Human Leukocyte Antigen Alleles (HLA-A, HLA-B, and HLA-DRB1) are associated with Acute Lymphoblastic Leukemia (ALL) : A Case-Control Study in a Sample of Iranian Population

Objective: This study seeks to elucidate the association between HLA-A, HLA-B, and HLA-DRB1 alleles and their relative risk contributions to ALL within an Iranian cohort. Methods: Utilizing a robust case-control design, this research involved 71 ALL patients and 71 age and sex-matched healthy individuals. Genotyping of specified HLA alleles was performed using the advanced PCR-SSP technique. Results: Our findings reveal a marked increase in the prevalence of the HLA-DRB1*04 allele among patients diagnosed with ALL compared to the control group (P<0.027). Conversely, the alleles HLA-A*26 (P=0.025), HLA-A*33 (P=0.020), and HLA-DRB1*03 (P=0.035) were observed at significantly reduced frequencies within the patient population. Conclusion: Our findings highlight HLA-DRB1*04 as a potential genetic marker for increased susceptibility to ALL, while HLA-A*26, HLA-A*33, and HLA-DRB1*03 emerge as protective factors.


Introduction
Acute lymphoblastic leukemia (ALL), the most prevalent cancer among children, sees about 75% of leukemia cases globally [1,2].Despite high survival rates exceeding 85% for those without relapse, those experiencing relapse face severe prognoses.This positions ALL as a significant pediatric oncological challenge [3,4].
Emerging studies suggest a sophisticated interplay between genetics and environmental factors in ALL's development.The timing of viral exposures and subsequent immune responses are particularly critical.This context highlights the importance of the Human Leukocyte Antigen (HLA) system in genetic susceptibility to ALL.The HLA, or major histocompatibility complex, involves over 100 polymorphic genes on chromosome 6 that are pivotal in immune defense, making it a focal point in ALL research [5,6].
The HLA system, also known as the major histocompatibility complex (MHC), comprises a set of highly polymorphic genes located on chromosome 6.

Human Leukocyte Antigen Alleles (HLA-A, HLA-B, and HLA-DRB1) are associated with Acute Lymphoblastic Leukemia (ALL) : A Case-Control Study in a Sample of Iranian Population
This region spans approximately 4 million base pairs and contains over 100 genes, divided into three primary classes: Class I (HLA-A, -B, -C), Class II (HLA-DP, -DQ, -DR), and Class III [7,8].hese genes are essential for coordinating the immune response against pathogens by facilitating the formation of TCR/peptide/HLA complexes.Given its pivotal role in immune mechanisms and its status as one of the most polymorphic gene clusters in the human genome, the HLA system has been extensively studied for its association with various diseases, including ALL [3].
Research into the association between HLA genes and ALL susceptibility continues to reveal a range of diverse and occasionally contradictory outcomes [9].Investigations conducted in Eastern China have shown the significant role of the HLA-DRB114 locus in ALL, although no significant disparities were observed in the HLA-A and HLA-B loci between affected individuals and control groups [7].Further studies have identified the HLA-DRB104 allele as increasing susceptibility to childhood ALL among females, additionally affecting the age at which the disease manifests [1].  3 Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Science, Tehran, Iran. 4 Department of Biochemistry, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran. 5 Student Research Committee, School of Medicine, Kerman University of Medical Sciences, Kerman, Iran. 6Hematology-Oncology and Stem Cell Transplantation Research Center, Tehran University of Medical Sciences, Tehran, Iran. 7Cellular and Molecular Research Center, Gerash University of Medical Sciences, Gerash, Iran.*For Correspondence: ahmad.fatemi2@gmail.comFurther examination within pediatric ALL populations has noted the prognostic implications of the HLA-DRB111 allele.Research in Turkey highlighted several alleles such as A03 and DRB104 as predisposing factors, with the DRB104 allele specifically linked to an increased risk of ALL, in contrast to the standard risk associated with the DRB107 allele [3].More recent findings suggest that the MBL2 BB genotype, when combined with DRB107 or co-inherited with HLA-DRB104 and DRB107, could contribute to pediatric ALL, emphasizing the role of initial immune defense mechanisms [2].
The complexity of HLA interactions is further evidenced by findings suggesting a link between ALL and the HLA-A11 and DRB101 alleles, alongside a noted negative association with the DRB113 allele [10].Complementary research in Hong Kong has highlighted specific HLA markers in high-risk pediatric ALL cases, where HLA-B67 emerged as a male-specific marker linked to increased susceptibility to relapse, and HLA-A29 and HLA-B07 were identified as indicators of poor prognosis [6].Additionally, the predictive potential of HLA class II expression in determining the efficacy of treatment for adult T-cell leukemia/lymphoma has been discovered, further illustrating the critical role of HLA gene expression in cancer progression and treatment response [11].
This backdrop of varying global research outcomes sets the stage for our case-control study, which aims to deepen understanding of the associations between specific HLA alleles and ALL development within an Iranian population [5] By contextualizing our research within global findings, we aim to contribute uniquely to the broader genetic narrative of ALL.

