Julia Kowalczyk

Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency

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.

Thioredoxin Reductase 2 (TXNRD2) Mutation Associated With Familial Glucocorticoid Deficiency (FGD)

Context: Classic ACTH resistance, due to disruption of ACTH signaling, accounts for the majority of cases of familial glucocorticoid deficiency (FGD). Recently FGD cases caused by mutations in the mitochondrial antioxidant, nicotinamide nucleotide transhydrogenase, have highlighted the importance of redox regulation in steroidogenesis. Objective: We hypothesized that other components of mitochondrial antioxidant systems would be good candidates in the etiology of FGD. Design: Whole-exome sequencing was performed on three related patients, and segregation of putative causal variants confirmed by Sanger sequencing of all family members. A TXNRD2-knockdown H295R cell line was created to investigate redox homeostasis. Setting: The study was conducted on patients from three pediatric centers in the United Kingdom. Patients: Seven individuals from a consanguineous Kashmiri kindred, six of whom presented with FGD between 0.1 and 10.8 years, participated in the study. Interventions: There were no interventions. Main Outcome Measure: Identification and functional interrogation of a novel homozygous mutation segregating with the disease trait were measured. Results: A stop gain mutation, p.Y447X in TXNRD2, encoding the mitochondrial selenoprotein thioredoxin reductase 2 (TXNRD2) was identified and segregated with disease in this extended kindred. RT-PCR and Western blotting revealed complete absence of TXNRD2 in patients homozygous for the mutation. TXNRD2 deficiency leads to impaired redox homeostasis in a human adrenocortical cell line. Conclusion: In contrast to the Txnrd2-knockout mouse model, in which embryonic lethality as a consequence of hematopoietic and cardiac defects is described, absence of TXNRD2 in humans leads to glucocorticoid deficiency. This is the first report of a homozygous mutation in any component of the thioredoxin antioxidant system leading to inherited disease in humans.

Familial glucocorticoid deficiency: New genes and mechanisms.

Familial Glucocorticoid deficiency (FGD), in which the adrenal cortex fails to produce glucocorticoids, was first shown to be caused by defects in the receptor for ACTH (MC2R) or its accessory protein (MRAP). Certain mutations in the steroidogenic acute regulatory protein (STAR) can also masquerade as FGD. Recently mutations in mini chromosome maintenance-deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT), genes involved in DNA replication and antioxidant defence respectively, have been recognised in FGD cohorts. These latest findings expand the spectrum of pathogenetic mechanisms causing adrenal disease and imply that the adrenal may be hypersensitive to replicative and oxidative stresses. Over time patients with MCM4 or NNT mutations may develop other organ pathologies related to their impaired gene functions and will therefore need careful monitoring.

Oxidative stress and adrenocortical insufficiency.

Maintenance of redox balance is essential for normal cellular functions. Any perturbation in this balance due to increased reactive oxygen species (ROS) leads to oxidative stress and may lead to cell dysfunction/damage/death. Mitochondria are responsible for the majority of cellular ROS production secondary to electron leakage as a consequence of respiration. Furthermore, electron leakage by the cytochrome P450 enzymes may render steroidogenic tissues acutely vulnerable to redox imbalance. The adrenal cortex, in particular, is well supplied with both enzymatic (glutathione peroxidases and peroxiredoxins) and non-enzymatic (vitamins A, C and E) antioxidants to cope with this increased production of ROS due to steroidogenesis. Nonetheless oxidative stress is implicated in several potentially lethal adrenal disorders including X-linked adrenoleukodystrophy, triple A syndrome and most recently familial glucocorticoid deficiency. The finding of mutations in antioxidant defence genes in the latter two conditions highlights how disturbances in redox homeostasis may have an effect on adrenal steroidogenesis.

