Nonchromosomal birth defects and risk of childhood acute leukemia: An assessment in 15 000 leukemia cases and 46 000 controls from the Childhood Cancer and Leukemia International Consortium.

Although recent studies have demonstrated associations between nonchromosomal birth defects and several pediatric cancers, less is known about their role on childhood leukemia susceptibility. Using data from the Childhood Cancer and Leukemia International Consortium, we evaluated associations between nonchromosomal birth defects and childhood leukemia. Pooling consortium data from 18 questionnaire-based and three registry-based case-control studies across 13 countries, we used multivariable logistic regression models to estimate odds ratios (ORs) and 95% confidence intervals (CIs) for the association between a spectrum of birth defects and leukemia. Our analyses included acute lymphoblastic leukemia (ALL, n = 13 115) and acute myeloid leukemia (AML, n = 2120) cases, along with 46 172 controls. We used the false discovery rate to account for multiple comparisons. In the questionnaire-based studies, the prevalence of birth defects was 5% among cases vs 4% in controls, whereas, in the registry-based studies, the prevalence was 11% among cases vs 7% in controls. In pooled adjusted analyses, there were several notable associations, including (1) digestive system defects and ALL (OR = 2.70, 95% CI: 1.46-4.98); (2) congenital anomalies of the heart and circulatory system and AML (OR = 2.86, 95% CI: 1.81-4.52) and (3) nervous system defects and AML (OR = 4.23, 95% CI: 1.50-11.89). Effect sizes were generally larger in registry-based studies. Overall, our results could point to novel genetic and environmental factors associated with birth defects that could also increase leukemia susceptibility. Additionally, differences between questionnaire- and registry-based studies point to the importance of complementary sources of birth defect phenotype data when exploring these associations.

Identifying childhood leukemia with an excess of hematological malignancies in first-degree relatives in Brazil.

Background: Familial aggregation in childhood leukemia is associated with epidemiological and genomic factors. Albeit epidemiological studies on the familial history of hematological malignancies (FHHMs) are scarce, genome-wide studies have identified inherited gene variants associated with leukemia risk. We revisited a dataset of acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) patients to explore the familial aggregation of malignancies among their relatives. Methods: A series of 5,878 childhood leukemia (≤21 years of age) from the EMiLI study (2000-2019) were assessed. Lack of well-documented familial history of cancer (FHC) and 670 cases associated with genetic phenotypic syndromes were excluded. Leukemia subtypes were established according to World Health Organization recommendations. Logistic regression-derived odds ratios (ORs) and 95% confidence intervals (CIs) were performed and adjusted by age as a continuous variable, where ALL was the reference group for AML and conversely. The pedigree of 18 families with excess hematological malignancy was constructed. Results: FHC was identified in 472 of 3,618 eligible cases (13%). Ninety-six of the 472 patients (20.3%) had an occurrence of FHHMs among relatives. Overall, FHC was significantly associated with AML (OR, 1.36; 95% CI, 1.01-1.82; p = 0.040). Regarding the first-degree relatives, the OR, 2.92 95% CI,1.57-5.42 and the adjOR, 1.16 (1.03-1.30; p0.001) were found for FHC and FHHM, respectively. Conclusion: Our findings confirmed that AML subtypes presented a significant association with hematological malignancies in first-degree relatives. Genomic studies are needed to identify germline mutations that significantly increase the risk of developing myeloid malignancies in Brazil.

A Novel Constitutional t(3;8)(p26;q21) and ANKRD26 and SRP72 Variants in a Child with Myelodysplastic Neoplasm: Clinical Implications.

BACKGROUND: Childhood myelodysplastic neoplasm (cMDS) often raises concerns about an underlying germline predisposition, and its verification is necessary to guide therapeutic choice and allow family counseling. Here, we report a novel constitutional t(3;8)(p26;q21) in a child with MDS, inherited from the father, the ANKRD26 and SRP72 variants from the maternal origin, and the acquisition of molecular alterations during MDS evolution. CASE PRESENTATION: A 4-year-old girl showed repeated infections and severe neutropenia. Bone marrow presented hypocellularity with dysplastic features. The patient had a t(3;8)(p26;q21)c identified by G-banding and FISH analysis. The family nucleus investigation identified the paternal origin of the chromosomal translocation. The NGS study identified ANKRD26 and SRP72 variants of maternal origin. CGH-array analysis detected alterations in PRSS3P2 and KANSL genes. Immunohistochemistry showed abnormal p53 expression during the MDS evolution. CONCLUSION: This study shows for the first time, cytogenetic and genomic abnormalities inherited from the father and mother, respectively, and their clinical implications. It also shows the importance of investigating patients with constitutional cytogenetic alterations and/or germline variants to provide information to their family nucleus for genetic counseling and understanding of the pathogenesis of childhood MDS.

