Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches
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...
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| Format: | Article |
| Language: | English |
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BMC
2018-10-01
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| Series: | BMC Research Notes |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13104-018-3872-x |
| _version_ | 1811296698084884480 |
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| author | Laura J. Smyth Alexander P. Maxwell Katherine A. Benson Jill Kilner Gareth J. McKay Amy Jayne McKnight |
| author_facet | Laura J. Smyth Alexander P. Maxwell Katherine A. Benson Jill Kilner Gareth J. McKay Amy Jayne McKnight |
| author_sort | Laura J. Smyth |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-04-13T05:52:21Z |
| format | Article |
| id | doaj.art-908196f64a8448b888bdfc046782e716 |
| institution | Directory Open Access Journal |
| issn | 1756-0500 |
| language | English |
| last_indexed | 2024-04-13T05:52:21Z |
| publishDate | 2018-10-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Research Notes |
| spelling | doaj.art-908196f64a8448b888bdfc046782e7162022-12-22T02:59:43ZengBMCBMC Research Notes1756-05002018-10-011111810.1186/s13104-018-3872-xValidation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approachesLaura J. Smyth0Alexander P. Maxwell1Katherine A. Benson2Jill Kilner3Gareth J. McKay4Amy Jayne McKnight5Genetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastGenetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastGenetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastGenetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastGenetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastGenetic Epidemiology Research Group, Centre for Public Health, Queen’s University of BelfastAbstract 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.http://link.springer.com/article/10.1186/s13104-018-3872-xDiabetesEpigeneticEWASKidneyMethylationmicroRNA |
| spellingShingle | Laura J. Smyth Alexander P. Maxwell Katherine A. Benson Jill Kilner Gareth J. McKay Amy Jayne McKnight Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches BMC Research Notes Diabetes Epigenetic EWAS Kidney Methylation microRNA |
| title | Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches |
| title_full | Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches |
| title_fullStr | Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches |
| title_full_unstemmed | Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches |
| title_short | Validation of differentially methylated microRNAs identified from an epigenome-wide association study; Sanger and next generation sequencing approaches |
| title_sort | validation of differentially methylated micrornas identified from an epigenome wide association study sanger and next generation sequencing approaches |
| topic | Diabetes Epigenetic EWAS Kidney Methylation microRNA |
| url | http://link.springer.com/article/10.1186/s13104-018-3872-x |
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