| Objective To investigate the methylation status of GTPase regulator associated with the focal adhesion kinase (GRAF) gene promoter and its clinical correlation in patients with myeloid malignancies including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML).Methods The methylation-specific PCR (MSP) technique was established and used to detect the methylation status of GRAF promoter in the bone marrow mononuclear cells from the patients with MDS (n=68), AML (n=132) and CML (n=61); The EvaGreen real-time quantitative polymerase chain reaction (RQ-PCR) assay was established and performed to measure GRAF transcripts in 71 cases with AML and 21 with nonmalignant hematological diseases.Results GRAF promoter hypermethylation was found in patients with MDS, AML, CML but in none of control group.①GRAF promoter hypermethylation was found in 28 (41.2%) MDS cases. Correlation was not found between GRAF promoter hypermethylation and sex, age, hematologic parameters, chromosomal abnormalities of MDS patients. The significant difference was not observed in the frequencies of GRAF promoter hypermethylation among the patients with refractory anemia/refractory anemia with ringed siderobisasis (RA/RAS) (2/5, 40.0%), fractory cytopenia with multilineage dysplasia (RCMD) and RCMD with ringed blasts (RCMD-RS) (8/21,38.1%), RA with excess blasts-1 (RAEB-1) (5/14,35.7%), RAEB-2 (7/14,50.0%),5q-syndrome (1/3,33.3%), MDS-U(2/3,66.7%) and refractory anemia with excess blasts in transformation/acute myeloid leukemia (RAEBt/AML) (3/8, 37.5%) (P＞0.05). The correlation was not observed between GRAF promoter hypermethylation and WHO classification, FAB classification or IPSS grouping. Cox regression analysis revealed that there were four factors affecting survival time, including GRAF methylation, age, WBC, and IPSS groups (P=0.017,0.001,0.001 and＜0.001, respectively) except for sex, hemoglobin level and platelet counts (P＞0.05). The estimated 50% survival time of the methylated GRAF group and unmethylated group was 12 (95% confidence interval 5.3-18.7 months) and 26 months (95% confidence interval 10.8-41.1 months), respectively. There was significant difference between these two groups (χ2=7.942, P=0.005).②GRAF promoter hypermethylation was found in 87 (65.9%) AML cases. There was no correlation between GRAF promoter hypermethylation and the sex, age, WBC counts, platelet counts, chromosomal abnormalities of AML patients (P＞0.05). The hypermethylation frequencies of GRAF promoter in AML subtypes were as follows:63.0%(17/27, M1),57.1%(24/42, M2),72.2%(13/18, M3),84.0%(21/25, M4),46.2%(6/13, M5),85.7%(6/7, M6). The significant difference was observed in the frequencies of GRAF gene hypermethylation among patients with different subtypes (χ2= 11.143, P =0.049). The frequencies of GRAF gene hypermethylation in the M4 and M6 were significantly higher than that in other subtypes; The expression level of GRAF transcript was significantly lower in AML (0.01%-169.75%, median 3.82%) than that in controls (14.49%-126.85%, median 56.04%)(P＜0.05). GRAF transcript was significantly lower in patients with methylated GRAF than those without methylated GRAF (P=005). There was no correlation between the level of GRAF transcript and the sex, age, hematologic parameters, FAB subtypes and karyotypic groups (P＞0.05).③GRAF promoter hypermethylation was found in 34 (55.7%) CML cases. Correlation was not found between GRAF promoter hypermethylation and the age, sex, hemoglobin concentration, platelet counts, chromosomal abnormalities of CML patients (P＞0.05). The hypermethylation frequencies of GRAF promoter in CML patients at CP, AP and BC were 55.3%(21/38),50.0%(2/4), 57.9%(11/19), respectively. Correlation was also not found between the frequencies of GRAF promoter hypermethylation and different CML stages (P＞0.05).Conclusions GRAF gene hypermethylation might be involved in the development of myeloid malignancies including MDS, AML and CML, which was an adverse prognostic factor in MDS. |
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