FDXR variants cause adrenal insufficiency and atypical sexual development

Genetic defects affecting steroid biosynthesis cause cortisol deficiency and differences of sex development; among these defects are recessive mutations in the steroidogenic enzymes CYP11A1 and CYP11B, whose function is supported by reducing equivalents donated by ferredoxin reductase (FDXR) and ferredoxin. So far, mutations in the mitochondrial flavoprotein FDXR have been associated with a progressive neuropathic mitochondriopathy named FDXR-related mitochondriopathy (FRM), but cortisol insufficiency has not been documented. However, patients with FRM often experience worsening or demise following stress associated with infections. We investigated 2 female patients with FRM carrying the potentially novel homozygous FDXR mutation p.G437R with ambiguous genitalia at birth and sudden death in the first year of life; they presented with cortisol deficiency and androgen excess compatible with 11-hydroxylase deficiency. In addition, steroidogenic FDXR-variant cell lines reprogrammed from 3 patients with FRM fibroblasts displayed deficient mineralocorticoid and glucocorticoid production. Finally, Fdxr-mutant mice allelic to the severe p.R386W human variant showed reduced progesterone and corticosterone production. Therefore, our comprehensive studies show that human FDXR variants may cause compensated but possibly life-threatening adrenocortical insufficiency in stress by affecting adrenal glucocorticoid and mineralocorticoid synthesis through direct enzyme inhibition, most likely in combination with disturbed mitochondrial redox balance.

respectively).Molecular genetics of the most frequent steroidogenic disorders associated with ambiguous genitalia were performed, being negative for CYP21A2 (MIM: 613815), CYP11B1 (MIM: 610613) and POR (MIM: 124015).At the age of 5 months, she was hospitalized in Pediatric Intensive Care Unit due to acute respiratory failure after a viral infection.She developed a few episodes of central apnea, requiring invasive mechanical ventilation and presented severe hypoglycemia (1.2 mmol/l) with normal electrolytes.She was on treatment with stress dose steroids because of suspected adrenal insufficiency and antihypertensive treatment due to increased blood pressure.The cardiovascular examination was normal.Brain MRI showed delayed myelination, cerebral atrophy and thinning of the corpus callosum.An extensive metabolic screening was negative for an underlying cause.Muscle biopsy showed mild variation in the size of the muscle fibers without structural alterations or pathological deposits and no specific mild increase in cytochrome c oxidase activity without "red ragged" or cytochrome c oxidase-negative fibers.Respiratory chain in muscle and fibroblasts biopsy and mitochondrial DNA investigations were normal.Other neuromuscular studies including electromyography were normal.Progressively she presented a severe and irreversible encephalopathy with chronic respiratory failure, swallowing difficulties, and other abnormal neurological functions and finally she passed away at 9 months.Sibling B. The second girl of this family was born at 37-0/7 weeks by normal delivery in Equatorial Guinea, two years after her sister.Apgar scores was 9/10 and her birth weight was 2.840 g.Pregnancy was unremarkable and the mother was on carbamazepine treatment.Sibling B presented with global developmental delay with choreiform movements of head and upper limbs, bilateral hearing loss and blindness.She had undergone a genital surgery to repair her ambiguous genitalia in another country.
She was admitted at 4 months in a hospital in Spain because of an acute respiratory infection.Her weight was 7 Kg (-0.1 SD), height 59 cm (-1.5 SD) and head circumference 40 cm (-1.5 SD).Physical examination revealed systolic blood pressure in the normal to high range for age.Karyotyping, FISH SRY and microarrays were normal (46, XX).Initially, congenital adrenal hyperplasia (CAH) was suspected, but electrolytes and urinary sodium were normal.At birth 17-hydroxyprogesterone was 13.31 nmol/L.Blood tests including full blood count, hepatic profile, urea and electrolytes, growth, bone and thyroid profile and viral antibody testing were normal.Like her sister, ophthalmology review found bilateral cataracts and optic nerves atrophy and abdominal and pelvic ultrasounds revealed hydrocolpos with female internal genitalia.During her admission, she had few episodes of central apnea.MRI of brain showed hypomyelination and brain atrophy and electroencephalogram was normal.Metabolic screen including amino acids, organic acids, acylcarnitine and sterol profile, carbohydrate-deficient transferrin and transferrin isoform analysis, galactitol, galactonate and galactose-1-phosphate were normal.Ammonia, lactate, creatine kinase and blood gas were normal.
Mitochondrial enzyme studies in fibroblasts were also normal.Levels of testosterone, dehydroepiandrosterone sulfate, and 17-hydroxyprogesterone were slightly high (1.35 nmol/L, RR: 0.1-0.38 nmol/L; 5.5 μmol/L, RR: 0.13-1.68μmol/L; 11.8 nmol/L, RR: 0.57-4.81nmol/L; respectively) with normal androstenedione (4.43 nmol/L, RR: 0.03-6 nmol/L).Basal plasma cortisol was in the lower range (187.5 nmol/L) with high adrenocorticotropin levels (59.9 pmol/L).Luteinizing hormone and follicle stimulating hormone and estradiol were normal.Human chorionic gonadotrophin stimulation test showed an absent response 72 hours after administration.At 5 months she developed a severe respiratory depression with hypercapnia requiring invasive mechanical ventilation.Chest Xray, echocardiogram and electrocardiogram were normal.Vasoactive or inotropic drugs were not required.Progressively, she presented neurological impairment with fluctuating levels of consciousness, choreiform movements and absent trunk and tendon reflexes without sedation drugs.
Acute phase proteins were normal, with negative test for respiratory viruses and sterile blood culture.
Urine culture yielded Escherichia Coli and Citrobacter Koseri ESBLs, which were treated with antibiotics.Stool culture yielded Salmonella spp., interpreted as a past infection because of absent of signs of disease.Finally, after 14 days in pediatric intensive care unit she passed away.An autopsy was performed.Macroscopically, the only remarkable finding was a mild increase in adrenal glands conjoint weight (7.1 g; N 4.8 ±2.2 gr); histology presented mild signs of cytoplasmic vacuolization, possibly due to lipid deposition (Figure S3).Specifically, none of the findings usually associated with CAH, like depletion of the lipid-rich cells of the zona fasciculata, was found.Internal genitalia consisted of a uterus, Fallopian tubes, and ovaries; microscopic examination showed no anomalies.The ovarian cortex displayed a normal population of primordial follicles, with some of them maturing towards secondary and tertiary follicles.As for central nervous system, there was extensive astrogliosis and discrete vacuolization of the white matter, with mild lymphocytic inflammation in perivascular spaces and leptomeninges, and microglial activation.

