Genetic polymorphisms of inflammasome genes associated with pediatric acute lymphoblastic leukemia and clinical prognosis in the Brazilian Amazon

The immune system plays an important role in the control of cancer development. To investigate the possible association of inflammasome genes to childhood leukemia we performed a case-control study with 158 patients with acute lymphoblastic leukemia and 192 healthy individuals. The IL1B and IL18 genetic polymorphisms were genotyped by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and NLRP1, NLRP3 and P2RX7 were genotyped using Real Time quantitative PCR (qPCR). The IL1B C/T rs19644 genotype was associated with the risk of developing ALL (C/C vs. C/T + T/T OR: 2.48 [95% CI: 1.26–4.88, p = 0.006]; C/C vs C/T OR: 2.74 [95% CI: 1.37–5.51, p = 0.003]) and the NLRP1 A/T rs12150220 (OR: 0.37 [95% CI: 0.16–0.87, p = 0.023]) was associated with protection against infectious comorbidities. It was not found association between NLRP3 and P2RX7 polymorphisms and acute lymphoblastic leukemia in our study. Our results suggest that the inflammasome single-variant polymorphisms (SNVs) may play a role in the development and prognostic of childhood leukemia. However, this finds requires further study within a larger population in order to prove it.

www.nature.com/scientificreports/ low stimulation of the immune system in early childhood followed by a second response to infections, could be responsible for the dysregulation of the immune system and increase the chances of developing ALL 5 . However, the process for the development of ALL remains unknown 6 . The inflammasome complex constitutes components of innate immunity involved in inflammatory processes and has been associated with the development of autoimmune inflammatory diseases and several types of cancers 7,8 . In acute lymphoblastic leukemia, NLRP1a-induced pyroptosis in hematopoietic progenitor cells can prevent cell proliferation and differentiation, contributing to the proliferation of altered blasts that will trigger the disease. The dysregulation of the inflammasome complex can also influence the prognosis of patients, since studies report that the constitutive activation of NLRP3 seems to cleave the glucocorticoid receptor, this being the first line of treatment for ALL, and thus increase the number of relapses 9,10 . Besides, studies reported that the genetic variants of inflammasome related genes contribute to ALL pathogenesis and prognosis since CARD8 rs2043211 A/T and T/T genotypes were associated with susceptibility, lower white blood cell (WBC) count and T-cell immunophenotype. NF-κB-94 ins/del ATTG was associated with protection in susceptibility of ALL. In addition, IL1β rs16944 and IL18 rs1946518 seems to increase the mRNA expression of NLRP3 and secretion of downstream cytokines 11 .
Although inflammasomes are associated with several types of diseases, there are few studies that demonstrate the relationship between the SNVs involving IL1B (Interleukin 1 beta), IL18 (Interleukin 18), NLRP1 (NLR family pyrin domain containing 1), NLRP3 (NLR family pyrin domain containing 3) and P2RX7 (Purinergic receptor P2X7) genes and their susceptibility or influence on the prognosis of ALL patients. In this study, we described that variants of inflammasomes were associated with the risk of developing pediatric ALL, and the resulting clinical prognosis in these patients.

Results
Clinical and epidemiological baseline of the patients. Demographic, clinical and laboratory data of ALL patients and controls are shown in Table 1. The mean age among individuals in the control group and patients with ALL was 38 and 12, respectively. In addition, the male gender was predominant in both groups (66% and 63%). The immunophenotype B-ALL was predominant (85%) in this study. Regarding the presence of comorbidities, 45% had some type of comorbidity on diagnosis, the most frequent being infectious diseases (86%) (e.g., cytomegalovirus, toxoplasmosis, rubella, varicella, parasitic diseases, among others), followed by other comorbidities (14%) (e.g., Aplasia and Burkitt's lymphoma). Most patients relapsed during treatment (66%) and approximately 41% of ALL patients died during treatment. The hemoglobin average was 8.65 g/ dL, hematocrit 25.4 g/dL, leukocytes 4.720/mm 3 and platelets 39.000/mm 3 .
