In this study, we investigated the association between telomere length in peripheral blood mononuclear cells, not tumor cells, and the clinical characteristics of tumors at diagnosis. Patients with an extra-abdominal primary tumor, lower stage tumor, MYCN non-amplified tumor, or low-risk tumor had longer germline telomere length than other patients. These clinical features are usually associated with better prognosis, and thus, it was unsurprising that longer telomere length was associated with better EFS and was an independent prognostic factor for EFS in our multivariate analysis. This is the first study to elucidate the clinical significance of germline telomere length in patients with neuroblastoma. We cannot explain the reason for the association between germline telomere length and the clinical characteristics of tumors. However, our results suggest that germline genomic characteristics, including telomere length, might affect the clinical characteristics of tumors at diagnosis and the treatment response.
For this reason, we performed genome-wide genotyping with a high-density bead chip to identify the genetic factors related to telomere length. In the GWAS analysis, novel risk SNPs, including HIVEP3 and LRRTM4, were significantly associated with telomere length (Table 3). The most significant association was with rs10842679 (P = 4.7E-07). That marker was adjacent to the BHLHE41 (basic helix-loop-helix family member e41) gene. BHLHE41 is known to be a transcription factor implicated in cellular functions such as proliferation, differentiation, and tumorigenesis 27. It is possible that this marker is linked to genetic factors that directly or indirectly affect the expression of the BHLHE41 gene. Follow-up functional studies would be required to clarify the exact mechanism. Table 3 summarizes information about the genes with significant markers. Among them, many genes significant to the HIVEP3 gene were found. No direct functional association between neuroblastoma and the HIVEP3 gene has yet been reported. However, studies of other carcinomas have reported that they have higher levels of HIVEP3 and SOX9 messenger RNA expression than non-carcinoma cells 29. In particular, patients with HIVEP3 and SOX9 overexpression showed a lower survival rate. In this study, patients with the recessive allele of the HIVEP3 gene had long telomere lengths. Follow-up studies comparing therapeutic effects and survival rates according to the expression of the HIVEP3 gene and telomere length changes are needed to clarify how telomere length is related to the function of the HIVEP3 gene.
A gene-based analysis was performed using the VEGAS2 algorithm, and the CNTN4 gene showed the most significant association (P = 1.0E-06; Supplementary Table 1). The rs71311728 marker within the CNTN4 gene also showed a significant association in the GWAS analysis (P = 0.0001). The rs71311728 marker within CNTN4 gene is a new marker with no reported association with other cancers. The CNTN4 gene encodes contactin 4, a member of the immunoglobulin superfamily 30. The functions of these proteins are suggested to involve synaptic plasticity. In addition, they are known to be involved in axon growth, guidance, and fasciculation 31. Although the details remain unclear, the effect of CNTN4 on the development of nerve endings and the development of neuroblastoma should be examined in future functional studies. To investigate the biological functions of the significant genes we identified, we used VEGAS2 to perform a gene ontology analysis. The results are summarized in Supplementary Table 2. The statistically significant GO categories (empirical P < 0.00005) were cadherin binding, cell to cell pathway, cell leading edge, neurite morphogenesis, cell part morphogenesis, and neuron development.
This is the first GWAS study to investigate an association with telomere length in neuroblastoma patients, and we found that the CNTN4 gene is associated with changes in telomere length in Korean neuroblastoma patients. Our number of samples was insufficient due to the rarity of neuroblastoma. However, the new genes and markers discovered through this study will contribute to other GWAS studies that can be conducted in various ethnicities in the future.
In conclusion, we found that longer germline telomere length is associated with favorable prognostic factors at diagnosis and eventually a better EFS in patients with neuroblastoma. In addition, the GWAS demonstrated that genetic markers and genes related to telomere length were associated with neurite morphogenesis and neuron development in neuroblastoma. A further study with a larger cohort of patients and functional studies are needed.