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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. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (P450) oxidoreductase (POR) donates electrons to all microsomal cytochrome P450s, including drug-metabolizing and steroidogenic enzymes. Severe POR mutations cause skeletal malformations and disordered steroidogenesis. The POR polymorphism A503V is found on approximately 28% of human alleles and decreases activities of CYP3A4 and steroidogenic CYP17, but not the activities of steroidogenic CYP21 or drug-metabolizing CYP1A2 and CYP2C19. CYP2D6 metabolizes about 25% of clinically used drugs; we assessed the capacity of POR variants to support the activities of human CYP2D6.N-27 forms of wildtype (WT), Q153R, A287P, R457H and A503V POR, and WT CYP2D6 were expressed in Escherichia coli. POR proteins in bacterial membranes were reconstituted with purified CYP2D6. Support of CYP2D6 was measured by metabolism of EOMCC (2H-1-benzopyran-3-carbonitrile,7-(ethoxy-methoxy)-2-oxo-(9Cl)), dextromethorphan and bufuralol. Michaelis constant (Km) and maximum velocity (Vmax) were determined in three triplicate experiments for each reaction; catalytic efficiency is expressed as Vmax/Km.Compared with WT POR, disease-causing POR mutants A287P and R457H supported no detectable CYP2D6 activity with EOMCC, but A287P supported approximately 25% activity with dextromethorphan and bufuralol. Q153R had increased function with CYP2D6 (128% with EOMCC, 198% with dextromethorphan, 153% with bufuralol). A503V supported decreased CYP2D6 activity: 85% with EOMCC, 62% with dextromethorphan and 53% with bufuralol.POR variants have different effects depending on the substrate metabolized. Disease-causing POR mutations R457H and A287P had poor activities, suggesting that diminished drug metabolism should be considered in affected patients. The common A503V polymorphism impaired CYP2D6 activities with two commonly used drugs by 40-50%, potentially explaining some genetic variation in drug metabolism. [ABSTRACT FROM AUTHOR]

CYP3A4 receives electrons from P450 oxidoreductase (POR) to metabolize about 50% of clinically used drugs. There is substantial inter-individual variation in CYP3A4 catalytic activity that is not explained by CYP3A4 genetic variants. CYP3A4 is flexible and distensible, permitting it to accommodate substrates varying in shape and size. To elucidate the mechanisms of variability in CYP3A4 catalysis, we examined the effects of genetic variants of POR, and explored the possibility that substrate-induced conformational changes in CYP3A4 differentially affect the ability of POR variants to support catalysis.We expressed human CYP3A4 and four POR variants (Q153R, A287P, R457H, A503 V) in bacteria, reconstituted them in vitro and measured the Michaelis constant and maximum velocity with testosterone, midazolam, quinidine and erythromycin as substrates.POR A287P and R457H had low activity with all substrates; Q153R had 76-94% of wild-type (WT) activity with midazolam and erythromycin, but 129-150% activity with testosterone and quinidine. The A503 V polymorphism reduced the CYP3A4 activity to 61-77% of WT with testosterone and midazolam, but had nearly WT activity with quinidine and erythromycin.POR variants affect CYP3A4 activities. The impact of a POR variant on catalysis by CYP3A4 is substrate-specific, probably because of substrate-induced conformational changes in CYP3A4. [ABSTRACT FROM AUTHOR]

Steroid 21-hydroxylase deficiency accounts for about 95% of cases of congenital adrenal hyperplasia (CAH). Newborns are currently being screened for the classical forms of this disease throughout the United States and in 12 other countries. As such, it seems important to develop the best practice guidelines for treating not only infants and children, but affected adults as well. This report gives a brief overview of the most recent expert opinion and clinical practice guidelines for CAH as formulated by The Endocrine Society Task Force. [ABSTRACT FROM AUTHOR]

Objective X-linked nephrogenic diabetes insipidus (XNDI), caused by mutations in the V2 vasopressin receptor (V2R), is clinically distinguished from central diabetes insipidus (CDI) by elevated serum vasopressin (AVP) levels and unresponsiveness to 1-desamino-8-d-arginine vasopressin (DDAVP). We report two infants with XNDI, and present the characterization and functional rescue of a novel V2R mutation. Patients Two male infants presented with poor growth and hypernatraemia. Both patients had measurable pretreatment serum AVP and polyuria that did not respond to DDAVP, suggesting NDI. However, both also had absent posterior pituitary bright spot on MRI, which is a finding more typical of CDI. Methods The AVPR2 gene encoding V2R was sequenced. The identified novel missense mutation was re-created by site-directed mutagenesis and expressed in HEK293 cells. V2R activity was assessed by the ability of transfected cells to produce cAMP in response to stimulation with DDAVP. Membrane localization of V2R was assessed by fluorescence microscopy. Results Patient 1 had a deletion of AVPR2; patient 2 had the novel mutation L57R. In transiently transfected HEK293 cells, DDAVP induced detectable but severely impaired L57R V2R activity compared to cells expressing wild-type V2R. Fluorescence microscopy showed that myc-tagged wild-type V2R localized to the cell membrane while L57R V2R remained intracellular. A nonpeptide V2R chaperone, SR121463, partially rescued L57R V2R function by allowing it to reach the cell membrane. Conclusions L57R V2R has impaired in vitro activity that can be partially improved by treatment with a V2R chaperone. The posterior pituitary hyperintensity may be absent in infants with XNDI. [ABSTRACT FROM AUTHOR]

