The frequency distribution of childhood leukemia, demography and KMT2A status, Brazil, 2000-2013.

	Cases n (%)	Controls n (%)	p Value
Acute Leukemia Subtype
ALL	164 (59.2)	-	-
AML	113 (40.8)	-
Sex
Male	147 (53.5)	144 (48.0)	0.211
Female	128 (46.5)	156 (52.0)
NI	2	0
Ethnicity
White	164 (60.3)	136 (49.0)	0.010
Non-white*	108 (39.7)	142 (51.0)
NI	5	22
KMT2A Status
KMT2A-r	103 (48.1)	-	-
KMT2A-WT	111 (51.9)	-
NI	63	-
Total	277 (48.0)	300 (52.0)	-

• ALL: Acute Lymphoblastic Leukemia; AML: Acute Myeloid Leukemia; NI: No Information available; KMT2A-r: KMT2A rearranged; KMT2A-WT: KMT2A wild type.
• (*) Comprises Brazilians with genetic admixture and self-defined skin color.

No significant association between the XRCC6 rs5751129 variant and EAL risk was observed in any analyzed parameter (^{Table S2}). The XRCC4 6869366 variant demonstrated a protective effect on KMT2A-r only among ALL cases under the dominant (OR = 0.30; CI = 0.09-1.00; p = 0.040) and additive (OR = 0.30; CI = 0.10-0.95; p = 0.040) models (^{Table S3}). However, the statistical significance of those associations was lost by adjusting OR by skin color and sex (dominant model: aOR = 0.30; CI = 0.08-1.01; p = 0.054 and additive model: aOR = 0.42; CI = 0.01-1.11; p = 0.050). However, for the XRCC4 rs28360071 an increased risk for ALL with KMT2A-r was associated with the heterozygous genotype (Codominant model; homozygous for insertion II vs. heterozygous ID: OR = 2.00; CI = 1.10-3.80; p = 0.039), even when adjusted (aOR = 2.23; CI = 1.17-4.25; p = 0.014) (Table 2). The results remained statistically significant after Bonferroni’s correction (p = 0.014 x 3 = 0.042).

Table 2
Genotype frequencies for XRCC4 rs28360071 in EAL subtypes and KMT2A status.

