Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency

Abstract

Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands.

Figure 1

Knockout or knockdown of NNT increases levels of mitochondrial ROS and apoptosis. A Adrenal zonation and steroidogenesis in 6NHsd and 6J mice. Wt and Nnt mutant mice showed similar patterns of CYP11A1 (red) and -B1 (green) staining, indicating no significant differences in zonation or steroidogenic capacity. The ZF cells in 6J mice however are slightly hyperplastic and disorganised being more densely packed and lacking the linear architecture seen in wt ZF. 40× magnification. B Increased apoptosis in 6J mice. Adrenals from 6J showed a higher number of caspase 3 positive cells (red) in zona fasciculata than 6NHsd. C Corticosterone levels in 6NHsd and 6J mouse serum stimulated with ACTH. Radioimmunoassay results revealed both basal and stimulated corticosterone levels were lower in 6J (p< 0.05). D Stable NNT knockdown (NNT-KD) in H295R cells. RT-PCR (i) and western blot analysis (ii) confirmed knockdown of NNT mRNA (71%) and protein (78%) levels in KD compared to SCR. E Detection of superoxide production by Mitosox. Quantitative analysis showed a significant increase (p<0.005) in superoxide production in NNT-KD relative to SCR cells. F Densitometric analysis showed a significant (p<0.001) increase in cleaved PARP between SCR and NNT-KD cells. Values are intensities of cleaved PARP relative to actin, n=12. G The GSH/GSSG ratio was lower in NNT-KD vs SCR cells (p<0.001). OS induced by the addition of 40μM menadione to the SCR cells reduced the ratio to 0.69±0.67.