Association of IL1B C/T rs19644 genotype with pediatric acute lymphoblastic leukemia. In Table 2, it is possible to observe the genotypic frequencies of all the SNVs under study. Among all the SNVs, IL1B rs19644 (p = ≤ 0.001), P2RX7 rs2230911 (p = 0.042), NLRP1 rs35865013 (p = 0.000) deviated from the Hardy-Weinberg balance. The IL1B C/T rs19644 genotype appears to be a risk factor for the development of ALL (C/C vs. C/T + T/ The NLRP1 A/T rs12150220 genotype is associated with protection against infectious comorbidities in pediatric ALL patients. In Table 3, it is possible to observe the genotype frequency of the SNVs under study in relation to comorbidities (infectious diseases), relapse and death. Logistic regression analysis was performed in order to investigate the association of genotypes with the variables under study. The NLRP1 A/T rs12150220 (OR: 0.37 [95% CI: 0.16-0.87, p = 0.023) was associated with protection against infectious comorbidities and this it also observed in multivariate analysis adjusted for age and sex (OR: 0.34 [95% CI: 0.16-0.73, p = 0.003). In this study, it was not observed association to relapse and death. Supplementary Table S2 summarizes the results from multivariate regression analysis for all the SNVs according to infectious comorbidities, relapse and death.
The case and control groups showed similar proportions of allele frequencies for each SNVs, and thus, no significant difference in allelic frequencies was found between both groups. The major allele frequencies for each polymorphism are shown in Supplementary Table S3.

Discussion
Inflammasomes are multimeric molecular complexes, formed in the cytoplasm in response to endogenous and exogenous stimuli that promote the activation of inflammatory caspases 12 . Over the years, inflammasomes have been linked to autoimmune 13,14 and inflammatory diseases 15 as well as several types of cancer 16 . Genetic variants of inflammasome related genes can contribute to ALL pathogenesis and prognosis as CARD8 and NF-κB 11 , however, there are few studies that demonstrate the role of others genes involved in pathway inflammasome in ALL.
To investigate the possible genetic contribution to childhood leukemia in the inflammasomes, we performed a study for the SNVs related to inflammasome genes. When the frequency of SNVs in the case and control group with other populations in the world was evaluated, we observed that all subjects presented sequences similar to the frequencies described for the Native South-American population, except NLRP1 rs35865013 A/G (A: 0.402 and G: 0.598), which presented a similar frequency to the population of South Asia 17 . The population of the Amazon Region has the highest high degree of inter-ethnic admixture due to the intense miscegenation process that occurred in the region and the strong indigenous influence on the population. Children with admixed www.nature.com/scientificreports/ ancestry have a higher risk of developing ALL due to the existence of genetic variations characteristic of Native South American. Native South American ancestry is predominantly found in the northern region of Brazil, where approximately 80% of the Amazon region is located 18 , and the literature has already described its association with susceptibility to ALL in children in the Brazilian Amazon 19 . In our study, the IL1B rs16944 polymorphism was associated with the risk of developing ALL. The IL1B gene is located on chromosome 2q14 and contains many single-nucleotide variants. IL1B rs16944 is located in the promoter region and T allele is associated with the increased of transcription activity and production of IL-1β cytokine [20][21][22] and increased mRNA expression of NLRP3 and ASC 11 . In the literature, this polymorphism has been associated with susceptibility or worse prognosis in individuals with autoimmune diseases and in several types of cancers 21,23 . In a study by Yin et al. [2016], it was shown that individuals with IL1B G/G rs16944 genotype are at risk of developing Myelodysplastic Syndrome (MDS) 24 . In addition, polymorphisms involving the IL-1β cytokine were associated with cytogenetic assessment of what would be considered a good prognosis in patients with acute myeloid leukemia (AML) (p = 0.043) 25 .
After infection or injury, an IL-1β is found at high medullary levels by monocytes and endothelial cells, and promotes myeloid differentiation through activation of the NF-kB pathway that results in the expansion of HSPCs 26 . Chronic exposure to IL-1β significantly impairs self-renewal and the ability of HSPCs to differentiate into lymphoid and erythroid lineage 27 . Therefore, chronic sustained inflammation may elicit the stem cell insult by inducing a state of chronic oxidative stress with elevated levels of reactive oxygen species (ROS) in the bone marrow, thereby creating a high-risk microenvironment for induction of genetic alterations due to the persistent inflammation-induced oxidative damage to DNA in hematopoietic cells 28 .
