The "backdoor pathway" of androgen synthesis in human male sexual development.

Mammalian sex determination (male versus female) is largely controlled by genes, whereas sex differentiation (development of reproductive structures) is largely controlled by hormones. Work in the 20th century indicated that female external anatomy was a "default" pathway of development not requiring steroids, whereas male genital development required testicular testosterone plus dihydrotestosterone (DHT) made in genital skin according to a "classic" pathway. Recent work added the description of an alternative "backdoor" pathway of androgen synthesis discovered in marsupials. Unique "backdoor steroids" are found in human hyperandrogenic disorders, and genetic disruption of the pathway causes disordered male sexual development, suggesting it plays an essential role. O'Shaughnessy and colleagues now show that the principal human backdoor androgen is androsterone and provide strong evidence that it derives from placental progesterone that is metabolized to androsterone in nontesticular tissues. These studies are essential to understanding human sexual development and its disorders.

A Novel Mitochondrial Complex of Aldosterone Synthase, Steroidogenic Acute Regulatory Protein, and Tom22 Synthesizes Aldosterone in the Rat Heart.

Aldosterone, which regulates renal salt retention, is synthesized in adrenocortical mitochondria in response to angiotensin II. Excess aldosterone causes myocardial injury and heart failure, but potential intracardiac aldosterone synthesis has been controversial. We hypothesized that the stressed heart might produce aldosterone. We used blue native gel electrophoresis, immunoblotting, protein crosslinking, coimmunoprecipitations, and mass spectrometry to assess rat cardiac aldosterone synthesis. Chronic infusion of angiotensin II increased circulating corticosterone levels 350-fold and induced cardiac fibrosis. Angiotensin II doubled and telmisartan inhibited aldosterone synthesis by heart mitochondria and cardiac production of aldosterone synthase (P450c11AS). Heart aldosterone synthesis required P450c11AS, Tom22 (a mitochondrial translocase receptor), and the intramitochondrial form of the steroidogenic acute regulatory protein (StAR); protein crosslinking and coimmunoprecipitation studies showed that these three proteins form a 110-kDa complex. In steroidogenic cells, extramitochondrial (37-kDa) StAR promotes cholesterol movement from the outer to inner mitochondrial membrane where cholesterol side-chain cleavage enzyme (P450scc) converts cholesterol to pregnenolone, thus initiating steroidogenesis, but no function has previously been ascribed to intramitochondrial (30-kDa) StAR; our data indicate that intramitochondrial 30-kDa StAR is required for aldosterone synthesis in the heart, forming a trimolecular complex with Tom22 and P450c11AS. This is the first activity ascribed to intramitochondrial StAR, but how this promotes P450c11AS activity is unclear. The stressed heart did not express P450scc, suggesting that circulating corticosterone (rather than intracellular cholesterol) is the substrate for cardiac aldosterone synthesis. Thus, the stressed heart produced aldosterone using a previously undescribed intramitochondrial mechanism that involves P450c11AS, Tom22, and 30-kDa StAR. SIGNIFICANCE STATEMENT: Prior studies of potential cardiac aldosterone synthesis have been inconsistent. This study shows that the stressed rat heart produces aldosterone by a novel mechanism involving aldosterone synthase, Tom22, and intramitochondrial steroidogenic acute regulatory protein (StAR) apparently using circulating corticosterone as substrate. This study establishes that the stressed rat heart produces aldosterone and for the first time identifies a biological role for intramitochondrial 30-kDa StAR.

Electron transfer by human wild-type and A287P mutant P450 oxidoreductase assessed by transient kinetics: functional basis of P450 oxidoreductase deficiency.

Cytochrome P450 oxidoreductase (POR) is a 2-flavin protein that transfers electrons from NADPH via its FAD and FMN moieties to all microsomal cytochrome P450 enzymes, including steroidogenic and drug-metabolizing P450s. Defects in the POR gene can cause POR deficiency (PORD), manifested clinically by disordered steroidogenesis, genital anomalies and skeletal malformations. We examined the POR mutant A287P, which is the most frequent cause of PORD in patients of European ancestry and partially disrupts most P450 activities in vitro. Flavin content analysis showed that A287P is deficient in FAD and FMN binding, although the mutation site is distant from the binding sites of both flavins. Externally added flavin partially restored the cytochrome c reductase activity of A287P, suggesting that flavin therapy may be useful for this frequent form of PORD. Transient kinetic dissection of the reaction of POR with NADPH and the reduction in cytochrome c by POR using stopped-flow techniques revealed defects in individual electron transfer steps mediated by A287P. A287P had impaired ability to accept electrons from NADPH, but was capable of a fast FMN → cytochrome c electron donation reaction. Thus the reduced rates of P450 activities with A287P may be due to deficient flavin and impaired electron transfer from NADPH.

