Disordered Electron Transfer: New Forms of Defective Steroidogenesis and Mitochondriopathy

Abstract

Most disorders of steroidogenesis, such as forms of congenital adrenal hyperplasia (CAH) are caused by mutations in genes encoding the steroidogenic enzymes and are often recognized clinically by cortisol deficiency, hyper- or hypo-androgenism, and/or altered mineralocorticoid function. Most steroidogenic enzymes are forms of cytochrome P450. Most P450s, including several steroidogenic enzymes, are microsomal, requiring electron donation by P450 oxidoreductase (POR); however, several steroidogenic enzymes are mitochondrial P450s, requiring electron donation via ferredoxin reductase (FDXR) and ferredoxin (FDX). POR deficiency is a rare but well-described form of CAH characterized by impaired activity of 21-hydroxylase (P450c21, CYP21A2) and 17-hydroxylase/17,20-lyase (P450c17, CYP17A1); more severely affected individuals also have the Antley-Bixler skeletal malformation syndrome and disordered genital development in both sexes, and hence is easily recognized. The 17,20-lyase activity of P450c17 requires both POR and cytochrome b₅ (b5), which promote electron transfer. Mutations of POR, b5, or P450c17 can cause selective 17,20-lyase deficiency. In addition to providing electrons to mitochondrial P450s, FDX, and FDXR are required for the synthesis of iron-sulfur clusters, which are used by many enzymes. Recent work has identified FDXR mutations in patients with visual impairment, optic atrophy, neuropathic hearing loss, and developmental delay, resembling the global neurologic disorders seen with mitochondrial diseases. Many of these patients have had life-threatening events or deadly infections, often without an apparent triggering event. Adrenal insufficiency has been predicted in such individuals but has only been documented recently. Neurologists, neonatologists, and geneticists should seek endocrine assistance in evaluating and treating patients with mutations in FDXR.

Steroidogenesis is the conversion of cholesterol into biologically active steroid hormones, (mineralocorticoids, glucocorticoids, androgens, estrogens, progestins), including conversion of a cholesterol precursor, 7-dehydrocholesterol, into biologically active 1,25-dihydroxyvitamin D. Most steroidogenic defects impair cortisol synthesis, with consequent pituitary hyperstimulation causing adrenal overgrowth, hence the term congenital adrenal hyperplasia (CAH), although not all defects in adrenal steroidogenesis cause adrenal overgrowth. Contemporary endocrinologists are attuned to the usual presenting features of 21-hydroxylase deficiency (21OHD, caused by mutations in CYP21A2), especially hyperandrogenism and salt loss. Human steroidogenesis is fairly well understood (1), but the clinical care of these disorders remains imperfect and evolving (2, 3) and important gaps in our understanding remain (4). New phenotypes have emerged beyond disorders of glucocorticoid and mineralocorticoid secretion and disorders of sexual development (DSD) associated with hyper- and hypo-androgenism. For example, among persons with salt-wasting 21OHD, about 10% will have CAH-X (5), which is 21OHD associated with the mild, “joint hypermobility” form of Ehlers-Danlos syndrome (EDS). This is because CYP21A2 gene deletions frequently extend into the overlapping TNX gene encoding Tenascin-X, an extracellular matrix protein whose homozygous deficiency causes a severe form of EDS (6) and whose haploinsufficiency causes the mild, “joint hypermobility” form of EDS (7). Cytochrome b₅ (b5) is familiar to endocrinologists for promoting the 17,20-lyase activity of P450c17 (CYP17A1), thus regulating the synthesis of androgen precursors (8, 9); but the principal action of b5 is the reduction of methemoglobin (10), and b5 deficiency causes DSD with methemoglobinemia (11, 12). Similarly, mutations in P450 oxidoreductase (POR) cause a form of CAH in which both 21-hydroxylase and 17-hydroxylase/17,20 lyase activities are impaired, but the diagnosis is usually first suspected because recessive POR deficiency also causes the Antley-Bixler skeletal malformation syndrome (13). POR serves to transfer electrons from reduced nicotinamide adenine dinucleotide (NADPH) to all cytochromes P450 located in endoplasmic reticulum (14, 15); the description of POR deficiency has drawn attention to the electron transfer proteins required by mitochondrial P450 enzymes: ferredoxin (FDX) and ferredoxin reductase (FDXR) (16). After years of searching, endocrine disorders associated with their mutations are emerging, further broadening the phenotypic spectrum of disorders of steroidogenesis. Here, we briefly review these factors and their disorders.