Chronic immune stimulation from infectious processes is a trigger for AML and MDS. The history of infectious diseases (tuberculosis, intestinal diseases, pneumonia, septicemia, pyelonephritis, sinusitis, nasopharyngitis,  29 . Thus, chronic inflammation can be identified as one of the triggers of hematological neoplasms. IL-18 is an important cytokine resulting from NLRP3 inflammasome activation, which is involved in the innate and acquired immune response. In multiple myeloma, the increase in serum IL-18 is associated with disease progression and lower chances of patient survival 30 . In this study, no association was found between IL18 polymorphism and ALL. However, in the study by Yalçin et al. on the Turkish population (2014), the G/C and C/C genotypes were associated with the risk of developing chronic myeloid leukemia, and the C/C genotype was associated with the risk of developing chronic lymphoid leukemia, which suggests a relationship between this polymorphism and the development of chronic leukemias characterized by the proliferation of mature cells, however, with loss of functionality 31 .  www.nature.com/scientificreports/ The NLRP1 inflammasome is expressed in hematopoietic progenitor cells and its activation results in a process of cell death which is dependent on Caspase 1 and is called pyroptosis. Some studies report that the prolonged cytopenia, induced by the activation of NLRP1 during infectious processes, ensures a proliferative advantage for the leukemic clone, as suggested by the Mel Greaves hypothesis on the development of ALL 32,33 . In chronic myeloid leukemia (CML), overexpression of NLRP1 gene is associated with the promotion of proliferation and reduction of apoptosis in CML cells, in addition to inducing resistance to imatinib 34 .
In this study, the NLRP1 A/T rs12150220 genotype was associated with protection against infectious diseases. In ALL, infections are present in 49% of patients on diagnosis 35 . Studies report that susceptibility to congenital toxoplasmosis is significantly associated with SNVs and involves the locus of the NLRP1 gene 36 , which strengthens the Mel Greaves hypothesis that genetic changes in the uterus followed by the acquisition of infections by common pathogens are involved in the development of ALL 5,37 .
NLRP3 is currently the best-studied member of the inflammasome family expressed in hematopoietic and lymphopoietic cells being responsible for the migration and spread of leukemic cells. The inflammatory process in leukemic patients promotes release of several chemoattractants and thus increases trafficking of leukemic cells and their spread within hematopoietic organs that contribute for the ALL development 38 . Paugh et al. (2016) reported that decreased methylation of the Caspase-1 promoter results in increased transcription and activation of NLRP3 and Caspase1, which cleaves the glucocorticoid receptors used in the treatment of ALL, what suggests their association with relapse episodes 10,39 . Besides, the NLRP3 expression is increased in patients with ALL compared to healthy individuals 40 but decreased in CML, what suggests different roles of inflammasome activity in acute and chronic leukemia. The A/A genotype NLRP3 rs35829419 was associated with risk of acute myeloid leukemia 25 but not when it observed CML patients 41 . In this study, we did not find association of NLRP3 SNVs, futher studies are necessary to better elucidate their role of NLRP3 in ALL.
The P2RX7 receptor is responsible for making NLRP3 sensitive to ATP, which is one of the main DAMPs released during inflammation and is highly expressed in tumor cells. Studies describe the high expression of P2RX7 in samples from ALL patients, especially those who relapse, as well as an association with dysregulation of the HSPCs' normal functioning, since it affects the ability of colony formation in vitro, which impairs the clonal expansion process observed in the HSPCs. However, the role of this mechanism in the development of ALL is still unknown 42 .
The P2RX7 polymorphisms cause a loss of receptor function, mainly in macrophages, and is responsible for a partial reduction of the channel and formation of pores in the membrane [43][44][45] . In chronic lymphoid leukemia, P2RX7 rs3751143 polymorphism appears to influence patient survival, and the A/C genotype is associated with longer survival (104 months) than the A/A genotype (72 months) 46,47 . In this study, we did not find association between P2RX7 polymorphisms and ALL. However, one of the main infectious comorbidities found in our patients was toxoplasmosis, an infection caused by Toxoplasma gondii. The P2RX7 receptor is an important mediator in the control of infection by Toxoplasma gondii, since it prevents its proliferation by stimulating the production of reactive oxygen species (ROS) and facilitates the acidification of parasitophorous vacuoles in macrophages infected by the parasite 48 . Loss of receptor function may be responsible for the susceptibility to Toxoplasma gondii infection in patients with acute lymphoblastic leukemia in this study, however, prospective studies are needed to confirm this relationship.
