1 Introduction

Cancer is becoming more frequent and lethal in both developed and developing countries, with global economic impacts (Siegel et al., 2021). Blood cancers account for nearly 10% of all newly diagnosed cancer cases in the world (Eid et al., 2021). The most common type of pediatric cancer and the most leading cause of cancer mortality in children is acute lymphoblastic leukemia (ALL) (Inaba et al., 2013). ALL affects both children and adults, with a peak of occurrence between 1 and 4 years (Malard and Mohty, 2020). While the most common type of leukemia malignancy among adults is acute myeloid leukemia (AML). It is characterized by the proliferation of myeloid blasts or progranulocytes that do not differentiate normally (Newell and Cook, 2021). Exogenous or endogenous exposures, genetic predisposition, and randomness all play a role in causation (Alkhouly et al., 2013; Inaba et al., 2013). Also, chromosome abnormalities and genetic alterations that impact lymphoid precursor cell formation and proliferation are directly involved in the abnormal proliferation. However, the genetic factors for these leukemia diseases remain unknown.

According to circumstantial evidence, the host immune system can actively inhibit or tolerate tumor development. The production of immunosuppressive cytokines or the modification of antigen-presenting molecules on neoplastic cells is commonly blamed for tumor escape from host immune surveillance (Alkhouly et al., 2013). HLA-G is a non-classical HLA molecule of the class Ib and is located between HLA-A and HLA-F located on chromosome 6p21.3. It has eight exons and seven introns (Castelli et al., 2008).

The HLA-G protein has special features including the reduced allelic polymorphism, restricted tissue expression, and the existence of seven expression isoforms that could be membranous (G1, G2, G3 and G4) or soluble proteins (G5, G6 and G7) (Paul et al., 2000).

HLA-G has been reported for its suppressive function as it decreases the human immune response through a variety of mechanisms, including the inhibition of natural killer (NK) cells and T lymphocytes' cytotoxic activities (Hofmeister and Weiss, 2003). HLA-G plays a key role in fetal maternal immune tolerance by inhibiting maternal immunity through its expression in trophoblast cells at fetus-maternal interface during pregnancy (Moreau et al., 1998). Thus, reduced placental expression of HLA-G has been associated with pathological conditions such as recurrent spontaneous abortion (Peng et al., 2008). Outside the placenta and in physiological conditions, HLA-G expression has been documented in few immune-privileged tissue, such as the thymus and the cornea (Le Discorde et al., 2003). However, an ectopic expression of HLA-G molecules has been associated with pathological conditions such as cancer, autoimmune and inflammatory diseases (Zaborek-Łyczba et al., 2021).

Many studies have been conducted on the HLA-G polymorphism and its functional association with human diseases. Thus far, 94 HLA-G alleles have been identified (IPD-IMGT/HLA, R, 3.47, Marsh, 2022; https://www.ebi.ac.uk/ipd/imgt/hla).

Polymorphism sites in the HLA-G gene's non-coding regions, particularly in the 3′ UTR exon 8, have been reported to influence its expression (Rizzo et al., 2014). The insertion or deletion of a 14-bp (5′-ATTTGTTCATGCCT-3′) polymorphism in the 3′ UTR of the HLA-G gene, also known as rs371194629, is one of the most important polymorphisms (Rousseau et al., 2003). It has been reported in the untranslated exon 8 and was involved in the stability and expression of mRNA. The Del allele stabilizes the mRNA with a consequent higher HLA-G expression, while the Ins allele destabilizes the mRNA and is linked with a lower levels of HLA-G expression (Svendsen et al., 2013). According to Chen et al. (2008), the Ins alleles was associated with lower HLA-G mRNA levels and Del allele with higher level. In recent years, a rising number of studies have investigated the relationship between HLA-G 14-bp Ins/Del polymorphism and malignancies in different populations (Adolf et al., 2021). In Saudi Arabia, few studies have reported the association of HLA-G 14 Ins/Del variant and cancer diseases including breast cancer and colorectal cancer have been reported (Al Omar and Mansour, 2019; Al Omar et al., 2015; Hassan et al., 2019). However, only a handful studies have been done on leukemia. In Saudi Arabia, so far, no studies had investigated the association between HLA-G and any leukemia diseases. In this study, we investigated the genotypic and allelic association of HLA-G 14 bp Ins/Del polymorphism with the two most frequent leukemia malignancies (ALL and AML).