ACTH Resistance: Genes and Mechanisms

ACTH resistance is a rare disorder typified by familial glucocorticoid deficiency (FGD), a genetically heterogeneous disease. Previously, genetic defects in FGD have been identified in the ACTH receptor gene (MC2R), its accessory protein (MRAP) and the steroidogenic acute regulatory protein gene (STAR). The defective mechanisms here are failures in ACTH ligand binding and/or receptor trafficking for MC2R and MRAP and, in the case of STAR mutations, inefficient cholesterol transport to allow steroidogenesis to proceed. Novel gene defects in FGD have recently been recognised in mini-chromosome maintenance-deficient 4 homologue (MCM4) and nicotinamide nucleotide transhydrogenase (NNT). MCM4 is one part of a DNA repair complex essential for DNA replication and genome stability, whilst NNT is involved in the glutathione redox system that protects cells against reactive oxygen species. The finding of mutations in these two genes implicates new pathogenetic mechanisms at play in FGD, and implies that the adrenal cortex is exquisitely sensitive to replicative and oxidative stresses.

Genetic characterisation of a cohort of children clinically labelled as GH or IGF1 insensitive: diagnostic value of serum IGF1 and height at presentation.

OBJECTIVE AND DESIGN GH insensitivity (GHI) encompasses growth failure, low serum IGF1 and normal/elevated serum GH. By contrast, IGF1 insensitivity results in pre- and postnatal growth failure associated with relatively high IGF1 levels. From 2008 to 2013, 72 patients from 68 families (45M), mean age 7.1 years (0.4-17.0) with short stature (mean height SDS -3.9; range -9.4 to -1.5), were referred for sequencing. METHODS As a genetics referral centre, we have sequenced appropriate candidate genes (GHR, including its pseudoexon (6Ψ), STAT5B, IGFALS, IGF1, IGF1R, OBSL1, CUL7 and CCDC8) in subjects referred with suspected GHI (n=69) or IGF1 insensitivity (n=3). RESULTS Mean serum IGF1 SDS was -2.7 (range -0.9 to -8.2) in GHI patients and 2.0, 3.7 and 4.4 in patients with suspected IGF1 insensitivity. Out of 69 GHI patients, 16 (23%) (19% families) had mutations in GH-IGF1 axis genes: homozygous GHR (n=13; 6 6Ψ, two novel IVS5ds+1 G to A) and homozygous IGFALS (n=3; one novel c.1291delT). In the GHI groups, two homozygous OBSL1 mutations were also identified (height SDS -4.9 and -5.7) and two patients had hypomethylation in imprinting control region 1 in 11p15 or mUPD7 consistent with Silver-Russell syndrome (SRS) (height SDS -3.7 and -4.3). A novel heterozygous IGF1R (c.112G>A) mutation was identified in one out of three (33%) IGF1-insensitive subjects. CONCLUSION Genotyping contributed to the diagnosis of children with suspected GHI and IGF1 insensitivity, particularly in the GHI subjects with low serum IGF1 SDS (<-2.0) and height SDS (<-2.5). Diagnoses with similar phenotypes included SRS and 3-M syndrome. In 71% patients, no diagnosis was defined justifying further genetic investigation.

Dominant-negative STAT5B mutations cause growth hormone insensitivity with short stature and mild immune dysregulation

Growth hormone (GH) insensitivity syndrome (GHIS) is a rare clinical condition in which production of insulin-like growth factor 1 is blunted and, consequently, postnatal growth impaired. Autosomal-recessive mutations in signal transducer and activator of transcription (STAT5B), the key signal transducer for GH, cause severe GHIS with additional characteristics of immune and, often fatal, pulmonary complications. Here we report dominant-negative, inactivating STAT5B germline mutations in patients with growth failure, eczema, and elevated IgE but without severe immune and pulmonary problems. These STAT5B missense mutants are robustly tyrosine phosphorylated upon stimulation, but are unable to nuclear localize, or fail to bind canonical STAT5B DNA response elements. Importantly, each variant retains the ability to dimerize with wild-type STAT5B, disrupting the normal transcriptional functions of wild-type STAT5B. We conclude that these STAT5B variants exert dominant-negative effects through distinct pathomechanisms, manifesting in milder clinical GHIS with general sparing of the immune system.Severe growth hormone insensitivity syndrome (GHIS) with immunodeficiency is caused by autosomal recessive mutations in STAT5B. Here the authors report heterozygous STAT5B mutations with dominant-negative effects, causing mild GHIS without immune defects.