Data availability

Data will be made available on request.

1 Introduction

Acute Lymphoblastic Leukemia (ALL) represents one of the most prevalent malignant disorders globally, characterized by a complex interplay of genetic and environmental factors. The hallmark of ALL is a rapid proliferation of abnormal leukocytes, which accumulate in the bone marrow and interfere with the production of normal white blood cells (Tebbi, 2021).

Research has consistently shown that ALL involves significant dysregulation of the immune system, particularly evident during early childhood development. Furthermore, polymorphisms in genes related to innate immunity are believed to play critical roles in initiating intrinsic biological changes that predispose individuals to cancer (Monlish et al., 2016).

A key component in understanding the pathogenesis of ALL is the role of Toll-like receptors (TLRs). These receptors, integral to the innate immune response, identify pathogen-associated and damage-associated molecular patterns. TLRs are not only crucial in immune response mechanisms but also in cancer biology. They are expressed across a variety of cells, including hematopoietic stem cells, progenitor cells, and effector immune cells, as well as endothelial cells. Their involvement in cancer development is well-documented, linking aberrant TLR signaling to a spectrum of hematological malignancies, including lymphoproliferative disorders and myelodysplastic syndromes, which possess a high risk of transformation into leukemia (Gao et al., 2019; Sallustio et al., 2019; Monlish et al., 2016).

Toll-like receptor 3 (TLR3) has been targeted as an immunotherapy for different types of cancer. Located in endosomes and plays a vital role in recognizing viral double-stranded RNA, a key step in viral infection responses, and triggers the production of type I interferon (Le Naour et al., 2020). Emerging evidence suggests that TLR3′s expression in tumor cells—or its activation within the tumor microenvironment—may paradoxically either inhibit or promote tumor progression (Zheng et al., 2021). Moreover, research indicates that genetic variations in TLR3 may affect the protein's expression and function, influencing susceptibility to cancer progression (Meng et al., 2020).

In this context, our case-control study has identified potential susceptibility genes that support a polygenic model of cancer predisposition based on genetic and gene expression variations. These insights could prove crucial for understanding tumor aggressiveness and developing prognostic markers for leukemia and other cancers.

2 Materials and methods

3 Results

3.5

3.5 Association of TLR3 relative mRNA expression and ALL

TLR3 mRNA expression was assessed in healthy and diseased subjects. In comparison to the control group, TLR3 mRNA expression was higher in ALL patients than in healthy individuals. In summary, the Δ Ct mean of TLR3 expression in the ALL patients were 8.541 ± 0.348, and for the healthy donors was 11.444 ± 0.482, P- value = 0.001 (Table 3). That was also confirmed by the 2^-ΔΔ Ct method (fold change: 8.9029 ± 1.7049, p-value: 0.0001) (Fig. 1).

Table 3 Change ALL in TLR3 expression in patients and healthy individuals.

	Control	Patient	${\chi }^2$	P- value
Δ Ct TLR3	11.444 ± 0.482	8.541 ± 0.348	17.3	0.001

Mood median test for TLR3, Data are shown as mean ± SE (standard Error).

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Fig. 1

TLR3 expression level in ALL and control. TLR3 level varied significantly between patients and controls (p = 0.001).

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4 Discussion

To date, no studies have explored the potential impact of TLR3 variants on the risk of blood cancers in the Saudi population. This study is the first to investigate several SNPs within TLR3 that have not been previously assessed in the context of leukemia. Initially, we analyzed the genotypic distribution of four TLR3 SNPs (rs3775296, rs5743312, rs3775291, and rs3775290) in Saudis and compared these distributions to those reported in other populations.

The minor allele (A) of rs3775296 in healthy Saudi individuals had a frequency of 0.08, the lowest recorded when compared to the global populations reported in the 1000 Genomes Project. In contrast, the major allele (C) had a frequency of 0.92, the highest among the surveyed groups. Notably, populations such as African Caribbeans in Barbados, Esan in Nigeria, and Japanese in Tokyo exhibited higher frequencies of the A allele.

