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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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Hypomethylation signature in inv(16) AML. Hypomethylation of MN1(A), SPARC(B), ST18(C) and DHRS3(D) regulatory regions in inv(16) patients compared to AML M4 without inv(16), other AML subtypes and healthy controls; asterisks correspond to statistically significant changes of expression in inv(16) patients versus other groups, ***P < 0.0001.
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Fig2: Hypomethylation signature in inv(16) AML. Hypomethylation of MN1(A), SPARC(B), ST18(C) and DHRS3(D) regulatory regions in inv(16) patients compared to AML M4 without inv(16), other AML subtypes and healthy controls; asterisks correspond to statistically significant changes of expression in inv(16) patients versus other groups, ***P < 0.0001.

Mentions: In summary, altogether 21 inv(16) AML, 15 non-inv(16) AML M4, 19 other AML (1 AML M0, 3 AML M1, 6 AML M2, 3 AML M3, 3 AML M5a, 2 AML M5b, 1 AML M6) and 10 healthy controls were examined. DNA methylation of individual regions (corresponding to individual amplicons) was expressed as an average DNA methylation of all CpGs in that particular region. Average levels of DNA methylation in assigned regulatory regions of MN1, SPARC, ST18 and DHRS3 were significantly lower for inv(16) versus non-inv(16) AML M4, other AML subtypes and healthy controls (P < 0.0001) (see Figure 2). Sequencing of FAM171A failed twice in all samples probably due to the low complexity of the amplicon (because of the long stretches of identical bases – homopolymers) introduced after bisulfite conversion and FAM171A was therefore excluded from further analysis. For MN1, both of the studied regions displayed lower levels of methylation (for MN1_region_2 hypomethylation graph see Additional file 4: Figure S2A). For SPARC, the second studied region (SPARC_region_2) had low DNA methylation levels in inv(16) as well as in other AML and healthy donors (see Additional file 4: Figure S2B). Therefore only methylation of region 1 has a potential impact on SPARC expression. The 454-pyrosequencing results point to the site-specific CBFB-MYH11 hypomethylation signature of genomic regions assigned to MN1, SPARC, ST18 and DHRS3.Figure 2


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)

Hypomethylation signature in inv(16) AML. Hypomethylation of MN1(A), SPARC(B), ST18(C) and DHRS3(D) regulatory regions in inv(16) patients compared to AML M4 without inv(16), other AML subtypes and healthy controls; asterisks correspond to statistically significant changes of expression in inv(16) patients versus other groups, ***P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig2: Hypomethylation signature in inv(16) AML. Hypomethylation of MN1(A), SPARC(B), ST18(C) and DHRS3(D) regulatory regions in inv(16) patients compared to AML M4 without inv(16), other AML subtypes and healthy controls; asterisks correspond to statistically significant changes of expression in inv(16) patients versus other groups, ***P < 0.0001.
Mentions: In summary, altogether 21 inv(16) AML, 15 non-inv(16) AML M4, 19 other AML (1 AML M0, 3 AML M1, 6 AML M2, 3 AML M3, 3 AML M5a, 2 AML M5b, 1 AML M6) and 10 healthy controls were examined. DNA methylation of individual regions (corresponding to individual amplicons) was expressed as an average DNA methylation of all CpGs in that particular region. Average levels of DNA methylation in assigned regulatory regions of MN1, SPARC, ST18 and DHRS3 were significantly lower for inv(16) versus non-inv(16) AML M4, other AML subtypes and healthy controls (P < 0.0001) (see Figure 2). Sequencing of FAM171A failed twice in all samples probably due to the low complexity of the amplicon (because of the long stretches of identical bases – homopolymers) introduced after bisulfite conversion and FAM171A was therefore excluded from further analysis. For MN1, both of the studied regions displayed lower levels of methylation (for MN1_region_2 hypomethylation graph see Additional file 4: Figure S2A). For SPARC, the second studied region (SPARC_region_2) had low DNA methylation levels in inv(16) as well as in other AML and healthy donors (see Additional file 4: Figure S2B). Therefore only methylation of region 1 has a potential impact on SPARC expression. The 454-pyrosequencing results point to the site-specific CBFB-MYH11 hypomethylation signature of genomic regions assigned to MN1, SPARC, ST18 and DHRS3.Figure 2

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