This study has some limitations. Despite the association of SNVs with ALL inflammasome and clinical data, the study population is small compared to other studies involving SNVs. Thus, prospective studies with a larger population are necessary in order to confirm the importance of the polymorphisms under study in ALL, including those in adult patients and rearrangements. The small sample size did not allow the comparison of the studied genotypes and alleles with the laboratory data. The absence of the analysis of gene expression and the determination of serum levels of proteins also limited the study, since they made it impossible to gain a better understanding of the influence of these molecules on the variables under study. Moreover, the control group was composed of blood donors. According to the Brazilian Ministry of Health, candidates of at least 16 years of age with the requirements of those responsible are eligible, however, the group with the highest adherence to donation are those over the age of 18, and these are the main components of our sample. However, the case group was composed of leukemic patients within the period of major incidence of disease (< 18 years). Although we have included this topic as a limitation of the study, we understand that when using children as a control group, false results could be formulated, since they could develop the disease after the study. While adults, supposedly would have had a longer time to develop the disease.
To our knowledge, this is the first study to describe the frequency of polymorphisms in the inflammasome genes in patients with acute lymphoblastic leukemia in the Amazon region. Inflammasomes are important complexes in the defense of the host and contribute to neoplastic development. Thus, the variant IL1B C/T rs16944 was associated with susceptibility to ALL in individuals from the Brazilian Amazon region. In addition, the variant NLRP1 A/T rs12150220 can promove protection from infectious diseases in acute lymphoblastic leukemia patients. However, future studies should be carried out in order to better elucidate the influence of these SNVs on the pathogenesis of ALL.

Materials and methods
Patients and sampling. In the case group, samples were included from 158 pediatric patients diagnosed with ALL according to the classification criteria of the World Health Organization (WHO) 49 , and who were treated at Fundação Hospitalar de Hematologia e Hemoterapia do Amazonas (HEMOAM). The patients had cryopreserved samples in the DNA library of the HLA typing laboratory of the HEMOAM, were < 18 years, of either gender or unrelated. Insufficient or low-concentration DNA samples, and patients with a history of bone marrow transplantation were excluded from the study. www.nature.com/scientificreports/ The healthy individuals (control group) consisted of 192 samples from blood donors of either gender, who donated at HEMOAM between January and December 2015 and agreed to participate in the research. In order to be considered healthy, all candidates were tested serologically for HIV, HCV, HBV, HBV, HTLV-1/2, Syphilis and Chagas disease and for HIV, HBV, HCV using the NAT HIV/HCV/HBV Kit. In addition, they were screened in interviews for diseases and other risk factors, according to the Brazilian Ministry of Health technical standards. Insufficient or low-concentration DNA samples and related candidates were excluded. Ethical issues. This study was approved by the Research Ethics Committee of the HEMOAM Foundation under protocol number 3.335.123/2019, CAAE 12615918.9.0000.0009. Prior inclusion of all patients and controls in the study, all the respective parents or legal guardians read and signed the informed consent form. This study was carried out in accordance with the guidelines of the Declaration of Helsinki and Resolution 466/12 of the Brazilian National Health Council for research involving human beings.
Biological sample and data collection. Approximately 4 mL of peripheral blood was obtained from ALL patients in remission using venipuncture in tubes with a vacuum system containing EDTA (BD Vacutainer EDTA K2®). From the control group, approximately 12 mL of peripheral blood were collected by venipuncture in tubes with a vacuum system containing EDTA, Sodium Citrate (BD Vacutainer Citrate Tube®) and with Separator Gel (Gel BD SST® II Advance) for complete blood count, biochemical tests and serology, respectively. In addition, demographic (age, gender), laboratory (blood count, immunophenotype) and clinical (comorbidities [infectious diseases and others], relapse and death), data were obtained from searches of medical records in the medical and statistical care system (SAME), iDoctor system and statistics sector of the HEMOAM.
Infections serologically tested as IgG + and IgM + (cytomegalovirus, toxoplasmosis, rubella, varicella, parasitic diseases, among others) were considered as infectious comorbidities according to Silva-Júnior et al. (2019) 50 . Aplasia, Systemic Arterial Hypertension (SAH), Diabetes Mellitus and Down Syndrome were included in the group "Others". In addition, patients who relapsed after induction therapy (35th day of treatment) were used as a relapse criterion. Death that occurred within 5 years after diagnosis was considered.
PCR reactions were performed according to the protocol described by Bhat