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Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

Simo-Riudalbas L, Diaz-Lagares A, Gatto S, Gagliardi M, Crujeiras AB, Matarazzo MR, Esteller M, Sandoval J - PLoS ONE (2015)

Bottom Line: Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein.Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.

ABSTRACT

Introduction and results: Immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.

Conclusions: In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

No MeSH data available.


Related in: MedlinePlus

Genome-wide DNA methylation profiles in ICF patients and control samples.(A) Histograms shows bimodal distribution pattern of DNA methylation profiles in ICF patients and normal donors. The frequency of CpGs according to DNA methylation levels are depicted in the graph. (B) Table showing number of average poorly methylated (methylation levels beta<0.33) and average highly methylated (methylation levels Beta>0.66). (C) Scatter plot represents comparison of DNA methylation levels of total CpG sites using the Infinium 450K DNA methylation assay. Green triangle selects hypomethylated area for ICF patients compared to controls. (D) Box plot displaying the distribution of Beta-values of total CpG sites of ICF versus healthy control donors. Normality was tested using the Shapiro-Wilk test and significance was evaluated with the Mann-Whitney U test and is indicated by three asterisks *** (p<0.001).
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pone.0132517.g001: Genome-wide DNA methylation profiles in ICF patients and control samples.(A) Histograms shows bimodal distribution pattern of DNA methylation profiles in ICF patients and normal donors. The frequency of CpGs according to DNA methylation levels are depicted in the graph. (B) Table showing number of average poorly methylated (methylation levels beta<0.33) and average highly methylated (methylation levels Beta>0.66). (C) Scatter plot represents comparison of DNA methylation levels of total CpG sites using the Infinium 450K DNA methylation assay. Green triangle selects hypomethylated area for ICF patients compared to controls. (D) Box plot displaying the distribution of Beta-values of total CpG sites of ICF versus healthy control donors. Normality was tested using the Shapiro-Wilk test and significance was evaluated with the Mann-Whitney U test and is indicated by three asterisks *** (p<0.001).

Mentions: Since the majority of ICF patients deal with mutations within the DNMT3B catalytic domain expected to variably interfere with the methyltransferase activity of the protein, the primary goal of this study was to describe the global DNA methylation profile affected by ICF specific DNMT3B mutant alleles. Here, we characterized the methylome of one lymphoblastoid cell line and of untrasformed fibroblasts derived from two different compound heterozygous ICF patients with the DNMT3B mutations V699G/R54X and A603T/STP807ins (PT5 and GM08747, respectively) compared to three control LCLs (XX, MS) and fibroblasts 3674 derived from healthy donors. Using this strategy that includes ICF patients derived from different tissue types, the variability and interference due to tissue-specific genes, will be reduced. The analysis, by calculating first the averaged Beta values for each CpG from the three controls and average Beta values from the two ICF patients and later the delta values (average ICF-average controls), shows that ICFs globally contain more poorly methylated (βvalue<0.33) and less highly methylated CpGs (βvalue>0.66) compared to controls (Fig 1A). In this sense, the accumulated number of poorly methylated CpGs ranging Beta values from 0 to 0.33 of ICF patients is 215,227; while for controls decrease to 202,003 (Delta ICF-Control = +13,224). However, an opposite pattern is obtained for highly methylated CpGs ranging from Beta values 0.66 to 1. In this case, control donor showed 143,108 highly methylated CpGs compared to lower number for ICF patients 138,873 CpGs (Delta ICF-Control = -4,235) (Fig 1B). A more comprehensive representation is the scatter plot of the DNA methylation levels (βvalue) of ICF patients compared to controls showing a higher accumulation of hypomethylated CpGs in ICFs than in controls, see triangle area in Fig 1C. Confirming these results we observed, using a non-parametic Mann-withney U test after testing normality with the Shapiro-Wilk test, a significant decrease in methylation level in ICF samples compared to controls (Fig 1D). We provide individual histograms, scatter and box plots for all the hybridized samples. Individual methylation levels were consistent, although control 2 (XX) presented lower global levels than the other two controls (MS and GM03674) (S1 Fig). Therefore, our results are in agreement with previous studies reporting that ICF syndrome is a disease characterized by DNA hypomethylation and we further demonstrate that the combination of the specific DNMT3B variants A603T/STP807ins and V699G/R54X derives in a global loss of DNA methylation levels.


Genome-Wide DNA Methylation Analysis Identifies Novel Hypomethylated Non-Pericentromeric Genes with Potential Clinical Implications in ICF Syndrome.

Simo-Riudalbas L, Diaz-Lagares A, Gatto S, Gagliardi M, Crujeiras AB, Matarazzo MR, Esteller M, Sandoval J - PLoS ONE (2015)

