Simultaneous detection 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 an 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 related 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 HLA locus. Moreover, by the detection of differentially methylated positions in promoters of immune-related genes, we identified relevant pathways following stimulation of cells. Taken together, we present a method that can be utilized to study the interplay between genetic variance and epigenetic regulation in the HLA class II region, potentially, in a wide disease context.