Identification of genes underlying different methylation profiles in refractory anemia with excess blast and refractory cytopenia with multilineage dysplasia in myelodysplastic syndrome

Suee Lee; Hyuk-Chan Kwon; Sung-Hyun Kim; Sung Yong Oh; Ji Hyun Lee; Yeon-Su Lee; Daekwan Seo; Jin-Yeong Han; Hyo-Jin Kim

doi:10.5045/kjh.2012.47.3.186

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Original Article

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Korean J Hematol 2012; 47(3):

Published online September 25, 2012

https://doi.org/10.5045/kjh.2012.47.3.186

Identification of genes underlying different methylation profiles in refractory anemia with excess blast and refractory cytopenia with multilineage dysplasia in myelodysplastic syndrome

Suee Lee¹, Hyuk-Chan Kwon¹, Sung-Hyun Kim¹, Sung Yong Oh¹, Ji Hyun Lee¹, Yeon-Su Lee³, Daekwan Seo⁴, Jin-Yeong Han², and Hyo-Jin Kim^1*

Author Info. +

¹Department of Internal Medicine, Dong-A University Medical Center, Busan, Korea.

²Department of Laboratory Medicine, Dong-A University Medical Center, Busan, Korea.

³Cancer Genomics Branch, National Cancer Center, Goyang, Korea.

⁴Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.

Correspondence to : Correspondence to Hyo-Jin Kim, M.D., Ph.D. Department of Internal Medicine, Dong-A University College of Medicine, 26, Daeshingongwon-ro, Seo-gu, Busan 602-715, Korea. Tel: +82-51-240-2951, Fax: +82-51-246-5044, kimhj@dau.ac.kr

Received: March 25, 2012; Revised: June 20, 2012; Accepted: August 3, 2012

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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Abstract

Background

Myelodysplastic syndrome (MDS) is a preleukemic condition that transforms into acute myeloid leukemia. However, the genetic events underlying this transformation remain poorly understood. Aberrant DNA methylation may play a causative role in the disease and its prognosis. Thus, we compared the DNA methylation profiles in refractory anemia with excess blast (RAEB) to those in refractory cytopenia with multilineage dysplasia (RCMD).

Methods

Bone marrow samples were collected from 20 patients with primary MDS (9 with RAEB and 11 with RCMD), and peripheral blood samples were collected from 4 healthy controls. These samples were assessed using a commercial whole genome-wide methylation assay. Methylation-specific polymerase chain reaction (PCR) was used to detect the methylation of candidate gene promoters in RAEB and RCMD.

Results

Microarray data revealed significant hypermethylation in 69 genes within RAEB but not RCMD. Candidate genes were mapped to 5 different networks, and network 1 had the highest score due to its involvement in gene expression, cancer, and cell cycle. Five genes (GSTM5, BIK, CENPH, RERG, and ANGPTL2) were associated with malignant disease progression. Among them, the methylated promoter pairs of GSTM5 (55.5% and 20%), BIK (20% and 0%), and ANGPTL2 (44.4% and 10%) were observed more frequently in RAEB.

Conclusion

DNA methylation of GSTM5, BIK, and ANGPTL2 may induce epigenetic silencing and contribute to the increasing blasts and resulting MDS progression; however, the functions of these genes were not determined. Further study focusing on epigenetic silencing using various detection modalities is required.

Keywords Myelodysplastic syndrome, DNA methylation, GSTM5, ANGPTL2, BIK

Article

Original Article

Korean J Hematol 2012; 47(3): 186-193

Published online September 25, 2012 https://doi.org/10.5045/kjh.2012.47.3.186

Identification of genes underlying different methylation profiles in refractory anemia with excess blast and refractory cytopenia with multilineage dysplasia in myelodysplastic syndrome

Suee Lee¹, Hyuk-Chan Kwon¹, Sung-Hyun Kim¹, Sung Yong Oh¹, Ji Hyun Lee¹, Yeon-Su Lee³, Daekwan Seo⁴, Jin-Yeong Han², and Hyo-Jin Kim^1*

¹Department of Internal Medicine, Dong-A University Medical Center, Busan, Korea.

²Department of Laboratory Medicine, Dong-A University Medical Center, Busan, Korea.

³Cancer Genomics Branch, National Cancer Center, Goyang, Korea.

⁴Laboratory of Experimental Carcinogenesis, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA.

Correspondence to: Correspondence to Hyo-Jin Kim, M.D., Ph.D. Department of Internal Medicine, Dong-A University College of Medicine, 26, Daeshingongwon-ro, Seo-gu, Busan 602-715, Korea. Tel: +82-51-240-2951, Fax: +82-51-246-5044, kimhj@dau.ac.kr

Received: March 25, 2012; Revised: June 20, 2012; Accepted: August 3, 2012

Abstract

Background

Methods

Results

Conclusion

Keywords: Myelodysplastic syndrome, DNA methylation, GSTM5, ANGPTL2, BIK

Abstract

Fig 1.

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Figure 1.

Cluster pattern for all microarray gene data. Abbreviations: RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blast.

Blood Research 2012; 47: 186-193https://doi.org/10.5045/kjh.2012.47.3.186

Fig 2.

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Figure 2.

Hierarchical clustering analysis. No noteworthy hypermethylated gene cluster patterns were identified.

Blood Research 2012; 47: 186-193https://doi.org/10.5045/kjh.2012.47.3.186

Fig 3.

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Figure 3.

Network 1 gene expression, cancer, and cell cycle. The 16 candidate genes are denoted in red and burrowed with white genes to form a disease network construct.

Blood Research 2012; 47: 186-193https://doi.org/10.5045/kjh.2012.47.3.186

Fig 4.

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Figure 4.

Methylation-specific PCR results on GSTM5, ANGPTL2, BIK, and RERG genes from bone marrow samples. Patients were designated numbers 1-10 (RCMD) and 11-19 (RAEB). The PCR products were analyzed by electrophoresis on a 2% agarose gel. Abbreviations: N, normal peripheral blood; H, Human leukemic HL-60 cells; M, amplified products used as primers for the methylated sequence; U, amplified products used as primers for the unmethylated sequence.

Blood Research 2012; 47: 186-193https://doi.org/10.5045/kjh.2012.47.3.186

Table 1 . Methylated allele primer sequences and optimized annealing temperatures for methylation-specific PCR..

Abbreviations: F, forward primer; R, reverse primer..

Table 2 . Patient characteristics (N=20)..

Abbreviations: MDS, myelodysplastic syndrome; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blast; IPSS, international prognostic scoring system; BM, bone marrow; WPSS, WHO classification-based prognostic scoring system..

Table 3 . Top networks based on IPA analysis..

Table 4 . Statistically different gene methylation in network 1..

Abbreviations: RCMD, refractory cytopenia with multilineage dysplasia; RAEB, refractory anemia with excess blast..