Potential role of increased oxygenation in altering perinatal adrenal steroidogenesis.

BACKGROUND: At birth, the large fetal adrenal involutes rapidly, and the patterns of steroidogenesis change dramatically; the event(s) triggering these changes remain largely unexplored. Fetal abdominal viscera receive hypoxic blood having a partial pressure of oxygen of only ~2 kPa (20-23 mm Hg); perinatal circulatory changes change this to adult values (~20 kPa). We hypothesized that transition from fetal hypoxia to postnatal normoxia participates in altering perinatal steroidogenesis. METHODS: We grew midgestation human fetal adrenal cells and human NCI-H295A adrenocortical carcinoma cells in 2% O2, then transitioned them to 20% O2 and quantitated steroidogenic mRNAs by quantitative PCR and microarrays. RESULTS: Transitioning fetal adrenal cells from hypoxia to normoxia increased mRNAs for 17α-hydroxylase/17,20 lyase (P450c17), 3ß-hydroxysteroid dehydrogenase (3ßHSD2), and steroidogenic acute regulatory protein (StAR). We repeated the protocol with NCI-H295A cells acclimated to hypoxia for 15 d, quantitating 31,255 transcripts by microarray. Using an arbitrary 1.5-fold difference, 1 d of normoxia increased 4 transcripts and decreased 56, whereas 2 d of normoxia increased 62 transcripts and decreased 105. P450c17, 3ßHSD2, and StAR were ranked among the top eight increased transcripts. CONCLUSION: These data suggest that the hypoxic/normoxic transition at birth contributes to perinatal changes in adrenal steroidogenesis.

Phosphorylation of human cytochrome P450c17 by p38α selectively increases 17,20 lyase activity and androgen biosynthesis.

Cytochrome P450c17, a steroidogenic enzyme encoded by the CYP17A1 gene, catalyzes the steroid 17α-hydroxylation needed for glucocorticoid synthesis, which may or may not be followed by 17,20 lyase activity needed for sex steroid synthesis. Whether or not P450c17 catalyzes 17,20 lyase activity is determined by three post-translational mechanisms influencing availability of reducing equivalents donated by P450 oxidoreductase (POR). These are increased amounts of POR, the allosteric action of cytochrome b5 to promote POR-P450c17 interaction, and Ser/Thr phosphorylation of P450c17, which also appears to promote POR-P450c17 interaction. The kinase(s) that phosphorylates P450c17 is unknown. In a series of kinase inhibition experiments, the pyridinyl imidazole drugs SB202190 and SB203580 inhibited 17,20 lyase but not 17α-hydroxylase activity in human adrenocortical HCI-H295A cells, suggesting an action on p38α or p38ß. Co-transfection of non-steroidogenic COS-1 cells with P450c17 and p38 expression vectors showed that p38α, but not p38ß, conferred 17,20 lyase activity on P450c17. Antiserum to P450c17 co-immunoprecipitated P450c17 and both p38 isoforms; however, knockdown of p38α, but not knockdown of p38ß, inhibited 17,20 lyase activity in NCI-H295A cells. Bacterially expressed human P450c17 was phosphorylated by p38α in vitro at a non-canonical site, conferring increased 17,20 lyase activity. This phosphorylation increased the maximum velocity, but not the Michaelis constant, of the 17,20 lyase reaction. p38α phosphorylates P450c17 in a fashion that confers increased 17,20 lyase activity, implying that the production of adrenal androgens (adrenarche) is a regulated event.

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation.

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.

Distinguishing deficiencies in the steroidogenic acute regulatory protein and the cholesterol side chain cleavage enzyme causing neonatal adrenal failure.

