Methylation in chronic kidney disease and its association with microRNAs: Analysis via Microarray, Sanger and Next Generation Sequencing: Oral Presentation

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Abstract

This study was designed to identify differential methylation affecting microRNAs (miRNAs) in patients with chronic kidney disease (CKD). Comparative analyses between traditional Sanger and next generation sequencing techniques were then conducted.

DNA samples from 255 patients with CKD (cases) were compared to 152 individuals without renal disease (controls) for differences in quantitative DNA methylation levels. Following stringent quality control procedures and correction for multiple testing, data was analysed from the HumanMethylation450K BeadChip array (Illumina) which assessed 485,577 sites. Extracting miRNA genes revealed 2,249 methylation sites within miRNAs, and five miRNAs associated with CKD (MIR940, MIR34A, MIR429, MIR141, and MIR329-2) were prioritised for follow-up. These results were supported by replication analysis of an independent cohort of CKD cases and controls (n=400) using the 450K platform.

Bidirectional Sanger sequencing was completed using carefully designed fragments for both genomic and bisulphite treated DNA flanking each miRNA. This approach determined 36 methylated CpG sites and 13 known SNPs, in the 23 cases and 23 controls analysed, but did not provide validation of methylation levels.

Next generation sequencing, using the Ion Chef, OneTouch2, and Torrent® Personal Genome Machine™ (Life Technologies®) was employed for fine-mapping of SNPs and methylated sites in the target region, providing qualitative validation of 450K results. Forty-six individuals with both blood-derived and cell-line derived DNA were resequenced for approximately 500bp surrounding each miRNA.

We have developed an effective technical protocol for validation and replication of EWAS studies, additionally confirming blood and cell-line may be useful sources of derived DNA for epigenetic studies.
Original languageEnglish
Pages (from-to)189
Number of pages1
JournalThe Ulster Medical Journal
Volume83
Issue number3
Publication statusPublished - Sep 2014
Event17th Meeting of the Irish Society of Human Genetics - St James’s Hospital, Dublin, Ireland
Duration: 05 Sep 2014 → …

Bibliographical note

Oral presentation. Abstracts for the 17th Meeting of the Irish Society of Human Genetics. S12

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