Dehydroepiandrosterone (DHEA) sulfotransferase, known as SULT2A1, converts the androgen precursor DHEA to its inactive sulfate ester, DHEAS [corrected], thereby preventing the conversion of DHEA to an active androgen. SULT2A1 requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) for catalytic activity. We have identified compound heterozygous mutations in the gene encoding human PAPS synthase 2 (PAPSS2) in a girl with premature pubarche, hyperandrogenic anovulation, very low DHEAS levels, and increased androgen levels. In vitro coincubation of human SULT2A1 and wild-type or mutant PAPSS2 proteins confirmed the inactivating nature of the mutations. These observations indicate that PAPSS2 deficiency is a monogenic adrenocortical cause of androgen excess.

Context:P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available.Objective:The objective of the study was to establish genotype-phenotype correlations in a large PORD cohort.Design:The design of the study was the clinical, biochemical, and genetic assessment including multiplex ligation-dependent probe amplification (MLPA) in 30 PORD patients from 11 countries.Results:We identified 23 P450 oxidoreductase (POR) mutations (14 novel) including an exonic deletion and a partial duplication detected by MLPA. Only 22% of unrelated patients carried homozygous POR mutations. p.A287P was the most common mutation (43% of unrelated alleles); no other hot spot was identified. Urinary steroid profiling showed characteristic PORD metabolomes with variable impairment of 17α-hydroxylase and 21-hydroxylase. Short cosyntropin testing revealed adrenal insufficiency in 89%. DSD was present in 15 of 18 46,XX and seven of 12 46,XY individuals. Homozygosity for p.A287P was invariably associated with 46,XX DSD but normal genitalia in 46,XY individuals. The majority of patients with mild to moderate skeletal malformations, assessed by a novel scoring system, were compound heterozygous for missense mutations, whereas nearly all patients with severe malformations carried a major loss-of-function defect on one of the affected alleles.Conclusions:We report clinical, biochemical, and genetic findings in a large PORD cohort and show that MLPA is a useful addition to POR mutation analysis. Homozygosity for the most frequent mutation in Caucasians, p.A287P, allows for prediction of genital phenotype and moderate malformations. Adrenal insufficiency is frequent, easily overlooked, but readily detected by cosyntropin testing.

SummaryCongenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders, which are usually due to inactivating mutations in single enzymes involved in adrenal steroid biosynthesis. The characteristics of the biochemical and clinical phenotype depend on the specific enzymatic defect. In 21-hydroxylase and 11 β -hydroxylase deficiency only adrenal steroidogenesis is affected, whereas a defect in 3 β -hydroxysteroid dehydrogenase or 17 α -hydroxylase also involves gonadal steroid biosynthesis. Recently, mutations in the electron donor enzyme P450 oxidoreductase were identified as the cause of CAH with apparent combined 17 α -hydroxylase and 21-hydroxylase deficiency, thereby illustrating the impact of redox regulation enzymes on steroidogenesis. P450 oxidoreductase deficiency (ORD) has a complex phenotype including two unique features not observed in any other CAH variant, skeletal malformations and severe genital ambiguity in both sexes. Despite invariably low circulating androgens, females with ORD may present with virilized genitalia and mothers may suffer from virilization during pregnancy. This apparently contradictory finding may be explained by the existence of an alternative pathway in human androgen biosynthesis, with important implications for physiology and pathophysiology. This review discusses the biochemical and clinical presentation and the genetic and functional basis of the currently known CAH variants, with a specific focus on ORD.

Adrenarche reflects the maturation of the adrenal zona reticularis resulting in increased secretion of the adrenal androgen precursor DHEA and its sulphate ester DHEAS. Premature adrenarche (PA) is defined by increased levels of DHEA and DHEAS before the age of 8 years in girls and 9 years in boys and the concurrent presence of signs of androgen action including adult-type body odour, oily skin and hair and pubic hair growth. PA is distinct from precocious puberty, which manifests with the development of secondary sexual characteristics including testicular growth and breast development. Idiopathic PA (IPA) has long been considered an extreme of normal variation, but emerging evidence links IPA to an increased risk of developing the metabolic syndrome (MS) and thus ultimately cardiovascular morbidity. Areas of controversy include the question whether IPA in girls is associated with a higher rate of progression to the polycystic ovary syndrome (PCOS) and whether low birth weight increases the risk of developing IPA. The recent discoveries of two novel monogenic causes of early onset androgen excess, apparent cortisone reductase deficiency and apparent DHEA sulphotransferase deficiency, support the notion that PA may represent a forerunner condition for PCOS. Future research including carefully designed longitudinal studies is required to address the apparent link between early onset androgen excess and the development of insulin resistance and the MS.