Participants
The present case-control study enrolled 71 individuals diagnosed with ALL, in accordance with the 2016 World Health Organization diagnostic criteria, alongside an equivalent number of healthy, non-related participants matched by age and sex.Sample size determination utilizing the PGA program aimed for a statistical power of 80% with a two-sided alpha error set at 5%.Despite exhaustive efforts to collect all available samples, the calculated target sample size was not achieved [12].

Ethics statement
This project was approved by the ethical committee of Kerman University of Medical Sciences (Ethical approval code: IR.KMU.REC.1398.131).Before initiating the collection of samples, all participants formally acknowledged and consented to their involvement by signing informed consent forms, thus upholding the study's ethical standards.

DNA extraction
Genomic DNA was isolated from whole blood samples that had been treated with EDTA (Ethylene-Diamine-Tetra-Acetic-Acid), which served as an anticoagulant, utilizing the EZ1 DSP DNA Blood Kit (QIAGEN, Netherlands).This protocol employed magnetic-particle technology and required sample volumes of either 200 or 350 μL.The extraction process commenced with the addition of chaotropic salts to the lysates, enhancing the DNA's affinity for the kit's magnetic particles and allowing for efficient, single-step extraction.Subsequent magnetic separation facilitated the isolation of DNAbound particles.The samples were then subjected to a washing process using an elution buffer to eliminate residual impurities.The purity and concentration of the isolated DNA were quantitatively assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, Massachusetts, USA).Post-extraction, the DNA samples were preserved at -20°C to ensure their stability for subsequent genotyping analyses.

HLA Typing
HLA allele genotyping was performed using the sequence-specific primer-polymerase chain reaction (SSP-PCR) technique, leveraging low-resolution typing kits supplied by Olerup, Sweden.This technique capitalizes on oligonucleotide primers designed to accommodate 3'-end mismatches, ensuring efficient amplification when there is near-perfect match to the target DNA.Following amplification, the PCR products were resolved using agarose gel electrophoresis, which separates DNA fragments by size.These fragments were visualized, photographed, and analyzed to assess the allelic diversity.Figure 1 presents a typical result from the SSP-PCR analysis, illustrating the specificity and precision of this genotyping approach.

Statistical Analysis
In this study, the Pearson's Chi-square test was strategically applied to discern variations in allele frequencies between the subject groups.Advanced multivariate logistic regression analyses were then conducted to probe the relationship between individual alleles and the risk of acute lymphoblastic leukemia (ALL), incorporating the derivation of odds ratios (ORs) and 95% confidence intervals (CIs) to enhance predictive accuracy.These statistical analyses were adeptly handled using the SPSS 22 software (IBM/SPSS Inc., New York, USA), configured to ensure rigorous data integrity.Statistical significance was established at a P-value of 0.05 or less, adhering to stringent analytical standards.

Study samples
In this research, the demographic and clinical profiles of 71 healthy individuals (47 males and 24 females, average age 23.26 ± 12.5) were rigorously analyzed alongside those of 71 patients diagnosed with ALL (48 males and 23 females, average age 21.63 ± 13 years), as detailed in Table 1.Advanced statistical matching was employed to ensure comparability between groups, with the cohorts demonstrating no significant differences in age and gender (P-values of 0.12 and 0.72, respectively).This methodological precision enhances the reliability of subsequent findings, setting a robust foundation for exploring the specific impacts of ALL, independent of

HLA-A allelic associations with ALL
In the analysis of HLA-A alleles among both healthy subjects and patients with ALL, distinctive differences in statistical significance were observed.Notably, the alleles HLA-A*26 and HLA-A*33 demonstrated a correlation with decreased risk of ALL, exhibiting p-values of 0.025 and 0.02, respectively.Conversely, other HLA-A alleles did not show significant associations with ALL risk.Detailed data on the frequency of HLA-A alleles and their correlations with ALL are systematically presented in Table 2.

