P04.03.A LONG READ SEQUENCING IDENTIFIES COMPLEX NF2 STRUCTURAL VARIATION IN MENINGIOMA

Abstract BACKGROUND Human meningiomas are most commonly driven by biallelic inactivation of the NF2 gene, typically through a combination of NF2 genomic mutation and allelic loss of chromosome 22q. However, approximately 30-40% of individuals with 22q loss do not exhibit definite NF2 mutations by exome-based sequencing. Long-read sequencing enables the accurate detection of long-range structural variations (SVs). In this study, we aimed to identify cryptic genomic alterations that complete the biallelic inactivation of 22q in selected meningioma cases using PacBio long-read sequencing. MATERIAL AND METHODS From March 2023 to December 2024, we conducted Revio long-read sequencing (Pacific Biosciences, CA, USA) on five patients presenting with 22q deletion but lacking detectable NF2 mutations via conventional panel sequencing. Tumor locations included the frontal convexity (n = 1), parietal convexity (n = 1), frontal skull base (n = 2), and sphenoid wing (n = 1). Two tumors were diagnosed as WHO grade 2 atypical meningiomas, while the remaining three were classified as WHO grade 1. High-fidelity (HiFi) sequencing reads were aligned to the T2T-CHM13 human reference genome using pbmm2 from the SMRTtools suite. SVs were identified using Sniffles (v2.5.2), followed by manual inspection and genomic reconstruction. Gel electrophoresis with appropriate primers near the breakends were performed for all cases. RESULTS We identified heterogenous structural variations within NF2 for all cases - one case harbored simple 50 base pair tandem duplication, two atypical meningiomas exhibited translocation of chr22-chr15 and chr22-chr1. Two additional cases demonstrated complex structural variations characterized by multi-chromosomal, long-range insertions within the NF2 locus—patterns suggestive of chromopexy, which are typically undetectable using short-read sequencing technologies. Notably, one of the complex events involved the insertion of a long-range alpha satellite region (ASR) from the centromere of chromosome 22 into the NF2 gene, enabled by the use of long-read sequencing combined with a complete human genome reference resolved at centromere and telomere levels (T2T-CHM13). All structural variations were validated through gel electrophoresis and fluorescence in situ hybridization (FISH). CONCLUSION Complex genomic rearrangements involving NF2 represent a significant driver event in meningioma development. Long-read sequencing demonstrates a powerful advantage in detecting SVs with precise breakpoint resolution, which are often missed by panel- or exome-based sequencing methods.