Expression profiling of FDXR, FDX1 and FDX2 in reprogrammed, induced adrenal-like cells (iALC)
RNA was extracted from wild-type and variant iALC using TRI Reagent (Sigma) and the Direct-zol miniprep RNA kit (Zymo Research), following the manufacturer's protocol.RNA was reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific).Gene expression analysis was performed by real-time quantitative PCR using the PowerUp SYBR Master Mix and the QuantStudio 1 thermocycler (Thermo Fisher Scientific) and target gene specific primers.
Technical triplicates were used to minimize variability.Transcripts encoding beta actin were used as internal control, and data were expressed using the 2 (−ΔΔCt) method.

In silico analysis
We built a homology model of human FDXR and analyzed the sequence and structural changes caused by mutations in FDXR indicated in Figure 3. Multiple cross-species sequence alignment showed that the mutations identified in our index patients and iPSC cells are in highly conserved residues, indicating that these amino acids may be significant to the structure or function of the protein (Figure 3B).In addition, the evolutionary conservation analysis indicated that all patient mutations are found in highly conserved amino acids (data now shown).The sequence conservation results were confirmed quantitatively by PolyPhen-2 analysis, which indicated that the G437R amino acid change was probably damaging, with a score of 0.975 (sensitivity: 0.76; specificity: 0.96), and the R386W mutation was also predicted to be damaging, with a score of 1.0 (sensitivity: 0.00; specificity: 1.00) (Table S2).
We then analyzed the impact on structural stability of mutated FDXR proteins compared to WT FDXR (Figure 3C and D).The G437 residue is located within the predicted ferredoxin reductase-type FADbinding domain, based on computer-generated annotations derived from UniProt sequences of human FDXR.The replacement of G437 by arginine is predicted to disrupt multiple interactions with R435 and I441 residues (Table S2), perturbing the structural stability of the enzyme.The same destabilizing effect was predicted by DynaMut (-0.99 kCal/mol), mCSM (-0.93kCal/mol) and DUET (-1.23 kCal/mol).A calculation of vibrational entropy energy difference between WT and G437R mutant proteins showed a ∆∆Svib of -0.6 kCal -1 .mol.K -1 indicating a reduction in molecular flexibility of the mutant protein.Disruption to the structural stability of the enzyme is expected to lead to a change in interaction with FDX and other partner proteins (1) and would therefore impact the activities of mitochondrial cytochromes P450 that require FDX as their redox partner (2).Based on the computerpredicted annotation of the human FDXR in UniProt, the R386 residue is in a flexible loop within the NADP binding region (Figure 3A).Stability calculations predicted that the replacement of R386 by W also disrupts multiple atomic interactions within FDXR (D424, M420, Y394, M388, N382) (Table S2).
Consequently, an increased rigidity in structure of the R386W mutant FDXR was predicted (Figure 3D).
All prediction methods indicated a slight increase in protein stability (DUET -0.17 kCal/mol, mCSM -0.23 kCal/mol, and DynaMut 0.1 kCal/mol) (Table S2).Calculation of vibrational entropy energy difference between WT and R386W mutant proteins showed a ∆∆Svib of -0.06 kCal -1 .mol.K -1 indicating a slight decrease in molecular flexibility (Table S2).Among the other mutations discussed in this report, F51L and R193H were predicted to cause increased flexibility in parts of the protein and P74L and R155W were predicted to have increased rigidity in overall structure (Figure 3D).

Figure S1. Adrenal-like cells from FDXR patients display lower production of all three steroid categories and are reprogrammed less efficiently than control cells. (A) Representative micrographs of reprogrammed patients' and control individual's fibroblasts at day 20 following differentiation start
in induced pluripotent cells.Acquisition was carried out using a 20x objective; scale bar = 150 um.(B) Steroid amounts in culture media secreted by reprogrammed fibroblasts from FDXR patients compared to control values (representing steroid amounts of reprogrammed fibroblasts from a nonaffected individual).Steroids are split among three graphs according to their belonging to a specific steroid class.BQL, Below Quantification Level, indicates the samples in which steroid levels were not measurable above the lowest quantification limit using LC-MS.Asterisks reflect discoveries found using a multiple unpaired t-test assuming individual variance for each steroid.(C) Concentrations as raw values of the endpoint or critical steroids for each pathway using a linear scale.(D) Transcript levels for the normalizer GAPDH calculated with the 2 -∆Ct method for each reprogrammed cell line.(E) shows the total levels of steroids detected using LC-MS within each cell line.Statistical analysis was conducted using a one-way ANOVA analysis followed by a Dunnett's multiple comparisons test.