GATA2 variants in patients with non-tuberculous mycobacterial or fungal infections without known immunodeficiencies.

INTRODUCTION: Haploinsufficiency of the hematopoietic transcription factor GATA2 is associated with a broad spectrum of diseases, including infection susceptibility and neoplasms. We aimed to investigate GATA2 variants in patients with non-tuberculous mycobacterial (NTM) and/or fungal infections (FI) without known immunodeficiencies. METHOD: We performed GATA2 genotyping in patients with NTM and/or FI. RESULTS: Twenty-two patients were enrolled (seventeen FI, four NTM and one with both infections). The pathogenic variant NG_029334.1:g.16287C>T was found in one patient (4.5%) and two asymptomatic offsprings. We also found the likely-benign variant NG_029334.1:g.12080G>A (rs2335052), the benign variant NG_029334.1:g.16225C>T (rs11708606) and the variant of uncertain significance NG_029334.1:g.16201G>A (rs369850507) in 18.2%, 27.3%, and 4.5% of the cases, respectively. Malignant diseases were additionally diagnosed in six patients. CONCLUSION: Although detected in 45.4% of the patients, most GATA2 variants were benign or likely benign. Identifying a pathogenic variant was essential for driving both the patient's treatment and familial counseling. Pathogenic variants carriers should receive genetic counseling, subsequent infection prevention measures and malignancies surveillance. Additionally, case-control genotyping should be carried out in Brazil to investigate whether the observed variants may be associated with susceptibility to opportunistic infections and/or concurrent neoplasms.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia.

INTRODUCTION: This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). METHODS: A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. RESULTS: We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p = 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. CONCLUSION: The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

The Interplay of Cesarean-Section Delivery and First-Birth Order as Risk Factors in Acute Lymphoblastic Leukemia.

BACKGROUND: Childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been associated with early-life exposures, including birth by cesarean section (C-section), and a deficit of social exposure (first child). These exposures as proxies for microbiome acquisition in infancy are essential to prime the immune system and restrain later dysregulated immune responses that can trigger ALL in susceptible individuals. We tested risk factors pertaining to immune stimulation that may impact BCP-ALL development. METHODS: Cases comprised 1,126 children (0-12 years) with ALL (BCP-ALL: 78.5%) from the EMiLI study group in Brazil (2002-2020). Age- and sex-matched controls (n = 2,252) were randomly selected from healthy children whose mothers participated in the National Placental and Umbilical Cord Blood Bank donation. Multiple logistic regression was run fitted and adjusted for selected covariates models. RESULTS: C-section delivery was associated with increased risk for ALL [odds ratio (OR) ALL: 1.10; 95% confidence intervals (CI), 1.04-1.15; ORBCP-ALL: 1.09; 95% CI, 1.03-1.14], as well as being the firstborn child. Interaction analysis showed a significant effect of first birth on the observed C-section associations (P < 0.0001). Indeed, high-risk children, namely, firstborn children delivered via C-section were at increased risk for ALL (OR: 2.33; 95% CI, 2.40-4.84) compared with non-first, vaginally born children. An increased risk was found for firstborn children delivered by C-section and non-breastfed with ALL (ORALL: 2.32; 95% CI, 1.27-4.24; ORBCP-ALL: 2.37; 95% CI, 1.18-4.76). CONCLUSIONS: Our observations are in accord with the prediction that exposures determining microbiome composition and adrenal pathway in infancy contribute to the risk of BCP-ALL. IMPACT: These findings encourage the exploration of potential preventive interventions. See related commentary by Wiemels and Gallant, p. 292.

Interaction between birth characteristics and CRHR1, MC2R, NR3C1, GLCCI1 variants in the childhood lymphoblastic leukemia risk.

