Congenital adrenal hyperplasia owing to 3 beta-hydroxysteroid dehydrogenase deficiency.

Recent advances in the genetics of the family of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) enzymes have helped in the understanding of the molecular basis and hormonal phenotype of bona fide 3 beta-HSD deficiency congenital adrenal hyperplasia (CAH). This article revisits the clinical spectra of 3 beta-HSD deficiency disorders, pathophysiology of 3 beta-HSD deficiency CAH, and updates genotype findings and diagnostic hormonal criteria for bona fide classic and nonclassic 3 beta-HSD deficiency CAH. The delta-5 steroid abnormality for the nonclassic 3 beta-HSD deficiency CAH, proven by genotype study, is substantially greater than the hormonal criteria for the disorder published before the advent of molecular information on the gene encoding adrenals and gonads in humans. In hyperandrogenic children and women, the pathogenic mechanism of a subtle abnormality in adrenal 3 beta-HSD activity, determined by modestly elevated ACTH stimulated delta-5 steroid levels, which led to the diagnosis of mild nonclassic 3 beta-HSD deficiency in the past, is outside of the type II 3 beta-HSD gene which encodes adrenals and gonads in humans and remains to be further explored.     

Late-onset adrenal steroid 3 beta-hydroxysteroid dehydrogenase deficiency. I. A cause of hirsutism in pubertal and postpubertal women

To investigate the adrenal cause of hyperandrogenism in peri- and postpubertal hirsute women, baseline and ACTH-stimulated serum concentrations of delta 5-17-hydroxypregnenolone (delta 5-17P), dehydroepiandrosterone (DHEA) and its sulfate, 17-hydroxyprogesterone (17-OHP), cortisol, delta 4-androstenedione, and testosterone were determined in 116 women with hirsutism or acne of peri- and postpubertal onset with or without menstrual abnormalities. The results were compared with the same steroid concentrations in 30 normal age-matched women. Sixteen of the 116 women with hirsutism whose ACTH-stimulated 17-OHP levels (mean +/- SD, 5404 +/- 3234 ng/dl; normal, 334 +/- 194) were markedly elevated while their ratios of delta 5-17P to 17-OHP (0.4 +/- 0.2; normal, 3.4 +/- 1.5) were low were diagnosed as having nonclassical symptomatic 21-hydroxylase deficiency. Seventeen other hirsute women, including 3 siblings, had very high responses of delta 5-17P (2276 +/- 669 ng/dl; normal, 985 +/- 327) and DHEA (2787 +/- 386 ng/dl; normal, 1050 +/- 384) to ACTH stimulation, with significantly elevated ratios of delta 5-17P to 17-OHP (11 +/- 2.0; normal, 3.4 +/- 1.5) and DHEA to delta 4-androstenedione (7.5 +/- 2.3; normal, 4.6 +/- 1.5). In these hirsute women, the morning serum delta 5-17P and DHEA concentrations were elevated, had a diurnal variation, and were suppressed with dexamethasone administration. We propose that partial adrenal 3 beta-hydroxysteroid dehydrogenase deficiency is the cause of hirsutism in these women. This may represent an allelic variant at the genetic locus for 3 beta-hydroxysteroid dehydrogenase deficiency similar to that reported for symptomatic nonclassical 21-hydroxylase deficiency producing peripubertal excess androgen syndrome.     

Congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase deficiency

Deficiency of 3beta-hydroxysteroid dehydrogenase belongs to less frequent types of congenital adrenal hyperplasia. It is a deficiency of the enzyme converting pregnenolon to progesteron, 17-OH-pregnenolon to 17-OH-progesteron and dehydroepiandrosteron to androstendion in the adrenal glands and gonads. Exact prevalence of forms with clinical symptoms is known; however, it is less frequent compared to deficiency of 21-hydroxylase. Clinical manifestation is very variable, from serious salt disorder, ambiguous genitalia and precocious puberty to oligosymptomatic forms with late effects or asymptomatic forms. No routine genetic analysis in Czech republic is available. Diagnosis can be suspected through increased levels of some steroid hormones in basal condition; the final determinent indicator is the concentration of steroid hormones after stimulation by adrenocorticotropic hormone.     

