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 ~ -1374 bp, M2: -159 bp ~ 217 bp and M3: 1058 bp ~ 1524 bp), one region of GATA4 (M: -392 bp ~ 107 bp) and three regions of HAND1 (M1: -887 bp ~ -414 bp, M2: -436 bp ~ 2 bp and M3: 37 bp ~ 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.