Impact of a novel homozygous mutation in nicotinamide nucleotide transhydrogenase on mitochondrial DNA integrity in a case of familial glucocorticoid deficiency

Abstract

BackgroundFamilial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder that is characterized by isolated glucocorticoid deficiency. Recently, mutations in the gene encoding for the mitochondrial nicotinamide nucleotide transhydrogenase (NNT) have been identified as a causative gene for FGD; however, no NNT activities have been reported in FGD patients carrying NNT mutations.MethodsClinical, biochemical and molecular analyses of lymphocytes from FDG homozygous and heterozygous carriers for the F215S NNT mutation were performed.ResultsIn this study, we described an FGD-affected Japanese patient carrying a novel NNT homozygous mutation (c.644T>C; F215S) with a significant loss-of-function (NNT activity = 31% of healthy controls) in peripheral blood cells' mitochondria. The NNT activities of the parents, heterozygous for the mutation, were 61% of the controls.ConclusionsOur results indicated that (i) mitochondrial biogenesis (citrate synthase activity) and/or mtDNA replication (mtDNA copy number) were affected at ≤ 60% NNT activity because these parameters were affected in individuals carrying either one or both mutated alleles; and (ii) other outcomes (mtDNA deletions, protein tyrosine nitration, OXPHOS capacity) were affected at ≤ 30% NNT activity as also observed in murine cerebellar mitochondria from C57BL/6J (NNT−/−) vs. C57BL/6JN (NNT+/+) substrains.General significanceBy studying a family affected with a novel point mutation in the NNT gene, a gene–dose response was found for various mitochondrial outcomes providing for novel insights into the role of NNT in the maintenance of mtDNA integrity beyond that described for preventing oxidative stress.

NNT mutations: a cause of primary adrenal insufficiency, oxidative stress and extra-adrenal defects

“…For this reason too, patients with NNT mutations need to be closely monitored. Two in vitro studies on the fibroblasts (12) and lymphocyte mitochondria (31) of patients homozygous for missense NNT mutations showed an increase in ROS levels, a decrease of ATP content, and impaired morphology of mitochondria with reduced mitochondrial mass and increased mitochondrial DNA deletion due to a lack of thymidylate biosynthesis (12,31). The results from those two studies suggest that all tissues can be injured, as do our results from the follow-up of patients with extra-adrenal defects.…”

The Contribution of Nicotinamide Nucleotide Transhydrogenase to Peroxide Detoxification Is Dependent on the Respiratory State and Counterbalanced by Other Sources of NADPH in Liver Mitochondria

“…It is noteworthy that this model allowed the study of liver mitochondria from mice that expressed mutated Nnt in heterozygosis, a genotype whose functional consequences are unknown but are probably relevant to the parents of patients with familiar glucocorticoid insufficiency linked to homozygous Nnt mutations (5,13). The congenic mouse model nearly eliminates potential genetic modifiers between mouse substrains (26); thus, phenotypic differences between Nnt genotypes can be directly attributed to this gene ϩ is reduced into NADPH during the oxidation of the substrates isocitrate (Isoc), malate (Mal), and glutamate (Glut) via IDH2, MEs, and GDH, respectively.…”

“…Homozygous mutations of Nnt that are linked to familiar glucocorticoid insufficiency have been documented in humans (5), whereas the C57BL/6J mouse substrains have been shown to exhibit metabolic abnormalities (6 -8) with major implications regarding their use as experimental models in basic research (9 -12). The phenotype of the Nnt mutation in heterozygotes is not clear in humans (13) or mice.…”

Nicotinamide Nucleotide Transhydrogenase as a novel treatment target in adrenocortical carcinoma

“…NCI-H295R (RRID:CVCL_0458) ACC cells (passage [10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25] were cultured under standard conditions using DMEM/Ham's F-12 medium (Gibco, Thermo Fisher) supplemented with 2.5% Nu serum (Corning), 1% penicillin-streptomycin (Gibco, Thermo Fisher) and 1% ITS+ universal cell culture premix (Corning). Cell line identity was confirmed through Short Tandem Repeat (STR) genetic analysis performed by the DNA Diagnostics Company (London, UK)…”