Steroid inhibitory effects upon human adrenal 3 beta-hydroxysteroid dehydrogenase activity

The inhibitory effects of varying concentrations of steroids upon 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta-HSD) kinetics were studied in human adrenal microsomes. Each enzyme assay was conducted in triplicate at five different concentrations of three substrates (dehydroepiandrosterone, pregnenolone, and 17OH-pregnenolone), using microsomes from at least three donors. Each steroid was screened for possible inhibition at concentrations of 10(-8) and 10(-6) M and then studied in more detail at five different concentrations. The type of inhibition and the inhibition constant (Ki) were determined by analysis of Lineweaver-Burk and Dixon plots, together with replots of the slopes from the Dixon plots. The mean Km (Michaelis-Menten constant) for the three substrates was 0.42 +/- 0.04 (SE) mumol/liter (n = 73). Each steroid tested, including delta 5-3 beta-hydroxysteroids, estrogens, and several delta 4-3-ketosteroids, with the exception of cortisol, caused significant inhibition of 3 beta-HSD activity, and in each case the steroid appeared to behave as a competitive inhibitor. In most cases the Ki value was approximately 10(-7) M. At micromolar concentrations several steroids, notably estrone and estradiol, caused almost total inhibition of adrenal 3 beta-HSD activity. Comparison of the calculated Ki values with available data concerning changes in intra-adrenal steroid concentrations during childhood suggests that these changes would be sufficient to cause a relative decline in 3 beta-HSD activity during adrenarche. Although postnatal circulating steroid concentrations would appear to be insufficient to influence adrenal steroidogenesis, the high serum levels of placental steroids during fetal life would be expected to cause marked 3 beta-HSD inhibition.     

Kinetic analysis of adrenal 3 beta-hydroxysteroid dehydrogenase activity during human development

Kinetic analyses of microsomal 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity in adrenal glands from 11 individuals, aged 1-60 yr, were carried out to determine whether changes in substrate or cofactor affinity (Km) or cellular content, as reflected in maximal velocity, could explain the changes in adrenal delta 5-3 beta-hydroxysteroid secretion that occur in late childhood and puberty. The Km values for the cofactor NAD+ were similar regardless which substrate, dehydroepiandrosterone (DHA), pregnenolone, or 17-hydroxypregnenolone (17OH-delta 5P), was used. The Km values for DHA (0.3 microM), pregnenolone (0.4 microM), and 17OH-delta 5P (0.3 microM) were similar and within the intraadrenal concentration ranges for DHA and 17OH-delta 5P previously reported. Each substrate was a competitive inhibitor for the others, with close similarity between affinity and inhibition constants. These observations point to the presence of a single 3 beta-HSD, rather than several substrate-specific variants. There was no change in substrate Km with age; the maximal velocity was lower (0.1-0.6 nmol/mg X min) in a single 1-yr-old infant than in later life, but there was no significant change (mean, 2.9-4.6 nmol/mg X min for the three substrates) between values at 12 and 60 yr. This suggests that ACTH-mediated induction of 3 beta-HSD may be low in infancy and higher in adults, while in vivo studies point to a reduction in actual 3 beta-HSD activity during this period. The likely explanation for this paradox between enzyme levels and final activity is that 3 beta-HSD is progressively inhibited during late childhood and puberty by rising intraadrenal concentrations of various delta 4-3-ketosteroids.     

Carboxyl-terminal mutations in 3beta-hydroxysteroid dehydrogenase type II cause severe salt-wasting congenital adrenal hyperplasia

INTRODUCTION: 3beta-Hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3beta-HSD protein is not known. OBJECTIVE: We studied the functional consequences of three novel C-terminal mutations in the 3beta-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3beta-HSD type II deficiency showing different degrees of under-virilization. METHODS AND RESULTS: In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3beta-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity. CONCLUSIONS: Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3beta-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3beta-HSD deficiency cannot be predicted from in vitro 3beta-HSD function alone.     