The steroidogenic acute regulatory protein (StAR) belongs to a family of 15 StAR-related lipid transfer (START) domain proteins termed StARD1-StARDI5. StAR (StARDI) induces adrenal and gonadal steroidogenesis by moving cholesterol from the outer mitochondrial membrane to the inner mitochondrial membrane by an unclear process that involves conformational changes that have been characterized as a molten globule transition. We expressed, purified, and assessed the activity and cholesterol-binding behavior of StARD1 and StARD3-D7, showing that StARD6 had activity equal to StARD1, whereas StARD4, D5, and D7 had little or no activity with adrenal mitochondria in vitro. Partial proteolysis examined by mass spectrometry suggests that StARD6 has a protease-sensitive C-terminus, similar to but smaller than that of StARD1. Experiments using urea denaturation, stopped-flow kinetics and measurements of mitochondrial membrane association suggests that StARDI and StARD6 both unfold and refold slowly with similar kinetic patterns. Isothermal titration calorimetry suggests that StARD6 interacts with mitochondrial membranes as well as or better than StARD I. Computational modeling of StARD6 suggests that it has a similar fold to StARD 1, with a hydrophobic sterol-binding pocket and a unique C-terminal extension. StARD6, which is expressed only in male germ-line cells, thus exhibits biological and biophysical properties that imply a role in steroidogenesis. [ABSTRACT FROM AUTHOR]

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and! or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17α-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17- the enzyme most closely associated with the hormonal phenotype-provided an excellent genotype!phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency. [ABSTRACT FROM AUTHOR]

Steroidogenic acute regulatory (STAR) protein facilitates import of cholesterol into adrenal and gonadal mitochondria where cholesterol is converted to pregnenolone, initiating steroidogenesis. StAR acts exclusively on the outer mitochondrial membrane (OMM) by unknown mechanisms. To identify StAR domains involved in membrane association, we reacted N-62 StAR with small unilamellar vesicles (SUVs) composed of lipids resembling the OMM. Solvent-exposed domains were digested with trypsin, Asp-N, or pepsin at different pH levels, and StAR peptides protected from proteolysis were identified by mass spectrometry. At pH 4 SUVs completely protected residues 259-282; at pH 6.5 this region was partially digested into 254-272, 254-273, and 254-274. Computer-graphic modeling of N-62 StAR indicated these peptides correspond to the C-terminal α4 helix and that residues Leu275, Thr263, and Arg272 in α4 form stabilizing interactions with Gln128, Asp150, and Asp106 in adjacent loops. CD spectroscopy of a 37-mer model of α4 (residues 247-287) indicated a random coil in aqueous buffer, but in 40% methanol the peptide was α-helical and achieved maximal α-helicity at pH 5.0 in the presence of SUVs. Reacting the 37-mer with diethyl pyrocarbamate incorporated into SUVs increased the number of modified residues. Thus the C-terminal α4 helix is critically involved in the membrane association of StAR with OMM lipids. The membrane association and the α-helical structure of the C terminus in the presence of OMM lipids are also pH-dependent. These results further support StAR undergoing a pH-dependent change in its conformation when interacting with the acidic phospholipid head groups of a membrane. [ABSTRACT FROM AUTHOR]

Combined partial deficiency of 17α-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase(POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients'phenotypes, but that assays of POR-supported 17α-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism. [ABSTRACT FROM AUTHOR]

Activation of vitamin D requires hepatic 25-hydroxylation and renal 1α-hydroxylation. Defects in renal P450c1α are well-described, but few patients with defective vitamin D 25-hydroxylation are reported. The cytochrome P450 enzymes CYP2D6 and CYP27 are potential 25-hydroxylases. We sequenced both genes in two reported families with hepatic 25-hydroxylase deficiency and found no mutations. 25-Hydroxylation occurs in both mitochondria and microsomes. The existence genes encoding distinct enzymes would provide genetic redundancy, explaining the rarity of apparent vitamin D 25-hydroxylase deficiency. [Copyright &y& Elsevier]

