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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.

Context: Disorders of steroidogenesis have been instrumental in delineating human steroidogenic pathways. Each genetic disorder seemed to correspond to a different steroidogenic activity, helping to identify several enzymes. Beginning in 1972, several patients have been reported as having "17,20 lyase deficiency," but there have been inconsistent genetic findings., Objective: This manuscript reviews the biochemistry, genetics, and clinical disorders of 17,20 lyase activity, which converts 21-carbon precursors of glucocorticoids to 19-carbon precursors of sex steroids., Findings: A single enzyme, cytochrome P450c17, catalyzes both 17α-hydroxylase activity and 17,20 lyase activity. The 17,20 lyase activity is especially sensitive to the activities of the accessory proteins P450 oxidoreductase and cytochrome b(5). The first cases of genetically and biochemically proven 17,20 lyase deficiency were reported in 1997, in which specific P450c17 mutations were identified that lost 17,20 lyase activity but not 17α-hydroxylase activity when assayed in vitro. Subsequent work identified other P450c17 mutations and mutations in the genes encoding P450 oxidoreductase and cytochrome b(5). Recently, the initially reported cases from 1972 were found to carry mutations in two aldo-keto reductases, AKR1C2 and AKR1C4. These AKR1C isozymes catalyze 3α-hydroxysteroid dehydrogenase activity in the so-called "backdoor pathway" by which the fetal testis produces dihydrotestosterone without the intermediacy of testosterone., Conclusions: 17,20 Lyase deficiency should be considered a syndrome with multiple causes, and not a single disease. Study of this very rare disorder has substantially advanced our understanding of the pathways, mechanisms, and control of androgen synthesis. Mutations in other, as-yet unidentified genes may also cause this phenotype.

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.

P450 oxidoreductase (POR) is a two-flavin protein that reduces microsomal P450 enzymes and some other proteins. Preparation of active bacterially expressed human POR for biochemical studies has been difficult because membrane-bound proteins tend to interact with column matrices. To reduce column-protein interactions and permit more vigorous washing, human POR lacking 27 N-terminal residues (N-27 POR) was modified to carry a C-terminal Gly3His6-tag (N-27 POR-G3H6). When expressed in Escherichia coli, N-27 POR-G3H6 could be purified to apparent homogeneity by a modified, single-step nickel-nitrilotriacetic acid affinity chromatography, yielding 31 mg POR per liter of culture, whereas standard purification of native N-27 POR required multiple steps, yielding 5 mg POR per liter. Both POR proteins had absorption maxima at 375 and 453 nm and both reduced cytochrome c with indistinguishable specific activities. Using progesterone as substrate for bacterially expressed purified human P450c17, the Michaelis constant for 17α-hydroxylase activity supported by N-27 POR or N-27 POR-G3H6 were 1.73 or 1.49 μm, and the maximal velocity was 0.029 or 0.026 pmol steroids per picomole P450 per minute, respectively. Using 17-hydroxypregnenolone as the P450c17 substrate, the Michaelis constant for 17,20 lyase activity using N-27 POR or N-27 POR-G3H6 was 1.92 or 1.89 μm and the maximal velocity was 0.041 or 0.042 pmol steroid per picomole P450 per minute, respectively. Thus, N-27 POR-G3H6 is equally active as native N-27 POR. This expression and purification system permits the rapid preparation of large amounts of highly pure, biologically active POR and may be generally applicable for the preparation of membrane-bound proteins.

P450 oxidoreductase (POR) is the flavoprotein that acts as the obligatory electron donor to all microsomal P450 enzymes, including those involved in hepatic drug metabolism as well as three steroidogenic P450 enzymes. The untranslated first exon of human POR was located recently, permitting analysis of human POR transcription. Expression of deletional mutants containing up to 3193 bp of the human POR promoter in human adrenal NCI-H295A and liver Hep-G2 cells located the proximal promoter at -325/-1 bp from the untranslated exon. Common human POR polymorphisms at -208 and -173 had little influence on transcription, but the polymorphism at -152 reduced transcription significantly in both cell lines. EMSA and supershift assays identified binding of Smad3/Smad4 between -249 and -261 and binding of thyroid hormone receptor-β (TRβ) at -240/-245. Chromatin immunoprecipitation showed that Smad3, Smad4, TRα, TRβ, and estrogen receptor-α were bound between -374 and -149. Cotransfection of vectors for these transcription factors and POR promoter-reporter constructs into both cell types followed by hormonal treatment showed that T(3) exerts major tropic effects via TRβ, with TRα, estrogen receptor-α, Smad3, and Smad4 exerting lesser, modulatory effects. T(3) also increased POR mRNA in both cell lines. Thyroid hormone also is essential for rat liver POR expression but acts via different transcription factor complexes. These are the first data on human POR gene transcription, establishing roles for TRβ and Smad3/4 in its expression and indicating that the common polymorphism at -152 may play a role in genetic variation in steroid biosynthesis and drug metabolism.

P450 oxidoreductase (POR) transports electrons from NADPH to all microsomal cytochrome P450 enzymes, including steroidogenic P450c17, P450c21 and P450aro. Severe POR mutations A287P (in Europeans) and R457H (in Japanese) cause the Antley-Bixler skeletal malformation syndrome (ABS) plus impaired steroidogenesis (causing genital anomalies), but the basis of ABS is unclear. We have characterized the activities of ∼40 POR variants, showing that assays based on P450c17 activities, but not cytochrome c assays, correlate with the clinical phenotype. The human POR gene is highly polymorphic: the A503V sequence variant, which decreases P450c17 activities to ∼60%, is found on ∼28% of human alleles. A promoter polymorphism (∼8% of Asians and ∼13% of Caucasians) at -152 reduces transcriptional activity by half. Screening of 35 POR variants showed that most mutants lacking activity with P450c17 or cytochrome c also lacked activity to support CYP1A2 and CYP2C19 metabolism of EOMCC (a fluorogenic non-drug substrate), although there were some remarkable differences: Q153R causes ABS and has ∼30% of wild-type activity with P450c17 but had 144% of WT activity with CYP1A2 and 284% with CYP2C19. The effects of POR variants on CYP3A4, which metabolizes nearly 50% of clinically used drugs, was examined with multiple, clinically relevant drug substrates, showing that A287P and R457H dramatically reduce drug metabolism, and that A503V variably impairs drug metabolism. The degree of activity can vary with the drug substrate assayed, as the drugs can influence the conformation of the P450. POR is probably an important contributor to genetic variation in both steroidogenesis and drug metabolism., (Copyright © 2010. Published by Elsevier Ireland Ltd.)