Ontogeny of 3beta-hydroxysteroid dehydrogenase expression in the rat adrenal gland as studied by immunohistochemistry

The enzyme 3beta-hydroxysteroid dehydrogenase plays a crucial role in the steroidogenic process in the adrenal gland. In the present study we tried to characterize its localization and developmental changes in the rat adrenal cortex during the postnatal period, using immunohistochemical methods. The development of the different zones evidenced specific particularities: the zona glomerulosa almost lacked 3beta-HSD in the first days after birth; then, 3beta-HSD increased, attaining a maximum around day 20 and afterwards it decreased again and remained less intense than the neighbouring zona fasciculata up until adulthood (65 days of age). The zona fasciculata was already intensely stained at birth and the expression of 3beta-HSD increased rapidly reaching a maximum after 2 weeks of life and that level was maintained from then on. The inner part of the zona fasciculata and the zona reticularis both of which develop postnatally were faintly immunostained before day 20. The expression of 3beta-HSD increased after that age to become approximately as intense as in the outer zona fasciculata and so remaining until day 90. The development of the zona glomerulosa was parallel to the secretion of aldosterone. The same did not occur with the zona fasciculata as the intensity of staining during the first 14 postnatal days was accompanied by very low levels of corticosterone.     

Effect of aging and adrenocorticotropin on adrenal delta 5-3 beta-hydroxysteroid dehydrogenase activity in male rats

Adrenal delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity was determined in male rate 4, 6, 12, 18 and 24 months of age. Mean (+/- SE) adrenal 3 beta-HSD concentration (microgram delta 4-androstenedione formed/minute/mg tissue), specific activity (microgram/minute/mg protein) and total content (microgram/minute/pair of adrenals) were less (p less than 0.001 to p less than 0.025) in male rats 12 months of age (0.222 +/- 0.010, 1.66 +/- 0.09 and 8.6 +/- 0.8, respectively) or older, than in males four months of age (0.372 +/- 0.011, 2.69 +/- 0.07 and 13.4 +/- 1.1, respectively). Subcutaneous administration of 10 IU adrenocorticotropin daily for a period of five days to male rats 24 months of age elevated adrenal weight by 50 percent and restored dehydrogenase activity to that of the young untreated animal. Therefore, adrenal function in male rats as determined by 3 beta-HSD activity declines with advancing age, but remains responsive to adrenocorticotropic stimulation.     

Nonsalt-losing congenital adrenal hyperplasia due to 3 beta-hydroxysteroid dehydrogenase deficiency with normal glomerulosa function

In studies of a 6-yr-old boy and his non-HLA identical 8-yr-old sister, we demonstrated 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency in the biosynthetic pathways of glucocorticoids and androgens, but not mineralocorticoids. The sister did not manifest abnormal genital development at birth, but developed premature adrenarche at the age of 4 yr, with clitoromegaly and advanced bone age. The brother had perineal hypospadias at birth and developed premature adrenarche at the age of 6 yr. In both siblings, baseline and ACTH-stimulated delta 5 steroids were markedly elevated. The baseline and ACTH-stimulated ratios of delta 5 to delta 4 steroids remained extremely high, and all steroids promptly suppressed with dexamethasone (DEX). Normal baseline PRA and serum and urinary aldosterone (Aldo) levels increased after stimulation with a low Na+ diet. Renal Na+ conservation was normal after dietary Na+ deprivation with and without DEX administration. The PRA to pH 1 Aldo ratio remained normal with normal and low Na+ diets, regardless of DEX administration, indicating normal glomerulosa function with renin stimulation. In both siblings, ACTH increased PRA and Aldo levels, maintaining the PRA to pH 1 Aldo ratio unchanged from the baseline value. In contrast, in control children, PRA was suppressed, while Aldo increased, resulting in a fall of the PRA to pH 1 Aldo ratio. The increase in PRA with exogenous ACTH in these siblings suggests there may be an ACTH-stimulable mineralocorticoid antagonist. During prolonged DEX administration, hCG administration caused a slight increase in 17-hydroxypregnenolone and dehydroepiandrosterone in both the siblings, while testosterone (T) rose poorly in the brother, and estradiol did not rise at all in the sister. These results suggest the possibility of a deficiency of 3 beta-HSD in the gonads as well as the adrenals. After [3H]dehydroepiandrosterone iv infusion, there was normal conversion to [3H]-conjugated testosterone glucuronide, suggesting the presence of normal peripheral 3 beta-HSD activity. We propose that in these siblings, there is a deficiency of 3 beta-HSD in the adrenal zona fasciculata and zona reticularis, whereas 3 beta-HSD activity is intact in the zona glomerulosa. In addition, in these siblings, 3 beta-HSD deficiency was present in the gonads, while peripheral 3 beta-HSD activity appeared to be intact. These cases demonstrate further the heterogeneity of congenital adrenal hyperplasia due to 3 beta-HSD deficiency.     