XRCC4 rs28360071	Control n (%)	ALL n (%)		AML n (%)
II	87 (30.3)	42 (26.9)		35 (31.5)
ID	134 (46.7)	85 (54.5)		50 (45.0)
DD	66 (23.0)	29 (18.6)		26 (23.4)
Genetic Model	p OR (CI)	p aOR (CI)	p OR (CI)	p aOR (CI)
Dominant	0.510 1.18 (0.80-1.82)	0.250 1.07 (0.75-1.55)	0.810 0.95 (0.60-1.52)	0.911 0.97 (0.59-1.58)
Recessive	0.330 0.80 (0.50-1.25)	0.247 0.90 (0.60-1.30)	1.000 1.02 (0.61-1.72)	0.830 0.94 (0.55-1.61)
II vs. ID	0.251 1.31 (0.83-2.10)	0.103 1.14 (0.80-1.70)	0.800 0.93 (0.60-1.54)	0.992 0.99 (0.58-1.68)
II vs. DD	0.773 0.91 (0.51-1.61)	0.931 0.94 (0.60-1.51)	1.000 1.00 (0.54-1.80)	0.837 0.93 (0.50-1.74)
Additive	0.671 1.11 (0.73-1.70)	0.379 0.82 (0.53-1.26)	0.910 0.95 (0.60-1.50)	0.870 1.04 (0.65-1.66)
KMT2A-WT
XRCC4 rs28360071	Control n (%)	ALL n (%)		AML n (%)
II	87 (30.3)	19 (30.6)		11 (25.0)
ID	134 (46.7)	28 (45.2)		21 (47.7)
DD	66 (23.0)	15 (24.2)		12 (27.3)
Genetic Model	p OR (CI)	p aOR (CI)	p OR (CI)	p aOR (CI)
Dominant	1.000 1.00 (0.54-1.80)	0.874 0.95 (0.51-1.74)	0.600 1.30 (0.63-2.70)	0.308 0.67 (0.31-1.44)
Recessive	0.900 1.10 (0.60-2.03)	0.937 0.97 (0.50-1.86)	0.600 1.30 (0.61-2.60)	5.053 0.83 (0.40-1.74)
II vs. ID	1.000 1.00 (0.50-1.90)	0.913 0.96 (0.50-1.85)	0.700 1.25 (0.60-2.70)	0.357 0.68 (0.30-1.54)
II vs. DD	1.000 1.04 (0.50-2.20)	0.850 0.92 (0.43-2.00	0.501 1.45 (0.60-3.50)	0.373 0.66 (0.26-1.64)
Additive	1.000 1.00 (0.60-1.80)	0.850 0.94 (0.52-1.69)	0.502 1.35 (0.70-2.70)	0.279 0.66 (0.31-1.39)
KMT2A-r
XRCC4 rs28360071	Control n (%)	ALL n (%)		AML n (%)
II	87 (30.3)	16 (20.8)		10 (43.5)
ID	134 (46.7)	49 (63.6)		9 (39.1)
DD	66 (23.0)	12 (15.6)		4 (17.4)
Genetic Model	p OR (CI)	p aOR (CI)	p OR (CI)	p aOR (CI)
Dominant	0.120 1.70 (0.91-3.04	0.057 1.81 (0.98-3.37)	0.242 0.60 (0.24-1.34)	0.333 0.64 (0.26-1.57)
Recessive	0.210 0.62 (0.32-1.21)	0.150 0.60 (0.30-1.19)	0.800 0.70 (0.23-2.15)	0.602 0.74 (0.24-2.28)
II vs. ID	0.039 2.00 (1.10-3.80)	0.014 2.23 (1.17-4.25)	0.331 0.60 (0.23-1.50)	0.434 0.67 (0.25-1.79)
II vs. DD	1.000 1.00 (0.44-2.23)	0.900 1.06 (0.46-2.45)	0.400 0.53 (0.16-1.80	0.393 0.58 (0.16-2.01)
Additive	0.210 1.50 (0.82-2.70)	0.115 0.61 (0.33-1.12)	0.171 0.60 (0.25-1.30)	0.276 1.60 (0.68-3.76)

• ALL: Acute Lymphoblastic Leukemia; AML: Acute Myeloid Leukemia; OR: Odds ratio; aOR: Odds ratio adjusted by skin color and sex; KMT2A-WT: KMT2A wild type; KMT2A-r: KMT2A rearranged; II: homozygous for insertion; ID: heterozygous; DD: homozygous for deletion.

There is a higher expression of XRCC4 in ALL patients

Due to the observed association of XRCC4 and ALL, XRCC4 mRNA expression levels in the bone marrow of patients was evaluated by qPCR. A higher expression of XRCC4 was found in ALL compared to AML (p = 0.025) (Fig. 1a). No differential expression was observed according to the three genotypes for the XRCC4 rs28360071 polymorphism (Fig. 1b), even when the data were stratified by KMT2A status. However, KMT2A-r samples seem to show slightly higher XRCC4 expression than that in KMT2A wild type (KMT2A-WT - KMT2A wild type) samples (p = 0.080) (Fig. 1c). Moreover, no differential expression was observed according to either genotypes or KMT2A status in both ALL and AML (^{Figure S1}).

Figure 1 -
Differential levels of XRCC4 mRNA in EAL bone marrow samples. (a) Comparison of the expression levels of XRCC4 in acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). (b) Differential levels of XRCC4 according to the genotypes for the XRCC4 rs28360071 polymorphism. (c) Comparison of XRCC4 expression levels in patients harboring KMT2A rearranged (KMT2A-r) and KMT2A wild type (KMT2A-WT). Experiments were performed in triplicates. Horizontal bars indicate median values.

