Introduction: Variants in genes that play a role in maintaining cellular redox homeostasis in adrenocortical cells may be associated with glucocorticoid deficiency, and it is unclear whether these cases may be associated with a wider phenotype. However, to date, only 1 case of a genetic variant in thioredoxin reductase type 2 (TXNRD2) in a South Asian kindred with familial glucocorticoid deficiency has been reported. Case Presentation: The index case was diagnosed with selective glucocorticoid deficiency at 10 years of age. He had a history of a small penis and a right undescended testis, which subsequently required an orchidopexy. The parents were of Pakistani origin and first cousins. The boy’s gonadal function was normal and autosomal recessive missense homozygous variants p.Val361Met;Val361Met in TXNRD2 were identified in him by whole-genome sequencing. Functional studies were performed using peripheral blood mononuclear cells from the patient, unaffected parents, and four age-matched healthy boys. Compared to the carriers and controls, the case had lower TXNRD2 protein on immunoblotting using anti-TXNRD2 antibody (1.3-fold), 95% CI: 1.8 (1.5–2.1), lower mRNA expression of TXNRD2 on quantitative RT-PCR (1.6-fold), 95% CI: 1.1 (0.7–1.4), and a lower glutathione:oxidized glutathione ratio (6.7-fold), 95% CI: 2.0 (1.6–2.4). Conclusions: In addition to confirming the critical role that TXNRD2 serves in maintaining adrenal function, by reporting the findings of atypical genitalia, this case further extends the phenotype.

Established Facts

  • Nicotinamide nucleotide transhydrogenase (NNT) and TXNRD2 genes play a role in maintaining cellular redox homeostasis in adrenocortical cells and have been identified as genetic causes of glucocorticoid deficiency with or without mineralocorticoid deficiency.

  • To date, a single report of familial glucocorticoid deficiency in a South Asian kindred has been described to be associated with a genetic variant in TXNRD2.

Novel Insights

  • This second report of isolated glucocorticoid deficiency with a homozygous missense variant in TXNRD2 strengthens the role of this protein in adrenocortical redox homeostasis.

  • In addition to selective glucocorticoid deficiency, our case also had micropenis and undescended testis, which have not been reported in the previous kindred with TXNRD2 variants.

Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterised by adrenocorticotropic hormone (ACTH) resistance and selective glucocorticoid deficiency. In some populations, such as in Irish traveller families, the prevalence has been reported to be around 1 in 200,000 [1, 2], but it is possible that the prevalence may be higher given that the condition can be associated with a spectrum of manifestations. Although in most cases, ACTH resistance is caused by genetic variants in the genes encoding the ACTH receptor (melanocortin 2 receptor [MC2R]) and the MC2R accessory protein, the molecular aetiology of ACTH resistance is very heterogeneous [3]. More recently, proteins involved in cellular redox balance have also been implicated and among these, gene variants in the nicotinamide nucleotide transhydrogenase (NNT) gene may account for about 7% among patient with primary adrenal insufficiency (PAI) after excluding the common causes of PAI such as congenital adrenal hyperplasia [4‒6]. NNT is integral for redox homeostasis and generates nicotinamide adenine dinucleotide phosphate (NADPH), which acts as the reducing agent for the thioredoxin and glutathione (GSH) systems to remove reactive oxygen species (ROS) [7]. Maintenance of thioredoxin and GSH in a reduced state requires mitochondrial reductase selenoprotein, TXNRD2. To date, only one variant, p.Y447*, in TXNRD2 has been associated with FGD in a South Asian kindred [8]. Here, we describe the second variant in this gene causing FGD in another South Asian family.

