Short Inverted Repeats as Mutational Hotspots and Putative Drivers of Genome Instability in Osteosarcoma

<b>Background/Objectives</b>: Short inverted repeats (SIRs) are abundant DNA motifs capable of forming secondary structures, such as hairpins and cruciforms, that can induce genome instability. However, their mutational consequences in cancer, particularly in osteosarcoma (OS), remain largely unexplored. <b>Methods</b>: In this study, we systematically identified over 5.2 million SIRs in the human genome and analyzed their mutational patterns across six common cancer types. <b>Results</b>: We found that increased small insertion and deletion (INDEL) density within SIR spacer regions represents a consistent feature across cancers, whereas elevated single nucleotide variant (SNV) and structural breakpoint density is cancer-type specific. Integrating whole-genome sequencing data from 13 OS patients, we found that both SNVs and INDELs are significantly enriched within SIR spacer regions in OS. Notably, genomic regions with higher SIR density tend to accumulate more somatic mutations, suggesting a link between SIR abundance and local genome instability. SIR-associated mutations frequently occur in oncogenes and tumor suppressor genes, including <i>TP53</i>, <i>NFATC2</i>, <i>MECOM</i>, <i>LRP1B</i>, <i>RB1</i>, <i>CNTNAP2</i>, and <i>PTPRD</i>, as well as in long non-coding RNAs. Mutational signature analysis further suggests that defective DNA mismatch repair and homologous recombination may act in concert with SIR-induced DNA structural instability to drive OS development. <b>Conclusions</b>: Our findings highlight SIRs as mutational hotspots and potential drivers of osteosarcoma pathogenesis.