The 3beta-hydroxysteroid dehydrogenase gene family of enzymes.

It now is apparent that a family of closely related genes encode for 3beta- hydroxysteroid dehydrogenase (3betaHSD). Studies on the regulation of these genes are in their infancy, but the regulation appears multifactorial. The various 3betaHSD genes are expressed principally in a tissue-specific manner likely involving separate mechanisms of regulation. To date, two human 3betaHSD genes and their products have been characterized; type I is expressed in placenta, sebaceous glands, and several other nonendocrine tissues, whereas the type II isoform is the principal 3betaHSD of adrenal cortex and gonads.     

Genetic variation in 11beta-hydroxysteroid dehydrogenase type 1 predicts adrenal hyperandrogenism among lean women with polycystic ovary syndrome

CONTEXT: Elevated adrenal androgen levels are common in polycystic ovary syndrome (PCOS), but the underlying pathogenetic mechanism is poorly understood. In the rare cortisone reductase deficiency, impaired regeneration of active cortisol from inert cortisone by 11beta-hydroxysteroid dehydrogenase (11beta-HSD1) results in compensatory activation of ACTH secretion and adrenal hyperandrogenism. 11beta-HSD1 deficiency may protect against obesity and its metabolic consequences because of impaired regeneration of cortisol in adipose tissue. OBJECTIVE: Our objective was to investigate a functional polymorphism in HSD11B1 (T-->G in the third intron rs12086634, which associates with lower 11beta-HSD1 activity) in PCOS with and without obesity. Design and Setting: We conducted a case-control study in lean and obese PCOS patients and controls at an academic hospital. PARTICIPANTS: Participants included 102 Caucasian PCOS patients and 98 controls comparable for age, weight, and race. MAIN OUTCOME MEASURES: We assessed genotype distribution and influence of genotypes on clinical, hormonal, and metabolic parameters. RESULTS: The G allele was significantly related to PCOS status (P = 0.041), and this association was mainly attributable to lean (P = 0.025), rather than obese (P = 0.424), PCOS patients. The G allele was associated with lower 0800-0830 h plasma cortisol (P < 0.001) and higher cortisol response to ACTH(1-24) (P < 0.001) in all women with PCOS and with higher dehydroepiandrosterone sulfate levels (P < 0.001), greater suppression of dehydroepiandrosterone sulfate by dexamethasone (P < 0.001), and lower fasting plasma low-density lipoprotein cholesterol (P = 0.002) levels in lean PCOS women. CONCLUSIONS: Genetic variation in 11beta-HSD1 contributes to enhanced cortisol clearance and compensatory adrenal hyperandrogenism in lean patients with PCOS but may be protective against obesity and some features of the metabolic syndrome.     

Histoenzymological studies of 3beta- and 17beta-hydroxysteroid dehydrogenases in the placentae and adrenal glands of bilaterally ovariectomized rats

The distribution and activities of 5-unsaturated-3beta- and 17beta-hydroxysteroid dehydrogenases (HSD) have been studied during the pregnancy of normal and ovariectomized rats on days 17, 19 and 21 post coitum. No hormonal substitution was provided after bilateral ovariectomy on day 15 post-coitum. 5-Unsaturated-3beta-HSD was characterized with pregnenolone, 17alpha-hydroxypregnenolone and dehydroepiandrosterone as substrates; oestradiol-17beta and testosterone were used for the investigation of 17beta-HSD activity. With these substrates, it was found that the placental and adrenal activities and distribution of 5-unsaturated-3beta- and 17beta-HSD did not differ in ovariectomized and control rats and it is suggested that increased placental or adrenal steroidogenesis does not supplement deficient ovarian function in order to maintain pregnancy. In the pregnant rat, the ovaries do not control the activities of 5-unsaturated-3beta- and 17beta-HSD in the placenta.     

