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Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.

Selmi C, Cavaciocchi F, Lleo A, Cheroni C, De Francesco R, Lombardi SA, De Santis M, Meda F, Raimondo MG, Crotti C, Folci M, Zammataro L, Mayo MJ, Bach N, Shimoda S, Gordon SC, Miozzo M, Invernizzi P, Podda M, Scavelli R, Martin MR, Seldin MF, Lasalle JM, Gershwin ME - Front Immunol (2014)

Bottom Line: We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays).Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes.In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center , Milan , Italy ; Division of Rheumatology, Allergy, and Clinical Immunology, University of California at Davis , Davis, CA , USA.

ABSTRACT
Primary biliary cirrhosis (PBC) is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ) twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n = 3 sets) and sisters of similar age (n = 8 pairs) discordant for disease. We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays). Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i) methylation profiles of 60 gene regions, (ii) CNV of 10 genes, and (iii) the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

No MeSH data available.


Related in: MedlinePlus

Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The red indicated an increased expression with a ΔCt value below the middle level and the green indicated a decreased expression with ΔCt value above the middle level.
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Figure 1: Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The red indicated an increased expression with a ΔCt value below the middle level and the green indicated a decreased expression with ΔCt value above the middle level.

Mentions: To provide additional support for our initial findings, we used RT-PCR to evaluate expression of each of the candidates that emerged from the DNA methylation (60), CNV (10), and expression studies (2), as well as previously reported GWAS in seven pairs of discordant sisters of similar age (Table 1) (7–9, 12, 13, 24, 25). Our data assist analysis contained: Ct data, sample design, assay design, average of Ct values of replicates, ΔCt, normalized versus endogenous controls Ct values ± SD and fold change (RQ) files, which displays RQ min and RQ max for each sample. p-Value was calculated from ΔCt files. Data assist v3.0 software was used with results exported from real-time PCR and for relative quantification analysis. Graphic result in heat map visualized analyzed data (Figure 1). Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The neutral/middle expression was set as the median of all the ΔCt values from all samples, the red indicated an increase with a ΔCt value below the middle level and the green indicated a decrease with ΔCt value above the middle level. The heat map from all samples is represented in Figure 1. Among the entire set of candidate genes, we found five genes that were underexpressed in at least three of seven sibling pairs with FC < 0.5 (CXCR5, HLA-B, IFI44L, IFIT1, SMARCA1) and one overexpressed gene in at least three of seven pairs with an FC > 2 (IL6). Additional 11 genes showed a widely variable expression profile in each sibling pair (CD80, FAM104B, HLA-DQB1, HLA-DRB1, HLA-G, MTCP1, NHS, PIN4, PRPF38A, THSD7A, and TNFAIP2) (Table 3; Figure 2).


Genome-wide analysis of DNA methylation, copy number variation, and gene expression in monozygotic twins discordant for primary biliary cirrhosis.

Selmi C, Cavaciocchi F, Lleo A, Cheroni C, De Francesco R, Lombardi SA, De Santis M, Meda F, Raimondo MG, Crotti C, Folci M, Zammataro L, Mayo MJ, Bach N, Shimoda S, Gordon SC, Miozzo M, Invernizzi P, Podda M, Scavelli R, Martin MR, Seldin MF, Lasalle JM, Gershwin ME - Front Immunol (2014)

Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The red indicated an increased expression with a ΔCt value below the middle level and the green indicated a decreased expression with ΔCt value above the middle level.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3975093&req=5

Figure 1: Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The red indicated an increased expression with a ΔCt value below the middle level and the green indicated a decreased expression with ΔCt value above the middle level.
Mentions: To provide additional support for our initial findings, we used RT-PCR to evaluate expression of each of the candidates that emerged from the DNA methylation (60), CNV (10), and expression studies (2), as well as previously reported GWAS in seven pairs of discordant sisters of similar age (Table 1) (7–9, 12, 13, 24, 25). Our data assist analysis contained: Ct data, sample design, assay design, average of Ct values of replicates, ΔCt, normalized versus endogenous controls Ct values ± SD and fold change (RQ) files, which displays RQ min and RQ max for each sample. p-Value was calculated from ΔCt files. Data assist v3.0 software was used with results exported from real-time PCR and for relative quantification analysis. Graphic result in heat map visualized analyzed data (Figure 1). Heat map showed, for all case/control sibling pairs, genes expression in red/green color based on ΔCt values using Pearson’s correlation. The neutral/middle expression was set as the median of all the ΔCt values from all samples, the red indicated an increase with a ΔCt value below the middle level and the green indicated a decrease with ΔCt value above the middle level. The heat map from all samples is represented in Figure 1. Among the entire set of candidate genes, we found five genes that were underexpressed in at least three of seven sibling pairs with FC < 0.5 (CXCR5, HLA-B, IFI44L, IFIT1, SMARCA1) and one overexpressed gene in at least three of seven pairs with an FC > 2 (IL6). Additional 11 genes showed a widely variable expression profile in each sibling pair (CD80, FAM104B, HLA-DQB1, HLA-DRB1, HLA-G, MTCP1, NHS, PIN4, PRPF38A, THSD7A, and TNFAIP2) (Table 3; Figure 2).

Bottom Line: We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays).Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes.In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

View Article: PubMed Central - PubMed

Affiliation: Division of Rheumatology and Clinical Immunology, Humanitas Clinical and Research Center , Milan , Italy ; Division of Rheumatology, Allergy, and Clinical Immunology, University of California at Davis , Davis, CA , USA.

ABSTRACT
Primary biliary cirrhosis (PBC) is an uncommon autoimmune disease with a homogeneous clinical phenotype that reflects incomplete disease concordance in monozygotic (MZ) twins. We have taken advantage of a unique collection consisting of genomic DNA and mRNA from peripheral blood cells of female MZ twins (n = 3 sets) and sisters of similar age (n = 8 pairs) discordant for disease. We performed a genome-wide study to investigate differences in (i) DNA methylation (using a custom tiled four-plex array containing tiled 50-mers 19,084 randomly chosen methylation sites), (ii) copy number variation (CNV) (with a chip including markers derived from the 1000 Genomes Project, all three HapMap phases, and recently published studies), and/or (iii) gene expression (by whole-genome expression arrays). Based on the results obtained from these three approaches we utilized quantitative PCR to compare the expression of candidate genes. Importantly, our data support consistent differences in discordant twins and siblings for the (i) methylation profiles of 60 gene regions, (ii) CNV of 10 genes, and (iii) the expression of 2 interferon-dependent genes. Quantitative PCR analysis showed that 17 of these genes are differentially expressed in discordant sibling pairs. In conclusion, we report that MZ twins and sisters discordant for PBC manifest particular epigenetic differences and highlight the value of the epigenetic study of twins.

No MeSH data available.


Related in: MedlinePlus