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Genomewide DNA Methylation Analysis Identifies Novel Methylated Genes in Non– Small-Cell Lung Carcinomas  Rejane Hughes Carvalho, PhD, Jun Hou, PhD, Vanja.

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Presentation on theme: "Genomewide DNA Methylation Analysis Identifies Novel Methylated Genes in Non– Small-Cell Lung Carcinomas  Rejane Hughes Carvalho, PhD, Jun Hou, PhD, Vanja."— Presentation transcript:

1 Genomewide DNA Methylation Analysis Identifies Novel Methylated Genes in Non– Small-Cell Lung Carcinomas  Rejane Hughes Carvalho, PhD, Jun Hou, PhD, Vanja Haberle, MSc, Joachim Aerts, PhD, Frank Grosveld, PhD, Boris Lenhard, PhD, Sjaak Philipsen, PhD  Journal of Thoracic Oncology  Volume 8, Issue 5, Pages (May 2013) DOI: /JTO.0b013e ed2 Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

2 FIGURE 1 Methylation reads for selected DMRs. Methylation reads for selected differentially methylated regions (DMRs). UCSC genome browser representation of MethylCap-seq reads in eight DMRs for a paired sample: control lung (2231N) and tumor (2214T). Light green: region significantly differentially methylated; light red: region selected for methylation-specific polymerase chain reaction (MSP). y-Axis: number of reads ranging from 0 to the maximum number of reads in the highest peak in the selected window per sample. (A) DMRs in the BARX1 (1) gene spanning more than 3000 base pairs (bp), with 500 bp significant bin and 172 bp MSP fragment. (B) DMRs in the PRDM14 (2) gene spanning from 5′UTR to first exon (promoter region) with a 1000-bp bin and 194-bp MSP fragment. (C) DMRs in the DMRTA2 (3) gene with a 1000-bp significant bin and 155 bp MSP fragment. (D) DMRs in the ZIC4 (4) gene in the promoter and first exon, showing a 500-bp significant bin in the promoter and 209-bp MSP fragment. (E) DMRs in the promoter region of the GRIK2 (5) gene, with a 500-bp bin and 179-bp MSP fragment. (F) Methylation of the EN1 gene showing DMRs spanning more than 25 kbp, and 500-bp bin approximately 10 kbp upstream of the gene and three MSP fragments at 10 kbp upstream (6), 6 kbp upstream (7), and the EN1 promoter (8) (119, 192, and 214 bp, respectively). Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e ed2) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

3 FIGURE 2 Representation of methylation-specific polymerase chain reaction (MSP) screening. (A) Cropped gel image of three paired samples and controls for all DMRs used. Meth: totally methylated control; Unmeth: totally umethylated control; M: primer for methylated CpG; U: primer for unmethylated CpG. (B) Control lung samples show less intense amplification of PARP15 and CDKN2A MSP fragments than matching tumor samples. (C) Intensities of HIST1H3B and ANKRD1B MSP fragments in control lung samples are not significantly different from those observed in matching tumor samples. Arrows point to methylated primers in control lung and tumor samples. DMRs, and differentially methylated regions. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e ed2) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

4 FIGURE 3 Differences in amplification intensities for methylated primer in control lung and matching tumor samples. (A) Control lung samples show less intense amplification of PARP15 and CDKN2A methylation-specific polymerase chain reaction (MSP) fragments than matching tumors samples. (B) Intensities of HIST1H3B and ANKRD1B MSP fragments in control lung samples in are not significantly different those observed in matching tumor samples. Red arrows point to methylated primers in control lung and tumor samples. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e ed2) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

5 FIGURE 4 Intensities of MSP fragments. (A) Distribution of differentially methylated regions (DMRs) selected for tumor; blue: control lung sample; red: tumor sample; p value is less than for all DMRs. (B) Intensity distribution of putative adenocarcinoma (ADC)-specific DMRs in ADC versus squamous cell carcinoma (SCC) tumors. (C) Intensity distribution of putative SCC-specific DMRs in ADC versus SCC tumors. *p Value is less than 0.001; **p value is less than MSP, methylation-specific polymerase chain reaction. Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e ed2) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

6 FIGURE 5 Percentages of methylated DNA. (A) Distribution of differentially methylated regions (DMRs) selected for tumor; p value is less than (B) Distribution of the percentages of methylated DNA for putative adenocarcinoma (ADC)-specific DMRs in ADC versus squamous cell carcinoma (SCC) tumors. (C) Distribution of the percentages of methylated DNA in the samples for putative SCC-specific DMRs in ADC versus SCC tumors. *p value is less than 0.001; **p value is less than Journal of Thoracic Oncology 2013 8, DOI: ( /JTO.0b013e ed2) Copyright © 2013 International Association for the Study of Lung Cancer Terms and Conditions

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