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) is a common cause of hyponatremia. We describe two infants whose clinical and laboratory evaluations were consistent with the presence of SIADH, yet who had undetectable arginine vasopressin (AVP) levels. We hypothesized that they had gain-of-function mutations in the V2 vasopressin receptor (V2R). DNA sequencing of each patient's V2R gene (AVPR2) identified missense mutations in both, with resultant changes in codon 137 from arginine to cysteine or leucine. These novel mutations cause constitutive activation of the receptor and are the likely cause of the patients' SIADH-like clinical picture, which we have termed “nephrogenic syndrome of inappropriate antidiuresis.” N Engl J Med 2005;352:1884-90. [ABSTRACT FROM AUTHOR]

Background: The Ehlers–Danlos syndrome is a heritable connective-tissue disorder caused by defects in fibrillar-collagen metabolism. Mutations in the type V collagen genes account for up to 50 percent of cases of classic Ehlers–Danlos syndrome, but many other cases are unexplained. We investigated whether the deficiency of the tenascins, extracellular-matrix proteins that are highly expressed in connective tissues, was associated with the Ehlers–Danlos syndrome. Methods: We screened serum samples from 151 patients with the classic, hypermobility, or vascular types of the Ehlers–Danlos syndrome; 75 patients with psoriasis; 93 patients with rheumatoid arthritis; and 21 healthy persons for the presence of tenascin-X and tenascin-C by enzyme-linked immunosorbent assay. We examined the expression of tenascins and type V collagen in skin by immunohistochemical methods and sequenced the tenascin-X gene. Results: Tenascin-X was present in serum from all normal subjects, all patients with psoriasis, all patients with rheumatoid arthritis, and 146 of 151 patients with the Ehlers–Danlos syndrome. Tenascin-X was absent from the serum of the five remaining patients with Ehlers–Danlos syndrome, who were unrelated. Tenascin-X deficiency was confirmed in these patients by analysis of skin fibroblasts and by immunostaining of skin. The expression of tenascin-C and type V collagen was normal in these patients. All five of these patients had hypermobile joints, hyperelastic skin, and easy bruising, without atrophic scarring. Tenascin-X mutations were identified in all tenascin-X–deficient patients; one patient had a homozygous tenascin-X gene deletion, one was heterozygous for the deletion, and three others had homozygous truncating point mutations, confirming a causative role for tenascin-X and a recessive pattern of inheritance. Conclusions: Tenascin-X deficiency causes a clinically distinct, recessive form of the Ehlers–Danlos syndrome. This finding indicates that factors other than the collagens or collagen-processing enzymes can cause the syndrome and suggests a central role for tenascin-X in maintaining the integrity of collagenous matrix. (N Engl J Med 2001;345:1167-75.) [ABSTRACT FROM AUTHOR]

Background: Congenital lipoid adrenal hyperplasia results in severe impairment of steroid biosynthesis in the adrenal glands and gonads that is manifested both in utero and postnatally. We recently found mutations in the gene for the steroidogenic acute regulatory protein in four patients with this syndrome, but it was not clear whether all patients have such mutations or why there is substantial clinical variation in these patients. Methods: We directly sequenced the gene for steroidogenic acute regulatory protein in 15 patients with congenital lipoid adrenal hyperplasia from 10 countries. Identified mutations were confirmed and recreated in expression vectors, transfected into cultured cells, and assayed for the presence and activity of steroidogenic acute regulatory protein. Results: Fifteen different mutations in the gene for steroidogenic acute regulatory protein were found in 14 patients; the mutation Gln258Stop was found in 80 percent of affected alleles from Japanese and Korean patients, and the mutation Arg182Leu was found in 78 percent of affected alleles from Palestinian patients. We developed diagnostic tests for these and eight other mutations. Thirteen of the 15 mutations were in exons 5, 6, or 7, and all rendered the steroidogenic acute regulatory protein inactive in functional assays. Some mutants with amino acid replacements were capable of normal mitochondrial processing, indicating that the activity of steroidogenic acute regulatory protein is not associated with its translocation into mitochondria. Steroidogenic cells lacking the protein retained low levels of steroidogenesis. This explains the secretion of some steroid hormones by the ovaries after puberty before affected cells accumulate large amounts of cholesterol esters. Conclusions: The congenital lipoid adrenal hyperplasia phenotype is the result of two separate events, an initial genetic loss of steroidogenesis that is dependent on steroidogenic acute regulatory protein and a subsequent loss of steroidogenesis that is independent of the protein due to cellular damage from accumulated cholesterol esters. (N Engl J Med 1996;335:1870-8.) [ABSTRACT FROM AUTHOR]

is not a required field for Obituaries., (© 2025 S. Karger AG, Basel.)