Steroidogenesis

The first enzymatic step in steroidogenesis is the conversion of cholesterol to pregnenolone in mitochondria, catalyzed by the cholesterol side-chain cleavage enzyme, P450scc (CYP11A1), encoded by the CYP11A1 gene (Fig. 1). Expression of CYP11A1 renders a cell “steroidogenic,” and the amount of P450scc produced determines a cell's steroidogenic capacity (17). In adrenal and gonadal steroidogenic cells that produce abundant steroids, mitochondrial cholesterol entry is facilitated by the steroidogenic acute regulatory protein (StAR) (18), which acts on the outer mitochondrial membrane to increase cholesterol import (19). In the placenta, brain, and skin, cholesterol enters mitochondria in steroidogenic cells without the action of StAR. This “StAR-independent steroidogenesis” is incompletely understood; it may involve mitochondrial entry of hydroxysterols, which are freely diffusible into mitochondria (18), or other proteins may substitute for StAR, such as placental MLN64 (20-22). Pregnenolone may be converted to progesterone by 3β-hydroxysteroid dehydrogenase, type 2 (3βHSD2), which is found in both the endoplasmic reticulum (ER) and mitochondria (23), where it appears to be located in the intermembranous space (24). Progesterone may be converted to glucocorticoids, mineralocorticoids, androgens, and estrogens by downstream enzymes, including cytoplasmic 17α-hydroxylase/17,20-lyase (P450c17, CYP17A1), 21-hydroxylase (P450c21, CYP21A2), and aromatase (P450aro, CYP19A1) and by mitochondrial 11β-hydroxylase (P450c11β, CYP11B1) and aldosterone synthase (P450c11AS, CYP11B2). The expression of these enzymes differs in different steroidogenic cell types, resulting in different steroidogenic pathways in different cell types (1).

Figure 1.

Simplified steroidogenic pathway. Only the human cytochrome P450 enzymes are shown; for figures showing all steroidogenic enzymes and other pathways, see (1). The “microsomal” (type 2) steroidogenic P450 enzymes are: P450c17 (17α-hydroxylase/17,20-lyase; CYP17A1), P450c21 (21-hydroxylase, CYP21A1), and P450aro (aromatase, CYP19A1); these 3 enzymes require electron donation from P450 oxidoreductase (POR). The 17,20-lyase activity of P450c17 is minimal in the absence of cytochrome b5 (b₅). The mitochondrial (type 1) steroidogenic P450 enzymes are: P450scc (cholesterol side-chain cleavage enzyme, CYP11A1), P450c11β (11β-hydroxylase, CYP11B1), and P450c11AS (aldosterone synthase, CYP11B2); these 3 enzymes require electron donation via ferredoxin (FDX) and ferredoxin reductase (FDXR). CYP11B2 catalyzes the 3 terminal steps (11-hydroxylation, 18-hydroxylation, and 18-methyl oxidase activity) in the production of aldosterone (Aldo) from deoxycorticosterone (DOC); each of these steps requires a pair of electrons donated from NADPH via FDX and FDXR.

Cytochromes P450

There are 2 classes of human P450 enzymes: type 1 P450 enzymes in the mitochondria and type 2 P450 enzymes in the ER. The human genome has genes for 57 cytochrome P450s; 7 are type 1 and 50 are type 2 (25, 26). Among human steroidogenic enzymes, 5 are type 1 P450s: P450scc, P450c11β, P450c11AS, vitamin D 1α-hydroxylase (P450c1α, CYP27B1), and vitamin D 24-hydroxylase (P450c24, CYP24A1). Four other human steroidogenic enzymes are P450 type 2 P450s: P450c17, P450c21 (CYP21A2), P450aro (CYP19A1), and the principal vitamin D 25-hydroxylase (CYP2R1) (1); vitamin D 25-hydroxylation can also be catalyzed by CYP27A1 in mitochondria (27, 28) and CYP4A22 in the ER (29). Both types of P450 require that electrons from NADPH reach the heme iron of the P450, where catalysis occurs. To reach the P450 enzymes in the mitochondria or ER, electrons from NADPH must travel via different electron-transport chains (Fig. 2) (14).

Figure 2.