2 Materials and methods

2.4

2.4 Genotyping of the 14-bp Ins/Del

The 14-bp Ins/Del polymorphism (rs371194629) in exon 8 (3′ UTR) of the HLA-G gene (Fig. 1) was genotyped using the primers GE14HLAG:5′-GTGATGGGCTGTTTAAAGTGTCACC-3′ and RHG4: 5′-GGAAGGAATGCAGTTCAGCATGA-3′ (Hviid et al., 1999). Polymerase Chain Reaction (PCR) was done in a volume of 20 μl containing 1 μl of DNA, 10 ul of PCR GoTaq® Master Mixes 2X (Promega, India), 0.5 ul of each primer (10 pmol) and 8 µl of RNase-free water. The PCR reactions were performed in a T100^TM Thermocycler (Bio-Rad, Singapore). For amplification, the following thermocycling conditions were used: an initial denaturation phase at 95 °C (5 min), followed by 34 cycles of 94 °C (30 s), 60 °C (30 s), 72 °C (1 min), and 72 °C (5 min) as a final extension. The amplicons, which are either 210 bp in length for the 14-bp Del allele, or 224 bp in length for the 14-bp Ins allele, were migrated in a 3% agarose gel stained with ethidium bromide. The gel was visualized and photographed in a BioDocAnalyze (Biometra GmbH, Germany).

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Fig. 1

Polymorphism in the HLA-G gene in 3′ UTR. The 14-bp Ins/Del in exon 8.

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For DNA sequencing, six randomly selected samples including the three genotypes were sequenced using the same reverse primer used in PCR reaction. The PCR products were purified using EXO-SAP (New England Biolabs) and sequenced using the BigDye Terminator v.3.1 Sequencing Kit (Applied Biosystems, CA, USA) in an ABI Prism 3730 XL Genetic Analyzer (Applied Biosystems).

3 Results

The 14 bp Ins/Del HLA-G 3′UTR polymorphism was successfully genotyped for all the 243 patients with acute leukemia, including 145 ALL and 98 AML and 115 healthy controls. Fig. 2A shows a gel image of the genotyping result of the 14 bp polymorphism. Six PCR-genotyped samples from each group were selected for sequencing. Fig. 2B shows an example of electropherograms of the three HLA-G 14 bp Ins/Del genotypes, that confirm the classical genotyping methods. Allele frequencies of the Del and Ins are reported in Table 1. The genotype frequency of the HLA-G polymorphism was in Hardy-Weinberg equilibrium (HWE) for controls, AML and ALL patients. For the genetic and allelic association, we have tested five genetic models (allelic, codominant, dominant, recessive, overdominant and additive). Results are reported in Table 1. The allele distribution between control group and ALL and AML, did not show any significant difference. The Del allele appears slightly higher for all tested groups than the Ins allele, with a frequencies similar to those reported for many populations. For all the tested models of association, no significant differences were observed between controls and the two leukemia cancers.

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Fig. 2

Analysis of the14bp Ins/Del polymorphism using gel electrophoresis (A). For each patient and control. In the gel, Lanes 1, 5, 6, 8–12 are heterozygous; Lane 2 homozygous Ins/Ins and lanes 3, 4 and 7 are homozygous Del/Del. (B) sequencing of the 3′ UTR fragment, showing the electropherogram at the level of the 14 bp Ins/Del. B1, profile sequence of a homozygous Del/Del; B2, Profile of homozygous Ins/Ins and B3, profile of heterozygous Del/Ins.

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Furthermore, we assessed the possible association between HLA-G 14 bp Ins/Del and the risk to ALL and AML by stratified analysis based on gender (Table 2) and age (Table 3). Once again, for both leukemia diseases, no significant association was observed with the studied polymorphism.

In order to assess the relationship between HLA-G gene expression and ALL diseases, we analyzed the HLA-G mRNA expression level in 12 ALL and 12 control individuals using relative quantitative RT-PCR. Fig. 3 shows the diagram of the mean fold of expression for healthy control and ALL patients.. In the control group, the level of HLA-G is two times higher than in patients. However, the difference did not reach significance (P = 0.22).

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Fig. 3

Relative expression of HLA-G from blood samples of ALL patients compared to healthy individuals.