Significantly, TLR3 rs3775296 displayed a distinct allele distribution between ALL patients and healthy controls, particularly in individuals diagnosed after the age of 18, indicating a more than ninefold increase in ALL risk associated with this allele in the Saudi population. This SNP's association with various diseases has been sporadically reported. For instance, a study by Fan et al. (2019) linked rs3775296 to an increased risk of breast cancer initiation and progression, while Wicherska-Pawłowska et al. (2022) found no significant differences in allele or genotype frequencies of this SNP in acute myeloid leukemia patients.

We concluded that rs3775296, located in the 5′-untranslated region of TLR3, could potentially affect amino acid residues and impact the gene's role in the innate immune system. These genetic variations might influence gene expression in response to inflammatory cytokines, leading to transcriptional modifications of TLR3 that could reduce it signaling efficiency and contribute to the development of cancer. Additionally, the minor allele (T) of rs5743312 in healthy Saudis was observed at a frequency of 0.09, comparable to frequencies in African, Mexican, and Peruvian populations but higher than those in Chinese and Japanese populations. Variant genotypes of the intronic TLR3 rs5743312 were significantly associated with an increased risk of developing ALL, particularly in males and adult patients. This finding aligns with research by Fan et al. (2019), which also noted an association between rs5743312 and breast cancer risk. A meta-analysis by Wang et al. (2015) further supported a link between rs5743312 and a heightened cancer risk overall.

Conversely, our results indicated no significant association between TLR3 rs3775291 polymorphisms and ALL risk, although a marginal correlation with increased susceptibility to ALL was noted. Consistent with earlier studies, this SNP did not alter TLR3 expression levels or its intracellular localization, though it negatively impacted dsRNA recognition and binding (Ma et al., 2016).

In TLR3 rs3775290, the presence of one copy of the mutant allele T provides resistance against the development of ALL. The frequency of the variant allele T for the investigated SNPs in healthy Saudis was 0.31, similar to that reported for the 1000 Genomes Project in various populations, such as Vietnamese, British (in England and Scotland), and Finnish (in Finland) (1000 Genomes Project Consortium, 2015). Conversely, many previous studies have reported a significant association between the nonsynonymous rs3775290 polymorphism and the increased risk of other types of cancer or diseases. A meta-analysis conducted by Cheng et al. (2014) revealed that the rs3775290 polymorphism, present in exon 4 of the TLR3 gene, might increase the risk of cancer. Additionally, individuals heterozygous at rs3775290 exhibited an increased relative risk of cervical cancer in Tunisian women (Zidi et al., 2018), although this relationship was not detected in India (Pandey et al., 2011). In a study by Hamdy et al. (2018), TLR3 rs3775290 was genotyped in a group of Egyptians infected with chronic hepatitis C virus; this study found that the CC genotype was a risk genotype for chronic hepatitis C virus persistence, a result also observed in the Tunisian population (Sghaier et al., 2017). However, Etokebe et al. (2009) reported no association between TLR3 rs3775290 and rs5743312 with breast cancer, prostate cancer risk (Mandal et al., 2012), and bladder cancer (Singh et al., 2013). Similarly, variant genotypes of TLR3 rs3775290 have not been correlated with susceptibility to multiple sclerosis in various ethnic populations (Deeba et al., 2019).