Diagrams of the cell biology of electron transfer to P450 enzymes. A, Microsomal (type 2) P450 enzymes. Both POR and the P450 are bound to the cytoplasmic aspect (CYTO) of the endoplasmic reticulum (ER). POR has 2 “wings,” one containing a flavin adenine dinucleotide (FAD) and one containing a flavin mononucleotide (FMN). NADPH interacts with and donates a pair of electrons to the FAD moiety. Electron receipt by the FAD elicits a conformational change, permitting the isoalloxazine rings of the FAD and FMN moieties to come sufficiently close together to permit the electrons to move from the FAD to the FMN. Electron receipt by the FMN permits the POR protein to revert to its original, open conformation; this permits the FMN domain to “dock” with the redox-partner binding site of the P450 by charge-charge interactions. The electrons reach the iron atom of the heme group of the P450, mediating catalysis. For some P450 reactions, notably the 17,20-lyase activity of human P450c17, cytochrome b₅ promotes increased activity. B, Mitochondrial (type 1) P450 enzymes. Both ferredoxin reductase (FDXR) and the P450 are bound to the inner mitochondrial membrane, but ferredoxin (FDX) is not. NADPH donates a pair of electrons to the FAD moiety of FDXR; which then donates them to the 2Fe2S center of FDX (represented by the ball-and-stick glyph). The same surface of FDX interacts with both the FAD of FDXR and the redox-partner binding site of the P450 by electrostatic interactions. FDX thus acts as an indiscriminate electron-shuttling protein that can support the catalysis of any available type 1 P450. The electrons reach the heme iron of the P450 permitting catalysis.

Electron Transfer to Microsomal P450s

All human type 2 P450 enzymes, including steroidogenic P450c17, P450c21, P450aro, and CYP2R1, as well as the other 46 microsomal P450s involved in drug/xenobiotic metabolism and synthesis of eicosanoids and leukotrienes, receive electrons from NADPH via P450 oxidoreductase (POR) (25). The activity of POR is not limited to P450s; POR also donates electrons to other enzymes, such as squalene monooxygenases, fatty acid elongase, heme oxygenase, b5, and to some small molecule substrates (15). POR has 2 distinct domains connected by a hinge; one domain contains a flavin adenine dinucleotide (FAD) moiety, the other contains a flavin mononucleotide (FMN) moiety (30). Before interacting with NADPH, POR is in an open conformation. When the electrons from NADPH are transferred to the FAD moiety, POR undergoes a conformational change that brings the FAD and FMN close together, permitting the electrons to travel to the FMN; this then returns POR to its open state. The FMN domain then interacts with the redox-partner binding site of the P450, permitting electron transfer to the heme iron in the P450, which then mediates catalysis (31) (Fig. 2A). Each P450 reaction cycle requires a pair of electrons, which are transferred from POR one at a time, but it remains unclear if both electrons are transferred by the same POR molecule or whether 2 separate interaction and electron transfer steps occur involving either the same or 2 different POR molecules (32).

POR Deficiency

Because POR participates in so many essential biochemical processes, it was initially thought that POR deficiency would be lethal, and POR-knockout mice die during fetal development (33, 34). However, human POR deficiency “only” disrupts steroidogenesis and also causes a skeletal malformation syndrome (13, 35). The associated skeletal malformations, termed Antley-Bixler syndrome (ABS), apparently results from impaired activity of CYP26B1, a type 2 P450 that degrades retinoic acid (36). The bony derangements in ABS may include craniosynostosis, brachycephaly, radio-ulnar or radio-humeral synostosis, bowed femora, arachnodactyly, midface hypoplasia, proptosis, and choanal stenosis, with different patients manifesting somewhat different features (35). The ABS phenotype can be seen with either recessive mutations in POR or dominant, gain-of-function mutations in fibroblast growth factor receptor 2 (FGFR2) (35). Thus, recessive ABS with abnormal steroids and DSD in either sex is caused by mutations in POR, and dominant ABS without disordered steroidogenesis is caused by mutations in FGFR2. Defective signaling by hedgehog proteins secondary to POR-associated defects in cholesterol synthesis may also play a role. More than 200 mutations causing POR amino acid changes have been reported in over 250 patients, affecting various P450 enzymes to differing degrees, explaining the great clinical and hormonal variability in POR deficiency (37). More severe POR mutations cause the ABS phenotype, but mutations retaining ∼20% to 40% of activity may cause disturbed steroidogenesis without ABS (37), this may be considered “nonclassic POR deficiency.” The roles of these mutations in clinical disease, drug metabolism, and POR transcriptional regulation have been reviewed elsewhere (15, 16, 38) and will not be discussed here.