Context: The cholesterol side-chain cleavage enzyme (P450scc), encoded by the CYP11A1 gene, converts cholesterol to pregnenolone to initiate steroidogenesis. Genetic defects in P450scc cause a rare autosomal recessive disorder that is clinically indistinguishable from congenital lipoid adrenal hyperplasia (lipoid CAH). Nonclassic lipoid CAH is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function., Objective: We describe two siblings with hormonal findings suggesting nonclassic lipoid CAH, who had a P450scc mutation that retains partial function., Patients and Methods: A 46,XY male presented with underdeveloped genitalia and partial adrenal insufficiency; his 46,XX sister presented with adrenal insufficiency. Hormonal studies suggested nonclassic lipoid CAH. Sequencing of the StAR gene was normal, but compound heterozygous mutations were found in the CYP11A1 gene. Mutations were recreated in the F2 plasmid expressing a fusion protein of the cholesterol side-chain cleavage system. P450scc activity was measured as Vmax/Km for pregnenolone production in transfected COS-1 cells., Results: The patients were compound heterozygous for the previously described frameshift mutation 835delA and the novel missense mutation A269V. When expressed in the P450scc moiety of F2, the A269V mutant retained 11% activity of the wild-type F2 protein., Conclusions: There is a broad clinical spectrum of P450scc deficiency. Partial loss-of-function CYP11A1 mutation can present with a hormonal phenotype indistinguishable from nonclassic lipoid CAH.

Context: Macronodular adrenocortical hyperplasia classically presents with progressive hypercortisolemia and Cushing syndrome. We describe a 29-yr-old man with massive macronodular adrenocortical hyperplasia without hypercortisolemia but rather markedly elevated and nonsuppressible production of dehydroepiandrosterone (DHEA) and its sulfate (DHEAS)., Objective: To characterize the clinical and molecular features of this case and to determine whether the tissue biochemically resembles the zona reticularis or fetal adrenal., Setting: University clinic, hospital, and laboratories., Design: Static and dynamic blood and urine testing were performed preoperatively. Tissue was studied by light microscopy, immunoblot, RNA microarray, and enzyme assay., Participant: A 29-yr-old man with incidentally discovered bilateral adrenal enlargement., Intervention: Bilateral adrenalectomy., Main Outcome Measures: Molecular studies compared with control samples., Results: Hypercortisolism and 21-hydroxylase deficiency were excluded. DHEA, DHEAS, and 17-hydroxypregnenolone were markedly elevated and did not suppress with dexamethasone 2 mg/d for 4 d. Homogenates of the adrenals demonstrated high 17-hydroxylase, good 17,20-lyase, and low or absent 21-hydroxylase and 3β-hydroxysteroid dehydrogenase activities. Immunoblots confirmed robust expression of cytochrome P450c17 and AKR1C3 but not P450c21. Microarray analysis demonstrated high CYP11A1 and CYP17A1 expression but low or absent HSD3B1, HSD3B2, and CYP21A2 expression. Expression of mRNA for cytochrome b(5) (CYB5A) and AKR1C3, markers of the zona reticularis, were markedly elevated., Conclusion: This is the first case of macronodular hyperplasia of the adrenal zona reticularis confirmed with studies of enzyme activity, mRNA expression, and protein identification. We speculate that this condition can be clinically silent in men but might cause severe hyperandrogenemia in women.

Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

Context: Inactivating mutations of the calcium-sensing receptor (CaSR) cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Most mutations are clustered in the N-terminal and Cys-rich regions of the extracellular domain (ECD) and seven-transmembrane domain. Disease-causing mutations are uncommon in the C terminus of ECD., Objective: The aim of the study was to characterize the CaSR mutations causing neonatal severe hyperparathyroidism in a consanguineous family., Methods: Parathyroid glands from the index patient were stained for CaSR protein. The CaSR gene was sequenced, mutations were recreated in CaSR cDNA, and HEK293 cells were transfected with the CaSR mutant expression vector. Cellular CaSR targeting was detected by immunoblotting and immunocytochemistry; CaSR activity was assayed by inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry., Results: Immunocytochemistry showed reduced intracellular CaSR in patient parathyroids. An in-frame homozygous deletion/insertion mutation, c.1031 > 1034 (delACAAinsT), replaced His344-Asn345 with a single Leu in CaSR loop III. The mutant reduced cell surface expression of CaSR in transfected HEK293 cells. Inositol phosphate accumulation, MAPK activation, and single-cell microfluorimetry revealed blunted signaling responses of the mutant receptor to changes in extracellular Ca(2+) concentration., Conclusion: Deletion of His344-Asn345 in the ECD loop III region affects cell surface targeting of CaSR in transfected cells and in affected parathyroid glands. Absence of conserved Asn345 may interfere with CaSR folding or glycosylation, leading to poor protein targeting to the cell membrane. This loss-of-function mutant indicates that the ECD loop III is required for CaSR activity.

Objective: We developed clinical practice guidelines for congenital adrenal hyperplasia (CAH)., Participants: The Task Force included a chair, selected by The Endocrine Society Clinical Guidelines Subcommittee (CGS), ten additional clinicians experienced in treating CAH, a methodologist, and a medical writer. Additional experts were also consulted. The authors received no corporate funding or remuneration., Consensus Process: Consensus was guided by systematic reviews of evidence and discussions. The guidelines were reviewed and approved sequentially by The Endocrine Society's CGS and Clinical Affairs Core Committee, members responding to a web posting, and The Endocrine Society Council. At each stage, the Task Force incorporated changes in response to written comments., Conclusions: We recommend universal newborn screening for severe steroid 21-hydroxylase deficiency followed by confirmatory tests. We recommend that prenatal treatment of CAH continue to be regarded as experimental. The diagnosis rests on clinical and hormonal data; genotyping is reserved for equivocal cases and genetic counseling. Glucocorticoid dosage should be minimized to avoid iatrogenic Cushing's syndrome. Mineralocorticoids and, in infants, supplemental sodium are recommended in classic CAH patients. We recommend against the routine use of experimental therapies to promote growth and delay puberty; we suggest patients avoid adrenalectomy. Surgical guidelines emphasize early single-stage genital repair for severely virilized girls, performed by experienced surgeons. Clinicians should consider patients' quality of life, consulting mental health professionals as appropriate. At the transition to adulthood, we recommend monitoring for potential complications of CAH. Finally, we recommend judicious use of medication during pregnancy and in symptomatic patients with nonclassic CAH.

Context: Nonclassic congenital lipoid adrenal hyperplasia (lipoid CAH) is a recently recognized disorder caused by mutations in the steroidogenic acute regulatory protein (StAR) that retain partial function. Affected individuals can present with a phenotype of late onset adrenal insufficiency with only mild or minimally disordered sexual development., Objectives: The aim was to delineate the clinical spectrum of StAR mutations and correlate phenotype with StAR activity., Patients: Four patients had nonclassic/atypical lipoid CAH. Adrenal insufficiency was manifested at birth in two patients and at 11 months and 4 yr in the other two. Three were 46,XY with underdeveloped genitalia., Methods: The StAR gene was sequenced, mutations were recreated in expression vectors, and StAR activity was measured as pregnenolone production in COS-1 cells cotransfected with the cholesterol side-chain cleavage system. StAR mutants were expressed as N-62 StAR in bacteria, and purified proteins were tested for activity with isolated steroidogenic mitochondria and for cholesterol-binding capacity., Results: DNA sequencing identified mutations on all alleles. Missense mutations were R188C, G221D, L260P, and F267S; we also tested R192C described by others. The respective activities of R188C, R192C, G221D, L260P, and F267S were 8.0, 39.4, 2.4, 3.1, and 6.1% of wild-type in transfected cells, and 12.8, 54.8, 6.3, 1.8, and 9.5% with isolated mitochondria. Cholesterol binding capacities of R188C, R192C, G221D, L260P, and F267S were 6.7, 55.3, 10.2, 4.6, and 20.9%. These data are correlated to the three-dimensional structure of StAR., Conclusions: There is a broad clinical spectrum of StAR mutations; StAR activities in vitro correlate well with clinical phenotypes.

Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity is cAMP-dependent serine/threonine phosphorylation of P450c17. Identifying the responsible kinase(s) and the P450c17 residues that undergo phosphorylation has been challenging, partly because of difficulties in preparing biochemically useful amounts of pure, catalytically active P450c17. We describe a modified strategy for preparing P450c17 in which the traditional carboxy-terminal 4xHis tag is replaced by 3xGly6xHis. This construct permits more rotational freedom of the protein when bound to the nickel affinity column, reducing steric associations between the protein and the column, and permitting a single-step chromatographic purification to apparent homogeneity. Using this vector, we explored P450c17 phosphorylation by mutagenesis of Ser and/or Thr residues to Asp or Glu to mimic the approximate size and charge of phospho-Ser or phospho-Thr. This strategy did not identify Ser and/or Thr site(s) that increase the ratio of lyase to hydroxylase activity, suggesting that the regulatory phosphorylation strategy of human P450c17 is very complicated. Although previous work has excluded protein kinase A (PKA) as the responsible kinase, the cAMP-inducible nature of the phosphorylation-associated increase in lyase activity suggests that PKA may play a role, possibly as a priming kinase. Using our novel vector and a series of mutations, we identified the P450c17 site phosphorylated by PKA as Ser258.

Patients with rare and complex diseases such as congenital adrenal hyperplasia (CAH) often receive fragmented and inadequate care unless efforts are coordinated among providers. Translating the concepts of the medical home and comprehensive health care for individuals with CAH offers many benefits for the affected individuals and their families. This manuscript represents the recommendations of a 1.5 day meeting held in September 2009 to discuss the ideal goals for comprehensive care centers for newborns, infants, children, adolescents, and adults with CAH. Participants included pediatric endocrinologists, internal medicine and reproductive endocrinologists, pediatric urologists, pediatric surgeons, psychologists, and pediatric endocrine nurse educators. One unique aspect of this meeting was the active participation of individuals personally affected by CAH as patients or parents of patients. Representatives of Health Research and Services Administration (HRSA), New York-Mid-Atlantic Consortium for Genetics and Newborn Screening Services (NYMAC), and National Newborn Screening and Genetics Resource Center (NNSGRC) also participated. Thus, this document should serve as a "roadmap" for the development phases of comprehensive care centers (CCC) for individuals and families affected by CAH.

Context: P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility., Objective: We describe four patients with POR deficiency and identify and characterize the activities of their mutations. A 46,XY male with micropenis and two 46,XX female infants with genital ambiguity presented with skeletal malformations, and a 46,XX adolescent presented with primary amenorrhea, elevated 17alpha-hydroxyprogesterone, and low sex steroids., Methods: The coding regions of the POR gene were sequenced, and the identified mutations were recreated in human POR cDNA expression vectors lacking 27 N-terminal residues. POR and human P450c17 were expressed in bacteria. POR activity was measured by four assays: reduction of cytochrome c, oxidation of reduced nicotinamide adenine dinucleotide phosphate, and support of the 17alpha-hydroxylase and 17,20 lyase activities of P450c17., Results: All four patients were compound heterozygotes for POR mutations, including five novel mutations: L577R, N185K, delE217, and frameshift mutations 1363delC and 697-698insGAAC. N185K and delE217 lacked measurable activity in the assays based on P450c17 but retained partial activity in the assays based on cytochrome c. As assessed by V(max)/Km, L577R supported 46% of 17alpha-hydroxylase activity but only 27% of 17,20 lyase activity. Computational modeling of these novel mutants revealed the structural basis for their reduced or absent activities., Conclusion: These patients illustrate the broad clinical spectrum of POR deficiency, including amenorrhea and infertility as the sole manifestation. POR assays based on P450c17 correlate well with hormonal and clinical phenotypes.

Context: Combined 17alpha-hydroxylase/17,20-lyase deficiency is a rare autosomal recessive form of congenital adrenal hyperplasia presenting with hypertension and sexual infantilism. This disorder is caused by defects in P450c17, encoded by the CYP17A1 gene., Objective: We describe a 14-yr-old female with clinical and hormonal features of 17alpha-hydroxylase/17,20-lyase deficiency and identify and characterize the activities of her CYP17A1 mutations., Methods: The coding regions of the CYP17A1 gene were amplified by PCR and sequenced. Mutations were recreated in P450c17 cDNA expression vectors; activities in transfected COS-1 cells were assayed by conversion of radiolabeled precursor steroids. One mutant was also expressed in Escherichia coli, and the reduced adsorption spectrum was measured., Results: The patient carried the previously described mutation R96W and the novel missense mutation H373D. Neither mutant had detectable activity when expressed in COS-1 cells. Membrane preparations from E. coli expressing the H373D mutant vector produced an absorption peak at 420 nm, whereas the wild-type produced a peak at 450 nm, suggesting that the H373D mutation interferes with protein folding., Conclusion: The novel P450c17 mutation H373D abolished enzyme activity because of protein misfolding. These data indicate an important role for this residue in P450c17 activity.

Catalysis by all 50 Type II (microsomal) P450 enzymes, including steroidogenic P450c17, P450c21, and P450aro and hepatic drug-metabolizing enzymes requires electron donation from P450 oxidoreductase (POR). POR knockout mice are embryonic lethal, but human POR mutations cause a complex disorder of steroidogenesis. Disorders of hepatic drug metabolism in human POR deficiency have not yet been described. To understand the potential contribution of POR to pharmacogenetics, we sequenced the POR gene in 842 normal persons from 4 ethnic groups. We detected 140 single nucleotide sequence variants of which 43 were in >1% of alleles, including 15 missense mutants; this brings the total of known POR missense mutants to 35. A503V was found on 28% of alleles, varying from 19% in African Americans to 37% in Chinese Americans. We expressed all 35 missense mutants in E. coli and assayed their activities to: oxidize NADPH, reduce cytochrome c, support the 17alpha-hydroxylase and 17,20 lyase activities of bacterially expressed human P450c17, and support the metabolism of fluorogenic EOMCC by bacterially expressed human CYP1A2 and CYP2C19. These data show that there are great differences in the activities of some POR mutants depending on the electron recipient assayed; for example, Q153R causes severely impaired steroid biosynthesis in human patients and in vitro, but is a gain-of-function mutant with CYP1A2 and 2C19. A503V reduces both activities of P450c17 in half, but had no effect on CYP1A2 or 2C19. POR variants are a previously unappreciated source of genetic variation in patterns of steroid synthesis and drug metabolism.