Context:Isolated 17,20 lyase deficiency is commonly defined by apparently normal 17α-hydroxylase activity but severely reduced 17,20 lyase activity of the bifunctional enzyme cytochrome P450 (CYP) enzyme 17A1 (CYP17A1), resulting in sex steroid deficiency but normal glucocorticoid and mineralocorticoid reserve. Cytochrome b5 (CYB5A) is thought to selectively enhance 17,20 lyase activity by facilitating the allosteric interaction of CYP17A1 with its electron donor P450 oxidoreductase (POR).Objective:We investigated a large consanguineous family including three siblings with 46,XY disorder of sex development (DSD) presenting with isolated 17,20 lyase deficiency.Design:We investigated the clinical and biochemical phenotype, conducted genetic analyses, and functionally characterized the identified CYB5A mutation in cell-based CYP17A1 coexpression assays.Results:All three siblings presented with 46,XY DSD, sex steroid deficiency, normal mineralocorticoids and glucocorticoids, and a urine steroid metabolome suggestive of isolated 17,20 lyase deficiency. CYP17A1 and POR sequences were normal, but we detected a homozygous CYB5A missense mutation (g.28,400A→T; p.H44L). Functional in vitro analysis revealed normal CYP17A1 17α-hydroxylase activity but severely impaired 17,20 lyase activity. In silico analysis suggested the disruption of CYB5A heme binding by p.H44L.Conclusion:We have identified the first human CYB5A missense mutation as the cause of isolated 17,20 lyase deficiency in three individuals with 46,XY DSD. Detailed review of previously reported cases with apparently isolated 17,20 lyase deficiency due to mutant CYP17A1 and POR reveals impaired 17α-hydroxylase activity as assessed by steroid metabolome analysis and short cosyntropin testing. This suggests that truly isolated 17,20 lyase deficiency is observed only in individuals with inactivating CYB5A mutations.

In addition to nonclassic 21-hydroxylase deficiency and steroid-secreting tumors, nonclassic 11OHD should be considered as an important differential diagnosis in patients with unexplained hyperandrogenism without 46,XX disorder of sex development. Nonclassic 11OHD is likely to be missed when relying on measuring standard steroid hormone panels. This diagnosis needs to be established early in life to avoid long-term health problems such as short stature, hyperandrogenism-related metabolic complications, potentially severe arterial hypertension, and cardiovascular consequences.

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.

Androgen biosynthesis in the human fetus proceeds through the adrenal sex steroid precursor dehydroepiandrosterone, which is converted to testosterone in the gonads, followed by further activation to 5α-dihydrotestosterone in genital skin, thereby facilitating male external genital differentiation. Congenital adrenal hyperplasia due to P450 oxidoreductase deficiency results in disrupted dehydroepiandrosterone biosynthesis, explaining undervirilization in affected boys. However, many affected girls are born virilized, despite low circulating androgens. We hypothesized that this is due to a prenatally active, alternative androgen biosynthesis pathway from 17α-hydroxyprogesterone to 5α-dihydrotestosterone, which bypasses dehydroepiandrosterone and testosterone, with increased activity in congenital adrenal hyperplasia variants associated with 17α-hydroxyprogesterone accumulation. Here we employ explant cultures of human fetal organs (adrenals, gonads, genital skin) from the major period of sexual differentiation and show that alternative pathway androgen biosynthesis is active in the fetus, as assessed by liquid chromatography–tandem mass spectrometry. We found androgen receptor expression in male and female genital skin using immunohistochemistry and demonstrated that both 5α-dihydrotestosterone and adrenal explant culture supernatant induce nuclear translocation of the androgen receptor in female genital skin primary cultures. Analyzing urinary steroid excretion by gas chromatography–mass spectrometry, we show that neonates with P450 oxidoreductase deficiency produce androgens through the alternative androgen pathway during the first weeks of life. We provide quantitative in vitro evidence that the corresponding P450 oxidoreductase mutations predominantly support alternative pathway androgen biosynthesis. These results indicate a key role of alternative pathway androgen biosynthesis in the prenatal virilization of girls affected by congenital adrenal hyperplasia due to P450 oxidoreductase deficiency.