Associations of NEFL gene polymorphisms with neuroblastoma risk in Chinese children from Jiangsu Province

Introduction

Neuroblastoma is the most prevalent extracranial solid tumor in children (1). It originates from neural crest cells, precursors to the sympathetic nervous system, and is frequently detected in the adrenal medulla or along the sympathetic ganglia (2). This type of tumor is characterized by a broad spectrum of clinical and biological heterogeneity, with cases spanning from spontaneous regression to highly aggressive, treatment-resistant forms that are resistant to treatment (3). Although progress has been made in understanding and managing neuroblastoma, early detection remains a major hurdle owing to the nonspecific nature of initial symptoms. Treatment strategies include a combination of surgery, chemotherapy, radiotherapy, and immunotherapy (4,5). However, the prognosis for high-risk patients remains poor, with a 5-year survival rate of approximately 50% (5). While some cases have favorable outcomes, the long-term complications caused by the disease and its intensive treatment protocols, along with a high risk of recurrence, impose a heavy burden on patients, families, and healthcare systems (6-8). These challenges reveal the urgent need for a more comprehensive understanding of the disease etiology and the advancement of more effective prevention and treatment options.

Both environmental and genetic factors influence neuroblastoma risk. Certain environmental exposures, such as maternal smoking or alcohol use during pregnancy, may modestly increase the risk. Moreover, hereditary susceptibilities are strongly related to neuroblastoma. Syndromes such as Beckwith-Wiedemann syndrome and neurofibromatosis type 1 predispose patients to the disease (9). The candidate gene approach has detected many neuroblastoma-contributing single nucleotide polymorphism (SNP) in various genes involved in fundamental cellular processes (10-14). Furthermore, genome-wide association studies (GWASs) have identified several neuroblastoma susceptibility loci, including variants in the HACE1, HSD17B12, CASC15, BARD1, LMO1, DUSP12, and LIN28B genes (15-20). Despite these advances, many biologically crucial genetic variants remain undiscovered and may be overlooked in GWASs because of stringent thresholds designed to minimize false positives. Exploring novel genetic factors may reveal critical mechanisms involved in neuroblastoma initiation and progression and facilitate the development of more precise screening tools and personalized interventions for this complex pediatric malignancy.

The NEFL gene encodes a critical component of the neurofilament protein family, which forms an essential part of the neuronal cytoskeleton and maintains structural stability and axonal integrity in neurons (21). NEFL abnormalities have been linked to neurological disorders, such as Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, contributing to neuronal degeneration and functional impairments (21-23). Altered NEFL expression has been implicated in tumorigenesis, metastasis, and tumor progression in various brain cancers (24-28), including neuroblastoma (28). However, direct evidence connecting NEFL to neuroblastoma pathogenesis is currently limited and warrants further investigation. The effects of genetic variations in NEFL, particularly SNPs, on neuroblastoma susceptibility have also been investigated across North American, Italian, and Chinese cohorts with unequivocal results (28,29). The association between causal genetic variants and disease risk may be impacted by the unique evolutionary histories, demographic events, and environmental pressures that shape the genetic architecture of each population (30). Cross-population validation is indispensable for confirming whether the identified SNP associations are restricted to a single population or applicable to other populations, including the current study population. Therefore, this study aimed to validate the associations between NEFL polymorphisms (rs11994014 G>A, rs2979704 T>C, and rs1059111 A>T) and neuroblastoma susceptibility in an eastern China child cohort of 402 cases and 473 controls. Investigating NEFL SNPs and their associations with neuroblastoma could provide valuable insights into the molecular underpinnings of this disease and help identify at-risk populations. Understanding these genetic influences could also facilitate the development of novel strategies for early detection, risk assessment, and targeted therapy, addressing an unmet need in neuroblastoma research and clinical management. We present this article in accordance with the STROBE reporting checklist (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-611/rc).

Methods

Ethics approval

Prior to participation, informed consent was obtained from the patients’ parents or legal guardians. The study was approved by the Institutional Review Board of the Children’s Hospital of Nanjing Medical University (approval No. 202112141-1) and was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Study cohort

This study involved 402 patients diagnosed with neuroblastoma and 473 healthy controls (Table S1) (31,32). The eligibility criteria for cases included children diagnosed with neuroblastoma, with pathological confirmation obtained prior to any treatments. Cases were recruited from Jiangsu Province in southern China. The sources of case ascertainment were hospitals and medical centers within the region where neuroblastoma patients were treated. Healthy controls were drawn from the same regions and timeframes as the cases and matched for age and sex to minimize confounding effects.