The index case initially presented with a right undescended testis and small penis and subsequently had undergone right orchidopexy and circumcision around 1 year of age. Endocrine investigations at 1.9 years included a human chorionic gonadotrophin stimulation test using 1,500 units human chorionic gonadotrophin on three consecutive days, which showed a rise in serum testosterone from <0.5 nmol/L at baseline to 9.9 nmol/L on Day 4 and a rise in dihydrotestosterone from 0.42 nmol/L to 1.31 nmol/L. A luteinising hormone-releasing hormone stimulation test performed at the same age showed a rise in luteinising hormone (LH) from <0.2 U/L to 12.0 U/L and a rise in follicle-stimulating hormone from 1.6 U/L to 5.2 IU/L. Serum anti-Müllerian hormone level was 872 pmol/L (age-matched reference range 230–1,300 pmol/L). Detailed urinary steroid analysis by gas chromatography-mass spectroscopy (GC-MS) did not reveal any abnormalities. At the age of 10 years, the boy represented to the endocrine clinic with a 12–18 month history of fatigue and a 6-month history of increased pigmentation. Preliminary investigations that were performed prior to referral included thyroid function tests on two occasions over a period of 3 months, which had revealed a thyroid stimulating hormone (TSH) of 9.15 mU/L and 8.54 mU/L (0.35–5.00 mU/L) and a free thyroxine (fT4) of 9.8 pmol/L and 10.0 pmol/L (9.0–21.0 pmol/L). For a year prior to presentation, he had been receiving homeschooling because of frequent upper respiratory tract infections, with difficulty in recovering from these. The family was of Pakistani origin and the parents were first cousins. His mother had been diagnosed with a nonprogressive inflammatory arthritis at the age of 13 years and a history of hypothyroidism was reported in a maternal uncle and hyperthyroidism in maternal grandmother. A history of a small phallus had also been reported in the maternal grandfather who deceased. His two sisters were healthy and one older brother had been diagnosed with a mild dysplastic pulmonary valve without any obstructive symptoms.

At 10.3 years of age, the index case had a height standard deviation score of 0.49 and a weight standard deviation score of −0.13 and had generalised hyperpigmentation, especially on the buccal mucosa, elbows, and old scars. His blood pressure was 106/60 mm Hg without any evidence of postural hypotension. Genital examination showed a small penis, measuring 3 cm, right scrotal testis of about 3–4 mL, and a left testis which was palpable in the upper scrotal region. No enlargement of the thyroid gland was evident. Assessment of adrenal function revealed a plasma ACTH of 1,070 mU/L (<20 mU/L), undetectable cortisol despite ACTH stimulation, aldosterone of <130 pmol/L (130–600), and a renin concentration of 26.5 mIU/L (<125 mIU/L). Serum urea, creatinine, and electrolytes were normal and a urine steroid analysis by GC-MS did not identify a defect in steroid metabolism. Adrenal autoantibodies were negative. Thyroid function test continued to reveal a raised TSH of 11.42 mU/L and a normal fT4 of 9.9 pmol/L. The serum thyroid peroxidase (TPO) antibody titre was <1.0 U/mL (<6.0 U/mL) and thyroid receptor antibody titre was 1.1 U/L (0–1.9 U/L). The patient was started on hydrocortisone replacement therapy and noticed a marked improvement in his fatigue levels. Following 5 months of replacement, his plasma ACTH had reduced to 265 mU/L. His TSH at this point was 5.7 mU/L with a fT4 of 12.3 pmol/L.

Genetic Analysis and Results

Following his initial presentation when his gonadal function was investigated, the patient had undergone genetic analysis using a 56 DSD gene next-generation sequencing panel [9] and this had identified a heterozygous variant of uncertain significance of CYP11B1 (NM_000497.3:c.[1182C>G];[=] p.[(Asn394Lys)];[(=)]. Following his presentation of PAI, a 12-gene panel for congenital adrenal hypoplasia and selective glucocorticoid deficiency that included AAAS, AIRE, CDKN1C, CYP11A1, MC2R, MRAP, NNT, NR0B1, NR5A1, SAMD9, STAR, and TBX19 was also explored and did not identify any abnormalities. However, on whole-genome sequencing, homozygous missense variants of TXNRD2 were identified from the index case and unaffected heterozygous parents by Trio analysis (NM_006440.5:c.1081G>A;1081G>A, p.Val361Met;Val361Met). This variant was absent from ExAC and gnomAD. Amino acid 361 lies within the pyridine nucleotide-disulphide oxidoreductase-binding domain of TXNRD2. In addition, in silico analysis suggested that the substitution by methionine at that position may reduce the stability of the protein’s 3D tertiary structure, causing loss of function. However, this is not a highly conserved amino acid throughout evolution and there was inconsistent evidence on other in silico predictions, with SIFT analysis indicating the substitution is “deleterious” but PolyPhen-2 and LoFtool both indicating it as “possibly damaging.”

