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Cytosine methylation changes in enhancer regions of core pro-fibrotic genes characterize kidney fibrosis development

Yi-An Ko1, Davoud Mohtat1, Masako Suzuki2, Ae Seo Deok Park1, Maria Concepcion Izquierdo1, Sang Youb Han1, Hyun Mi Kang1, Han Si1, Thomas Hostetter6, James M Pullman3, Melissa Fazzari24, Amit Verma5, Deyou Zheng2, John M Greally2* and Katalin Susztak1*

Author Affiliations

1 Renal Electrolyte and Hypertension Division, Perelman School of Medicine, University of Pennsylvania United States,, 415 Curie Blvd, Philadelphia, PA 19104, USA

2 Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA

3 Department of Pathology, Montefiore Medical Center, Bronx, NY 10461, USA

4 Department of Epidemiology and Population Health, Albert Einstein College of Medicine, Bronx, NY 10461, USA

5 Department of Oncology, Albert Einstein College of Medicine, Bronx, NY 10461, USA

6 Case Western Reserve University, Cleveland, Ohio 44106, USA

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Genome Biology 2013, 14:R108  doi:10.1186/gb-2013-14-10-r108

Published: 7 October 2013

Additional files

Additional file 1: Table S1:

Demographic, clinical and histological characteristics of the samples.

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Additional file 2: Figure S1:

Principal component analysis of the transcript levels in the original dataset show no significant differences based on diabetes status of the samples. Dark red circles indicate CKD gene expression data points, light red circles indicate DKD data points, light blue diabetic control data points and dark blue control data points.

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Additional file 3: Table S2:

List of differentially methylated regions in CKD. List of differentially methylated loci, P-values, genomic location (hg18), and their methylation levels in individual samples, with the nearest annotated transcript to each DMR listed.

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Additional file 4: Figure S2:

MassArray-based confirmation of cytosine methylation levels. Absolute methylation values are plotted on the y-axis while relative methylation values from the HELP dataset are shown on the x-axis. Each plot represents methylation values from one human kidney tissue (HK). We ran each sample with nine different primer sets that represent low, intermediate and highly methylated regions.

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Additional file 5: Table S3:

External validation of HELP DMRs using the Illumina Infinium 450K platform of 87 DKD samples. The CKD (HELP) DMRs were assigned to the nearest RefSeq genes and the methylation differences for these RefSeq genes were extracted from the Infinium 450K arrays. The probes showing differential methylation are listed here. Multiple probes presented for the HELP DMRs were also differentially methylated in the Infinium 450K arrays. The number of gene-based, unique overlapping DMRs was 1,061.

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Additional file 6: Figure S3:

MassArray-based confirmation and external validation of the differentially methylated loci. (A,D) Average HpaII/MspI methylation ratio of DMRs on the HELP array in control (blue) and CKD kidneys (red). The original data can be found in Additional file 3. (B,E) MassArray Epityper-based absolute methylation level of the locus for control (blue) and CKD kidneys (red). Note that one HELP probe represents multiple CpG sites. (C,F) Methylation difference between DKD and control for this region in the external validation dataset. This dataset was generated using the Illumina Infinium 450K arrays from 66 control and 21 DKD microdissected kidney samples. The original data can be found in Additional file 7). (A-C) Changes in the Dermatopontin gene (DPT); (D-F) changes in the Down syndrome cell adhesion molecule (DSCAM) locus.

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Additional file 7: Figure S4:

RefSeq annotation of the DMRs. The number of probes on the Roche-NimbleGen customized array, DMRs, hypo- or hypermethylated DMRs in each Refseq-based annotation groups. Relative enrichment ratio of the DMR compared with the representation of the different elements on the methylation microarray.

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Additional file 8: Table S4:

Correlations of DMR and transcript levels. DMRs from the HELP assay and its corresponding transcripts (Affymetrix arrays) are listed. There were multiple DMRs for some of the transcripts and they are all listed. Chromosomal location, methylation level, gene expression differences, and P-values are included in the table.

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Additional file 9: Table S5:

CKD DMRs observed in decitabine-treated cells. CKD DMR loci showing statistically significant differences in HKC8 cells treated with 0.5 μM decitabine. The CKD DMRs were analyzed using HELP assays, while the decitabine-treated cells were analyzed using Infinium arrays.

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