The splicing process of XRCC4 could be altered due to rs28360071

In silico analysis performed by HSF predicted three changes on intron 3, caused by the deletion of 30 base pairs and activation of an intronic cryptic donor site, an alteration of a splicing silencer site, and the creation of a splicing enhancer site. From those changes, the former was interpreted by the software as having a potential impact on splicing with a high score (^{Figure S2}). We have performed direct sequencing of XRCC4 in bone marrow cDNA samples, and no structural alterations in cDNA lengths were observed, regardless of the XRCC4 rs28360071 genotype status (^{Figure S3}).

Discussion

In the present study, the correlation between genetic polymorphisms in NHEJ genes and the risk for EAL was evaluated. No correlation was found between the overall risk for EAL with KMT2A-WT and XRCC4 (rs6869366 and rs28360071) or XRCC6 (rs5751129) variants included here.

The XRCC6 rs5751129 SNP is associated with decreased transcript and protein expression levels (Hsu et al., 2013). Individuals carrying the *C allele had lower XRCC6 mRNA expression (Chang et al., 2012; Hsu et al., 2013). Regarding acute childhood leukemia (0-18 years old), patients carrying the heterozygous genotype had a significantly increased risk of developing the disease (Pei et al., 2013). In the present work, no association between rs5751129 and EAL was found, regardless KMT2A status.

Wu and colleagues found an increased risk of leukemia related to both XRCC4 variants among Taiwanese children (Wu et al., 2010). These authors found that *G and deleted alleles of XRCC4 rs6869366 and rs28360071, respectively, are associated with a higher risk for childhood leukemia. The present study demonstrates that the *T allele of XRCC4 rs6869366 has a protective effect against ALL, although the association was not significant after p-value adjustment. Another study showed that XRCC4 variants are associated with an increased risk for ALL, particularly in ALL cases with chromosomal translocations and hyperdiploidy, both alone and in conjunction with exposure to ionizing radiation (Chokkalingam et al., 2011).

Here, an increased risk for ALL with KMT2A-r was found to be associated with the ID genotype of XRCC4 rs28360071. These data suggest an influence of the 30-bp deletion allele over the XRCC4 protein or its repair. Other studies have also shown significant associations between XRCC4 variants and childhood ALL with ETV6-RUNX1 [t(12; 21)] translocations (Chokkalingam et al., 2011). To examine whether the deletion allele could be related to gene expression levels, XRCC4 mRNA expression was assessed by qPCR, showing higher expression in ALL than in AML samples. Although not statistically significant, KMT2A-r samples showed higher gene expression than that of KMT2A-WT samples. Chiou and colleagues studied the expression of the genes related to NHEJ in samples from children with pediatric ALL (Chiou et al., 2007). These studies also showed an increased expression of XRCC4 mRNA. We did not find any variation of XRCC4 expression associated with XRCC4 rs28360071 genotypes, suggesting that the deletion allele could influence the risk for EAL by another mechanism. In this sense, the use of an in silico tool pointed out that the deleted allele could potentially implicate XRCC4 splicing, through activation of a cryptic donor site on intron 3. Cryptic splice sites are dormant or less used splicing sites found both in exons and introns that can be activated by mutations. When activated, these sites could be efficiently used by the splicing machinery, possibly resulting in genetic disorders (Buratti et al., 2007; Wang and Cooper, 2007). In the presence of the 30 bp deletion in intron 3 of XRCC4, the activation of a cryptic donor splicing site may lead to preferential use of this donor site in detriment of the original one. As a consequence, this event can lead to the inclusion of part of intron 3 into the mature mRNA, which could cause protein structure modifications.

Additionally, the defective mRNA may also include a premature stop codon, which could even deflagrate degradation of the mRNA before translation by nonsense-mediated decay, as previously reported (Nicholson and Muhlemann, 2010; Santos-Rebouças et al., 2011). As no changes in the splicing process were observed by XRCC4 cDNA sequencing, we hypothesize that the influence of the rs28360071 polymorphism on the development of EAL might occur by another biological mechanism. We are not able to determine the impact of this polymorphism on the biology of the protein and how it implicates on NHEJ repair capacity with the data we showed here. Future studies are needed to investigate the molecular mechanisms underlying the phenotypic impact caused by rs28360071.