Cytochrome b₅

There are 2 human genes encoding cytochrome b₅ (b5). CYB5A on chromosome 18q22.3, produces 2 alternatively spliced mRNAs. Exons 1 to 4 encode the 98-amino acid, soluble, cytosolic form found mainly in erythrocytes, where it reduces methemoglobin to hemoglobin. Exons 1 to 3 plus 5 and 6 encode the 134 amino acid form found in the ER; this is the predominant form in most cells, delivering electrons to microsomal desaturases that synthesize fatty acids, and is the overwhelmingly predominant and possibly only form of b5 involved in steroidogenesis (39-41). CYB5B on chromosome 16q22.1 encodes a 146-amino acid form found on the outer mitochondrial membrane and is widely expressed, including in steroidogenic tissues (41). Cytochrome b5 has a heme-binding domain and a structural core, from which the C-terminal membrane-anchoring helix extends. Cytochrome b5 can augment some P450 activities; apparently acting as an alternative donor of the second electron in the P450 cycle (42). However, some of the actions of b5 can be observed with apo-b5, which lacks a heme group and hence cannot transfer electrons (43). With human P450c17, b5 selectively stimulates 17,20-lyase activity but has negligible effects on 17-hydroxylase activity (8, 9).

Cytochrome b5 appears to enhance the interaction of P450c17 and POR, allosterically promoting more efficient electron transfer but not directly participating in electron transfer, as apo-b5 was as effective as holo-b5. This mechanism could account for b5 having no effect on the Km (Michaelis constant) of P450c17, while increasing the Vmax (maximum velocity) of the 17,20-lyase reaction (8, 9). Similarly, excess POR increases 17,20-lyase activity in the absence of b5 (44, 45), and mutations in the POR binding site of P450c17 selectively reduce 17,20-lyase activity (46, 47). Nevertheless, in recent work where the heme of b5 is replaced with a heme containing manganese rather than iron, this Mn-b5 was incapable of electron transfer and did not promote 17,20-lyase activity, even though the Mn-b5 interacted with the P450c17 (48, 49). Thus, current data suggest that b5 promotes 17,20-lyase activity by acting as an alternative electron donor. The 17,20-lyase activity of P450c17 can also be increased by phosphorylation of P450c17 on serine or threonine residues (50, 51), apparently catalyzed by p38α, a cAMP-dependent mitogen-activated protein kinase (52); the mechanism of this effect remains undetermined.

Because the reduction of methemoglobin is the principal physiologic role of b5, and methemoglobinemia is typically caused by deficiency of cytochrome b5 reductase, methemoglobinemia is a predictable consequence of b5 deficiency. The first report of b5 deficiency was in a patient with methemoglobinemia and DSD, but studies of steroidogenesis apparently were not done (10, 53). Since then, a small number of reports have described b5 deficiency in patients with apparent isolated 17,20-lyase deficiency, sometimes associated with methemoglobinemia (11, 12, 54). Most cases have been associated with 46,XY DSD, but normal development and fertility were reported with methemoglobinemia in a 24-year-old woman homozygous for b5 Y35X, which suggests possible compensation by CYB5B (55).

Electron Transfer to Mitochondrial P450s

Mitochondrial, type 1 P450 enzymes receive electrons from NADPH via an electron transfer chain consisting of ferredoxin reductase (FDXR), which is loosely associated with the inner mitochondrial membrane, and ferredoxin (FDX) in the mitochondrial matrix (Fig. 2B) (1, 26). A pair of electrons from NADPH is accepted by FDXR (also termed adrenodoxin reductase), a 54-kDa flavoprotein encoded by the FDXR gene on chromosome 17q24 (56, 57). The flavin adenine dinucleotide (FAD) moiety of FDXR donates the electrons to the iron-sulfur (Fe-S) moiety of the 14 kDa FDX (also termed adrenodoxin). The same surface of FDX interacts sequentially with FDXR and with the recipient mitochondrial P450 (58). After FDX forms a 1:1 complex with FDXR it dissociates, then forms a 1:1 complex with the P450, thus acting as a diffusible electron shuttle (Fig. 2B). The human FDXR gene encodes 2 alternatively spliced mRNAs that can encode proteins of 491 or 497 amino acids (56, 57). Only the shorter protein is active in steroidogenesis (59); it is not known whether the longer form exerts an activity.