Functional Analysis Methods

Blood samples were collected from the case and his parents and analysed as part of the GIRD study (West of Scotland Ethics approval, 14/WS/0036) following informed consent from the parents. Excess blood samples from four age-matched healthy boys, recruited as part of the GO-VASC study, were used as controls and details of this study and its ethics approval are available at http://researchdata.gla.ac.uk/997/. To obtain peripheral blood mononuclear cells (PBMCs), total blood from EDTA-containing tubes was pipetted over a gradient solution of Ficol:Hipaque Plus (density 1.077 g/mL; Amersham Biosciences) and centrifuged at 400 g for 35 min at 20°C with the slowest acceleration and deceleration. PBMCs, identified as a cloudy layer, were removed with a Pasteur pipette and transferred to a 50 mL tube with ice cold phosphate-buffered serum (1.37 m NaCl, 27 mm KCLl Na2PO4 100 mm, KH2PO4 18 mm), which approximates 0.8% saline. This was centrifuged at 400 g for 10 min (full acceleration and deceleration). This step was repeated twice. The cell pellet was suspended in lysis reagent for all further experiments including RNA isolation. For mRNA expression studies, total RNA was extracted using Qiazol as per the manufacturer’s instructions (Qiagen, UK) from PBMCs, and cDNA was prepared by reverse transcription as per the manufacturer’s instructions (Thermo Fisher, UK) and synthesised on a Multi-Block PCR Thermal Cycler (Satellite 0.2 Thermo Cooler, Thermo Fisher, UK) under the following conditions: 10 min at 25°C (primer annealing); 120 min at 37°C (cDNA synthesis); 5 min at 85°C (reverse transcriptase inactivation), and maintenance at 4°C until thermal cycler termination. Target gene expression was identified using Qiagen QuantiTech primer assays (Qiagen, UK) and SYBR® Green (UK). Primers used are shown in Table 1. Transcript gene expression was normalised using the housekeeping human hypoxanthine-guanine phosphoribosyltranferase gene (Qiagen, UK). The 2δδCT method was then used to calculate relative gene expression. The results are expressed as fold increase. PBMC proteins were extracted and separated on precasted 4–12% NuPAGE gels (Thermo Fisher Scientific, UK). Semi-dry blotting (Power Blot, Thermo Fisher Scientific, UK) was performed on low-fluorescence PVDF membranes (Merck, UK). Membranes were blocked in 5% bovine serum albumin at room temperature. Membranes were incubated with primary antibodies overnight at 4°C and then with secondary antibodies for 1 h at room temperature. Fluorescent signals were measured using the Odyssey CLX, LI-COR scanner and analysed using ImageStudioLite software (LI-COR Biosciences, Lincoln, USA). GAPDH (1:10,000) was used as an internal housekeeping loading control protein. The results were normalised according to cellular protein levels. Antibodies used are shown in Table 2. GSH/oxidised glutathione (GSSG) ratios were calculated using the GSH/GSSG-Glo Assay (Promega, Madison, USA) according to the manufacturer’s instructions. PBMCs from the case and controls were plated on a 96-well plate and triplicate measurements of basal luminescence (relative light units) were measured using the Orion II luminometer (Titerek-Berthold, Germany). Comparison of variables between the case and control group was performed using the Crawford and Howell method [10], where a negative t value was deemed to reject the null hypothesis.

Table 1.

Primers used for mRNA expression studies

GeneForward primerReverse primer
Thioredoxin reductase 2 5′ – TTG​AGG​TCT​ATC​ACG​CCC-3′ 5′ – CTT​GAG​TAA​CTT​CGC​CTG​C – 3′ 
Human hypoxanthine-guanine phosphoribosyltranferase 5′ – TGA​CAC​TGG​CAA​AAC​AAT​GCA – 3′ 5′ – GGT​CCT​TTT​CAC​CAG​CAA​GCT – 3′ 
GeneForward primerReverse primer
Thioredoxin reductase 2 5′ – TTG​AGG​TCT​ATC​ACG​CCC-3′ 5′ – CTT​GAG​TAA​CTT​CGC​CTG​C – 3′ 
Human hypoxanthine-guanine phosphoribosyltranferase 5′ – TGA​CAC​TGG​CAA​AAC​AAT​GCA – 3′ 5′ – GGT​CCT​TTT​CAC​CAG​CAA​GCT – 3′ 

The details of the primers used in this study are as follows: Thioredoxin reductase 2 (TXNRD2): Product name: Hs_TXNRD2_vb.1_SG QuantiTect Primer Assay, GeneGlobe Id: QT01015014, and catalogue number: 249900.

Human hypoxanthine-guanine phosphoribosyltransferase (hHRPT): Product name: Hs_HPRT1_1_SG QuantiTect Primer Assay, GeneGlobe Id: QT00059066, and catalogue number: 249900.