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); but 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society.)

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 3β-hydroxysteroid dehydrogenase, 11β-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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 3β-hydroxysteroid dehydrogenase and 11β-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., (© 2022 S. Karger AG, Basel.)

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., (© 2022 S. Karger AG, Basel.)

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.

In May 2014, the National Institutes of Health (NIH) stated its intent to "require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells." Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sex and gender are used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the "observer effect") and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in addition to aneuploidy; chromosomal numbers can change as cells are passaged. Additionally, culture medium contains steroids, growth hormone, and insulin that might influence expression of various genes. These issues often are not taken into account, determined, or even considered. Issues pertaining to the "sex" of cultured cells are beyond the scope of this Statement. However, we will discuss the factors that influence sex and gender in both basic research (that using animal models) and clinical research (that involving human subjects), as well as in some areas of science where sex differences are routinely studied. Sex differences in baseline physiology and associated mechanisms form the foundation for understanding sex differences in diseases pathology, treatments, and outcomes. The purpose of this Statement is to highlight lessons learned, caveats, and what to consider when evaluating data pertaining to sex differences, using 3 areas of research as examples; it is not intended to serve as a guideline for research design., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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., Competing Interests: The authors have declared that no competing interests exist.

Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency (21OHD) has a worldwide incidence of 1 in 15-20,000. Affected individuals have adrenal insufficiency and androgen excess; the androgen excess begins during fetal life, typically resulting in 46,XX disordered sexual development. In 21OHD, 17-hydroxyprogesterone (17OHP), the steroid proximal to 21-hydroxylase, accumulates. Most industrialized countries have newborn screening programs that measure 17OHP; such screening has permitted rapid detection of newborns with 21OHD, saving lives previously lost to mineralocorticoid deficiency and salt wasting. However, newborn screening is plagued by false positives. 17OHP is above most "cutoff values" in the first 24 h of life, is high in otherwise normal premature infants, and in many term infants with physiologic stress from unrelated diseases. In addition, newborn 17OHP may be elevated in other forms of CAH, including 11-hydroxylase deficiency, 3β-hydroxysteroid dehydrogenase deficiency, and P450 oxidoreductase deficiency. In 21OHD, some of the accumulated intra-adrenal 17OHP is converted to 21-deoxycortisol (21-deoxy) by 11β-hydroxylase (CYP11B1); 21-deoxy is not elevated in premature infants or in other forms of CAH, and hence is a more specific marker for 21OHD. However, 21-deoxy assays have not been generally available until recently, hence experience is limited. We urge clinical investigators, commercial reference laboratories, and newborn screening programs to investigate replacing 17OHP with 21-deoxy as the analyte of choice for studies of 21OHD., (© 2019 The Author Published by S. Karger AG, Basel.)

Objective: To update the congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency clinical practice guideline published by the Endocrine Society in 2010., Conclusions: The writing committee presents updated best practice guidelines for the clinical management of congenital adrenal hyperplasia based on published evidence and expert opinion with added considerations for patient safety, quality of life, cost, and utilization.

Congenital adrenal hyperplasia (CAH) is a group of genetic disorders of adrenal steroidogenesis that impair cortisol synthesis, with compensatory increases in ACTH leading to hyperplastic adrenals. The term 'CAH' is generally used to mean 'steroid 21-hydroxylase deficiency' (21OHD) as 21OHD accounts for about 95% of CAH in most populations; the incidences of the rare forms of CAH vary with ethnicity and geography. These forms of CAH are easily understood on the basis of the biochemistry of steroidogenesis. Defects in the steroidogenic acute regulatory protein, StAR, disrupt all steroidogenesis and are the second-most common form of CAH in Japan and Korea; very rare defects in the cholesterol side-chain cleavage enzyme, P450scc, are clinically indistinguishable from StAR defects. Defects in 3β-hydroxysteroid dehydrogenase, which also causes disordered sexual development, were once thought to be fairly common, but genetic analyses show that steroid measurements are generally unreliable for this disorder. Defects in 17-hydroxylase/17,20-lyase ablate synthesis of sex steroids and also cause mineralocorticoid hypertension; these are common in Brazil and in China. Isolated 17,20-lyase deficiency can be caused by rare mutations in at least three different proteins. P450 oxidoreductase (POR) is a co-factor used by 21-hydroxylase, 17-hydroxylase/17,20-lyase and aromatase; various POR defects, found in different populations, affect these enzymes differently. 11-Hydroxylase deficiency is the second-most common form of CAH in European populations but the retention of aldosterone synthesis distinguishes it from 21OHD. Aldosterone synthase deficiency is a rare salt-losing disorder. Mild, 'non-classic' defects in all of these factors have been described. Both the severe and non-classic disorders can be treated if recognized., (© 2018 European Society of Endocrinology.)