Iron-Sulfur Proteins

Iron-sulfur clusters are prosthetic groups that are typically 2Fe-2S or 4Fe-4S, formed by reductive coupling of 2 2Fe-2S clusters (60), and are found in proteins that participate in electron transfer, such as FDX and several proteins in the mitochondrial respiratory chain complexes I, II, and III (61). The cellular processes producing such Fe-S clusters are highly conserved among eukaryotes, involving about 20 proteins that mediate 2 major steps: assembly of the Fe-S cluster on the scaffold protein ISCU (iron-sulfur cluster assembly enzyme), and transfer of the Fe-S cluster to a recipient protein (62-69). Reports of disorders of the assembly of Fe-S clusters and of the Fe-S proteins are increasingly reported in patients with neurologic and muscle diseases. The most widely studied protein participating in the assembly of Fe-S clusters is frataxin; expansion of a GAA repeat in intron 1 of its gene, FRA, causes Friedrich's ataxia (70). Mutations in ISCU affect assembly of Fe-S clusters in aconitase and succinate dehydrogenase, thus disrupting the Krebs cycle, primarily in skeletal muscle, causing exercise-induced lactic acidosis, muscle weakness, and, rarely, rhabdomyolysis (71-74).

Ferredoxins (FDX1 and FDX2)

Mitochondrial ferredoxins carry electrons bound to a 2Fe-2S cluster. Ferredoxin was first identified in bacteria in the 1960s (75). In 1986, the amino acid sequences of bovine adrenal “adrenodoxin” and liver “hepatoredoxin” were found to be identical (76), suggesting there was only one mammalian ferredoxin, but that report, published in Russian, was not widely seen. Bovine adrenal “adrenodoxin” cDNA was cloned in 1985 (77), and human adrenal “adrenodoxin” (78) and placental “ferredoxin” (79) cDNAs were cloned in 1988; the 2 human sequences were identical, showing that the same gene was expressed in both tissues. Also in 1988, a single adrenodoxin gene was cloned (80) and located to chromosome 11q22 (81), which is predominantly, but not exclusively, expressed in steroidogenic tissues. That adrenodoxin/ferredoxin is now termed FDX1, encoded by the FDX1 gene. The role of FDX1 in steroidogenesis suggests that FDX1 mutations would be predicted to disrupt steroidogenesis similarly to CYP11A1 (P450scc) mutations, but a human FDX1 mutation has not (yet) been reported. Deletion of the related zebrafish fdx1b gene led to defective synthesis of cortisol and androgens (82, 83), but there are important differences in human and zebrafish steroidogenesis, hence the zebrafish results may not indicate the effects of a human FDX1 mutation.

Yeast Yah1, the homolog of human FDX1, participates in synthesis of Fe-S clusters, but knockdown of FDX1 did not affect Fe-S cluster synthesis. Instead, the related FDX2 gene (on chromosome 19p13.2), (formerly termed FDX1L), encodes FDX2, which supports Fe-S cluster synthesis but has little or no role in reducing mitochondrial P450s (84). The amino acid sequences of human FDX1 and FDX2 are 43% identical and 69% similar, and these FDX proteins have very similar three-dimensional structures (85), but their gene sequences are sufficiently different that FDX2 was not detected in studies to determine the chromosomal location of FDX1 (81). Both FDX1 and FDX2 participate in the synthesis of Fe-S clusters (65, 86), but FDX2 is more important for this activity, especially in the central nervous system, where FDX2 is well-expressed and very little FDX1 is found. FDX1 is abundantly expressed in steroidogenic tissues whereas FDX2 is not, indicating that FDX1 is the principal form of ferredoxin involved in steroidogenesis (84). Nevertheless, it is unclear to what degree, if any, FDX1 and FDX2 might be able to substitute for one another in clinical situations where one is deficient.