This present study demonstrated a statistically significant difference between the mean levels of TLR3/GAPDH mRNA expression in the blood of patients with ALL compared with normal cases. Although TLR3 is located in endocytic compartments inside the cell, increasing evidence has shown that TLR3 is also expressed in tumor cells. TLR3 pattern recognition is associated with dsRNA, which has been proposed as an endogenous ligand for its activation. dsRNA released from injured cells could initiate paracrine activation of TLR3, thus inducing tumor cell growth and facilitating evasion from immune surveillance, suggesting that TLR3 activation could play an antagonistic role in tumor progression (Zheng et al., 2021). Importantly, the expression and functionality of TLR3 receptors in ALL have rarely been investigated. Our results follow other data published in 2008 by Jiang and his colleagues; (Jiang et al., 2008) they screened various human and murine tumor cell lines and found that all tested tumor cell lines expressed TLR3 at different levels, suggesting that the ligation of TLR3 can initiate signaling cascades and result in the activation of downstream effector molecules. These molecules include NF-κB, p38, JNK, and IFN regulatory factors (IRFs), which may be involved in tumor growth and apoptosis. The results shown here are also in line with previous findings in other types of cancer. Oblak & Jerala (2011) and González-Reyes et al. (2011), indicated that TLR3, TLR4, and TLR9 expression levels were significantly and positively associated with tumor size, stage, metastasis, and tumoral aggressiveness in breast cancer. Therefore, TLR3 may be applied as a promising therapeutic target for breast cancer. Additionally, González-Reyes et al. (2011) conducted the same experiment in prostate carcinoma and found high expression of TLR3, TLR4, and TLR9 in comparison with benign prostate. Furthermore, Fernandez-Garcia et al. (2014) revealed that gastric carcinomas show an increased level of TLR3, TLR4, and TLR9 expression, and this high expression of TLR3 was found to be significantly correlated with poor outcomes in gastric cancer patients.

In contrast, Sánchez-Cuaxospa et al. (2016) found that low expression of TLR3 in lymphocytes (T, B, and NK cells), monocytes, and dendritic cells from pediatric patients with ALL compared to the healthy group. However, Abdi et al. (2013) reported that primary cells from multiple myeloma patients showed low expression of TLR2, TLR3, and TLR5, while all human myeloma cell lines exhibited robust expression of TLR3. Thus, this may be due to the influence of the tissue origin, which may have a potential impact on determining the TLR expression pattern. Furthermore, Eiró and his colleagues (2012) observed that TLR2, TLR3, TLR4, and TLR5 expression was absent in colorectal carcinoma patients. Therefore, TLRs overexpression can have diverse biological significance in the context of the complex relationship between inflammation and cancer development.

While this study primarily employs traditional statistical methods to analyze the association between TLR3 polymorphisms and ALL susceptibility, incorporating machine learning techniques such as artificial neural networks (ANNs) represent a promising avenue for future research. ANNs could potentially enhance the predictive accuracy of genetic susceptibility to ALL by learning complex patterns and interactions within large genomic datasets.

ANNs have been effectively used in various genetic studies to model nonlinear relationships and interactions that are not easily captured by conventional statistical approaches. For instance, studies like Sekban et al. (2024) and Garg et al. (2022a,b) have demonstrated the utility of ANN and other machine learning methods in predicting material properties and biological responses, suggesting a similar approach could be applied to genetic data in leukemia research.

Future studies could explore using ANNs to integrate genetic markers with clinical data (e.g., age, gender, and medical history) to develop a more comprehensive predictive model of ALL risk. This approach might uncover new insights into the genetic architecture of ALL and improve the precision of risk stratification in clinical settings.

5 Conclusion

In conclusion, our study identifies and quantifies the association of specific SNPs in the TLR3 gene with ALL in the Saudi population. We found that the rs5743312 C/T polymorphism is associated with an increased risk of ALL, while the rs3775290 C/T polymorphism is inversely associated, suggesting a protective effect against the development of ALL. These findings enhance our understanding of the genetic basis of ALL and could guide future research into more effective, personalized treatment approaches.

CRediT authorship contribution statement

Rasha Alonaizan: Writing – review & editing, Project administration, Funding acquisition. Fadwa M Alkhulaifi: Writing – original draft, Methodology, Formal analysis, Data curation. Ahmed rady: Validation, Data curation. Suliman Alomar: Writing – review & editing, Supervision, Conceptualization.