Mutations of the CYP21A2 gene encoding adrenal 21-hydroxylase cause congenital adrenal hyperplasia (CAH). The CYP21A2 gene is partially overlapped by the TNXB gene, which encodes an extracellular matrix protein called Tenascin-X (TNX). Mutations affecting both alleles of TNXB cause a severe, autosomal recessive form of Ehlers-Danlos syndrome (EDS). Rarely, patients with severe, salt-wasting CAH have deletions of CYP21A2 that extend into TNXB, resulting in a "contiguous gene syndrome" consisting of CAH and EDS. Heterozygosity for TNXB mutations causing haploinsufficiency of TNX may be associated with the mild "hypermobility form" of EDS, which principally affects small and large joints. Studies of patients with salt-wasting CAH found that up to 10% had clinical features of EDS, associated joint hypermobility, haploinsufficiency of TNX and heterozygosity for TNXB mutations, now called "CAH-X." These patients have joint hypermobility and a spectrum of other comorbidities associated with their connective tissue disorder, including chronic arthralgia, joint subluxations, hernias, and cardiac defects. Other disorders are beginning to be associated with TNX deficiency, including familial vesicoureteral reflux and neurologic disorders. Further work is needed to delineate the full spectrum of TNX-deficient disorders, with and without associated CAH., (© 2018 S. Karger AG, Basel.)

The hypothalamic-pituitary-adrenal (HPA) axis is central to homeostasis, stress responses, energy metabolism, and neuropsychiatric function. The history of this complex system involves discovery of the relevant glands (adrenal, pituitary, hypothalamus), hormones (cortisol, corticotropin, corticotropin-releasing hormone), and the receptors for these hormones. The adrenal and pituitary were identified by classical anatomists, but most of this history has taken place rather recently, and has involved complex chemistry, biochemistry, genetics, and clinical investigation. The integration of the HPA axis with modern neurology and psychiatry has cemented the role of endocrinology in contemporary studies of behavior., (© 2018 S. Karger AG, Basel.)

