Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches

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Abstract

OBJECTIVES: Altered DNA methylation and microRNA profiles are associated with diabetic kidney disease. This study compared different sequencing approaches to define the genetic and epigenetic architecture of sequences surrounding microRNAs associated with diabetic kidney disease.

RESULTS: We compared Sanger and next generation sequencing to validate microRNAs associated with diabetic kidney disease identified from an epigenome-wide association study (EWAS). These microRNAs demonstrated differential methylation levels in cases with diabetic kidney disease compared to controls with long duration of type 1 diabetes and no evidence of kidney disease (Padjusted < 10-5). Targeted next generation sequencing analysis of genomic DNA and matched cell-line transformed DNA samples identified four genomic variants within the microRNAs, two within miR-329-2 and two within miR-429. Sanger sequencing of genomic DNA replicated these findings and confirmed the altered methylation status of the CpG sites identified by the EWAS in bisulphite-treated DNA. This investigation successfully fine-mapped the genetic sequence around key microRNAs. Variants have been detected which may affect their methylation status and methylated CpG sites have been confirmed. Additionally, we explored both the fidelity of next generation sequencing analysis and the potential efficacy of cell-line transformed DNA samples in place of finite patient samples in discovery genetic and epigenetic research.

Original languageEnglish
Article number767
JournalBMC Research Notes
Volume11
Issue number1
DOIs
Publication statusPublished - 29 Oct 2018

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