FDX2 has been implicated in several neurological diseases (87, 88), including Friedreich's ataxia and Parkinson's disease (89). Specific role(s) for FDX2 in these disorders remain uncertain, but 2 studies reported mitochondrial muscle myopathy with or without optic atrophy and reversible leukoencephalopathy (MEOAL; OMIM #251900) in patients with FDX2 mutations. A 15-year-old girl born to consanguineous parents had normal psychomotor development to age 12, then had episodes of proximal muscle weakness, myoglobinuria, lactic acidosis, and increased serum creatine kinase; a homozygous missense mutation was identified by whole exome sequencing in the initiation codon of the FDX1L (FDX2) gene (90). FDX2 protein was essentially undetectable in a muscle biopsy or cultured fibroblasts, and activities of aconitase and respiratory complexes I, II, and III, all of which have Fe-S clusters, were impaired. Six similar patients from 2 families presented variably from early childhood to adulthood with nonprogressive optic atrophy, muscle weakness, cramps, and myalgia, often associated with exercise, infection, or low temperature; other studies, including muscle biopsies, implicated disordered mitochondrial function (91). Patient DNA was homozygous for FDX2 missense mutations, and RNA and protein blotting studies suggested the mutant FDX2 protein was unstable. Thus, mutations in FDX2 appear to cause neurologic impairments, apparently related to impaired synthesis of Fe-S clusters, yielding global mitochondrial dysfunction. Studies of steroidogenesis were not done in these patients.

Ferredoxin Reductase

In addition to FDX1, FDX2, and other proteins, the synthesis of Fe-S clusters requires FDXR (64, 67-69). Early studies showed low levels of ferredoxin reductase (FDXR) mRNA in all tissues, although expression in adrenal and testis was about 100-fold greater (92). Because both FDX1 and FDX2 play a role in the biogenesis of Fe-S centers and there is only one FDXR gene (57), one might predict that FDXR mutations would also affect Fe-S synthesis and result in a phenotype similar to that of FDX2 deficiency (ie, MEOAL), but also with impaired steroidogenesis. Knockdown of the genes for FDX1, FDX2, or FDXR in human cell lines reduced Fe-S cluster synthesis and impaired several enzymes that rely on Fe-S clusters for activity, and also depleted cytosolic iron, causing mitochondrial iron overload (65, 86). Thus, interference with FDX1, FDX2, or FDXR, disrupts the synthesis and/or assembly of Fe-S clusters and disrupts intracellular iron homeostasis.

Animal Models of FDXR Deficiency

Long before human FDXR mutations were sought, the first report of a genetic defect in FDXR was the identification of the dare mutation and gene in Drosophila melanogaster (91). The dare mutant was identified in a screen of mutations that affected olfactory-driven learning and memory; this Drosophila mutant phenotype was named “dare” from defective in avoidance of repellants. When the responsible gene was cloned and sequenced, it was found to be Drosophila FDXR; as FDXR was then generally termed adrenodoxin reductase, the name “dare” was repurposed as Drosophila  adrenodoxin reductase (93). Insect development and metamorphosis, including sexual development and cuticle (exoskeleton) development, depend on the steroid hormone ecdysone; the developmental arrest characteristic of the dare phenotype could be rescued by feeding mutant larvae 20-hydroxyecdysone. In situ hybridization showed that dare expression was confined to steroidogenic tissues: the parathoracic cells of the larval “ring gland” and the ovary, where FDXR is required for egg chamber development (94). In adult flies, timed dare mutations caused degeneration of the adult nervous system. This dare was the first gene/factor discovered in the insect pathway from cholesterol to ecdysone, the so-called “Halloween pathway” now known to include both microsomal and mitochondrial P450 enzymes, as well as short-chain dehydrogenases—much like human steroidogenic pathways, but without cleavage of the side chain of cholesterol (95-97).

Mice are the most widely used mammalian model for human biology and disease. Fairfield and 38 collaborators used ethyl-nitrosourea mutagenesis to create 172 mouse (C57BL/6J) mutants and performed exome sequencing to identify mutations in 91 of the strains produced; one strain was homozygous for the Fdxr mutation R389Q (98). The principal phenotype in these animals was described as “behavioral; neurological.” These mice have decreased visual acuity, a progressive gait disorder, and Fdxr activity that is reduced to between ∼33% and 50% of normal, varying with the tissue assessed (99). Mouse Fdxr R389Q corresponds to human R392Q, now known to be a cause of FDXR deficiency (99).

