ESPE Abstracts

Background: The mitochondrial enzyme nicotinamide nucleotide transidrogenase (NNT) is essential in the antioxidant defense mechanisms and appears to be essential to the stages of adrenal steroidogenesis that occur inside the mitochondria.

Aim: To characterize how mutations in NNT gene impair adrenal steroidogenesis resulting in familial glucocorticoid deficiency (FGD).

Methods: Genomic DNA of a 1.8 years old boy with FGD was evaluated by whole exome sequencing (WES). Candidate genetic variants were analyzed in silico and confirmed by Sanger sequencing. Genotype-phenotype association of a new homozygous loss-of-function NNT variant with FGD was assessed in vitro. Several antioxidant mechanisms were evaluated under basal and oxidative induction conditions (100uM H202 by 5h) in transient cultures using leukocyte cells from the patient, the heterozygous family members and wild-type (WT) controls. Several mitochondrial parameters including reactive oxygen species (ROS) intracellular production (DCFDA), reduced glutathione (GSH; GSH-Glo Assay) and mitochondrial mass (Mitotraker) were analyzed. To ascertain its specific role adrenal cortex, siRNA NNT gene knockdown was performed in H295 adrenocortical cells to evaluate the effects on mitochondrial parameters and cortisol secretion (RIA).

Results: WES revealed the homozygous NNT p.G866D pathogenic variant in the affected patient. Family pedigree analysis confirmed the segregation of this variant with FGD in homozygosis. Heterozygous careers (consanguineous parents and a young brother) were asymptomatic. Compared to wild-type (WT), both under basal and oxidative stress conditions, homozygous p.G866D NNT mononuclear leukocytes exhibited increased ROS production (P=0,02 and P=0,0001), decreased GSH levels (P<0,01 and P<0,001, r) and decreased mitochondrial mass (P=0,01 and P=0,001), respectively. Specific knockdown of the NNT gene in the H295 adrenal cells as shown by reduction of 55% and 50% in its RNAm protein levels did not change cell viability or cortisol secretion in basal condition. However, after 24h of treatment with 10uM forskolin, a potent stimulator of steroidogenesis, there was a marked decrease of cortisol production (P<0.0001).

Conclusion: Loss-of-function NNT mutations impair antioxidants mechanisms and affects the glutathione reductase systems, resulting in accumulation of reative oxygen species. NNT impairment in adrenal cells decreases cortisol secretion. Altogether, these data explain the occurrence of adrenal insufficiency and confirm the genotype-phenotype association in patients carrying pathogenic NNT mutations.