ESPE2023 Rapid Free Communications Adrenals and HPA Axis (6 abstracts)
Lack of NAD(P)+ transhydrogenase activity in patients with primary adrenal insufficiency due to NNT mutations
Annelise Francisco 1 , Ayse Mine Yilmaz Goler 2 , Claudia Daniele Carvalho Navarro 1 , Asan Onder 3 , Melek Yildiz 4 , Yasemin Kendir Demirkol 5 , Betul Karademir Yilmaz 2 , Tuba Seven Menevse 6 , Tulay Guran 6 & Roger Frigério Castilho 1
1Department of Pathology, School of Medical Sciences, University of Campinas, Campinas, Brazil. 2Marmara University Faculty of Medicine, Department of Biochemistry, Faculty of Medicine, Genetic and Metabolic Diseases Research Center, Istanbul, Turkey. 3Medeniyet University Goztepe Training and Research Hospital, Department of Pediatric Endocrinology and Diabetes, Istanbul, Turkey. 4Istanbul University, Istanbul Faculty of Medicine, Department of Pediatric Endocrinology and Diabetes, Istanbul, Turkey. 5Umraniye Training and Research Hospital, Pediatric Genetic Diseases, Istanbul, Turkey. 6Marmara University Faculty of Medicine, Department of Pediatric Endocrinology and Diabetes, Istanbul, Turkey
Background: Mutations in the nicotinamide nucleotide transhydrogenase (NNT) gene are a rare cause of primary adrenal insufficiency (PAI), as well as cardiomyopathies and functional impairment of the gonads.
Objective: Despite the description of different NNT mutations in homozygosis and compound heterozygosis in PAI patients, it remains to be clarified to which extent the function and expression of the mature protein are compromised.
Design: Blood samples from healthy controls, patients with previous diagnosis of NNT mutation-driven PAI (n=5) and their parents were analyzed for NAD(P)+ transhydrogenase (NNT) activity and expression.
Methods: NNT activity was assessed by its reverse reaction assay standardized for digitonin-permeabilized peripheral blood mononuclear cells (PBMC). The enzymatic assay was validated in PBMC samples from a mice model of NNT absence. Additionally, the PBMC samples were evaluated for NNT expression by western blot and RT-qPCR, and for mitochondrial oxygen consumption.
Results: NNT activity was undetectable (<4% of healthy controls) in PBMC samples from patients, independent of the pathogenic genetic variant. In their parents, NNT activity was approximately half of the healthy controls. NNT mature protein expression was lower in patients than in the control groups, while mRNA levels widely varied among genotypes. Moreover, NNT mutations did not impair the mitochondrial bioenergetic function in PBMC.
Conclusions: The manifestation of PAI in NNT-mutated patients is associated with a complete impairment of NNT activity. Evaluation of NNT activity can be useful to characterize disease-causing NNT mutations.
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