Until the mid-1980s studies of steroidogenesis largely depended on identifying steroid structures and measuring steroid concentrations in body fluids. The molecular biology revolution radically revolutionized studies of steroidogenesis with the cloning of known steroidogenic enzymes, by identifying novel factors, and delineating the genetic basis of known and newly discovered diseases. Unfortunately, this dramatic success has led many young research-oriented endocrinologists to regard steroidogenesis as a 'solved area'. However, many important and exciting questions remain, especially concerning the mechanisms of cholesterol delivery to the steroidogenic machinery, the biochemistry of androgen synthesis, the regulation and biological role of adrenarche, fetal adrenal development and involution, the roles of steroids made in 'extraglandular' cells, and the search for genetic disorders. This review outlines some of these questions, but this list is necessarily incomplete., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Douglas M. (Doug) Stocco is Professor Emeritus at Texas Tech University Health Sciences Center in Lubbock, TX, and is internationally renowned for his work characterizing the steroidogenic acute regulatory protein, StAR. Stocco's laboratory isolated and cloned StAR from mouse Leydig MA-10 cells, collaborated on the demonstration that StAR mutations cause congenital lipoid adrenal hyperplasia, and delineated much of what is known about the intracellular pathways that regulate its production. This work resolved a decades-long quest to identify the mechanism underlying the acute regulation of steroidogenesis., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Steroidogenesis begins with cellular internalization of low-density lipoprotein particles and subsequent intracellular processing of cholesterol. Disorders in these steps include Adrenoleukodystrophy, Wolman Disease and its milder variant Cholesterol Ester Storage Disease, and Niemann-Pick Type C Disease, all of which may present with adrenal insufficiency. The means by which cholesterol is directed to steroidogenic mitochondria remains incompletely understood. Once cholesterol reaches the outer mitochondrial membrane, its delivery to the inner mitochondrial membrane is regulated by the steroidogenic acute regulatory protein (StAR). Severe StAR mutations cause classic congenital lipoid adrenal hyperplasia, characterized by lipid accumulation in the adrenal, adrenal insufficiency, and disordered sexual development in 46,XY individuals. The lipoid CAH phenotype, including spontaneous puberty in 46,XX females, is explained by a two-hit model. StAR mutations that retain partial function cause a milder, non-classic disease characterized by glucocorticoid deficiency, with lesser disorders of mineralocorticoid and sex steroid synthesis. Once inside the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side-chain cleavage enzyme, P450scc, encoded by the CYP11A1 gene. Rare patients with mutations of P450scc are clinically and hormonally indistinguishable from those with lipoid CAH, and may also present as milder non-classic disease. Patients with P450scc defects do not have the massive adrenal hyperplasia that characterizes lipoid CAH, but adrenal imaging may occasionally fail to distinguish these, necessitating DNA sequencing., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vitamin D, an inactive secosteroid pro-hormone, is produced by the action of ultraviolet light on 7-dehydrocholesterol in the skin. The active hormone, 1,25(OH) 2 D is produced by sequential 25-hydroxylation in the liver, principally by CYP2R1, and 1α-hydroxylation in the kidney by CYP27B1. Mutations in CYP27B1 cause 1α-hydroxylase deficiency, also known as vitamin D dependent rickets type I or hereditary pseudo-vitamin D deficient rickets; very rare mutations in CYP2R1 can cause 25-hydroxylase deficiency. Both deficiencies cause hypocalcemia, secondary hyperparathyroidism, severe rickets in infancy, and low serum concentrations of 1,25(OH) 2 D; both disorders respond to hormonal replacement therapy with calcitriol. The inactivation of vitamin D is principally initiated by its 23- and 24-hydroxylation by CYP24A1. Mutations in CYP24A1 can cause both severe neonatal hypercalcemia and a less severe adult hypercalcemic syndrome. Other pathways of vitamin D metabolism are under investigation, notably its 20-hydroxylation by the cholesterol side-chain cleavage enzyme, CYP11A1., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A single enzyme, microsomal P450c17, catalyzes the 17α-hydroxylase activity needed to make cortisol and the subsequent 17,20 lyase activity needed to produce the 19-carbon precursors of sex steroids. The biochemical decision concerning whether P450c17 stops after 17α-hydroxylation or proceeds to 17,20 lyase activity is largely dependent on three post-translational factors. First, 17,20 lyase activity is especially sensitive to the molar abundance of the electron-transfer protein P450 oxidoreductase (POR). Second, cytochrome b5 strongly promotes 17,20 lyase activity, principally by acting as an allosteric factor promoting the interaction of P450c17 with POR, although a minor role as an alternative electron-transfer protein has not been wholly excluded. Third, the serine/threonine phosphorylation of P450c17 itself promotes 17,20 lyase activity, again apparently by promoting the interaction of P450c17 with POR. The principal kinase that phosphorylates P450c17 to confer 17,20 lyase activity appears to be p38α (MAPK14), which increases the maximum velocity of the 17,20 lyase reaction, while having no effect on the Michaelis constant for 17,20 lyase or any detectable effect on the 17α-hydroxylase reaction. Other kinases can also phosphorylate P450c17, but only p38α has been shown to affect its enzymology. Understanding the mechanisms regulating 17,20 lyase activity is essential for the understanding of hyperandrogenic disorders such as premature, exaggerated adrenarche and the polycystic ovary syndrome, and also for the design of selective 17,20 lyase inhibitors for use in hyperandrogenic states and in sex-steroid dependent cancers., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Prenatal treatment of congenital adrenal hyperplasia by administering dexamethasone to a woman presumed to be carrying an at-risk fetus remains a controversial experimental treatment. Review of data from animal experimentation and human trials indicates that dexamethasone cannot be considered safe for the fetus. In animals, prenatal dexamethasone decreases birth weight, affects renal, pancreatic beta cell and brain development, increases anxiety and predisposes to adult hypertension and hyperglycemia. In human studies, prenatal dexamethasone is associated with orofacial clefts, decreased birth weight, poorer verbal working memory, and poorer self-perception of scholastic and social competence. Numerous medical societies have cautioned that prenatal treatment of adrenal hyperplasia with dexamethasone is not appropriate for routine clinical practice and should only be done in Institutional Review Board approved, prospective clinical research settings with written informed consent. The data indicate that this treatment is inconsistent with the classic medical ethical maxim to 'first do no harm'., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Context: Mutations of the CYP24A1 gene encoding the 24-hydroxylase (24OHase) that inactivates metabolites of vitamin D can cause hypercalcemia in infants and adults; in vitro assays of 24OHase activity have been difficult., Objective: We sought an alternative assay to characterize a CYP24A1 mutation in a young adult with bilateral nephrolithiasis and hypercalcemia associated with ingestion of excess vitamin D supplements and robust dairy intake for 5 years., Methods: CYP24A1 exons were sequenced from leukocyte DNA. Wild-type and mutant CYP24A1 cDNAs were expressed in JEG-3 cells, and 24OHase activity was assayed by a two-hybrid system., Results: The CYP24A1 missense mutation L409S was found on only one allele; no other mutation was found in exons or in at least 30 bp of each intron/exon junction. Based on assays of endogenous 24OHase activity and of activity from a transiently transfected CYP24A1 cDNA expression vector, JEG-3 cells were chosen over HepG2, Y1, MA10, and NCI-H295A cells for two-hybrid assays of 24OHase activity. The apparent Michaelis constant, Km(app), was 9.0 ± 2.0 nM for CYP24A1 and 8.6 ± 2.2 nM for its mutant; the apparent maximum velocity, Vmax(app), was 0.71 ± 0.055 d(-1) for the wild type and 0.22 ± 0.026 d(-1) for the mutant. As assessed by Vmax/Km, the L409S mutant has 32% of wild-type activity (P = .0012)., Conclusions: The two-hybrid system in JEG-3 cells provides a simple, sensitive, quantitative assay of 24OHase activity. Heterozygous mutation of CYP24A1 may cause hypercalcemia in the setting of excessive vitamin D intake, but it is also possible that the patient had another, unidentified CYP24A1 mutation on the other allele.

