A loss-of-function mutation p.T256M in NDRG4 is implicated in the pathogenesis of pulmonary atresia with ventricular septal defect (PA/VSD) and tetralogy of Fallot (TOF)

Abstract

Pulmonary atresia with ventricular septal defect (PA/VSD) is a rare congenital heart disease (CHD) characterized by a lack of luminal continuity and blood flow from either the right ventricle or the pulmonary artery, together with VSDs. The prevalence of PA/VSD is about 0.2% of live births and approximately 2% of CHDs. PA/VSD is similar to tetralogy of Fallot (TOF) in terms of structural and pathological characteristics. The pathogenesis of these two CHDs remains incompletely understood. It was previously reported that N-myc downstream-regulated gene (NDRG)4 is required for myocyte proliferation during early cardiac development. In the present study, we enrolled 80 unrelated patients with PA/VSD or TOF and identified a probably damaging variant p.T256M of NDRG4. The p.T256M variant impaired the proliferation ability of human cardiac myocytes (hCM). Furthermore, the p.T256M variant resulted in G1 and G2 arrest of hCM, followed by an increase in p27 and caspase-9 expression. Our results provide evidence that the p.T256M variant in NDRG4 is a pathogenic variant associated with impaired hCM proliferation and cell-cycle arrest and likely contributes towards the pathogenesis of PA/VSD and TOF.

Keywords: NDRG4; PA/VSD; TOF; cardiac myocytes; p27; proliferation.

Fig. 1

Characteristics of the mutation c.767C>T in NDRG4. (A) Sequence chromatograms of NDRG4 variants in patients. Arrows indicate heterozygous nucleotide changes. (B) Homology analysis of the 256T position of NDRG4 protein across different species.

Fig. 2

mRNA and protein expression levels of NDRG4 in human embryos. (A) The mRNA expression levels of NDRG4 and ISL1 in the same samples were investigated in human embryonic hearts (n = 3). (B–G) Immunohistochemistry of NDRG4 in human embryos at CS13. (B–D) Negative control. (E–G) WT NDRG4. ISL1, Islet‐1; OFT, outflow tract; A, atrium; V, ventricle. Scale bar in (B, E) = 50 μm, Scale bar in (C, D, F, G) = 20 μm.

Fig. 3

mRNA abundance and protein expression level of NDRG4. hCM was transfected with blank vector (control), as well as WT and variant plasmids of NDRG4, and mRNA and protein expression was analyzed by (A) qRT‐PCR and (B) western blotting (n = 4). Data are the mean ± SEM.

Fig. 4

Mutant NDRG4 protein impaired the regulation of cell proliferation in hCM. hCM was transfected with blank vector (control), as well as WT and variant plasmids of NDRG4. (A) Cell proliferation was detected by the CCK‐8 assay at each of the indicated time points (0, 12, 24, 48 and 72 h); two‐way analysis of variance. (B–E) Cell‐cycle distribution was detected using a flow cytometer (n = 4, *P < 0.05, **P < 0.01); unpaired two‐tailed Student's t‐test. Data are the mean ± SEM.

Fig. 5

mRNA and protein expression of P27. Blank vector (control), as well as WT and variant plasmids of NDRG4, were transfected into hCM and harvested. (A) qRT‐PCR analysis of cell‐cycle progression markers and apoptotic markers (n = 4–5, *P < 0.05, **P < 0.01); unpaired two‐tailed Student's t‐test. (B) Western blot analysis of P27 (n = 3); unpaired two‐tailed Student's t‐test. Data are the mean ± SEM.

Fig. 6

Subcellular localization of P27. Blank vector (control), as well as WT and variant plasmids of NDRG4, were transfected into hCM and harvested. (A–I) Representative images of immunofluorescence staining of P27 proteins in control (A–C), WT (D–F) and variant (G–I) groups (n = 3). p27. Scale bar = 20 μm.