Long-read Oxford Nanopore sequencing reveals complex rearrangements and regulatory disruption in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) is a rare malignancy characterised by extensive structural genomic alterations and a low burden of recurrent single nucleotide variants. However, the full spectrum and functional impact of structural variation (SVs) remain incompletely understood because short-read sequencing has limitation ability to resolve complex genomic rearrangements. Here, we performed integrated short-read and long-read whole-genome sequencing on tumour-normal pairs from three MPM patients, together with RNA sequencing and nanopore-derived promoter methylation profiling. Long-reads sequencing substantially improved SV detection, identifying 61-156 novel SVs per sample, including complex rearrangements and breakpoint-resolved events affecting cancer-associated genes. Complex SV clusters consistent with chromoplexy and chromothripsis were observed and frequently involved oncogenes. Integration with transcriptomics data showed that several SVs-affected genes, including WEE1 and GPC6, exhibited increased expression independent of gene dosage. Promoter methylation analysis revealed a conserved bimodal methylation landscape across tumours and a significant inverse relationship with gene expression. SV-associated genes showed coordinated promoter hypermethylation and transcriptional activation, suggesting that SVs may influence gene regulation through epigenetic mechanisms. Survival analysis using the TCGA-MESO cohort further showed that elevated expression of WEE1 and GPC6 was associated with poorer overall survival. Together, these findings highlight the value of long-read sequencing for uncovering functionally and clinically relevant structural variation in MPM.