MicroRNAs and methylation in chronic kidney disease: fine mapping and analysis by Sanger and next generation sequencing

Differential DNA methylation and miRNAs are associated with common complex diseases including chronic kidney disease (CKD). This study sought to identify differential methylation affecting miRNA genes in uraemic patients using a case-control approach followed by validation, replication, and fine mapping of top-ranked loci. Initial association analysis was carried out for 255 CKD cases and 152 unaffected controls with stringent quality control and correction for multiple testing. Quantitative DNA methylation was assessed at 485,577 sites across the methylome by the Infinium® HumanMethylation 450K BeadChip array (Illumina); single-CpG level results were extracted for 2,249 sites in miRNA genes across the genome. Five miRNA genes (P<10-5) were selected for follow-up; MIR940, MIR34A, MIR429, MIR141, and MIR329-2. Bidirectional Sanger sequencing of bisulfite treated DNA immediately flanking prioritised CpG sites in 23 cases and 23 controls revealed 36 methylated CpG sites. Sequencing of genomic DNA revealed 13 known SNPs flanking the CpG sites of interest. Validation of methylation levels and fine mapping using 46 individuals with both blood-derived and cell-line derived DNA was conducted using the Ion Chef, OneTouch2, and Torrent® Personal Genome Machine™ (Life Technologies®), revealing multiple known and novel SNPs. Replication using an independent case-control group (n=400) on the 450K platform revealed additional support. Epigenetic features including DNA methylation and miRNAs may cause or accelerate renal injury. This study has identified several miRNA genes, with differential methylation profiles, associated with CKD and enabled us to form a comparison of specific experimental approaches for validation, fine-mapping and replication of epigenome wide association studies.