Selection of SNPs and genotyping

Three NEFL SNPs (rs11994014 G>A, rs2979704 T>C, and rs1059111 A>T) that were significantly associated with neuroblastoma in Caucasians were chosen for this study. Genotyping was performed in the laboratory following a standard protocol published previously (33,34). The TIANamp Blood DNA Kit (TIANGEN Biotech Co. Ltd., Beijing, China) was used to extract total DNA from peripheral blood samples. After confirming the quality of the DNA samples in terms of purity and concentration, the samples were prepared and preloaded into 386-well plates. TaqMan real-time polymerase chain reaction (PCR) assays with specific probes were conducted on an ABI Q6 instrument (Applied Biosystems, Foster City, CA, USA). Unaware of the case or control status of the samples, laboratory personnel performed the genotyping in a blinded manner. Stringent quality control measures were applied to ensure the reliability of the genotyping results.

Statistical analysis

Hardy-Weinberg equilibrium (HWE) for the SNPs in the control group was tested via the goodness-of-fit χ² test to confirm that the observed genotype distribution matched the expected frequencies. Both univariate and multivariate logistic regression analyses were performed to determine whether specific SNPs were significantly associated with neuroblastoma susceptibility. Furthermore, stratified analyses were conducted on the basis of sex, age, clinical stage, and site of origin to examine potential differences in these associations across subgroups. Statistical analyses were carried out via SAS software (SAS Institute Inc., Cary, NC, USA). The threshold for statistical significance was set at a two-sided P value of less than 0.05.

Results

Overall analysis

Genotypic analyses were conducted for three NEFL polymorphisms (rs11994014 G>A, rs2979704 T>C, and rs1059111 A>T) in all participants (Table 1). In the control group, the genotype frequencies for all assessed SNPs conformed to HWE, supporting the validity of the genetic data and ensuring the robustness of subsequent association analyses.

We performed both univariate and multivariate logistic regression analyses to assess the genotype distributions of rs11994014 G>A, rs2979704 T>C, and rs1059111 A>T in neuroblastoma patients and controls. Various genetic models have been adopted, including homozygous, heterozygous, additive, dominant, and recessive models. However, none of the SNPs showed significant associations with neuroblastoma risk in the overall analysis. These findings revealed that rs11994014 GG/GA, rs2979704 TT/TC, and rs1059111 AT/TT were risk genotypes. We subsequently dichotomized all the participants according to the number of risk genotypes they carried. However, the results indicated no meaningful alteration in neuroblastoma risk for children with three risk genotypes relative to those with 0–2 risk genotypes (Table 1).

Stratified analysis

Stratification allows the examination of potential interactions between genetic factors and demographic or clinical characteristics. Given the potential influence of factors such as age, sex, site of origin, and clinical stage on genetic associations, we conducted stratified analyses to explore whether these factors could reveal more specific relationships between NEFL polymorphisms and neuroblastoma risk. Stratified analyses focused specifically on the association between rs11994014 G>A and the number of risk genotypes carried by participants. However, these stratified analyses did not yield statistically significant results. This finding suggests that, within this cohort, the associations between these NEFL polymorphisms and neuroblastoma susceptibility may not be influenced by these stratifying factors (Table 2).

Discussion

This study was performed to verify the association between neuroblastoma risk and three neuroblastoma susceptibility loci in the NEFL gene (rs11994014 G>A, rs2979704 T>C, and rs1059111 A>T) previously identified in Caucasian populations via an independent cohort from eastern China. Despite rigorous statistical analyses, including overall and stratified approaches, no significant associations were found between the selected NEFL polymorphisms and neuroblastoma risk in our study population.

Our results were different from those of the original study, which indicated that the NEFL rs11994014 G>A polymorphism was associated with decreased neuroblastoma risk in a European American population [minor allele frequency (MAF) =0.22] and an Italian population (MAF =0.30) (28). Moreover, rs2979704 T>C and rs1059111 A>T reduce neuroblastoma risk in the Italian population but are not associated with neuroblastoma risk in the European American population (28). Overall, the minor alleles rs11994014-A, rs2979704-C, and rs1059111-A were associated with a reduced risk of developing neuroblastoma in the combined study population of European Americans and Italians (28). However, our findings were in line with those of a previous study in a southern Chinese population, which revealed that none of the three NEFL SNPs were significantly associated with the risk of developing neuroblastoma in the present eastern Chinese cohort despite differences in MAFs [e.g., rs11994014 (G>A): 0.40 vs. 0.35] (29). SNPs identified in one population may have different MAFs or linkage disequilibrium (LD) patterns than those in another population. For example, an SNP significantly associated with neuroblastoma in a European American population and an Italian population might be less/or more frequent or exhibit weaker/or stronger LD with causal variants in a Chinese Han population. These differences affect its detectability, biological relevance, the statistical power of studies, the generalizability of findings, and the strength and direction of associations. In addition, gene-environment interactions influencing neuroblastoma risk may also differ between populations, further complicating direct extrapolation of findings. Moreover, even within the same ethnic group, such as the Chinese Han population, regional differences, such as those between southern China and eastern China, may influence the association between SNPs and neuroblastoma susceptibility. These variations can arise from historical migrations, genetic drift, environmental exposures, or cultural practices that shape the genetic structure and gene-environment interactions unique to each region (30). For example, allele frequencies and LD patterns of SNPs may vary regionally, affecting the strength and direction of associations. Therefore, verifying the association results in diverse regional populations increases the validity of the current findings and their applicability to broader Chinese Han populations.

