Estinto, Gilda (2025) Functional impact of synonymous variants in cancer predisposition genes and neuroblastoma pathogenesis. [Tesi di dottorato]

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Abstract

Background: Although synonymous single-nucleotide variants (sSNVs) do not alter amino acid sequences, they can influence gene function through effects on splicing, RNA structure, transcription factor or miRNA binding, and codon usage. However, their contribution to cancer predisposition remains poorly understood. Aim: To systematically evaluate the impact of rare synonymous variants in neuroblastoma (NB), a pediatric cancer originating from aberrant neural crest cell development. Methods: Whole-exome sequencing data from 724 NB cases and 2,858 controls were analyzed to identify rare variants (MAF < 0.01) in 138 pediatric cancer predisposition genes (CPGs). Functional characterization integrated conservation metrics (PhastCons, GERP, PhyloP), splicing predictions (SpliceAI ≥ 0.2), pathogenicity scoring (EPEL), transcriptional regulatory assessment using ATAC-seq from NB cell lines, and codon optimality analysis. Results: Rare synonymous and spliceogenic synonymous variants, similar to missense variants, showed a significant depletion in CPGs in NB compared with controls (p < 2 × 10⁻¹⁶), indicating strong evolutionary constraint on essential cancer genes. Despite this global constraint, a subset of sSNVs displayed functional enrichment patterns: six CPGs carrying only sSNVs were enriched in NTRK and growth-factor receptor signaling, whereas 14 genes harboring only missense variants were enriched in DNA repair pathways, suggesting distinct biological roles. Synonymous variants in CPGs occurred at more conserved sites than in non-CPGs (PhyloP p = 0.031; PhastCons p = 0.014). Spliceogenic sSNVs (SpliceAI ≥ 0.8) were significantly enriched in NB cases (p = 0.020, OR = 4.9), involving ACD, SMARCA4, and EPAS1. Eight-six predicted pathogenic sSNVs were significantly overrepresented in NB-specific open chromatin regions (OR = 3.35, p = 0.0049). These sSNVs were predicted to alter the TF-binding motifs for RELB, MAFK, FOXA2, ZFX, POU2F2, IKZF1, and GMEB1, while codon usage analysis revealed a shift toward non-optimal codons, suggesting reduced translational efficiency. Conclusions: This integrative analysis reveals that rare synonymous variants are not functionally silent but act under evolutionary constraint, influencing splicing, transcriptional regulation, and translation. These findings highlight a previously underappreciated layer of germline variation contributing to neuroblastoma predisposition and support the systematic inclusion of synonymous variants in cancer predisposition analyses to uncover hidden regulatory mechanisms in oncogenesis.

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