Background: The incidence rate of childhood acute lymphoblastic leukemia (ALL) differs worldwide, and the interplay between hemostasis actors and the maladaptive responses to environmental exposures has been explored. It has been proposed that endogenous cortisol, induced by different triggers, would eliminate pre-leukemic clones originated in utero. Herein, we tested if the interaction between CRHR1rs242941 C>A, MC2Rrs1893219 A>G, NR3C1rs41423247 G>C, and GLCCI1rs37972 C>T (players in glucocorticoid secretion) and birth characteristics would be associated with ALL risk. Methods: Children aged <10 years were enrolled within the EMiLI project (period: 2012 to 2020). The study had three steps: (1) observational analysis of birth characteristics (n = 533 cases and 1,603 controls); (2) genotyping to identify single-nucleotide variants (n = 756 cases and 431 controls); and (3) case-only to test gene-environment interactions (n = 402 cases). Genetic syndromes were exclusion criteria. The controls were healthy children. The distribution of the variables was assessed through Pearson's chi-square test. Logistic regression (LR) tests were run fitted and adjusted for selected covariate models to estimate the association risk. Formal interaction analysis was also performed. Genotyping was tested by qPCR with TaqMan probes (NR3C1) or by high-resolution melting (MC2R and GLCCI1). Hardy-Weinberg equilibrium (HWE) was accessed by the chi-square test. The genotype-risk association was tested in co-dominant, dominant, and recessive models. The gene-environment interaction odds ratio (iOR) was assessed in case-only. Results: Low birthweight, C-section, and low maternal schooling were associated with increased risk for ALL, adjOR 2.11, 95% CI, 1.02-4.33; adjOR 1.59, 95% CI, 1.16-2.17; and adjOR 3.78, 95% CI, 2.47-5.83, respectively, in a multiple logistic regression model. MC2R rs1893219 A>G was negatively associated with ALL (AG: OR = 0.68; 95% CI = 0.50-0.94 and GG: OR = 0.60; 95% CI = 0.42-0.85), while for GLCCI1 rs37972 C>T, TT was positively associated with ALL (OR = 1.91; 95% CI = 1.21-3.00). The combination of genotypes for MC2R (AA) and GLCCI1 (TT) increased ALL risk (OR = 2.61; 95% CI = 1.16-5.87). In a multiplicative interaction, MC2R rs1893219 A>G was associated with children whose mothers had less than 9 years of schooling (iOR = 1.99; 95% CI = 1.11-1.55). Conclusion: Our study has demonstrated a significant association between MC2R rs1893219 A>G (reduced risk) and GLCCI1 rs37972 C>T variants (increased risk) and childhood ALL susceptibility. Based on this evidence, genes controlling the HPA axis activity may play a role in leukemogenesis, and further investigation is needed to substantiate our findings.

Multiparametric flow cytometry directing the evaluation of CRLF2 rearrangements and JAK2 status in pediatric B cell precursor acute lymphoblastic leukemia

Asbtract Introduction This study aimed to determine whether cytokine receptor-like factor 2 (CRLF2) antigen expression evaluated using multiparametric flow cytometry (MFC) could predict the genotype of CRLF2 and Janus kinase 2 (JAK2) status for application in the diagnosis of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Methods A total of 321 BCP-ALL bone marrow samples were collected, 291 at diagnosis and 13 at first relapse, while 17 samples were excluded due to low cellular viability. The CRLF2 antigen expression was evaluated using flow cytometry (percentage of positivity and median fluorescence intensity [MFI]). The CRLF2 transcript levels were assessed via quantitative reverse transcription polymerase chain reaction using SYBR Green. The CRLF2 rearrangements (CRLF2-r) were identified using the CRLF2 break-apart probe via fluorescence in situ hybridization. Sanger sequencing was performed to identify the JAK2 exon 16 mutations. Results We observed that 60 of the 291 cases (20.6%) presented CRLF2 antigen positivity, whereas the CRLF2 transcript overexpression was found in 19 of 113 cases (16.8%). The JAK2 mutation was found in four out of 116 cases (3.4%), all of which had CRLF2 ≥10% of positive cells and intermediate or high MFI (p < 0.0001). In addition, in the 13 cases with the CRLF2-r, a positive correlation was found with the CRLF2 antigen intermediate (61.5%) MFI (p= 0.017). Finally, the CRLF2-positive antigen was identified in the BCP-ALL subclones. Conclusion The identification of the CRLF2 antigen using the MFC, based on the percentage of positivity and MFI values, is a useful tool for predicting JAK2 mutations and CRLF2-r.