Table 2.

Primary and secondary antibodies used for immunoblotting experiments

NameManufacturerDilutionHost species
GAPDH Abcam, UK 1:1,000 Rabbit 
TXNRD2 Sigma Aldrich, UK 1:500 Rabbit 
Anti-rabbit Alexa Fluor 488 Life Technologies 1:10,000 Goat 
NameManufacturerDilutionHost species
GAPDH Abcam, UK 1:1,000 Rabbit 
TXNRD2 Sigma Aldrich, UK 1:500 Rabbit 
Anti-rabbit Alexa Fluor 488 Life Technologies 1:10,000 Goat 

Catalogue numbers of GAPDH, TXNRD2, and anti-rabbit Alexa Fluor 488 used in this study are ab9485, HPA003323, and 710369, respectively.

Functional Analysis Results

Compared to the heterozygote carriers and controls, the case displayed lower mRNA expression of TXNRD2 (1.6 fold, t = −1.1), 95% CI: 1.1 (0.7–1.4) (Fig. 1a), a lower GSH/GSSG ratio (6.7 fold, t = −4.3), 95% CI: 2.0 (1.6–2.4) (Fig. 1b), and lower TXNRD2 protein levels (1.3 fold, t = −0.9), 95% CI: 1.8 (1.5–2.1) (Fig. 1c). The TNXRD2 protein level and mRNA expression were only marginally reduced compared to the much greater reduction in the GSH/GSSG ratio, suggesting that the missense variant was primarily leading to marked dysfunction of the mutant protein rather than protein loss as seen in previous cases [8].

Fig. 1.

Results of functional analysis in a case compared to healthy controls. aTXNRD2 mRNA expression on quantitative RT-PCR. b GSH/GSSG ratio. c TXNRD2 protein level. d Lysed PBMC extract from homozygous patient, heterozygous carriers, and 4 age-matched controls were immunoblotted with anti-TXNRD2 antibody. n = 1 case, 6 controls (parents of case and 4 age-matched boys). Results are shown as mean and 95% CI.

Fig. 1.

Results of functional analysis in a case compared to healthy controls. aTXNRD2 mRNA expression on quantitative RT-PCR. b GSH/GSSG ratio. c TXNRD2 protein level. d Lysed PBMC extract from homozygous patient, heterozygous carriers, and 4 age-matched controls were immunoblotted with anti-TXNRD2 antibody. n = 1 case, 6 controls (parents of case and 4 age-matched boys). Results are shown as mean and 95% CI.

Close modal

Maintenance of redox homeostasis by eliminating mitochondrial superoxides is important for adrenal steroidogenesis. NNT generates high concentration of NADPH for thioredoxin and GSH pathways and these contribute to the reduction of peroxiredoxin 3, the most important superoxide-eliminating enzyme in the mitochondria of adrenocortical cells [11, 12]. The loss of function in the NNT gene leads to reduced NADPH production, causing deficiencies in antioxidant defences, decreased integrity of mitochondrial DNA, and reduced mitochondrial mass [13]. TXNRD2 deficiency, which decreases the capacity of the thioredoxin system, will result in higher levels of ROS and subsequently impact the GSH system, causing an altered GSH/GSSG ratio, a marker of oxidative stress [8]. Until now, there has only been one report of TXNRD2 deficiency associated with a stop mutation, p.Y447* in TXNRD2, causing FGD [8]. We report another case with selective glucocorticoid deficiency with a homozygous loss-of-function TXNRD2 variant and clear functional evidence of impaired redox homeostasis leading to a defect of adrenal steroidogenesis [14, 15]. The demand for a higher rate of cortisol production compared to aldosterone production predisposes the zona fasciculata to higher levels of oxidative stress, manifested as selective glucocorticoid deficiency in individuals with NNT or TXNRD2 variants. Combined glucocorticoid and mineralocorticoid deficiency has also been reported in individuals with NNT mutations with mineralocorticoid deficiency present either at the onset or during the follow-up period [2, 16]. In contrast to NNT mutations, our case and previous case report with TXNRD2 mutation cases exhibited exclusively glucocorticoid deficiency without any clinical symptoms of mineralocorticoid deficiency [8]. As TXNRD2 is widely expressed in various tissues in humans including the heart, testes, and thyroid [8], individuals with TXNRD2 deficiency are potentially at risk of developing extra-adrenal features. In addition to selective glucocorticoid deficiency, our case also had micropenis and undescended testis, which have not previously been reported in patients with TXNRD2 variants. However, testicular disorders including cryptorchidism have been reported as extra-adrenal features in patients with selective glucocorticoid or combined mineralocorticoid/glucocorticoid deficiency due to NNT mutations [2, 17]. On detailed examination of the gonadal function, the patient did not show any abnormalities. In humans, two heterozygous inactivating mutations in TXNRD2 have been described in 3/227 patients with dilated cardiomyopathy [18]. Echocardiogram was performed in our patient and his heterozygous parents after the TXNRD2 variant was identified and revealed no abnormalities.