Human FDXR Deficiency

From 2017 to 2023, at least 77 patients from more than 50 families were described carrying 59 different biallelic (homozygous or compound heterozygous) FDXR mutations causing a generalized mitochondriopathy, variably presenting with optic atrophy, retinal dystrophy, neuropathic hearing loss, developmental delay, mild movement disorders, and even Leigh syndrome with infantile-onset encephalopathy and death (99-110); this disorder has been termed FDXR-related mitochondriopathy (FRM) (110). Most families with FRM were not known to be related to one another, but the frequent identification of specific mutations in some studies suggested there were local founder effects or unknown consanguinity. Among 13 families (26 alleles) in a study published in 2017, 11 alleles carried the missense mutation R392W (99), and among 8 families (16 alleles) in a study published in 2021, 5 alleles carried P372H (104); however, neither of these mutations was found in any other report, indicating that they are not mutational “hot spots.” Another study screened 2186 patients with hereditary optic neuropathy: 1126 had apparent mitochondrial inheritance, with mitochondrial defects found in 199, and 1680 had apparent autosomal disease, with mutations found in 451. Among those with autosomal disease, mutations in only 10 genes accounted for 434/451 (96%) of those patients, and 5/434 of these patients had mutations in FDXR; all of whom also had deafness, and most were < 10 years old (109). In a recent study, FDXR R386W was found in one or both alleles in ∼25% of affected individuals; most were Hispanic, many of Mexican heritage. The genome databases MCPS and gnomAD reported allele frequencies of 0.0027 and 0.001274, respectively for Indigenous Mexican and Latino/Admixed American population, indicating carrier frequencies of 1/185 and 1/394 in the Indigenous and Admixed Mexican populations, respectively (110), thus FDXR deficiency may be more common in this population. All patients who have been reported on to date had at least one allele that retained partial activity; the same situation is found among patients with POR deficiency: no patient has been reported with wholly null mutations on both alleles. These observations suggest that homozygosity or compound heterozygosity for null alleles for either POR or FDXR may be lethal in embryonic or fetal life.

Cell culture studies are consistent with the observations from human genetics. Knockdown of FDXR in HeLa cells and in human K562 erythroid cells leads to iron overload (86); similarly, primary cultures of fibroblasts from patients with FRM had reduced FDXR activity and increased production of reactive oxygen species (99). Thus clinical, genetic, biochemical, and cell biologic data show that FDXR deficiency causes a generalized mitochondrial disorder, FRM, primarily seen in the central nervous system, that shares many features with FDX2 deficiency and other mitochondrial disorders. However, from a clinical endocrine perspective, the most remarkable feature of these reports was the absence of studies assessing clinical adrenal function or addressing the obligatory role of FDXR in steroid hormone synthesis; adrenal insufficiency had been predicted in these patients (4, 16).

A very recent study has now shown such adrenal insufficiency in 2 severely affected siblings with FRM, DSD, and hypertension, with a steroid pattern suggesting 11-hydroxylase deficiency (111). This study also showed that the previously reported mice carrying the Fdxr R389Q mutation also had impaired adrenal function, with impaired corticosterone synthesis, indicative of defective FDXR-supported activity of 11β-hydroxylase (CYP11B1). Review of the previously published cases of FRM showed that 20/77 (26%) of these patients also had a history of severe, often life-threatening events or deadly infections suggestive of adrenal insufficiency (111). Only 3 steroidogenic P450 enzymes, the cholesterol side-chain cleavage enzyme, P450scc (CYP11A1), 11β-hydroxylase, P450c11β (CYP11B1), and aldosterone synthase, P450c11AS (CYP11B2), reside in mitochondria and require electron donation from FDX and FDXR (Fig. 1). P450scc is the rate-limiting enzyme in steroidogenesis (1), hence interference with its activity by FDXR deficiency must impair all steroidogenesis, but as 100-fold more cortisol is produced than aldosterone (1), glucocorticoid deficiency is the predictable result. However, cortisol synthesis also requires P450c11β, and indeed the 2 index FDXR-deficient patients had clear signs of 11β-hydroxylase deficiency (mineralocorticoid hypertension secondary to overproduction of deoxycorticosterone [DOC], and increased plasma 11-deoxycortisol) (111). FDXR is also required by P450c11AS, which catalyzes the last 3 steps in the production of aldosterone from DOC (1) (Fig. 1); impairment of P450c11AS might be expected to manifest with salt loss, but overproduction of DOC from impairment of P450c11β would mask aldosterone deficiency. The 2 FDXR-deficient infants did not survive long enough to permit investigation of their renin-angiotensin-aldosterone axis (111).