The present results are not similar to those reported by other groups studying pediatric acute leukemia. This discrepancy is likely due to the characteristic of the study population since the present work includes a unique series of children up to 24 months old, and most studies regarding childhood leukemia include children up to 18 years old. The disease development of EAL is related to a prenatal origin, resulting from in utero exposure to DNA damage agents. In contrast, the disease among older children can have a different etiology, including the acquisition of additional genetic aberrations during childhood. Since other studies do not analyze ALL and AML separately, a comparison of the results between studies should be performed carefully. Indeed, we must consider that the Brazilian population has unique miscegenation profile and is one of the most heterogeneous populations worldwide (Parra et al., 2003).

Herein, we assessed the influence of genetic polymorphisms in central elements of c-NHEJ on the development of EAL and KMT2A-r. Our hypothesis was based on the fact that maternal exposure to xenobiotic compounds generates DSBs on DNA of fetal hematological stem cells. Once DNA repair is not appropriate, chromosomal translocations could occur within KMT2A, thus driving and sustaining in utero leukemogenesis.

To the best of our knowledge, this study is the first to report polymorphisms in the NHEJ repair system and KMT2A-r in EAL. Although the risk for EAL was not related to XRCC6 rs5751129 or XRCC4 rs6869366 variants, the 30-bp deletion within the third intron of XRCC4 influenced the risk of ALL with KMT2A-r cases. Additional studies are necessary to evaluate the functional impact of these variants in the biology of leukemia.

Acknowledgments

This research was financially supported through a grant from the FAPERJ ≠E_26/110.169/2013; MSPO is a CNPq scholar ≠301594/2015-5. This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001. We would like to thank Bruno A. Gonçalves, Camilla G. Andrade, Caroline B. Blunk, Gisele M. Vasconcelos, Eugenia T. Granado and Denise N. de Oliveira for technical support. We would also like to thank the members of the Brazilian Collaborative Study Group of Infant Acute Leukemia who contributed for the present study: Eugênia T. Granado, Elda Noronha, Bruno Aguiar, Gisele M. Vasconcelos and Paulo Neto from the PHOP-CPq, INCa, Rio de Janeiro for thecnical support and the physicians who sent samples along with clinical data from the patients (Sidnei Epelman and Renato Guedes from Hospital Santa Marcelina, São Paulo, Maura Ikeda and Teresa Cristina Oliveira from Hospital Amaral Carvalho, São Paulo, Eloisa C. Fialho from Hospital Napoleão Laureano, Paraíba, Gustavo R. Neves from Hospital Sarina Rolin, São Paulo, Isis Maria Q. Magalhães, José Carlos Córdoba and Ricardo Camargo from Hospital da Criança de Brasília José Alencar, Brasília, Patricia C. de Brito and Anna Carolina S. Dias, Hospital Araújo Jorge, Goiás, Juliana T. Costa and Luciana N. S. Souza from Hospital Martagão Gesteira, Bahia, Marcelo S. Santos and Rosania Basegio from Centro de Tratamento Onco-hematológico Infantil de Mato Grosso do Sul, Mato Grosso do Sul and Teresa Cristina Cardoso-Fonseca from Santa Casa de Misericórdia de Itabuna, Bahia).The head of the group is Dr Maria S. Pombo-de-Oliveira.

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Internet Resourses

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Supplementary material

The following online material is available for this article:

Table S1 - Allele and genotype frequencies of XRCC6 rs5751129, XRCC4 rs6869366 and XRCC4 rs28360071 in cases and controls.

Table S2 - Genotype frequencies of XRCC6 rs5751129 in EAL subtypes and KMT2A status.

Table S3 - Genotype frequencies of XRCC4 rs6869366 in EAL subtypes and KMT2A status.

Figure S1 - Differential levels of XRCC4 mRNA in EAL bone marrow samples.

Figure S2 - In silico predictions for XRCC4 rs28360071 polymorphism (NM_022550.3:c.315+31090del30).

Figure S3 - Sequencing of bone marrow XRCC4 cDNA of patients with EAL.

Publication Dates

• Publication in this collection
  02 Dec 2020
• Date of issue
  2020

History

• Received
  26 May 2020
• Accepted
  20 Oct 2020