F8 inversions at Xq28 causing hemophilia A are associated with specific methylation changes: Implication for molecular epigenetic diagnosis

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. Particularly, for genomic inversions the disease causing mechanism may not be clear especially if the inversion border don't cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, mainly in an epigenetic context, may provide additional layer of information towards sequence specific regulation of gene expression in human diseases. Herein, we study one of such inversion hotspots at Xq28, which leads to the disruption of F8 gene and result in hemophilia A phenotype. To undermine the epigenetic consequence of this rearrangement, we evaluated DNA methylation levels of 12 CpG rich regions with the coverage of 550 Kb by using bisulfite-pyrosequencing and NGS based bisulfite re-sequencing enrichment assay. Our results show that inversion prone area harbors wide spread methylation changes at the studied regions, however only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (2 regions), Intron 22 inversion (2 regions); and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, 2 CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. The developed hemophilia A methylation inversion “HAMI” assay, provide advantage over conventional PCR based methods which could fails to detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in F8 (Xq28) region are associated with detectable methylation levels and can be used as a molecular diagnostic tool.