HLA-B allelic associations with ALL
The analysis of this study clearly delineates the absence of an association between the prevalence of HLA-B alleles and ALL incidence among both patients and healthy controls.Detailed in Table 3, these findings are systematically presented.

HLA-DRB1 allelic associations with ALL
The results of our investigation identified statistically significant differences in the frequencies of the HLA-DRB1*03 and DRB1*04 alleles between the healthy controls and individuals diagnosed with ALL, evidenced by p-values of 0.035 and 0.027, respectively.These findings, including the specific allele frequencies and their relationships with ALL incidence, are comprehensively detailed in Table 4.

Discussion
Genetic and environmental factors are intricately linked in the pathogenesis of ALL [1].While the precise biological mechanisms initiating ALL remain largely undefined, extensive research has illuminated the critical involvement of specific HLA alleles in the disease's onset and progression [6,13,14].Detailed analysis of these alleles in ALL patients promises to unlock valuable insights into prognosis, therapeutic response, and the likelihood of relapse [15,16,5].In this context, the current investigation is designed to rigorously assess the associations between HLA-A, HLA-B, and HLA-DRB1 alleles and the susceptibility to ALL within a distinct cohort of Iranian patients.Building upon prior research, our findings suggest that the HLA-DRB1*13 allele, which occurs less frequently among ALL patients, may exert a protective effect against the disease, while the HLA-DRB1*04 allele, which shows a moderate increase in prevalence among affected individuals, could be a susceptibility factor for childhood ALL [17].This study supports these observations by demonstrating a significant association between the frequencies of HLA-DRB1*03 and DRB1*04 alleles and the incidence of ALL.The results of this investigation elucidate that the presence of HLA-A*26 and HLA-A*33 alleles is significantly associated with the incidence of ALL.Comprehensive analyses have delineated a spectrum of HLA-A alleles, with A*33, A*01, A*03, and A*26 exhibiting protective effects, whereas A*24, A*31, A*23, A*30, A*68, and A*74 are identified as predisposing factors, collectively accounting for half of the alleles analyzed within populations at high risk for ALL [14].This nuanced understanding challenges and expands upon previous findings, such as those reported by William Klitz and collaborators, who posit that A*26 and A*33 alleles act as protective agents in the incidence of childhood ALL [27].
The current investigation found no significant correlation between HLA-B alleles and ALL.This lack of association is consistent with findings from Zhou et al., who also did not observe notable differences in HLA-B allele frequencies between ALL patients and a control group from Eastern China.In contrast, a comprehensive study in Northern China involving ALL patients and healthy controls reported a different pattern [7].This study identified a higher frequency of HLA-B*13:01 and B*40:02, along with a lower frequency of B*46:01 in ALL patients compared to the control population [28].
Several significant constraints have impacted the robustness of this study.Primarily, the small sample size restricts our ability to definitively conclude the associations between the identified HLA alleles and ALL.Despite over two years of diligent efforts to collect a comprehensive dataset, the research team was unable to secure a larger cohort.Additionally, the scope of the study was confined to a particular geographical region, which might have introduced a geographical bias affecting the generalizability of the findings.Considering the known variability of HLA alleles among different ethnic groups, it is imperative for future studies to expand their participant base to include a more diverse array of global populations.
In conclusion, our study identifies HLA-DRB1*04 as a potentially pivotal susceptibility factor for ALL, while HLA-A*26, HLA-A*33, and HLA-DRB1*03 emerge as likely protective elements.This delineation paves the way for groundbreaking approaches in the stratification of genetic risk factors, enabling more precise predictions of ALL susceptibility across various demographic landscapes.

Figure 1 .
Figure 1.Representative Result of PCR-SSP for HLA Typing.

Table 1
. Demographic and Clinical Characteristics of ALL Cases and Healthy Controls demographic variances.

Table 2 .
Association of HLA-A Alleles and Relative Risk of ALL Incidence (*P<0.05 is in bold).

Table 3 .
HLA-B allele Frequencies and association with Relative Risk of ALL Incidence.

Table 4 .
Corroborating our results, a seminal study from Turkey in 2002 identified a positive correlation between the HLA-DRB1*04 allele and ALL, Association of HLA-DRB1 Alleles and Relative Risk of ALL Incidence (*P<0.05 is in bold).