Long-read genomic analyses to elucidate hidden structural variations associated with MECP2 duplication syndrome

MECP2 duplication syndrome results from duplication of the MECP2 gene, encoding methyl-CpG-binding protein 2. Structural variations in this region can be detected by short-read next-generation sequencing, but resolving its precise genomic architecture remains challenging because of the involvement of complex and highly repetitive sequences. This study investigated the hidden structural variations using optical genome mapping and targeted long-read nanopore sequencing. We identified 14 breakpoints within the Xq28 regions encompassing MECP2 in four individuals from four families with MECP2 duplication syndrome. Combining the above methods enabled us to identify all the precise breakpoints, except for two inversions embedded within highly repetitive sequences. This also represents the most precise delineation to date of complex structural variants in MECP2 duplication syndrome. Notably, leveraging long nanopore reads (> 75 kb) allowed us to span low-copy repeat regions, including the approximately 72 kb J-group low-copy repeat which was difficult to be resolved, as well as GC-rich segments and dense clusters of short interspersed nuclear elements such as Alu, thus enhancing breakpoint-detection accuracy. We also detected previously underreported rare and complex rearrangement patterns. These findings highlight the power of integrating long-read sequencing with optical genome mapping for the delineation of complex genomic architectures, thus enhancing our understanding of the genomic structure underlying MECP2 duplication syndrome.