GATA2 variants in patients with non-tuberculous mycobacterial or fungal infections without known immunodeficiencies

ABSTRACT Introduction Haploinsufficiency of the hematopoietic transcription factor GATA2 is associated with a broad spectrum of diseases, including infection susceptibility and neoplasms. We aimed to investigate GATA2 variants in patients with non-tuberculous mycobacterial (NTM) and/or fungal infections (FI) without known immunodeficiencies. Method We performed GATA2 genotyping in patients with NTM and/or FI. Results Twenty-two patients were enrolled (seventeen FI, four NTM and one with both infections). The pathogenic variant NG_029334.1:g.16287C>T was found in one patient (4.5%) and two asymptomatic offsprings. We also found the likely-benign variant NG_029334.1:g.12080G>A (rs2335052), the benign variant NG_029334.1:g.16225C>T (rs11708606) and the variant of uncertain significance NG_029334.1:g.16201G>A (rs369850507) in 18.2%, 27.3%, and 4.5% of the cases, respectively. Malignant diseases were additionally diagnosed in six patients. Conclusion Although detected in 45.4% of the patients, most GATA2 variants were benign or likely benign. Identifying a pathogenic variant was essential for driving both the patient's treatment and familial counseling. Pathogenic variants carriers should receive genetic counseling, subsequent infection prevention measures and malignancies surveillance. Additionally, case-control genotyping should be carried out in Brazil to investigate whether the observed variants may be associated with susceptibility to opportunistic infections and/or concurrent neoplasms.

The genomic landscape of teenage and young adult T-cell acute lymphoblastic leukemia.

BACKGROUND: Treatment on risk adapted intensive pediatric protocols has improved outcome for teenagers and young adults (TYA) with T-cell acute lymphoblastic leukemia (T-ALL). Understanding the biology of disease in this age group and the genetic basis of relapse is a key goal as patients with relapsed/refractory disease have poor outcomes with conventional chemotherapy and novel molecular targets are required. This study examines the question of whether TYA T-ALL has a specific biological-molecular profile distinct from pediatric or adult T-ALL. METHODS: Genomic characterization was undertaken of a retrospective discovery cohort of 80 patients aged 15-26 years with primary or relapsed T-ALL, using a combination of Genome-Wide Human SNP Array 6.0, targeted gene mutation and promoter methylation analyses. Findings were confirmed by MLPA, real-time quantitative PCR, and FISH. Whole Exome Sequencing was performed in 4 patients with matched presentation and relapse to model clonal evolution. A prevalence analysis was performed on a final data set of 1,792 individual cases to identify genetic lesions with age specific frequency patterns, including 972 pediatric (1-14 years), 439 TYA (15-24 years) and 381 adult (≥25 years) cases. These cases were extracted from 19 publications with comparable genomic data identified through a PubMed search. RESULTS: Genomic characterization of this large cohort of TYA T-ALL patients identified recurrent isochromosome 7q i(7q) in our discovery cohort (n = 3). Prevalence analysis did not identify any age specific genetic abnormalities. Genomic analysis of 6 pairs of matched presentation - relapsed T-ALL established that all relapses were clonally related to the initial leukemia. Whole exome sequencing analysis revealed recurrent, targetable, mutations disrupting NOTCH, PI3K/AKT/mTOR, FLT3, NRAS as well as drug metabolism pathways. CONCLUSIONS: All genetic aberrations in TYA T-ALL occurred with an incidence similar or intermediate to that reported in the pediatric and adult literature, demonstrating that overall TYA T-ALL exhibits a transitional genomic profile. Analysis of matched presentation - relapse supported the hypothesis that relapse is driven by the Darwinian evolution of sub-clones associated with drug resistance (NT5C2 and TP53 mutations) and re-iterative mutation of known key T-ALL drivers, including NOTCH1.

Therapy-related acute myeloid leukemia with KMT2A-SNX9 gene fusion associated with a hyperdiploid karyotype after hemophagocytic lymphohistiocytosis.

Therapy-related acute myeloid leukemia (t-AML) following treatment with topoisomerase-II inhibitors has been increasingly reported. These compounds (e.g. etoposide) promote DNA damage and are associated with KMT2A rearrangements. They are widely used as first-line treatment in hemophagocytic lymphohistiocytosis (HLH). Here we describe a newborn who developed t-AML after HLH treatment. We provide detailed clinical, cytogenetic, and molecular characteristics of this patient, including the identification of a novel gene fusion - KMT2A-SNX9 - in t-AML. Considering the dismal outcome of this case, we discuss the side-effects of etoposide administration during HLH treatment in infants.

Pediatric Acute Promyelocytic Leukemia: Epidemiology, Molecular Features, and Importance of GST-Theta 1 in Chemotherapy Response and Outcome.