Thus, neonatologists, neurologists, and geneticists caring for these patients must become familiar with the life-threatening possibility of adrenal insufficiency when FRM is suspected, and seek endocrine assistance to evaluate this possibility, as treating adrenal insufficiency may prolong survival and quality of life. Future studies should include clinical investigation of adrenal reserve by performing adrenocorticotropic hormone (ACTH) stimulation tests with, at minimum, measurement of cortisol and 11-deoxycortisol; such ACTH testing should now be routine when a disorder of FDX or FDXR is suspected. Cell biologic studies of steroidogenesis and FDXR function are essential, but the logical approach of transfecting nonsteroidogenic cells with vectors expressing a mitochondrial P450 plus either wild-type or mutant FDXR was unreliable because of high background FDXR activity in cultured cells. Instead, induced pluripotent cells derived from FDXR-deficient patient fibroblasts that had been differentiated in vitro toward an adrenal-like lineage were needed to show the effects of FDXR mutations on steroidogenesis (111). We speculate that patients with less severe FDXR mutations that retain partial activity will have compensated adrenal insufficiency, as seen in the nonclassic forms of 21-hydroxylase deficiency (112), lipoid CAH (113), P450scc deficiency (114), and POR deficiency without ABS (13, 35, 38, 115). However, while the adrenal insufficiency of those nonclassic forms of CAH appears to be stable as the patients age, we speculate that the clinical findings in “nonclassic FDXR deficiency,” like many other mitochondriopathies, will worsen with age, requiring long-term monitoring of adrenal function.

Conclusions

Disorders of the factors that transfer electrons from NADPH to steroidogenic cytochrome P450 enzymes are a newly recognized group of disorders of steroidogenesis, presenting similarly to CAH and adrenal insufficiency. Mutations in P450 oxidoreductase were first described in 2004, and are now well-characterized clinically, genetically, and biochemically. Mutations in cytochrome b5 were first described in 2010, causing isolated 17,20-lyase deficiency, but this remains one of the rarest disorders in steroidogenesis. Mutations in FDXR were first reported in 2017, causing visual impairment, neuropathic hearing loss, and other features of mitochondriopathy, but the predictable steroidogenic consequences were not sought. Recent work has now reported adrenal insufficiency in patients with severe FDXR deficiency; additional careful clinical studies of adrenal and gonadal steroidogenesis in these patients is needed, and physicians caring for these patients must become aware of the possibility of potentially lethal adrenal insufficiency in these patients. Mutations of FDX2 (which does not appear to participate in steroidogenesis) are associated with neurologic disorders but not with adrenal insufficiency. Mutations in FDX1, which does participate in steroidogenesis have not (yet) been reported, but we predict that they will be found and will cause adrenal insufficiency. Finally, most genetic disorders—not just those affecting steroidogenesis—are first identified in more severely affected individuals, with milder cases and “nonclassical” disease generally being reported later; thus the severely affected FDXR-deficient patients initially reported (111) probably do not represent a “typical picture” of FDXR deficiency; astute physicians should be alert for milder forms of this disorder.

Disclosures

The authors report no conflict of interest.

Abbreviations

21OHD

21-hydroxylase deficiency

ABS

Antley-Bixler syndrome

b5

cytochrome b₅

CAH

congenital adrenal hyperplasia

DOC

deoxycorticosterone

DSD

disorder/difference of sexual development

EDS

Ehlers-Danlos syndrome

ER

endoplasmic reticulum

FAD

flavin adenine dinucleotide

FDX

ferredoxin

FDXR

ferredoxin reductase

FGFR2

fibroblast growth factor receptor 2

FMN

flavin mononucleotide

FRM

FDXR-related mitochondriopathy

ISCU

iron-sulfur cluster assembly enzyme

MEOAL

mitochondrial muscle myopathy with or without optic atrophy and reversible leukoencephalopathy

NADPH

reduced nicotinamide dinucleotide phosphate

POR

P450 oxidoreductase

StAR

steroidogenic acute regulatory protein

Data Availability

Data sharing is not applicable to this article as no datasets were generated or analyzed during the current study.