The enzymes and pathways of steroidogenesis are central to an understanding of adrenarche. The quantitative regulation of steroidogenesis occurs at the first step, the conversion of cholesterol to pregnenolone. Chronic quantitative regulation is principally at the level of transcription of the CYP11A1 gene encoding P450scc, which is the enzymatically rate-limiting step. Acute regulation is mediated by the steroidogenic acute regulatory protein (StAR), which facilitates the rapid influx of cholesterol into mitochondria, where P450scc resides. Qualitative regulation, which determines the type of steroid produced in a cell, is principally at the level of P450c17 (CYP17). In the absence of P450c17 in the zona glomerulosa, C21 deoxy steroids are produced, leading to the mineralocorticoid, aldosterone. In the presence of the 17alpha-hydroxylase but not the 17,20 lyase activity of P450c17 in the zona fasciculata, C21, 17-hydroxy steroids are produced, leading to the glucocorticoid, cortisol. When both the 17alpha-hydroxylase and 17,20 lyase activities of P450c17 are present in the zona reticularis, the androgen precursor DHEA is produced. The discrimination between 17alpha-hydroxylase and 17,20 lyase activities is regulated by two post-translational events, the serine phosphorylation of P450c17 and the allosteric action of cytochrome b5, both of which act to optimize the interaction of P450c17 with its obligatory electron donor, P450 oxidoreductase. In the adrenal zona reticularis, the abundant expression of P450 oxidoreductase and cytochrome b5, and the low expression of 3beta-hydroxysteroid dehydrogenase (HSD3B2) result in the production of the large amounts of DHEA that characterize adrenarche.

Context: 21-Hydroxylase deficiency (21OHD) is caused by CYP21A2 gene mutations disrupting the adrenal 21-hydroxylase, P450c21. CYP21A2 mutations generally correlate well with the 21OHD phenotype, but some children with severe CYP21A2 mutations have residual 21-hydroxylase activity. Some hepatic P450 enzymes can 21-hydroxylate progesterone, but their physiological relevance in modifying 21OHD is not known., Objective: We determined the ability of CYP2C19 and CYP3A4 to 21-hydroxylate progesterone and 17-hydroxyprogesterone (17OHP), determined the impact of the common P450 oxidoreductase (POR) variant A503V on these activities, and examined correlations between CYP2C19 variants and phenotype in patients with 21OHD., Methods: Bacterially expressed, N-terminally modified, C-His-tagged human P450c21, CYP2C19, and CYP3A4 were combined with bacterially expressed wild-type and A503V POR. The 21-hydroxylation of radiolabeled progesterone and 17OHP was assessed, and the Michaelis constant (Km) and maximum velocity (Vmax) of the reactions were measured. CYP2C19 was genotyped in 21OHD patients with genotypes predicting severe congenital adrenal hyperplasia., Results: Compared to P450c21, the Vmax/Km for 21-hydroxylation of progesterone by CYP2C19 and CYP3A4 were 17 and 10%, respectively. With both forms of POR, the Km for P450c21 was approximately 2.6 microm, the Km for CYP2C19 was approximately 11 microm, and the Km for CYP3A4 was approximately 110 microm. Neither CYP2C19 nor CYP3A4 could 21-hydroxylate 17OHP. The CYP2C19 ultrametabolizer allele CYP2C19 17 was homozygous in one of five patients with a 21OHD phenotype that was milder than predicted by the CYP21A2 genotype., Conclusions: CYP2C19 and CYP3A4 can 21-hydroxylate progesterone but not 17OHP, possibly ameliorating mineralocorticoid deficiency, but not glucocorticoid deficiency. Multiple enzymes probably contribute to extraadrenal 21-hydroxylation.

Context: Very few patients have been described with isolated 17,20-lyase deficiency who have had their mutations in P450c17 (17alpha-hydroxylase/17,20-lyase) proven by DNA sequencing and in vitro characterization of the mutations. Most patients with 17,20-lyase deficiency have mutations in the domain of P450c17 that interact with the electron-donating redox partner, P450 oxidoreductase (POR)., Objective: Our objective was to clarify the genetic and functional basis of isolated 17,20-lyase deficiency in familial cases who were previously reported as having 17,20-lyase deficiency., Patients: Four undervirilized males of an extended Bedouin family were investigated. One of these has previously been reported to carry mutations in the CYP17A1 gene encoding P450c17 causing isolated 17,20-lyase deficiency., Methods: Serum hormones were evaluated before and after stimulation with ACTH. Urinary steroid metabolites were profiled by gas chromatography-mass spectrometry. Exons 1 and 8 of CYP17A1 previously reported to harbor mutations in one of these patients and all 15 coding exons of POR were sequenced., Results: Gas chromatography-mass spectrometry (GC-MS) urinary steroid profiling and serum steroid measurements showed combined deficiencies of 17,20-lyase and 21-hydroxylase. Sequencing of exons 1 and 8 of CYP17A1 in two different laboratories showed no mutations. Sequencing of POR showed that all four patients were homozygous for G539R, a previously studied mutation that retains 46% of normal capacity to support the 17alpha-hydroxylase activity but only 8% of the 17,20-lyase activity of P450c17., Conclusion: POR deficiency can masquerade clinically as isolated 17,20-lyase deficiency.

Context: 21-hydroxylase deficiency (21OHD) is a common genetic disorder caused by mutations in the CYP21A2 gene, which encodes the adrenal 21-hydroxylase, microsomal P450c21. CYP21A2 gene mutations generally correlate well with impaired P450c21 enzymatic activity and the clinical findings in 21OHD, but occasional discrepancies between genotype and phenotype suggest the effects of modifier genes. Mutations in P450 oxidoreductase (POR), the protein that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate to all microsomal P450s, can ameliorate the 21OHD phenotype and, therefore, could be a modifier gene., Objectives: We sought to identify POR variants in patients with 21OHD having discordant phenotype and genotype, and to evaluate their effect on 21-hydroxylase activity., Patients and Methods: We determined the CYP21A2 genotypes of 313 Brazilian patients with 21OHD and correlated the genotype and phenotype. The POR gene was sequenced in 17 patients with discordant genotype and phenotype. Wild-type and A503V POR, and P450c21 were expressed in bacteria and reconstituted in vitro. Activities were assayed by conversion of [(14)C]progesterone to deoxycorticosterone and [(3)H]17-hydroxyprogesterone to 11-deoxycortisol, and assessed by thin layer chromatography and phosphorimaging., Results: The A503V POR variant was found in 10 of 30 alleles, the same ratio as in the normal population. There were no significant differences in Michaelis constant, maximum velocity and maximum velocity/Michaelis constant of 21-hydroxylase activity supported by wild-type and A503V POR., Conclusion: The only POR missense polymorphism found in atypical 21OHD patients was A503V. Although A503V reduces P450c17 enzymatic activity, it does not influence P450c21 activity, indicating that POR A503V does not modify the 21OHD phenotype.

Polycystic ovary syndrome (PCOS) is a common endocrinopathy affecting 4%-8% of reproductive-aged women. The syndrome is characterized by hyperandrogenemia and disordered gonadotropin secretion and is often associated with insulin resistance. However, rather than being one disease entity caused by a single molecular defect, PCOS under its current diagnostic criteria most likely includes a number of distinct disease processes with similar clinical phenotypes but different pathophysiologic mechanisms. The serine phosphorylation hypothesis can potentially explain two major features of PCOS--hyperandrogenemia and insulin resistance. Further defining the molecular mechanisms regulating androgen biosynthesis and insulin action in PCOS patients will permit a better understanding of the syndrome and may lead to the generation of novel specific pharmacologic therapies.