Previous studies have suggested a variation in the incidence of acute promyelocytic leukemia (APL) among the geographic regions with relatively higher percentages in the Latin American population. We aimed to explore the population burden of pediatric APL, gathering information from the population-based cancer registry (PBCR) and the diagnosis of APL obtained through incident cases from a hospital-based cohort. The homozygous deletion in glutathione S-transferases (GSTs) leads to a loss of enzyme detoxification activity, possibly affecting the treatment response. Mutations in the RAS pathway genes are also considered to be a key component of the disease both in the pathogenesis and in the outcomes. We have assessed mutations in a RAS-MAP kinase pathway (FLT3, PTPN11, and K-/NRAS) and GST variant predisposition risk in the outcome. Out of the 805 children and adolescents with acute myeloid leukemia (AML) who are registered in the PBCR, 35 (4.3%) were APL cases. The age-adjusted incidence rate (AAIR) was 0.03 per 100,000 person-years. One-hundred and sixty-three patients with APL were studied out of 931 AML cases (17.5%) from a hospital-based cohort. Mutations in FLT3, KRAS, and NRAS accounted for 52.1% of the cases. Patients with APL presented a 5-year probability of the overall survival (OS) of 67.3 ± 5.8%. A GST-theta 1 (GSTT1) null genotype conferred adverse prognosis, with an estimated hazard ratio of 2.8, 95% confidence interval (CI) 1.2-6.9. We speculate that the GSTT1 polymorphism is associated with therapeutics and would allow better OS of patients with APL with a GSTT1 null genotype.

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

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.

The multiple ways Wnt signaling contributes to acute leukemia pathogenesis.

WNT proteins constitute a very conserved family of secreted glycoproteins that act as short-range ligands for signaling with critical roles in hematopoiesis, embryonic development, and tissue homeostasis. These proteins transduce signals via the canonical pathway, which is ß-catenin-mediated and better-characterized, or via more diverse noncanonical pathways that are ß-catenin independent and comprise the planar cell polarity (PCP) pathway and the WNT/Ca++ pathways. Several proteins regulate Wnt signaling through a variety of sophisticated mechanisms. Disorders within the pathway can contribute to various human diseases, and the dysregulation of Wnt pathways by different molecular mechanisms is implicated in the pathogenesis of many types of cancer, including the hematological malignancies. The types of leukemia differ considerably and can be subdivided into chronic, myeloid or lymphocytic, and acute, myeloid or lymphocytic, leukemia, according to the differentiation stage of the predominant cells, the progenitor lineage, the diagnostic age strata, and the specific molecular drivers behind their development. Here, we review the role of Wnt signaling in normal hematopoiesis and discuss in detail the multiple ways canonical Wnt signaling can be dysregulated in acute leukemia, including alterations in gene expression and protein levels, epigenetic regulation, and mutations. Furthermore, we highlight the different impacts of these alterations, considering the distinct forms of the disease, and the therapeutic potential of targeting Wnt signaling.

MTHFR rs1801133 polymorphism is associated with increased risk of B-cell precursor lymphoblastic leukaemia with recurrent genetic aberrations of fetal origin.

BACKGROUND: Childhood acute lymphoblastic leukaemia (ALL) is a heterogeneous disease associated with multiple risk factors including genetic susceptibility. Polymorphisms in folate genes have been associated with a protective effect against ALL, although some studies contradict these findings. We aimed to test whether there is an association between the MTHFR rs1801133 variant and the occurrence of B-cell precursor ALL (BCP-ALL) taking in account molecularly distinct subtypes of fetal origin. METHODS: We performed a case-control genotyping study with 2067 samples, 1309 ALL and 758 controls, from children aged ≤ 15 years for MTHFR rs1801133 polymorphism. Risk associations were calculated by odds ratios estimated with unconditional logistic regression, adjusted for frequency-matched ethnic groups. RESULTS: Overall, MTHFR rs1801133 does not impact ALL risk in children with more than 6 years of age. A significant positive association for MTHFR rs1801133 variant was found for ALL with KMT2A-r in the dominant model (adj. OR, 1.48, 95 % CI, 1.01-2.17), while ETV6-RUNX1 and Hyperdiploid subgroups have shown a borderline effect (adj. OR, 1.33, 95 % CI, 0.99-1.78). CONCLUSIONS: The polymorphism MTHFR rs1801133 increased the risk of infant ALL in Brazilian population.