3 beta-hydroxysteroid dehydrogenase/isomerase in the fetal zone and neocortex of the human fetal adrenal gland

The fetal zone of the human fetal adrenal (HFA) gland is established to have decreased 3 beta-hydroxysteroid dehydrogenase/delta 4-5 isomerase (3 beta HSD) activity compared to the neocortex or definitive zone. 3 beta HSD activity, however, can be induced in primary cell culture through treatment with ACTH. Therefore, the HFA with two distinct steroidogenic zones with differences in 3 beta HSD activity as well as the capacity to increase 3 beta HSD activity in response to ACTH provides an excellent model to study the regulation of this enzyme. The presence of 3 beta HSD in the fetal and neocortex zones of the HFA was examined using a polyclonal antibody raised against purified human placental microsomal 3 beta HSD. After homogenates of the fetal and neocortical zones of the HFA were electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel and immunoblotted, the presence of the 3 beta HSD protein with a molecular size of 45 kDa could be demonstrated only in the neocortical zone. ACTH treatment (greater than 2 days) of fetal and neocortical zone explant cultures produced increases in cortisol secretion associated with the respective levels of immunodetectable 3 beta HSD protein. Cortisol and dehydroepiandrosterone sulfate were the respective principal steroid products of neocortical and fetal zone explants. After ACTH treatment, immunodetectable 3 beta HSD was induced to a greater magnitude in the neocortex. These findings provide evidence that the lack of 3 beta HSD activity in the fetal zone, previously considered to be the result of the presence of an endogenous inhibitor, is due to an absence of the protein in this portion of the gland. The lack or minimal expression of 3 beta HSD in the fetal zone of HFA may be due to the action (or lack thereof) of a tissue-specific factor regulating the synthesis of 3 beta HSD.     

Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.

Transient expression in nonsteroidogenic mammalian cells of the rat wild type I and type II 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) cDNAs shows that the encoded proteins, in addition to being able to catalyze the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into the corresponding delta 4-3-ketosteroids, interconvert 5 alpha-dihydrotestosterone (DHT) and 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol). When homogenate from cells transfected with a plasmid vector containing type I 3 beta-HSD is incubated in the presence of DHT using NAD+ as cofactor, a somewhat unexpected metabolite is formed, namely 5 alpha-androstanedione (A-dione), thus indicating an intrinsic androgenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity of this 3 beta-HSD isoform. Although the relative Vmax of 17 beta-HSD activity is 14.9-fold lower than that of 3 beta-HSD activity, the Km value for the 17 beta-HSD activity of type I 3 beta-HSD is 7.97 microM, a value which is in the same range as the conversion of DHT into 3 beta-diol which shows a Km value of 4.02 microM. Interestingly, this 17 beta-HSD activity is highly predominant in unbroken cells in culture, thus supporting the physiological relevance of this "secondary" activity. Such 17 beta-HSD activity is inhibited by the classical substrates of 3 beta-HSD, namely pregnenolone (PREG), dehydroepiandrosterone (DHEA), delta 5-androstene-3 beta,17 beta-diol (delta 5-diol), 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol) and DHT, with IC50 values of 2.7, 1.0, 3.2, 6.2, and 6.3 microM, respectively. Although dual enzymatic activities have been previously reported for purified preparations of other steroidogenic enzymes, the present data demonstrate the multifunctional enzymatic activities associated with a recombinant oxidoreductase enzyme. In addition to its well known 3 beta-HSD activity, this enzyme possesses the ability to catalyze DHT into A-dione thus potentially controlling the level of the active androgen DHT in classical steroidogenic as well as peripheral intracrine tissues.     

Stabilization and assay of a 17beta-hydroxysteroid dehydrogenase in the serum of pregnant women.

Up to the present the activity of the 17beta-hydroxysteroid dehydrogenase (17beta-HSD) in the blood of pregnant women has not been accurately measured because of its great instability. The enzyme was stabilized by mixing fresh serum with an equal volume of potassium phosphate buffer (100 mM, PH 7.4), containing 40% (v/v) glycerol, 40 muM oestradiol-17beta (Oe2) and 0.4% (v/v) ethanol. Under these conditions the 17beta-HSD was stable for several days at -20, +4 and +20 degrees C and resistant to heat denaturation at 60 degrees C. The Km-value for Oe2 with NAD or NADP as cosubstrate was 5 X 10(-6) or 2 X 10(-6) M, respectively. In the presence of NAD, Oe2 was oxidized twice as rapidly as with NADP. The Km-value for NAD was 10(-5) M. Under the conditions of assay [3H]oestrone formation was linear with time (3 h) and with protein concentration. Serum enzyme activities in normal pregnancies (between 20 and 60 muU/ml serum at the end of gestation) were nearly twice as high as those reported by other investigators. In normal single and twin births highest activities were obtained at the expulsion of the placenta which in all cases was followed by a rapid decrease in activity.