Data_Sheet_1_F8 Inversions at Xq28 Causing Hemophilia A Are Associated With Specific Methylation Changes: Implication for Molecular Epigenetic Diagnosis.PDF

Diverse DNA structural variations (SVs) in human cancers and several other diseases are well documented. For genomic inversions in particular, the disease causing mechanism may not be clear, especially if the inversion border does not cross a coding sequence. Understanding about the molecular processes of these inverted genomic sequences, in a mainly epigenetic context, may provide additional information regarding sequence-specific regulation of gene expression in human diseases. Herein, we study one such inversion hotspot at Xq28, which leads to the disruption of F8 gene and results in hemophilia A phenotype. To determine 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 next-generation sequencing (NGS)-based bisulfite re-sequencing enrichment assay. Our results show that this inversion prone area harbors widespread methylation changes at the studied regions. However, only 5/12 regions showed significant methylation changes, specifically in case of intron 1 inversion (two regions), intron 22 inversion (two regions) and one common region in both inversions. Interestingly, these aberrant methylated regions were found to be overlapping with the inversion proximities. In addition, two CpG sites reached 100% sensitivity and specificity to discriminate wild type from intron 22 and intron 1 inversion samples. While we found age to be an influencing factor on methylation levels at some regions, covariate analysis still confirms the differential methylation induced by inversion, regardless of age. The hemophilia A methylation inversion “HAMI” assay provides an advantage over conventional PCR-based methods, which may not detect novel rare genomic rearrangements. Taken together, we showed that genomic inversions in the F8 (Xq28) region are associated with detectable changes in methylation levels and can be used as an epigenetic diagnostic marker.

人类癌症及其他多种疾病中存在的多样化DNA结构变异（SVs）已有充分记载。其中，针对基因组倒位而言，其致病机制尚不明确，尤其是当倒位边界未跨越编码序列时。若能在表观遗传背景下解析这类倒位基因组序列的分子过程，或可为人类疾病中基因表达的序列特异性调控机制提供新的见解。本研究聚焦于Xq28染色体区域的一处倒位热点，该位点可破坏F8基因，进而引发血友病A表型。为明确该基因组重排的表观遗传后果，我们采用亚硫酸氢盐焦磷酸测序以及基于下一代测序（NGS）的亚硫酸氢盐重测序富集检测法，对覆盖550kb的12个CpG富集区域的DNA甲基化水平进行了评估。结果显示，该易倒位区域在我们研究的范围内存在广泛的甲基化改变。不过，仅12个区域中的5个出现了显著的甲基化变化，具体为内含子1倒位相关的2个区域、内含子22倒位相关的2个区域，以及两种倒位共有的1个区域。有趣的是，这些异常甲基化区域与倒位侧翼区域存在重叠。此外，有2个CpG位点可实现100%的灵敏度和特异性，用以区分野生型样本与内含子1倒位、内含子22倒位样本。尽管我们发现年龄会对部分区域的甲基化水平产生影响，但协变量分析仍证实，倒位所诱导的差异甲基化不受年龄因素的干扰。本研究中的血友病A甲基化倒位（HAMI）检测法相较于传统基于PCR的方法具有优势，后者无法检测到新型罕见基因组重排。综上，我们证实F8基因（Xq28区域）的基因组倒位与可检测到的甲基化水平变化相关，且可作为表观遗传诊断标志物。