OBJECTIVES: To determine the genetic basis of disordered steroidogenesis in Kuwaiti siblings. STUDY DESIGN: Two siblings (46,XX and 46,XY) had normal female external genitalia and severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Abdominal ultrasonography showed normal size adrenal glands, suggesting cholesterol side chain cleavage enzyme (P450scc) deficiency. The CYP11A1 gene encoding P450scc and the STAR gene encoding the steroidogenic acute regulatory protein (StAR) were directly sequenced from leukocyte DNA. RESULTS: All exons and intron/exon boundaries of the CYP11A1 gene were normal; the STAR gene was homozygous for a novel 14-base deletion/frameshift in exon 4 (g.4643_4656del), so that no functional protein could be produced. Both parents and an unaffected sibling were heterozygous; zygosity was confirmed with a BsmF1 restriction fragment length polymorphism. CONCLUSIONS: Unlike most patients with StAR deficiency, our patients did not have the massive adrenal hyperplasia typical of congenital lipoid adrenal hyperplasia. The distinction between StAR and P450scc deficiency may require gene sequencing.

Prenatal treatment of congenital adrenal hyperplasia: risks outweigh benefits.

Prenatal treatment of congenital adrenal hyperplasia by administering dexamethasone to a woman presumed to be carrying an at-risk fetus has been described as safe and effective in several reports. A review of data from animal experimentation and human trials indicates that first-trimester dexamethasone decreases birthweight; affects renal, pancreatic beta cell, and brain development; increases anxiety; and predisposes to adult hypertension and hyperglycemia. In human studies, first-trimester dexamethasone is associated with orofacial clefts, decreased birthweight, poorer verbal working memory, and poorer self-perception of scholastic and social competence. Numerous medical societies have cautioned that prenatal treatment of congenital adrenal hyperplasia with dexamethasone should only be done in prospective clinical research settings with institutional review board approval, and therefore is not appropriate for routine community practice.

Mutant P450 oxidoreductase causes disordered steroidogenesis with and without Antley-Bixler syndrome.

Deficient activities of multiple steroidogenic enzymes have been reported without and with Antley-Bixler syndrome (ABS), but mutations of corresponding cytochrome P450 enzymes have not been found. We identified mutations in POR, encoding P450 oxidoreductase, the obligate electron donor for these enzymes, in a woman with amenorrhea and three children with ABS, even though knock-out of POR is embryonically lethal in mice. Mutations of POR also affect drug-metabolizing P450 enzymes, explaining the association of ABS with maternal fluconazole ingestion.

History of Adrenal Research: From Ancient Anatomy to Contemporary Molecular Biology.

The adrenal is a small, anatomically unimposing structure that escaped scientific notice until 1564 and whose existence was doubted by many until the 18th century. Adrenal functions were inferred from the adrenal insufficiency syndrome described by Addison and from the obesity and virilization that accompanied many adrenal malignancies, but early physiologists sometimes confused the roles of the cortex and medulla. Medullary epinephrine was the first hormone to be isolated (in 1901), and numerous cortical steroids were isolated between 1930 and 1949. The treatment of arthritis, Addison's disease, and congenital adrenal hyperplasia (CAH) with cortisone in the 1950s revolutionized clinical endocrinology and steroid research. Cases of CAH had been reported in the 19th century, but a defect in 21-hydroxylation in CAH was not identified until 1957. Other forms of CAH, including deficiencies of 3ß-hydroxysteroid dehydrogenase, 11ß-hydroxylase, and 17α-hydroxylase were defined hormonally in the 1960s. Cytochrome P450 enzymes were described in 1962-1964, and steroid 21-hydroxylation was the first biosynthetic activity associated with a P450. Understanding of the genetic and biochemical bases of these disorders advanced rapidly from 1984 to 2004. The cloning of genes for steroidogenic enzymes and related factors revealed many mutations causing known diseases and facilitated the discovery of new disorders. Genetics and cell biology have replaced steroid chemistry as the key disciplines for understanding and teaching steroidogenesis and its disorders.

Effect of P450 oxidoreductase variants on the metabolism of model substrates mediated by CYP2C9.1, CYP2C9.2, and CYP2C9.3.

