Simultaneous identification of DNA variation and methylation at HLA class II locus and immune gene promoters using targeted SureSelect Methyl-Sequencing
The Human Leukocyte Antigen (HLA) locus associates with a variety of complex diseases, particularly autoimmune and inflammatory conditions. The HLA-DR15 haplotype, for example, confers the major risk for developing Multiple Sclerosis in Caucasians, pinpointing important role in the etiology of this chronic inflammatory disease of the central nervous system. In addition to the protein-coding variants that shape the functional HLA-antigen-T cell interaction, recent studies suggest that the levels of HLA molecule expression, that are epigenetically controlled, also play a role in disease development. However, deciphering the exact molecular mechanisms of the HLA association has been hampered by the tremendous genetic complexity of the locus and a lack of robust approaches to investigate it. Here we developed a method to specifically enrich the genomic DNA from the HLA class II locus (chr6:32,426,802-34,167,129) and proximal promoters of 2,157 immune-relevant genes, utilizing the Agilent RNA-based SureSelect Methyl-Seq Capture method, followed by sequencing to detect genetic and epigenetic variation. We demonstrated successful simultaneous detection of the genetic variation and quantification of DNA methylation levels in the HLA locus. Moreover, by utilizing differentially methylated positions in promoters of immune-related genes, we detected relevant pathways following stimulation of cells. Taken together, we present a method that can be utilized to study the impact of the interplay of genetic and epigenetic regulation in HLA class II region on cellular states in a wide context of HLA haplotypes and diseases. Overall design: THP-1 (DNA-IFN?/LPS, DNA- IFN?), THP-1 (BS-IFN?/LPS and BS-IFN?) and THP-1 (EM-IFN?/LPS and EM-IFN?)