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4 Discussion

The prevalence of leukemia in Saudi Arabia is increasing every year. The number of cases passed from 297,000 to 437,033 between 1990 and 2018. The number of new cases in 2020 was 27885, while it was 24,485 in 2018 (Bawazir et al., 2019; Ferlay et al., 2021). Even though it is a result of screening methods progress, development of the immune therapy methods is necessary in order to improve leukemia control and treatment. HLA-G expression in both tumor and non-tumor cells is one of the mechanisms by which malignant cells escape detection by the host immune system (Amiot et al., 2011). This non-classical HLA-class I molecule was considered as an immunosuppressive checkpoint molecule, associated with immune evasion of malignant cells and was therefore inspected as a potential target for immunotherapy (Jasinski-Bergner et al., 2022). The level of expression of the HLA-G in some pathologic sites was associated in most of cases with unfavorable prognosis (Imani et al., 2018). While the relatively low polymorphism reported in coding or non-coding HLA-G molecule compared to classical HLA-genes, the most relevant polymorphisms were located within the regulatory untranslated regions of the gene, including the 3′ and 5′UTRs. Studies on the association between HLA-G polymorphism in coding and non-coding regions with cancer, infectious or autoimmune diseases have confirmed, in most of them, the biological effect features of this polymorphism (Attia et al., 2020). Controversial results in associations were reported according to the disease and the ethnicity which could be attributed to the etiological processes of cancer, including gene-to-gene or gene-environment interactions, sample sizes, and the age of disease onset. Several studies have found that certain HLA-G gene variants are linked to cancer development (Yie et al., 2007; Zheng et al., 2021). One of the most interesting dimorphic site, was the 14 bp Del/Ins in the 3′UTR region, as it was associated with HLA-G mRNA stability and alternative splicing (Hviid et al., 2003; Rousseau et al., 2003). In this case control study, we have investigated relationships between the allelic and genotypic 14 bp Ins/Del polymorphism of the HLA-G and two main types of acute leukemia; ALL and AML. We have explored different genetic models in this study, however our results showed lack of any association between alleles or genotypes of the HLA-G 14 bp Ins/Del polymorphism and ALL and AML. To the best of our knowledge and instead of the large number of case/ control studies that have explored the association between the HLA-G 14 bp polymorphism and solid cancer diseases. This is the first case/control study exploring the association between HLA-G 14 bp Ins/Del polymorphism and leukemia diseases. In a cohort study, including only patients with chronic lymphoid leukemia. Roberta et al. (2014) showed that the Del/Del genotype was associated with higher expression of HLA-G and reduced overall survival, compared to those with other genotypes and the presence of HLA-G molecules in this leukemia environment, creates a favourable setting for CLL expansion. The authors confirmed that in the presence of Del/Del patients have a poorer overall survival than those sharing either Ins/Del or Ins/Ins genotypes (Roberta et al., 2014). However, in another study, Własiuk et al. (2013) noted that higher levels of HLA-G on the surface and soluble are observed in AML patients especially on the surface of dendritic cells and CD4+ T cells and this high level of expression is not associated with any HLA-G 14 bp genotypes. In addition, we have performed an extensive analysis for the association between this HLA-G polymorphism and AML or ALL in subgroups according to age and gender. For all these analyses, we did not find any association considering these parameters. Considering that the functional feature of the HLA-G molecules are mostly associated with the level of expression, we have studied the mRNA expression and compared between ALL and healthy control. Our results did not show significant difference between these two groups, instead of the higher level among the control group. Unfortunately, we failed to obtain mRNA for AML patients. To compare with our results, we did not find in bibliography any case control study for HLA-G 14 bp genetic association and gene expression for ALL or AML diseases using healthy controls as reference group.

However, some studies have explored the relationships between these two aspects of HLA-G and some clinical and cellular parameters of variant leukemia. In some studies, the circulating blasts from CML and AML have been reported to express or to secrete HLA-G and the level of expression was associated with higher blast cells in the bone marrow and relapse (Caocci et al., 2017). Moreover, HLA-G expression has been suggested as predictive tumor biomarker to track disease progression or as a therapeutic target to improve immune responses to leukemia (Locafaro et al., 2014). Maki et al. (2008) pointed out that blocking HLA-G1 with a particular antibody enhanced the sensitivity of CLL samples to NK-mediated death, suggesting that HLA-G protects CLL cells from NK-mediated killing and so may contribute to their immunological escape in vivo. On the other hand, some studies reported opposite results. In one of these studies, patients with AML, CML, ALL, and CLL were selected for their HLA-G expression and they found that the HLA-G molecule was not expressed in newly separated human leukemia cells and hence plays no role in their immunological escape (Poláková et al., 2003). In the secreted forms of HLA-G, Giannopoulos et al. (2008) did no found differences in HLA-G mRNA expression or levels in the plasma of patients.

In conclusion, although our study did not show a significant association between HLA-G 14 bp polymorphism or gene expression with AML or ALL in the Saudi population, further investigations including large number of subjects with more clinical data are necessary to state for this association.