OBJECTIVES: CYP2C9 is a microsomal cytochrome P450 that receives electrons from P450 oxidoreductase (POR) to metabolize about 15% of clinically used drugs. Similar to many P450 enzymes, CYP2C9 is polymorphic, with the hypomorphic *2 and *3 variants accounting for about 20% of White alleles. POR is also polymorphic, with the amino acid sequence variant A503V accounting for 19-37% of alleles in different populations. We aimed to understand how polymorphisms in these two interacting proteins might affect drug metabolism. METHODS: We assayed the activities of CYP2C9.1, CYP2C9.2, and CYP2C9.3 to metabolize diclofenac, flurbiprofen, and tolbutamide using a wild type or one of four POR variants (Q153R, A287P, R457H, and A503V). Human CYP2C9 and POR variants were expressed in bacteria, purified, and reconstituted in vitro and the Michaelis constant and maximum velocity were measured with each CYP2C9/POR combination and each substrate. RESULTS: With wild-type POR, the CYP2C9 activities were CYP2C9.1>CYP2C9.2>>CYP2C9.3 with all three substrates. Both the common A503V polymorphism and the rare Q153R variant showed modest increases in activity with all three CYP2C9 isoforms and all three substrates. This is in contrast to previous studies in which A503V showed a modest loss of function with CYP1A2, CYP2C19, CYP2D6, CYP3A4, and CYP17A1. The disease-causing POR variants A287P and R457H had a very low or unmeasurable activity with all CYP2C9 isoforms and all substrates, which is consistent with their low activities with other CYPs. CONCLUSION: POR variants affect CYP2C9 activities. The impact of a POR variant on catalysis varies with the isoform of CYP2C9 and the assay substrate.

Prenatal treatment of congenital adrenal hyperplasia-not standard of care.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency is a common autosomal recessive disorder due to mutations in the CYP21A2 gene. Since genetic counselors play a crucial role in educating families about inherited disorders, they need to have thorough knowledge regarding the pathophysiology of CAH especially the effects on the fetus, the complex genetics of this disorder, and the controversies surrounding experimental prenatal dexamethasone treatment. Affected female fetuses may have varying degree of virilization of the external genitalia. Starting in the 1980's, supraphysiologic glucocorticoid treatment was used to decrease the virilization of the external genitalia of affected female fetuses. However, recent clinical observations, animal studies and greater awareness of the details of human fetal adrenal physiology raise concerns regarding the safety of this prenatal treatment. We review the pathophysiology of CAH, the safety and ethical considerations of prenatal dexamethasone treatment and the views of multiple medical societies that conclude that this experimental therapy should only be done in prospective trials approved by ethical review boards.

A Brief History of the Pediatric Endocrine Society (PES).

The Pediatric Endocrine Society (PES) was initially established in 1972 as the Lawson Wilkins Pediatric Endocrine Society (LWPES), by some of Wilkins' former fellows. As the society grew from its 37 founding members and Dr. Wilkins' influence faded, the name of the society was changed in 2010 and now counts about 1,500 members, mostly from the US and Canada. Pediatric endocrine training programs headed by (LW)PES members have welcomed fellows from throughout the world, many of whom have gone on to leadership positions in their home countries. Starting in 1981, the (LW)PES has collaborated with pediatric endocrine societies around the world in quadrennial meetings, fostering collaborations, transfer of ideas, devising joint practice guidelines, and enjoying one another's fellowship and counsel. The PES presently has committees and special interest groups concerned with all aspects of pediatric endocrinology, assuring that our clinical and academic resources reflect both breadth and depth. To celebrate our 50th anniversary, selected members have written the historical manuscripts featured in this special issue of Hormone Research in Pediatrics. These historical reviews delve into the origins of our specialty, sometimes deep into antiquity, provide useful background information, and illustrate the kinds of intellectual struggles that have led to the development of contemporary pediatric endocrinology, worldwide.

A Brief History of Congenital Adrenal Hyperplasia.