Background: Supraphysiological "stress dosing" is generally given to adrenally insufficient patients undergoing operative procedures and/or general anesthesia. However, the normal responses of cortisol to surgery are poorly documented, especially in small children. Recent studies in adults suggest that massive glucocorticoid dosing is not needed, especially in minimally invasive surgery., Objective: We sought to characterize the normal cortisol secretion rate in healthy children undergoing minimally and moderately invasive urological procedures., Design and Setting: This was a prospective observational study conducted at a tertiary referral center., Patients: Thirty healthy children, ages 5 months to 6 years, were studied undergoing elective urological procedures., Methods: Procedures were performed by a single surgeon; anesthesia was by a standard protocol. Sera were obtained at 5 points: iv catheter placement, intubation, 50% completion of surgery, anesthesia reversal, and 1 hour postoperative. Cortisol and cortisone were quantitated by liquid chromatography-tandem mass spectrometry., Results: Group mean cortisol values ranged from 4.21 to 5.71 μg/dL across the 5 time points; none of these mean values differed significantly (P < .05). There were no differences according to age, time of procedure, caudal anesthesia, and moderate vs minimally invasive procedures; 3 patients had higher values. There was a modest diminution in cortisone across the 5 time points., Conclusions: Minimal and moderately invasive urological procedures do not result in a cortisol stress response in healthy children. Peak cortisol levels were seen 1 hour postoperatively. These data suggest that current guidelines for stress dosing in adrenally insufficient patients substantially exceed physiological requirements during minimally invasive procedures.

Following development of the fetal bipotential gonad into a testis, male genital differentiation requires testicular androgens. Fetal Leydig cells produce testosterone that is converted to dihydrotestosterone in genital skin, resulting in labio-scrotal fusion. An alternative 'backdoor' pathway of dihydrotestosterone synthesis that bypasses testosterone has been described in marsupials, but its relevance to human biology has been uncertain. The classic and backdoor pathways share many enzymes, but a 3α-reductase, AKR1C2, is unique to the backdoor pathway. Human AKR1C2 mutations cause disordered sexual differentiation, lending weight to the idea that both pathways are required for normal human male genital development. These observations indicate that fetal dihydrotestosterone acts both as a hormone and as a paracrine factor, substantially revising the classic paradigm for fetal male sexual development., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

The adrenal is a small gland that escaped anatomic notice until the 16th century, and whose essential role in physiology was not established until the mid 19th century. Early studies were confounded by failure to distinguish the effects of the cortex from those of the medulla, but advances in steroid chemistry permitted the isolation, characterization and synthesis of many steroids by the mid 20th century. Knowledge of steroid structures, radiolabeled steroid conversions, and the identification of accumulated urinary steroids in diseases of steroidogenesis permitted a generally correct description of the steroidogenic pathways, but one confounded by the failure to distinguish species-specific differences. The advent of cloning technologies and molecular genetics rapidly corrected and clarified the understanding of steroidogenic processes. Our laboratory in San Francisco was one of several contributing to this effort, focusing on human steroidogenic enzymes, the genetic disorders in their biosynthesis and the transcriptional and post-translational mechanisms regulating enzyme activity., (Copyright © 2012 Walter L. Miller. Published by Elsevier Ireland Ltd.. All rights reserved.)

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., (Copyright © 2013 Mosby, Inc. All rights reserved.)

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&#95;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., (Copyright © 2013 Mosby, Inc. All rights reserved.)