A heterozygous variant of uncertain significance of CYP11B1 (NM_000497.3:c.[1182C>G];[=]p.[(Asn394Lys)];[(=)] had been identified in this patient. Although heterozygous mutations may not be the direct cause of the disease, they could still impact protein expression or activity. Additionally, about 40% of the total electron flow from NADPH was found to be directed toward the production of ROS during steroid hydroxylation by CYP11B1 [15]. Therefore, it is possible that heterozygous mutations in the CYP11B1 gene may act as a disease modifier. However, no abnormal urinary steroid precursors specific to CYP11B1 deficiency were found by GC-MS in this patient, suggesting no alteration in steroid 11β-hydroxylase function. In addition, we did not measure the level of thioredoxin reductase activity, which may have further strengthened the findings of this study. However, we believe that the altered GSH/GSSG ratio was sufficient evidence to suggest functional impairment of TXNDR2.

Our case also had transient subclinical hypothyroidism, which has not been previously reported in patients with TXNRD2 mutations. However, hypothyroidism has been previously reported in patients with selective glucocorticoid deficiency or combined mineralocorticoid and glucocorticoid deficiency due to NNT mutations [2]. The thyroid gland is highly susceptible to oxidative stress because of the exposure to H2O2, a substrate of TPO for thyroid hormone synthesis. Previous studies have also demonstrated that the function of thyroid cells can be disrupted when the thyroid cells are exposed to ROS by the inhibition of TPO activity and iodide organification [19, 20]. However, the thyroid function of our patient returned to normal after glucocorticoid replacement therapy. Reversible subclinical hypothyroidism in the presence of severe adrenal insufficiency has been previously reported and it is possible that this may explain the transient abnormalities of thyroid function in this case [21‒24].

In conclusion, this second report of selective glucocorticoid deficiency with a homozygous missense variant in TXNRD2 strengthens the role of this gene in adrenocortical redox homeostasis. In addition to selective glucocorticoid deficiency, our case also had micropenis and undescended testis, which have not been reported in the previous kindred with p.Y447* TXNRD2 variant. The significance of the mild thyroid dysfunction remains unclear and careful long-term follow-up of the mineralocorticoid status, as well as cardiac, testicular, and thyroid functions are warranted in this patient.

We are grateful to the teams at Glasgow Polyomics, Edinburgh Genomics, and the West of Scotland Centre for Genomic Medicine for performing DNA sequencing work.

This study protocol was reviewed and approved by the West of Scotland Ethics Committee approval number 14/WS/0036 and R&D permission (GN14KH079) were obtained for whole-genome sequencing and for data storage and analysis. Written informed consent was obtained from the parents for genetic analysis and publication of the details of the medical case.

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

The next-generation sequencing was facilitated by grants from the Wellcome Trust ISSF and Scottish Genome Partnership. This Scottish Genomes Partnership is funded by the Chief Scientist Office of the Scottish Government Health Directorates [SGP/1] and the Medical Research Council Whole Genome Sequencing for Health and Wealth Initiative (MC/PC/15080). S.P. is funded by the Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. A.K.L.-H. is funded by an NES/CSO Clinical Lectureship.

S.P. contributed to the clinical data collection and the functional studies and reviewed and wrote the manuscript. A.K.L.-H. contributed to functional studies analysis and wrote the manuscript. M.M. contributed to the functional studies analysis and revised the manuscript. L.A.M. and R.P. contributed to initial conceptualisation on the functional studies and revised the manuscript. R.M. and E.S.T. provided genetic variant information, contributed to initial conceptualisation on functional studies, and revised the manuscript. S.F.A. contributed to initial conceptualisation of the project and wrote, revised, and finalised the manuscript.

All data analysed during this study are included in this article. Further enquiries can be directed to the corresponding author.

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