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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

PBX3 expression levels associated with DNA methylation of its downstream located regulatory region. DNA methylation levels in AML patients with upregulated (up), downregulated (down) and normal levels of PBX3 expression demonstrating the impact of PBX3 methylation on expression; upregulation and downregulation was defined as a change in expression of at least one order of magnitude as well as 2-fold from healthy donors’ average PBX3 expression; asterisks correspond to statistically significant changes of methylation, ***P < 0.0001, **P = 0.002.
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Fig5: PBX3 expression levels associated with DNA methylation of its downstream located regulatory region. DNA methylation levels in AML patients with upregulated (up), downregulated (down) and normal levels of PBX3 expression demonstrating the impact of PBX3 methylation on expression; upregulation and downregulation was defined as a change in expression of at least one order of magnitude as well as 2-fold from healthy donors’ average PBX3 expression; asterisks correspond to statistically significant changes of methylation, ***P < 0.0001, **P = 0.002.

Mentions: We measured the expression of PBX3 mRNA in 123 AML diagnostic samples and 15 healthy controls (for a graph see Additional file 6: Figure S3). 24% of AML had down- and 22% upregulated PBX3 expression (with a minimum change in the expression of more than 2-fold and at the same time of more than one order of magnitude from healthy donors’ PBX3 average expression). 454 pyrosequencing established the role of DNA methylation in down- and upregulation of PBX3 in 30 AML patients at diagnosis (all of them with blast count ≥ 60%; Figure 5). Elevated levels of PBX3 were connected with hypomethylation of a regulatory region (median methylation level 0.25, range 0.15 – 0.36; P < 0.0001), whereas decreased levels of expression with hypermethylation (median methylation level 0.51, range 0.31 – 0.98; P = 0.002). Control samples of healthy donors and AML samples with normal PBX3 expression displayed intermediate levels of methylation (median 0.35, range 0.19 – 0.51). These results demonstrate the link between PBX3 expression and DNA methylation levels of the PBX3 regulatory region located downstream of the PBX3 annotated CpG island.Figure 5


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)

PBX3 expression levels associated with DNA methylation of its downstream located regulatory region. DNA methylation levels in AML patients with upregulated (up), downregulated (down) and normal levels of PBX3 expression demonstrating the impact of PBX3 methylation on expression; upregulation and downregulation was defined as a change in expression of at least one order of magnitude as well as 2-fold from healthy donors’ average PBX3 expression; asterisks correspond to statistically significant changes of methylation, ***P < 0.0001, **P = 0.002.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig5: PBX3 expression levels associated with DNA methylation of its downstream located regulatory region. DNA methylation levels in AML patients with upregulated (up), downregulated (down) and normal levels of PBX3 expression demonstrating the impact of PBX3 methylation on expression; upregulation and downregulation was defined as a change in expression of at least one order of magnitude as well as 2-fold from healthy donors’ average PBX3 expression; asterisks correspond to statistically significant changes of methylation, ***P < 0.0001, **P = 0.002.
Mentions: We measured the expression of PBX3 mRNA in 123 AML diagnostic samples and 15 healthy controls (for a graph see Additional file 6: Figure S3). 24% of AML had down- and 22% upregulated PBX3 expression (with a minimum change in the expression of more than 2-fold and at the same time of more than one order of magnitude from healthy donors’ PBX3 average expression). 454 pyrosequencing established the role of DNA methylation in down- and upregulation of PBX3 in 30 AML patients at diagnosis (all of them with blast count ≥ 60%; Figure 5). Elevated levels of PBX3 were connected with hypomethylation of a regulatory region (median methylation level 0.25, range 0.15 – 0.36; P < 0.0001), whereas decreased levels of expression with hypermethylation (median methylation level 0.51, range 0.31 – 0.98; P = 0.002). Control samples of healthy donors and AML samples with normal PBX3 expression displayed intermediate levels of methylation (median 0.35, range 0.19 – 0.51). These results demonstrate the link between PBX3 expression and DNA methylation levels of the PBX3 regulatory region located downstream of the PBX3 annotated CpG island.Figure 5

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