The NEFL is a critical component of the neurofilament triplet and is essential for the structural integrity and function of neuronal cells. Neurofilaments are intermediate filaments involved in maintaining neuronal cytoskeleton stability and facilitating intracellular transport (35). These processes are fundamental in rapidly growing and differentiating neural cells, such as those implicated in neuroblastoma, a malignancy arising from neural crest progenitors. Dysregulation of NEFL expression or function may contribute to impaired differentiation and increased proliferation, which are key hallmarks of tumorigenesis (26,27). Clinically, patients with higher NEFL expression in their primary neuroblastoma specimens had improved overall survival (28). NEFL overexpression specifically suppressed the growth and invasion and enhanced the differentiation of neuroblastoma cells (28). The NEFL rs2979704 and rs1059111 polymorphisms potentially function by affecting the binding of transcription factors or microRNAs. Functional analyses revealed that the rs1059111 polymorphism has a cis effect on NEFL expression, with the A allele of the SNP rs1059111 leading to increased expression of NEFL in comparison to the T allele (28). Despite their biological importance, the three NEFL SNPs analyzed in this study did not show significant associations with neuroblastoma risk. Several factors may explain these findings. First, the specific polymorphisms analyzed might not directly affect NEFL expression or function but could be in LD with other genetic variants with subtle effects, making their contribution challenging to detect in this sample size. Moreover, NEFL may not be centrally involved in the pathways driving neuroblastoma development and progression, thereby limiting its direct association with susceptibility. It is also important to consider the role of gene-environment interactions, which were not assessed in this study. Environmental exposures, such as prenatal toxins, infections, or parental lifestyle factors, could interact with genetic predispositions to modulate neuroblastoma risk. Without accounting for such interactions, the true contribution of NEFL SNPs to disease susceptibility might remain obscured. Additionally, epigenetic modifications influenced by environmental factors may alter NEFL expression, further complicating its association with neuroblastoma.

Several limitations in this study should be considered. First, the relatively moderate sample size might have constrained our ability to detect associations with small effect sizes and potentially underestimated the true impact of the NEFL gene SNPs on neuroblastoma risk. Second, the investigation was conducted exclusively in a single ethnic population, which limits the applicability of these findings to other populations with different genetic compositions. Third, while NEFL has been studied in the context of other neurological and cancer-related conditions, its role in neuroblastoma remains largely unexplored. The limited data on NEFL expression and its functional significance in neuroblastoma tissues hinder a comprehensive understanding of its biological relevance. Finally, this study focused solely on a single gene, preventing the exploration of potential interactions between genes or between genetic and environmental factors. Since neuroblastoma is a multifactorial disease, future studies incorporating gene-gene and gene-environment interaction analyses are crucial for a more comprehensive understanding of its pathogenesis.

Conclusions

Our study revealed a lack of association between polymorphisms in the NEFL gene and neuroblastoma susceptibility in children in an eastern Chinese cohort. Future research involving larger, ethnically diverse cohorts may be needed to confirm these observations and perform gene-environment interaction analyses to better understand their role in neuroblastoma pathogenesis.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-611/rc

Data Sharing Statement: Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-611/dss

Peer Review File: Available at https://tp.amegroups.com/article/view/10.21037/tp-2024-611/prf

Funding: This study was supported by grants from the Beijing Research Ward Excellence Program, BRWEP (No. BRWEP2024W102090107), and the Guangzhou Science and Technology Project (No. SL2024A04J02095).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tp.amegroups.com/article/view/10.21037/tp-2024-611/coif). J.H. serves as an unpaid editorial board member of Translational Pediatrics from July 2024 to June 2025. The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Prior to participation, informed consent was obtained from the patients’ parents or legal guardians. The study was approved by the Institutional Review Board of the Children’s Hospital of Nanjing Medical University (approval No. 202112141-1) and was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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