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CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

Hájková H, Fritz MH, Haškovec C, Schwarz J, Šálek C, Marková J, Krejčík Z, Dostálová Merkerová M, Kostečka A, Vostrý M, Fuchs O, Michalová K, Cetkovský P, Beneš V - J Hematol Oncol (2014)

Bottom Line: Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression.We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis.Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data.

Methods: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes.

Results: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients.

Conclusions: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

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Related in: MedlinePlus

Inv(16) methylation and expression cluster. (A) Hierarchal DNA methylation clustering of CpGs (n = 2 010 310) using the correlation as a distance measure and Ward’s method (AML_1 to AML_14 – AML patients; CD34_pool – healthy control’s CD34+ pool) indicating CBFB-MYH11 methylation cluster (in ellipse), other molecular abnormalities did not form clusters neither clinical characteristics did it; (B) Hierarchal gene expression clustering of altogether 8884 genes with a detection P value ≤ 0.05 in all 14 AML patients (AML_1 to AML_14) and 4 CD34+ healthy control cells confirmed inv(16) cluster (in ellipse).
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Fig1: Inv(16) methylation and expression cluster. (A) Hierarchal DNA methylation clustering of CpGs (n = 2 010 310) using the correlation as a distance measure and Ward’s method (AML_1 to AML_14 – AML patients; CD34_pool – healthy control’s CD34+ pool) indicating CBFB-MYH11 methylation cluster (in ellipse), other molecular abnormalities did not form clusters neither clinical characteristics did it; (B) Hierarchal gene expression clustering of altogether 8884 genes with a detection P value ≤ 0.05 in all 14 AML patients (AML_1 to AML_14) and 4 CD34+ healthy control cells confirmed inv(16) cluster (in ellipse).

Mentions: The hierarchal clustering analysis was done with the R package Pvclust [10] using the correlation as a distance measure and Ward’s method. Only positions of autosomes (i.e. excluding mitochondria and sex chromosome calls) with coverage at least 10 and available for all samples (n = 2 010 310) were included in the analysis. From the clinical and molecular characteristics, only CBFB-MYH11 patients (samples 1 and 2) clustered together. In Figure 1A, clustering of all CpGs is shown (for separate clustering of CpGs inside or outside CpG islands see the Additional file 1: Figure S1A and S1B). None of the other molecular abnormalities formed clusters and we did not observe any effect of clinical status of AML (de novo, therapy-related, or AML with myelodysplasia-related changes). One of the inv(16) sample was derived from BM (sample 1) while the other (sample 2) from PB. Importantly, this had no effect on their clustering consistency. We investigated this uniformity between the results from BM and PB in more details using 454 pyrosequencing in a larger number of patients (see below).Figure 1


CBFB-MYH11 hypomethylation signature and PBX3 differential methylation revealed by targeted bisulfite sequencing in patients with acute myeloid leukemia.

Hájková H, Fritz MH, Haškovec C, Schwarz J, Šálek C, Marková J, Krejčík Z, Dostálová Merkerová M, Kostečka A, Vostrý M, Fuchs O, Michalová K, Cetkovský P, Beneš V - J Hematol Oncol (2014)

Inv(16) methylation and expression cluster. (A) Hierarchal DNA methylation clustering of CpGs (n = 2 010 310) using the correlation as a distance measure and Ward’s method (AML_1 to AML_14 – AML patients; CD34_pool – healthy control’s CD34+ pool) indicating CBFB-MYH11 methylation cluster (in ellipse), other molecular abnormalities did not form clusters neither clinical characteristics did it; (B) Hierarchal gene expression clustering of altogether 8884 genes with a detection P value ≤ 0.05 in all 14 AML patients (AML_1 to AML_14) and 4 CD34+ healthy control cells confirmed inv(16) cluster (in ellipse).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197269&req=5

Fig1: Inv(16) methylation and expression cluster. (A) Hierarchal DNA methylation clustering of CpGs (n = 2 010 310) using the correlation as a distance measure and Ward’s method (AML_1 to AML_14 – AML patients; CD34_pool – healthy control’s CD34+ pool) indicating CBFB-MYH11 methylation cluster (in ellipse), other molecular abnormalities did not form clusters neither clinical characteristics did it; (B) Hierarchal gene expression clustering of altogether 8884 genes with a detection P value ≤ 0.05 in all 14 AML patients (AML_1 to AML_14) and 4 CD34+ healthy control cells confirmed inv(16) cluster (in ellipse).
Mentions: The hierarchal clustering analysis was done with the R package Pvclust [10] using the correlation as a distance measure and Ward’s method. Only positions of autosomes (i.e. excluding mitochondria and sex chromosome calls) with coverage at least 10 and available for all samples (n = 2 010 310) were included in the analysis. From the clinical and molecular characteristics, only CBFB-MYH11 patients (samples 1 and 2) clustered together. In Figure 1A, clustering of all CpGs is shown (for separate clustering of CpGs inside or outside CpG islands see the Additional file 1: Figure S1A and S1B). None of the other molecular abnormalities formed clusters and we did not observe any effect of clinical status of AML (de novo, therapy-related, or AML with myelodysplasia-related changes). One of the inv(16) sample was derived from BM (sample 1) while the other (sample 2) from PB. Importantly, this had no effect on their clustering consistency. We investigated this uniformity between the results from BM and PB in more details using 454 pyrosequencing in a larger number of patients (see below).Figure 1

Bottom Line: Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression.We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis.Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Studying DNA methylation changes in the context of structural rearrangements and point mutations as well as gene expression changes enables the identification of genes that are important for disease onset and progression in different subtypes of acute myeloid leukemia (AML) patients. The aim of this study was to identify differentially methylated genes with potential impact on AML pathogenesis based on the correlation of methylation and expression data.

Methods: The primary method of studying DNA methylation changes was targeted bisulfite sequencing capturing approximately 84 megabases (Mb) of the genome in 14 diagnostic AML patients and a healthy donors' CD34+ pool. Subsequently, selected DNA methylation changes were confirmed by 454 bisulfite pyrosequencing in a larger cohort of samples. Furthermore, we addressed gene expression by microarray profiling and correlated methylation of regions adjacent to transcription start sites with expression of corresponding genes.

Results: Here, we report a novel hypomethylation pattern, specific to CBFB-MYH11 fusion resulting from inv(16) rearrangement that is associated with genes previously described as upregulated in inv(16) AML. We assume that this hypomethylation and corresponding overexpresion occurs in the genes whose function is important in inv(16) leukemogenesis. Further, by comparing all targeted methylation and microarray expression data, PBX3 differential methylation was found to correlate with its gene expression. PBX3 has been recently shown to be a key interaction partner of HOX genes during leukemogenesis and we revealed higher incidence of relapses in PBX3-overexpressing patients.

Conclusions: We discovered new genomic regions with aberrant DNA methylation that are associated with expression of genes involved in leukemogenesis. Our results demonstrate the potential of the targeted approach for DNA methylation studies to reveal new regulatory regions.

Show MeSH
Related in: MedlinePlus