Cytochrome P450c17 (P450c17) is the single enzyme that catalyzes steroid 17alpha-hydroxylase and 17,20 lyase activities and hence is the crucial decision-making step that determines the class of steroid made in a steroidogenic cell. Although both activities are catalyzed on a single active site, the ratio of these activities is regulated by posttranslational events. Serine phosphorylation of P450c17 increases 17,20 lyase activity by increasing the enzyme's affinity for its redox partner, P450 oxidoreductase. We searched for the relevant kinase(s) that phosphorylates P450c17 by microarray studies and by testing of kinase inhibitors. Microarrays show that 145 of the 278 known serine/threonine kinases are expressed in human adrenal NCI-H295A cells, only six of which were induced more than 2-fold by treatment with 8-Br-cAMP. Key components of the ERK1/2 and MAPK/ERK kinase (MEK)1/2 pathways, which have been implicated in the insulin resistance of PCOS, were not found in NCI-H295A cells, implying that these pathways do not participate in P450c17 phosphorylation. Treatment with various kinase inhibitors that probe the protein kinase A/phosphatidylinositol 3-kinase/Akt pathway and the calcium/calmodulin/MAPK kinase pathway had no effect on the ratio of 17,20 lyase activity to 17alpha-hydroxylase activity, appearing to eliminate these pathways as candidates leading to the phosphorylation of P450c17. Two inhibitors that target the Rho-associated, coiled-coil containing protein kinase (ROCK)/Rho pathway suppressed 17,20 lyase activity and P450c17 phosphorylation, both in NCI-H295A cells and in COS-1 cells transfected with a P450c17 expression vector. ROCK1 phosphorylated P450c17 in vitro, but that phosphorylation did not affect 17,20 lyase activity. We conclude that members of the ROCK/Rho pathway act upstream from the kinase that phosphorylates P450c17 in a fashion that augments 17,20 lyase activity, possibly acting to catalyze a priming phosphorylation.

Context: Mitochondrial cytochrome P450scc converts cholesterol to pregnenolone in all steroidogenic tissues. Although progesterone production from the fetally-derived placenta is necessary to maintain pregnancy to term, four patients with mutations in the gene encoding P450scc (CYP11A1), have been described, one in a 46,XX female and three in underandrogenized 46,XY individuals, all with primary adrenal failure., Objective: Our aim was to determine whether P450scc mutations might be found in other children and to explore genotype/phenotype correlations., Methods and Patients: We performed mutational analysis of CYP11A1 in individuals with 46,XY disorders of sex development and primary adrenal failure, followed by functional studies of P450scc activity and of P450scc RNA splicing., Results: Among nine 46,XY infants with adrenal failure and disordered sexual differentiation, two infants had compound heterozygous mutations in CYP11A1. One patient harbored the novel P450scc missense mutations L141W and V415E, which retained 38 and 0% activity, respectively. The other carried a CYP11A1 frameshift mutation c835delA (0% activity) and a splice site mutation [IVS3+(2-3)insT] that prevented correct splicing of P450scc mRNA., Conclusions: P450scc deficiency is a recently recognized disorder that may be more frequent than originally thought. The phenotypic spectrum ranges from severe loss-of-function mutations associated with prematurity, complete underandrogenization, and severe, early-onset adrenal failure, to partial deficiencies found in children born at term with clitoromegaly and later-onset adrenal failure. In contradistinction to congenital lipoid adrenal hyperplasia caused by steroidogenic acute regulatory protein mutations, adrenal hyperplasia has not been reported in any of the six patients with P450scc deficiency.

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

P450 oxidoreductase (POR) deficiency is an autosomal recessive disorder of steroidogenesis with multiple clinical manifestations. POR is the electron donor for all microsomal P450 enzymes, including the three steroidogenic enzymes P450c17 (17alpha-hydroxylase/17,20-lyase), P450c21 (21-hydroxylase), and P450aro (aromatase). Since the first description of POR mutations in 2004, about 50 patients have been reported. Serum steroid profiles indicate partial deficiencies in 21-hydroxylase, 17alpha-hydroxylase and 17,20-lyase. The 17-OH progesterone levels are elevated, as in 21-hydroxylase deficiency, while androgen levels are low; cortisol may be normal but is poorly responsive to adrenocorticotropic hormone. Most patients also have associated skeletal malfor- mations (craniosynostosis, radio-ulnar synostosis, midface hypoplasia, bowed femora) termed Antley-Bixler syndrome. Antley-Bixler syndrome with normal steroidogenesis is caused by autosomal dominant gain-of-function mutations in fibroblast growth factor receptor 2. Males with POR deficiency are often undervirilized, while females can be virilized. The prognosis for patients with POR deficiency appears to depend on the severity of the bony malformations and their timely treatment. The potential impact of POR mutations on drug metabolism by other hepatic P450 enzymes requires further investigation. Given the varied physical and biochemical phenotype of POR deficiency and the risk of adrenal insufficiency, clinicians should be alert to this potential diagnosis., ((c) 2008 S. Karger AG, Basel)

The enzymes and pathways of steroidogenesis are familiar to most endocrinologists, but the biochemistry and molecular biology of these processes are still being studied. This chapter outlines current knowledge about each enzyme. The quantitative regulation of steroidogenesis occurs at the first step, the conversion of cholesterol to pregnenolone. Chronic regulation is principally at the level of transcription of the gene for P450 side chain cleave (P450scc), which is the enzymatically rate-limiting step. Acute regulation is mediated by steroidogenic acute regulatory protein, which facilitates the rapid influx of cholesterol into mitochondria, where P450scc resides. Qualitative regulation, determining the class of steroid produced, is principally determined by P450c17. In the absence of P450c17 in the zona glomerulosa, C21 deoxy steroids are produced, leading to the mineralocorticoid aldosterone. In the presence of the 17alpha-hydroxylase but not the 17,20 lyase activity of P450c17 in the zona fasciculata, C21, 17-hydroxy steroids are produced, leading to the glucocorticoid cortisol. When both the 17alpha-hydroxylase and 17,20 lyase activities of P450c17 are present in the zona reticularis, the androgen precursor dehydroepiandrosterone is produced. The discrimination between 17alpha-hydroxylase and 17,20 lyase activities is regulated by two posttranslational events, the serine phosphorylation of P450c17 and the allosteric action of cytochrome b5, both of which act to optimize the interaction of P450c17 with its obligatory electron donor, P450 oxidoreductase.

Context: Vitamin D 1alpha-hydroxylase deficiency, also known as vitamin D-dependent rickets type 1, is an autosomal recessive disorder characterized by the early onset of rickets with hypocalcemia and is caused by mutations of the 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha-hydroxylase, CYP27B1) gene. The human gene encoding the 1alpha-hydroxylase is 5 kb in length, located on chromosome 12, and comprises nine exons and eight introns. We previously isolated the human 1alpha-hydroxylase cDNA and gene and identified 19 different mutations in 25 patients with 1alpha-hydroxylase deficiency. OBJECTIVES, PATIENTS, AND METHODS: We analyzed the 1alpha-hydroxylase gene of 10 patients, five from Korea, two from the United States, and one each from Argentina, Denmark, and Morocco, all from nonconsanguineous families. Each had clinical and radiographic features of rickets, hypocalcemia, and low serum concentrations of 1,25-dihydroxyvitamin D(3)., Results: Direct sequencing identified the responsible 1alpha-hydroxylase gene mutations in 19 of 20 alleles. Four novel and four known mutations were identified. The new mutations included a nonsense mutation in exon 6, substitution of adenine for guanine (2561G-->A) creating a stop signal at codon 328; deletion of adenine in exon 9 (3922delA) causing a frameshift; substitution of thymine for cytosine in exon 2 (1031C-->T) causing the amino acid change P112L; and a splice site mutation, substitution of adenine for guanine in the first nucleotide of intron 7 (IVS7+1 G-->A) causing a frameshift., Conclusions: Mutations in the 1alpha-hydroxylase gene previously were identified in 44 patients, to which we add 10 more. The studies show a strong correlation between 1alpha-hydroxylase mutations and the clinical findings of 1alpha-hydroxylase deficiency.

Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.

Context: P450 oxidoreductase (POR) deficiency is a disorder of steroidogenesis affecting the microsomal P450 enzymes that use POR as an electron donor. The clinical presentation is variable; patients can be asymptomatic or can present with genital anomalies and the Antley-Bixler syndrome, characterized by craniosynostosis and other bony anomalies. Obligately heterozygous parents are normal. Combined POR and 21-hydroxylase deficiencies have not been reported., Objective: The aim was to explore the manifestations of combined deficiencies of 21-hydroxylase and POR and to search for lesions in apparent manifesting POR heterozygotes., Patients and Methods: A newborn female had craniosynostosis, severe salt wasting, minimal virilization, grossly elevated 17OH-progesterone, and minimally elevated androgens. DNA encoding 21-hydroxylase, POR, and fibroblast growth factor receptor 2 was sequenced. For POR, the first untranslated exon (exon 1U), 5' flanking DNA, and most introns were sequenced in five apparent manifesting POR heterozygotes., Results: CYP21B mutations were found on both alleles, proving classical 21-hydroxylase deficiency. Fibroblast growth factor receptor 2 exons 8 and 10 were normal. A POR mutation, A287P, was found only on the maternal allele. Five previously reported patients had POR mutations found on only one allele, but their clinical characteristics were indistinguishable from patients with mutations on both alleles. Sequencing of exon 1U, 274 bp of POR 5' flanking DNA, and 12 of the 15 POR introns did not identify additional mutations affecting gene expression or splicing., Conclusion: Manifesting heterozygosity is a possible feature of POR deficiency and may ameliorate the findings in coexisting 21-hydroxylase deficiency.

Steroidogenic acute regulatory protein (StAR) stimulates adrenal and gonadal steroidogenesis by increasing the influx of cholesterol into mitochondria, where it is converted to pregnenolone to initiate steroidogenesis. StAR acts on the outer mitochondrial membrane where each molecule stimulates the mitochondrial import of several hundred molecules of cholesterol, but the precise mechanism of the action of StAR remains uncertain. StAR has a sterol-binding pocket that can accommodate one molecule of cholesterol. Direct assays show that StAR can bind cholesterol with stoichiometry approaching 1:1, and several disease-causing mutants with decreased or absent activity have correspondingly decreased cholesterol binding. We show that the StAR mutant R182L, which causes severe disease and is devoid of measurable activity in transfected cells or with isolated steroidogenic mitochondria, nevertheless, can bind as much [(14)C]- or NBD-cholesterol as wild-type StAR under equilibrium conditions and can transfer cholesterol between liposomes in vitro. Similarly, the artificial mutant S195A had 46.5% of the activity of wild-type StAR but bound cholesterol indistinguishably from wild-type. Competition assays showed that the rate of binding (t((1/2)on)) for R182L was only 36% of the wild-type and the rate of dissociation (t((1/2)off)) was 57% of wild-type, whereas the t((1/2)on) and t((1/2)off) for S195A and S195D were essentially the same for wild-type. These data indicate that cholesterol binding and transfer activities are distinct from its activity to induce steroidogenesis. StAR appears to act by other mechanisms in addition to cholesterol binding.

Cholesterol is the starting point for biosynthesis of steroids, oxysterols and bile acids, and is also an essential component of cellular membranes. The mechanisms directing the intracellular trafficking of this insoluble molecule have received attention through the discovery of the steroidogenic acute regulatory protein (StAR) and related proteins containing StAR-related lipid transfer domains. Much of our understanding of the physiology of StAR derives from studies of congenital lipoid adrenal hyperplasia, which is caused by StAR mutations. Multiple lines of evidence show that StAR moves cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. The precise mechanism by which StAR's action on the outer mitochondrial membrane stimulates the flow of cholesterol to the inner membrane remains unclear. When StAR interacts with protonated phospholipid head groups on the outer mitochondrial membrane, it undergoes a conformational change (molten globule transition) that opens and closes StAR's cholesterol-binding pocket; this conformational change is required for cholesterol binding, which is required for StAR activity. The action of StAR probably requires interaction with the peripheral benzodiazepine receptor.

The steroidogenic acute regulatory protein (StAR) mediates the acute steroidogenic response by moving cholesterol from the outer to inner mitochondrial membrane, but the mechanism of StAR's action has remained mysterious. We showed that StAR acts on the outer membrane, requires cholesterol binding, and requires the structural change previously described as a pH-dependent molten globule. The current model is that StAR's interaction with protonated phospholipid head groups on the outer mitochondrial membrane induces a molten globule transition needed for StAR to take up cholesterol. Recent data suggest a functional interaction between StAR and the peripheral benzodiazepine receptor (PBR). Whereas many models have suggested that StAR delivers cholesterol to PBR, we suggest that StAR removes cholesterol from the cholesterol-binding domain of PBR and delivers it to the inner mitochondrial membrane.

Context: Lipoid congenital adrenal hyperplasia is a severe disorder of adrenal and gonadal steroidogenesis caused by mutations in the steroidogenic acute regulatory protein (StAR). Affected children typically present with life-threatening adrenal insufficiency in early infancy due to a failure of glucocorticoid (cortisol) and mineralocorticoid (aldosterone) biosynthesis, and 46,XY genetic males have complete lack of androgenization and appear phenotypically female due to impaired testicular androgen secretion in utero., Objective: The objective of this study was to investigate whether nonclassic forms of this condition exist., Patients and Methods: Sequence analysis of the gene encoding StAR was undertaken in three children from two families who presented with primary adrenal insufficiency at 2-4 yr of age; the males had normal genital development. Identified mutants were tested in a series of biochemical assays., Results: DNA sequencing identified homozygous StAR mutations Val187Met and Arg188Cys in these two families. Functional studies of StAR activity in cells and in vitro and cholesterol-binding assays showed these mutants retained approximately 20% of wild-type activity., Conclusions: These patients define a new disorder, nonclassic lipoid congenital adrenal hyperplasia, and represent a new cause of nonautoimmune Addison disease (primary adrenal failure).

