DNA methylation status of NKX2-5, GATA4 and HAND1 in patients with tetralogy of fallot

Background: NKX2-5, GATA4 and HAND1 are essential for heart development, however, little is known regarding their epigenetic regulation in the pathogenesis of tetralogy of fallot (TOF). Methods: Methylation levels were measured in three regions of NKX2-5 (M1: -1596 bp similar to -1374 bp, M2: -159 bp similar to 217 bp and M3: 1058 bp similar to 1524 bp), one region of GATA4 (M: -392 bp similar to 107 bp) and three regions of HAND1 (M1: -887 bp similar to -414 bp, M2: -436 bp similar to 2 bp and M3: 37 bp similar to 398 bp) using the Sequenom MassARRAY platform. QRT-PCR was used to analyze NKX2-5 and HAND1 mRNA levels in the right ventricular myocardium of TOF patients. Results: TOF patients had a significantly higher NKX2-5_M3 median methylation level than controls (41.65% vs. 22.18%; p = 0.0074; interquartile range [IQR]: 30.46%-53.35%, N = 30 and 20.07%-24.31%, N = 5; respectively). The HAND1_M1 median methylation level was also significantly higher in TOF patients than controls (30.05% vs. 17.54%; p = 0.0054; IQR: 20.77%-40.89%, N = 30 and IQR: 14.69%-20.64%; N = 6; respectively). The methylation statuses of NKX2-5_M1, NKX2-5_M2, GATA4_M, HAND1_M2 or HAND1_M3 were not significantly different in TOF patients compared to controls. The methylation values for NKX2-5_M3 were negatively correlated with mRNA levels (r = -0.463, p = 0.010, N = 30) and there was a significant association between HAND1_M1 methylation status and mRNA levels (r = -0.524, p = 0.003, N = 30) in TOF patients. Conclusions: Aberrant methylation statuses of the NKX2-5 gene body and HAND1 promoter regions are associated with the regulation of gene transcription in TOF patients and may play an important role in the pathogenesis of TOF.