The adrenal has played a major role in the history of pediatric endocrinology. Cases of congenital adrenal hyperplasia (CAH) were reported in the 19th century, leading to the understanding that the adrenal influenced sexual phenotypes as well as being mysteriously required for survival. Numerous adrenal steroids were isolated in the early 20th century, and bioassays eventually distinguished glucocorticoids, mineralocorticoids, and androgens. Treatment of CAH with cortisone in 1950 by Wilkins and by Bartter and Albright revolutionized clinical endocrinology and launched a productive era of pediatric adrenal research. Through careful clinical studies, Wilkins established the contemporary approach to treating CAH. Alfred Bongiovanni identified defective 21-hydroxylation in CAH in 1957, followed by deficiencies of 3ß-hydroxysteroid dehydrogenase and 11ß-hydroxylase. P450 enzymes were described in 1962-1964, and 21-hydroxylation was the first activity ascribed to a P450. Accurate assays for 17OH-progesterone in newborns and in response to ACTH permitted the diagnosis of CAH in children and families. Application of the techniques of molecular genetics elucidated genetic and biochemical bases of these disorders from 1984 to 2004. Pediatric endocrinologists played central roles in identifying the genes responsible for both common and rare forms of congenital adrenal hyperplasia and determining their most appropriate treatments.

Rickets, Vitamin D, and Ca/P Metabolism.

Rickets was a major public health problem dating from Roman times, and medical descriptions of rickets date from the 17th century. Sniadecki first advocated treatment by exposure to sunshine in 1822; contemporaneously, several British physicians advocated use of cod liver oil. Both approaches were successful. Work in 1924 showed that exposure to UV light endowed fats and other foods with antirachitic properties. Vitamins D2 and D3, the antirachitic agent in cod liver oil, were, respectively, produced by UV radiation of ergosterol and 7-dehydrocholesterol. Calcitriol (1,25[OH]2D3) was identified as the biologically active form of vitamin D in the early 1970s. The vitamin D 25-hydroxylase, 24-hydroxylase, and 1α-hydroxylase were cloned in the 1990s and their genetic defects were soon delineated. The vitamin D receptor was also cloned and its mutations identified in vitamin D-resistant rickets. Work with parathyroid hormone (PTH) began much later, as the parathyroids were not identified until the late 19th century. In 1925, James B. Collip (of insulin fame) identified PTH by its ability to correct tetany in parathyroidectomized dogs, but only in the 1970s was it clear that only a small fragment of PTH conveyed its activity. Congenital hypoparathyroidism with immune defects was described in 1968, eventually linked to microdeletions in chromosome 22q11.2. X-linked hypophosphatemic rickets was reported in 1957, and genetic linkage analysis identified the causative PHEX gene in 1997. Autosomal dominant hypophosphatemic rickets similarly led to the discovery of FGF23, a phosphate-wasting humoral factor made in bone, in 2000, revolutionizing our understanding of phosphorus metabolism.

Congenital Adrenal Hyperplasia-Current Insights in Pathophysiology, Diagnostics, and Management.

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders affecting cortisol biosynthesis. Reduced activity of an enzyme required for cortisol production leads to chronic overstimulation of the adrenal cortex and accumulation of precursors proximal to the blocked enzymatic step. The most common form of CAH is caused by steroid 21-hydroxylase deficiency due to mutations in CYP21A2. Since the last publication summarizing CAH in Endocrine Reviews in 2000, there have been numerous new developments. These include more detailed understanding of steroidogenic pathways, refinements in neonatal screening, improved diagnostic measurements utilizing chromatography and mass spectrometry coupled with steroid profiling, and improved genotyping methods. Clinical trials of alternative medications and modes of delivery have been recently completed or are under way. Genetic and cell-based treatments are being explored. A large body of data concerning long-term outcomes in patients affected by CAH, including psychosexual well-being, has been enhanced by the establishment of disease registries. This review provides the reader with current insights in CAH with special attention to these new developments.

Early steps in steroidogenesis: intracellular cholesterol trafficking.