Purpose of Review: P450 oxidoreductase deficiency--a newly described form of congenital adrenal hyperplasia--typically presents a steroid profile suggesting combined deficiencies of steroid 21-hydroxylase and 17alpha-hydroxylase/17,20-lyase activities. These and other enzymes require electron donation from P450 oxidoreductase. The clinical spectrum of P450 oxidoreductase deficiency ranges from severely affected children with ambiguous genitalia, adrenal insufficiency and the Antley-Bixler skeletal malformation syndrome to mildly affected individuals with polycystic ovary syndrome. We review current knowledge of P450 oxidoreductase deficiency and its broader implications., Recent Findings: Since the first report in 2004, at least 21 P450 oxidoreductase mutations have been reported in over 40 patients. The often subtle manifestations of P450 oxidoreductase deficiency suggest it may be relatively common. P450 oxidoreductase deficiency, with or without Antley-Bixler syndrome, is autosomal recessive, whereas Antley-Bixler syndrome without disordered steroidogenesis is caused by autosomal dominant fibroblast growth factor receptor 2 mutations. In-vitro assays of P450 oxidoreductase missense mutations based on P450 oxidoreductase-supported P450c17 activities provide excellent genotype/phenotype correlations. The causal connection between P450 oxidoreductase deficiency and disordered bone formation remains unclear., Summary: P450 oxidoreductase mutations cause combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase. Individuals with an Antley-Bixler syndrome-like phenotype presenting with sexual ambiguity or other abnormalities in steroidogenesis should be analyzed for P450 oxidoreductase deficiency.

The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal alpha-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM.

Microsomal P450 enzymes, which metabolize drugs and catalyze steroid biosynthesis require electron donation from NADPH via P450 oxidoreductase (POR). POR knockout mice are embryonically lethal, but we found recessive human POR missense mutations causing disordered steroidogenesis and Antley-Bixler syndrome (ABS), a skeletal malformation syndrome featuring craniosynostosis. Dominant mutations in exons 8 and 10 of fibroblast growth factor receptor 2 (FGFR2) cause phenotypically related craniosynostosis syndromes and were reported in patients with ABS and normal steroidogenesis. Sequencing POR and FGFR2 exons in 32 patients with ABS and/or hormonal findings suggesting POR deficiency showed complete genetic segregation of POR and FGFR2 mutations. Fifteen patients carried POR mutations on both alleles, four carried POR mutations on 1 allele, nine carried FGFR2/3 mutations on one allele and no mutation was found in three patients. The 34 affected POR alleles included 10 with A287P, 7 with R457H, 9 other missense mutations and 7 frameshifts. These 11 missense mutations and 10 others identified by database mining were expressed in E. coli, purified to apparent homogeneity, and their catalytic capacities were measured in four assays: reduction of cytochrome c, oxidation of NADPH, and support of the 17alpha-hydroxylase and 17,20 lyase activities of human P450c17. As assessed by Vmax/Km, 17,20 lyase activity provided the best correlation with clinical findings. Modeling human POR on the X-ray crystal structure of rat POR shows that these mutant activities correlate well with their locations in the structure. POR deficiency is a new disease, distinct from the craniosynostosis syndromes caused by FGFR mutations.

Sex steroid synthesis requires the 17,20 lyase activity of P450c17, which is enhanced by cytochrome b5, acting as an allosteric factor to promote association of P450c17 with its electron donor, P450 oxidoreductase. Cytochrome b5 is preferentially expressed in the fetal adrenal and postadrenarchal adrenal zona reticularis; the basis of this tissue-specific, developmentally regulated transcription of the b5 gene is unknown. We found b5 expression in all cell lines tested, including human adrenal NCI-H295A cells, where its mRNA is reduced by cAMP and phorbol ester. Multiple sites, between -83 and -122 bp upstream from the first ATG, initiate transcription. Deletional mutagenesis localized all detectable promoter activity within -327/+15, and deoxyribonuclease I footprinting identified protein binding at -72/-107 and -157/-197. DNA segments -65/-40, -114/-70 and -270/-245 fused to TK32/Luc yielded significant activity, and mutations in their Sp sites abolished that activity; electrophoretic mobility shift assay (EMSA) showed that Sp3, but not Sp1, binds to these Sp sites. Nuclear factor 1 (NF-1) and GATA-6, but not GATA-4 bind to the NF-1 and GATA sites in -157/-197. In Drosophila S2 cells, Sp3 increased -327/Luc activity 58-fold, but Sp1 and NF-1 isoforms were inactive. Mutating the three Sp sites ablated activity without or with cotransfection of Sp1/Sp3. In NCI-H295A cells, mutating the three Sp sites reduced activity to 39%; mutating the Sp, GATA, and NF-1 sites abolished activity. In JEG-3 cells, GATA-4 was inactive, GATA-6 augmented -327/Luc activity to 231% over the control, and steroidogenic factor 1 augmented activity to 655% over the control; these activities required the Sp and NF-1 sites. Transcription of cytochrome b5 shares many features with the regulation of P450c17, whose activity it enhances.

Cytochrome P450 enzymes catalyze the degradation of drugs and xenobiotics, but also catalyze a wide variety of biosynthetic processes, including most steps in steroidogenesis. The catalytic rate of a P450 enzyme is determined in large part by the rate of electron transfer from its redox partners. Type I P450 enzymes, found in mitochondria, receive electrons from reduced nicotinamide adenine dinucleotide (NADPH) via the intermediacy of two proteins-ferredoxin reductase (a flavoprotein) and ferredoxin (an iron/sulfur protein). Type I P450 enzymes include the cholesterol side-chain cleavage enzyme (P450scc), the two isozymes of 11-hydroxylase (P450c11beta and P450c11AS), and several vitamin D-metabolizing enzymes. Disorders of these enzymes, but not of the two redox partners, have been described. Type II P450 enzymes, found in the endoplasmic reticulum, receive electrons from NADPH via P450 oxidoreductase (POR), which contains two flavin moieties. Steroidogenic Type II P450 enzymes include 17alpha-hydroxylase/17,20 lyase (P450c17), 21-hydroxylase (P450c21), and aromatase (P450aro). All P450 enzymes catalyze multiple reactions, but P450c17 appears to be unique in that the ratio of its activities is regulated at a posttranslational level. Three factors can increase the degree of 17,20 lyase activity relative to the 17alpha-hydroxylase activity by increasing electron flow from POR: a high molar ratio of POR to P450c17, serine phosphorylation of P450c17, and the presence of cytochrome b(5), acting as an allosteric factor to promote the interaction of POR with P450c17. POR is required for the activity of all 50 human Type II P450 enzymes, and ablation of the Por gene in mice causes embryonic lethality. Nevertheless, mutation of the human POR gene is compatible with life, causing multiple steroidogenic defects and a skeletal dysplasia called Antley-Bixler syndrome.