Genome-wide DNA methylation profiles in ICF patients and control samples.(A) Histograms shows bimodal distribution pattern of DNA methylation profiles in ICF patients and normal donors. The frequency of CpGs according to DNA methylation levels are depicted in the graph. (B) Table showing number of average poorly methylated (methylation levels beta<0.33) and average highly methylated (methylation levels Beta>0.66). (C) Scatter plot represents comparison of DNA methylation levels of total CpG sites using the Infinium 450K DNA methylation assay. Green triangle selects hypomethylated area for ICF patients compared to controls. (D) Box plot displaying the distribution of Beta-values of total CpG sites of ICF versus healthy control donors. Normality was tested using the Shapiro-Wilk test and significance was evaluated with the Mann-Whitney U test and is indicated by three asterisks *** (p<0.001).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4498748&req=5

pone.0132517.g001: Genome-wide DNA methylation profiles in ICF patients and control samples.(A) Histograms shows bimodal distribution pattern of DNA methylation profiles in ICF patients and normal donors. The frequency of CpGs according to DNA methylation levels are depicted in the graph. (B) Table showing number of average poorly methylated (methylation levels beta<0.33) and average highly methylated (methylation levels Beta>0.66). (C) Scatter plot represents comparison of DNA methylation levels of total CpG sites using the Infinium 450K DNA methylation assay. Green triangle selects hypomethylated area for ICF patients compared to controls. (D) Box plot displaying the distribution of Beta-values of total CpG sites of ICF versus healthy control donors. Normality was tested using the Shapiro-Wilk test and significance was evaluated with the Mann-Whitney U test and is indicated by three asterisks *** (p<0.001).
Mentions: Since the majority of ICF patients deal with mutations within the DNMT3B catalytic domain expected to variably interfere with the methyltransferase activity of the protein, the primary goal of this study was to describe the global DNA methylation profile affected by ICF specific DNMT3B mutant alleles. Here, we characterized the methylome of one lymphoblastoid cell line and of untrasformed fibroblasts derived from two different compound heterozygous ICF patients with the DNMT3B mutations V699G/R54X and A603T/STP807ins (PT5 and GM08747, respectively) compared to three control LCLs (XX, MS) and fibroblasts 3674 derived from healthy donors. Using this strategy that includes ICF patients derived from different tissue types, the variability and interference due to tissue-specific genes, will be reduced. The analysis, by calculating first the averaged Beta values for each CpG from the three controls and average Beta values from the two ICF patients and later the delta values (average ICF-average controls), shows that ICFs globally contain more poorly methylated (βvalue<0.33) and less highly methylated CpGs (βvalue>0.66) compared to controls (Fig 1A). In this sense, the accumulated number of poorly methylated CpGs ranging Beta values from 0 to 0.33 of ICF patients is 215,227; while for controls decrease to 202,003 (Delta ICF-Control = +13,224). However, an opposite pattern is obtained for highly methylated CpGs ranging from Beta values 0.66 to 1. In this case, control donor showed 143,108 highly methylated CpGs compared to lower number for ICF patients 138,873 CpGs (Delta ICF-Control = -4,235) (Fig 1B). A more comprehensive representation is the scatter plot of the DNA methylation levels (βvalue) of ICF patients compared to controls showing a higher accumulation of hypomethylated CpGs in ICFs than in controls, see triangle area in Fig 1C. Confirming these results we observed, using a non-parametic Mann-withney U test after testing normality with the Shapiro-Wilk test, a significant decrease in methylation level in ICF samples compared to controls (Fig 1D). We provide individual histograms, scatter and box plots for all the hybridized samples. Individual methylation levels were consistent, although control 2 (XX) presented lower global levels than the other two controls (MS and GM03674) (S1 Fig). Therefore, our results are in agreement with previous studies reporting that ICF syndrome is a disease characterized by DNA hypomethylation and we further demonstrate that the combination of the specific DNMT3B variants A603T/STP807ins and V699G/R54X derives in a global loss of DNA methylation levels.

Bottom Line: Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein.Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B.

View Article: PubMed Central - PubMed

Affiliation: Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain.

ABSTRACT

Introduction and results: Immunodeficiency, centromeric instability and facial anomalies syndrome (ICF) is a rare autosomal recessive disease, characterized by severe hypomethylation in pericentromeric regions of chromosomes (1, 16 and 9), marked immunodeficiency and facial anomalies. The majority of ICF patients present mutations in the DNMT3B gene, affecting the DNA methyltransferase activity of the protein. In the present study, we have used the Infinium 450K DNA methylation array to evaluate the methylation level of 450,000 CpGs in lymphoblastoid cell lines and untrasformed fibroblasts derived from ICF patients and healthy donors. Our results demonstrate that ICF-specific DNMT3B variants A603T/STP807ins and V699G/R54X cause global DNA hypomethylation compared to wild-type protein. We identified 181 novel differentially methylated positions (DMPs) including subtelomeric and intrachromosomic regions, outside the classical ICF-related pericentromeric hypomethylated positions. Interestingly, these sites were mainly located in intergenic regions and inside the CpG islands. Among the identified hypomethylated CpG-island associated genes, we confirmed the overexpression of three selected genes, BOLL, SYCP2 and NCRNA00221, in ICF compared to healthy controls, which are supposed to be expressed in germ line and silenced in somatic tissues.

Conclusions: In conclusion, this study contributes in clarifying the direct relationship between DNA methylation defect and gene expression impairment in ICF syndrome, identifying novel direct target genes of DNMT3B. A high percentage of the DMPs are located in the subtelomeric regions, indicating a specific role of DNMT3B in methylating these chromosomal sites. Therefore, we provide further evidence that hypomethylation in specific non-pericentromeric regions of chromosomes might be involved in the molecular pathogenesis of ICF syndrome. The detection of DNA hypomethylation at BOLL, SYCP2 and NCRNA00221 may pave the way for the development of specific clinical biomarkers with the aim to facilitate the identification of ICF patients.

No MeSH data available.


Related in: MedlinePlus