Steroid hormones are made from cholesterol, primarily derived from lipoproteins that enter cells via receptor-mediated endocytosis. In endo-lysosomes, cholesterol is released from cholesterol esters by lysosomal acid lipase (LAL; disordered in Wolman disease) and exported via Niemann-Pick type C (NPC) proteins (disordered in NPC disease). These diseases are characterized by accumulated cholesterol and cholesterol esters in most cell types. Mechanisms for trans-cytoplasmic cholesterol transport, membrane insertion, and retrieval from membranes are less clear. Cholesterol esters and "free" cholesterol are enzymatically interconverted in lipid droplets. Cholesterol transport to the cholesterol-poor outer mitochondrial membrane (OMM) appears to involve cholesterol transport proteins. Cytochrome P450scc (CYP11A1) then initiates steroidogenesis by converting cholesterol to pregnenolone on the inner mitochondrial membrane (IMM). Acute steroidogenic responses are regulated by cholesterol delivery from OMM to IMM, triggered by the steroidogenic acute regulatory protein (StAR). Chronic steroidogenic capacity is determined by CYP11A1 gene transcription. StAR mutations cause congenital lipoid adrenal hyperplasia, with absent steroidogenesis, potentially lethal salt loss, and 46,XY sex reversal. StAR mutations initially destroy most, but not all steroidogenesis; low levels of StAR-independent steroidogenesis are lost later due to cellular damage, explaining the clinical findings. Rare P450scc mutations cause a similar syndrome. This review addresses these early steps in steroid biosynthesis.

Genetics of P450 oxidoreductase: sequence variation in 842 individuals of four ethnicities and activities of 15 missense mutations.

P450 oxidoreductase (POR) is an electron-donating flavoprotein required for the activity of all microsomal cytochrome P450 enzymes. We sequenced 5,655 bp of the POR gene in a representative population of 842 healthy unrelated individuals in four ethnic groups: 218 African Americans, 260 Caucasian Americans, 179 Chinese Americans, and 185 Mexican Americans. One hundred forty SNPs were detected, of which 43 were found in >/=1% of alleles. Twelve SNPs were in the POR promoter region. Fifteen of 32 exonic variations altered the POR amino acid sequence; 13 of these 15 are previously undescribed missense variations. We found eight indels, only one of which was in the coding region. A previously described variant, A503V, was found on 27.9% of all alleles with some ethnic predilection (19.1% in African Americans, 26.4% in Caucasian Americans, 36.7% Chinese Americans, and 31.0% in Mexican Americans). We built cDNA expression vectors for the 13 previously undescribed missense variants, expressed each protein lacking 27 N-terminal residues in Escherichia coli, and assayed the apparent K(m) and V(max) of each in four assays: reduction of cytochrome c, oxidation of NADPH, 17alpha-hydroxylase activity of P450c17, and 17,20 lyase activity of P450c17. The catalytic activities of several missense mutants differed substantially in these assays, indicating that each POR mutant must be assayed separately with each potential target P450 enzyme. The activity of A503V was reduced to a modest but statistically significant degree in all four assays, suggesting that it may play an important role in interindividual variation in drug response.

Steroidogenic electron-transfer factors and their diseases.

Most steroidogenesis disorders are caused by mutations in genes encoding the steroidogenic enzymes, but work in the past 20 years has identified related disorders caused by mutations in the genes encoding the cofactors that transport electrons from NADPH to P450 enzymes. Most P450s are microsomal and require electron donation by P450 oxidoreductase (POR); by contrast, mitochondrial P450s require electron donation via ferredoxin reductase (FdxR) and ferredoxin (Fdx). POR deficiency is the most common and best-described of these new forms of congenital adrenal hyperplasia. Severe POR deficiency is characterized by the Antley-Bixler skeletal malformation syndrome and genital ambiguity in both sexes, and hence is easily recognized, but mild forms may present only with infertility and subtle disorders of steroidogenesis. The common POR polymorphism A503V reduces catalysis by P450c17 (17-hydroxylase/17,20-lyase) and the principal drugmetabolizing P450 enzymes. The 17,20-lyase activity of P450c17 requires the allosteric action of cytochrome b5, which promotes interaction of P450c17 with POR, with consequent electron transfer. Rare b5 mutations are one of several causes of 17,20-lyase deficiency. In addition to their roles with steroidogenic mitochondrial P450s, Fdx and FdxR participate in the synthesis of iron-sulfur clusters used by many enzymes. Disruptions in the assembly of Fe-S clusters is associated with Friedreich ataxia and Parkinson disease. Recent work has identified mutations in FdxR in patients with neuropathic hearing loss and visual impairment, somewhat resembling the global neurologic disorders seen with mitochondrial diseases. Impaired steroidogenesis is to be expected in such individuals, but this has not yet been studied.