The cholesterol side-chain cleavage enzyme, P450scc, initiates biosynthesis of all steroid hormones. Adrenal and gonadal P450scc expression requires steroidogenic factor-1 (SF1), but P450scc expression in human placental JEG-3 cells utilizes an SF1-independent element at -155/-131 that is inactive in adrenals and gonads. We previously cloned two transcription factors, long terminal repeat binding protein (LBP)-1b and LBP-9, from JEG-3 cells. In transient transfection assays, LBP-1b activated the -155/-131 element whereas LBP-9 suppressed its LBP-1b-stimulated expression. To assess the roles of these factors on the intact P450scc gene, we stably expressed LBP-1b or LBP-9 in JEG-3 cells. All cell lines stably expressing a fusion protein of LBP-1b and enhanced green fluorescent protein increased P450scc expression, but cell lines stably expressing LBP-9 fused to enhanced green fluorescent protein either increased or decreased P450scc expression. 8-Br-cAMP induced endogenous LBP-9, but not LBP-1b expression. Glutathione-S-transferase pull-down assays showed that LBP-1b and LBP-9 can dimerize with themselves and with each other; LBP-1b residues 300-540 and LBP-9 residues 300-479 were required for dimer formation. Glutathione-S-transferase pull-down assays, bandshifts, and transient transfection assays showed that TReP-132 (another factor that can bind to -155/-131) does not interact with either LBP-1b or LBP-9, or influence their ability to induce or suppress transcription from the -155/-131 element. Gal4 transactivation assays showed that transcriptional repression activity by LBP-9 requires residues 100-200. RNAi interference of either LBP-1b or LBP-9 mRNAs decreased P450scc expression. LBP-1b is an important SF1-independent transcriptional activator stimulating P450scc expression in human placental JEG-3 cells, whereas LBP-9 modulates the action of LBP-1b, exerting both positive and negative effects.

Hormone-induced steroid biosynthesis begins with the transfer of cholesterol from intracellular stores into mitochondria. Steroidogenic acute regulatory protein (StAR) and peripheral-type benzodiazepine receptor (PBR) have been implicated in this rate-determining step of steroidogenesis. MA-10 mouse Leydig tumor cells were treated with and without oligodeoxynucleotides (ODNs) antisense to PBR and StAR followed by treatment with saturating concentrations of human choriogonadotropin. Treatment with ODNs antisense but not missense for both proteins inhibited the respective protein expression and the ability of the cells to synthesize steroids in response to human choriogonadotropin. Treatment of the cells with either ODNs antisense to PBR or a transducible peptide antagonist to PBR resulted in inhibition of the accumulation of the mature mitochondrial 30-kDa StAR protein, suggesting that the presence of PBR is required for StAR import into mitochondria. Addition of in vitro transcribed/translated 37-kDa StAR or a fusion protein of Tom20 (translocase of outer membrane) and StAR (Tom/StAR) to mitochondria isolated from control cells increased pregnenolone formation. Mitochondria isolated from cells treated with ODNs antisense, but not missense, to PBR failed to form pregnenolone and respond to either StAR or Tom/StAR proteins. Reincorporation of in vitro transcribed/translated PBR, but not PBR missing the cholesterol-binding domain, into MA-10 mitochondria rescued the ability of the mitochondria to form steroids and the ability of the mitochondria to respond to StAR and Tom/StAR proteins. These data suggest that both StAR and PBR proteins are indispensable elements of the steroidogenic machinery and function in a coordinated manner to transfer cholesterol into mitochondria.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH, eventually destroying all adrenal and gonadal steroidogenesis. Lipoid CAH is caused by mutations in the steroidogenic acute regulatory protein (StAR), which facilitates the entry of cholesterol into mitochondria to initiate steroidogenesis. Patients with lipoid CAH typically present with a salt-losing crisis in the first 2 months of life, although presentation as late as 10 months with partial retention of StAR activity has been reported. We describe eight patients from six Saudi Arabian families who were first diagnosed at 1-14 months of age (median, 4-7 months; mean, 7 months). Five patients were 46,XY, and three were 46,XX. At presentation, all had hyponatremia, hyperkalemia, elevated ACTH, and low cortisol. Pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, testosterone, androstenedione, and dehydroepiandrosterone sulfate were all low in those patients in whom it was measured. DNA sequencing showed that one patient was homozygous for the StAR mutation M144R, and the other seven, from five apparently unrelated families, were homozygous for the StAR mutation R182H. Each mutation was recreated in a human StAR cDNA expression vector and found to be wholly inactive in a standard assay of COS-1 cells cotransfected with the cholesterol side-chain cleavage enzyme system. Thus, the loss of all assayable activity in vitro correlated poorly with the later onset of clinical symptoms in these patients. Lipoid CAH may present much later in life than previously thought.

Androgens and estrogens are primarily made from dehydroepiandrosterone (DHEA), which is made from cholesterol via four steps. First, cholesterol enters the mitochondria with the assistance of the steroidogenic acute regulatory protein (StAR). Mutations in the StAR gene cause congenital lipoid adrenal hyperplasia (lipoid CAH), a potentially lethal disease in which virtually no steroids are made. Lipoid CAH is common among Palestinian Arabs and people from eastern Arabia, and among Korean and Japanese people. Second, within the mitochondria, cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc; disorder of this enzyme is very rare, probably due to embryonic lethality. Third, pregnenolone undergoes 17alpha-hydroxylation by microsomal P450c17. 17alpha-Hydroxylase deficiency, manifesting as female sexual infantilism and hypertension, is rare except in Brazil. Finally, 17-OH pregnenolone is converted to DHEA by the 17,20 lyase activity of P450c17. The ratio of the 17,20 lyase to 17alpha-hydroxylase activity of P450c17 determines the ratio of C21 to C19 steroids produced. This ratio is regulated posttranslationally by at least three factors: the abundance of the electron-donating protein P450 oxidoreductase (POR), the presence of cytochrome b5 and the serine phosphorylation of P450c17. Mutations of POR are a new, recently described disorder manifesting as the Antley-Bixler skeletal dysplasia syndrome, and a form of polycystic ovary syndrome., (Copyright 2005 S. Karger AG, Basel)

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase is well-described, but patients' genes for these enzymes lack mutations. Recent work has identified mutations in the gene for P450 oxidoreductase (POR) in such patients. POR-deficient individuals have a broad range of disorders, from infants with congenital malformations to women with the polycysic ovary syndrome. POR transfers electrons to all microsomal P450 enzymes: its deficiency affects steroidogenesis, drug metabolism and other processes.

Objective: Adrenal crisis from salt-losing congenital adrenal hyperplasia (CAH) typically occurs in the first 2 weeks of life. We evaluated 3 infants with adrenal crisis who presented at 6 to 8 months of age with near-miss sudden infant death syndrome (SIDS)., Subjects: Three 46,XY phenotypic female infants presented near death at 6 to 8 months of age with adrenal crisis and unmeasurable steroid hormones consistent with congenital lipoid adrenal hyperplasia (lipoid CAH)., Methods: We sequenced genes potentially causing this phenotype: steroidogenic acute regulatory protein (StAR), the cholesterol side-chain cleavage enzyme, adrenodoxin reductase, adrenodoxin, and steroidogenic factor 1 (SF1). Site-directed mutagenesis and functional assays were performed for the missense mutation., Results: Hormonal values showed complete absence of adrenal and gonadal steroids. Patient 1 was a compound heterozygote for missense mutation R140P and an mRNA splice donor site mutation in the StAR gene. The R140P mutation was wholly inactive in vitro. Patient 2 was homozygous for a 7 base pair StAR deletion causing a frameshift. No mutations were found in Patient 3, suggesting a novel disease., Conclusions: Although genetic disorders of steroidogenesis typically present in the first month of life, some defects, especially those in StAR, can present in mid-infancy, when adrenal hyperplasias are rarely considered. Adrenal insufficiency is a subtle disorder that may cause cardiovascular collapse, causing unexplained infant death that resembles SIDS.