Context: The cholesterol side-chain cleavage enzyme P450scc, encoded by CYP11A1, converts cholesterol to pregnenolone to initiate steroidogenesis. P450scc deficiency can disrupt adrenal and gonadal steroidogenesis, resembling congenital lipoid adrenal hyperplasia clinically and hormonally; only 12 such patients have been reported previously., Objective: We sought to expand clinical and genetic experience with P450scc deficiency., Patients and Methods: We sequenced candidate genes in 7 children with adrenal insufficiency who lacked disordered sexual development. P450scc missense mutations were recreated in the F2 vector, which expresses the fusion protein P450scc-Ferredoxin Reductase-Ferredoxin. COS-1 cells were transfected, production of pregnenolone was assayed, and apparent kinetic parameters were calculated. Previously described P450scc mutants were assayed in parallel., Results: Four of five Bedouin children in one kindred were compound heterozygotes for mutations c.694C>T (Arg232Stop) and c.644T>C (Phe215Ser). Single-nucleotide polymorphism analysis confirmed segregation of these mutations. The fifth kindred member and another Bedouin patient presented in infancy and were homozygous for Arg232Stop. A patient from Fiji presenting in infancy was homozygous for c.358T>C (Arg120Stop). All mutations are novel. As assayed in the F2 fusion protein, P450scc Phe215Ser retained 2.5% of wild-type activity; previously described mutants Leu141Trp and Ala269Val had 2.6% and 12% of wild-type activity, respectively, and Val415Glu and c.835delA lacked detectable activity., Conclusions: Although P450scc is required to produce placental progesterone required to maintain pregnancy, severe mutations in P450scc are compatible with term gestation; milder P450scc mutations may present later without disordered sexual development. Enlarged adrenals usually distinguish steroidogenic acute regulatory protein deficiency from P450scc deficiency, but only DNA sequencing is definitive.

P450 oxidoreductase (POR) is the flavoprotein that transfers electrons from NADPH to microsomal cytochrome P450 enzymes and to some other proteins. Protocols for genotyping human POR for common polymorphisms are described. Expression in E. coli of recombinant human POR, its purification, and different methods of assessing the effect of amino-acid sequence variants of POR on the activity of various cytochromes P450 are also described.

Adrenal gonadal, placental and brain mitochondria contain several steroidogenic enzymes, notably the cholesterol side chain cleavage enzyme, P450scc, which is the enzymatic rate-limiting step in steroidogenesis which determines cellular steroidogenic capacity. Even before this step, the access of cholesterol to this enzyme system is both rate-limiting and the site of acute regulation via the steroidogenic acute regulatory protein (StAR) which interacts with a complex multi-component 'transduceosome' on the outer mitochondrial membrane (OMM). The components of the transduceosome include the 18 kDa translocator protein (TSPO), the voltage-dependent anion channel (VDAC-1), TSPO-associated protein 7 (PAP7, ACBD3 for acyl-CoA-binding-domain 3), and protein kinase A regulatory subunit 1α (PKAR1A). The precise fashion in which these proteins interact and move cholesterol from the OMM to P450scc, and the means by which cholesterol is loaded into the OMM, remain unclear. Human deficiency diseases have been described for StAR and for P450scc. Mitochondria also contain several 'downstream' steroidogenic enzymes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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.

P450c17 catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. P450c17 is expressed in human fetal and postnatal adrenals and gonads and in the developing mouse nervous system, but little is known about its expression in the human nervous system. We obtained portions of 9-, 10-, and 11-wk gestation human fetuses and delineated the pattern of expression of P450c17 in their peripheral nervous systems by immunocytochemistry using the P450c17 antiserum previously used to characterize P450c17 in the mouse brain. P450c17 was readily detected in the dorsal root ganglia (DRG) and spinal cord. Neural structures were identified with antisera to the cytoskeletal protein neural cell adhesion molecule; DRG were identified with antisera to the neuronal transcription factor BRN3A and neurotrophin receptor tropomyosin-receptor-kinase B. The identification of P450c17 was confirmed using commercial antisera directed against different domains of P450c17 and by using antisera immunodepleted with authentic human P450c17. We also found expression of the P450 cholesterol side-chain cleavage enzyme (P450scc) in the spinal cord and DRG. Expression of P450scc is limited to cell bodies; unlike P450c17, we never detected P450scc in fiber tracts. Catalysis by P450c17 requires electron donation from P450 oxidoreductase (POR). Dual-label immunohistochemistry detected P450c17 and POR colocalized in DRG bundles, but some fibers containing P450c17 lacked POR. These data suggest that neurosteroids synthesized via these two enzymes may act in the developing human nervous system. The expression of P450c17 in structures lacking POR means that P450c17 may not be steroidogenic in those locations, suggesting that P450c17 may have additional functions that do not require POR.