Steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-progesterone.

OBJECTIVE: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC-MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. METHODS: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2 gene analysis and 1-24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC-MS/MS. RESULTS: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2 mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC-MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS ≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. CONCLUSIONS: LC-MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.

Assessment of the 21-hydroxylase deficiency and the adrenal functions in young females with Turner syndrome.

There are few reports of an association between Turner syndrome (TS) and 21-hydroxylase deficiency. However, this association is more frequent in some populations. The aim of this study was to evaluate the incidence of 21-hydroxylase deficiency in patients with TS in our population. 21-hydroxylase deficiency was evaluated in 44 TS cases with 45X (n=20) and 24 mosaic cases. A standard dose adrenocorticotropic (ACTH) stimulation test (Synacthen, Novartis, Basel, Switzerland) was performed, and 17 hydroxyprogesterone (17OHP), dehydroepiandrosterone sulfate (DHEAS) and cortisol responses were evaluated. Patients with increased 17OHP responses in the stimulation test also underwent 21-hydroxylase gene analysis. The mean age was 14.6 +/- 4 (2.6-22.4); 37 patients were on growth hormone (GH) treatment. Nine patients were at prepubertal stage, whereas 35 were pubertal (24 on gonadal steroids and 11 spontaneously). Six patients were obese. Only one of our patients had a level of 7.5 ng/mL of 17OHP, and there was no mutation found in congenital adrenal hyperplasia (CAH) genetic analysis. In other cases, peak 17OHP levels were < or = 6 ng/mL. The mean peak 17OHP was 2.62 +/- 1.48 (1.19-7.5) ng/mL, the cortisol level was 37.6 +/- 8.43 (23.9-56.2) microg/dL and the DHEAS was 135.2+/- 87.3 (15-413) microg/dL. The increased mean basal and peak cortisol levels (20.5 +/- 10.2 and 37.6 +/- 8.4 microg/dL) were remarkable findings. Whereas basal cortisol was above 20 microg/dL in 38.7% of patients, exaggerated results up to 56.2 microg/dL were obtained in peak cortisol levels. The basal and peak 17OHP cortisol levels were not correlated with the presence of puberty, chromosome structure, gonadal steroid use, obesity or growth hormone use. This trial suggested that 21-hydroxylase deficiency was not common among patients with TS in our population. Adrenal function should be assessed, at least in the presence of clitoral enlargement in patients with TS, particularly if their karyotype does not contain a Y chromosome.

Metabolic profile and body composition in adult women with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

CONTEXT: The chronic, often supraphysiological glucocorticoid doses used in congenital adrenal hyperplasia (CAH) might increase morbidity in cardiovascular disease and diabetes. OBJECTIVE: Our aim was to assess risk factors for cardiovascular disease and diabetes in CAH women. SUBJECTS, METHODS, AND DESIGN: We compared 61 women, 18-63 yr, with CAH due to 21-hydroxylase deficiency with 61 age- and sex- matched controls. Twenty-seven were younger than 30 yr, and 34 were 30 yr or older. Anthropometry, fat and lean mass measured by dual-energy x-ray absorptiometry, serum lipids, insulin, and adrenocortical steroids were studied. MAIN OUTCOME MEASURE: Body composition and cardiovascular risk factors were the main outcome measures. RESULTS: Younger patients and controls had similar waist to hip ratio, lean and fat mass, and insulin. Older patients had higher waist to hip ratio, lean mass, and insulin than controls. Fat mass was similar to controls but higher than in younger patients. Lipid profiles were slightly more favorable in older patients than controls. Gestational diabetes was more common in patients (21% of pregnancies vs. 0, P < 0.026). Few older patients had hypertension, cardiovascular disease, or diabetes. Despite moderate glucocorticoid doses, most patients had suppressed androgens. CONCLUSIONS: No clear evidence of unfavorable cardiovascular risk factors were found. Increased fat mass and higher insulin levels were, however, found in patients older than 30 yr. High frequency of gestational diabetes is a risk marker for future diabetes. Lifelong follow-up, lifestyle modifications, and attempts to adjust and reduce the glucocorticoid doses seem important.

[Polycystic ovary syndrome and congenital adrenal hyperplasia: a different entity for comparable phenotypes?]. / Syndrome des ovaires potykystiques et syndrome adrénogenital: une entité différente pour un phenotype comparable?

The phenotypes of the polycystic ovarian syndrome (PCOS) and congenital adrenal hyperplasia syndrome (CAHS) present a number of similarities. The main symptoms of PCOS are obesity, menstrual disorders, hirsutism, and low fertility in which the pituitary and adrenal glands are spared. The CAHS is a group of various entities all characterised by different degrees of malfunction of the 21-hydroxylase (CYP21) enzyme. The consequences are a downfall of the levels of aldosterone and cortisol, and the hyperproduction of adrenal androgen hormones. It is capital to be able to recognise these 2 entities in terms of identification of high risk families because the female foetuses suffering from CAHS will undergo severe virilization of there genitals in utero, which can efficiently be prevented by a administration of corticotherapy to the mother throughout the pregnancy.

Serum 21-Deoxycortisol, 17-Hydroxyprogesterone, and 11-deoxycortisol in classic congenital adrenal hyperplasia: clinical and hormonal correlations and identification of patients with 11beta-hydroxylase deficiency among a large group with alleged 21-hydroxylase deficiency.

INTRODUCTION: 21-Hydroxylase deficiency (21OHD) is the most common cause of congenital adrenal hyperplasia, followed in frequency by 11beta-hydroxylase deficiency (11betaOHD). Although the relative frequency of 11betaOHD is reported as between 3 and 5% of the cases, these numbers may have been somewhat underestimated. MATERIALS AND METHODS: In 133 patients (89 females/44 males; 10 d-20.9 yr) with alleged classic 21OHD and five (three females/two males; 7.3-21 yr) with documented 11betaOHD, we measured serum 21-deoxycortisol (21DF), 17-hydroxyprogesterone (17OHP), and 11-deoxycortisol (S), 48 h after glucocorticoid withdrawal. We also studied 20 sex- and age-matched control subjects. Serum steroid levels were determined by RIA after HPLC purification. OBJECTIVES: The objectives of this study were to: 1) quantify 21DF in patients with congenital adrenal hyperplasia, 2) correlate hormonal with clinical data, and 3) identify possible misdiagnosed patients with 11betaOHD among those with 21OHD. RESULTS: In 21OHD, 17OHP (217-100,472 ng/dl) and 21DF (<39-14,105 ng/dl) were mostly elevated and positively correlated (r = 0.7202; P < 0.001). Except for higher 17OHP in pubertal patients, 17OHP and 21DF values were similar according to sex, disease severity, or prevailing glucocorticoid dose. One additional patient with 11betaOHD was detected (1%) and also one with apparent combined 11beta- and 21OHD. S levels were elevated in 11betaOHD and normal but significantly higher in 21OHD than in controls. CONCLUSION: To recognize patients with 21- and/or 11betaOHD, we recommend evaluation of 17OHP or 21DF and S. Also, 21DF may be useful to follow up pubertal patients with 21OHD. Because 1% of patients with alleged 21OHD may have 11betaOHD, its frequency seems underestimated, as per our experience in a Brazilian population.

Bioconversion of 16 alpha-hydroxyprogesterone to 16 alpha-hydroxylated corticosteroids by newborn rat adrenal cells in primary culture.

Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups. Minor metabolites resulted from 18-hydroxylation and 6 beta-hydroxylation of the substrate. The metabolism of 16 alpha-hydroxyprogesterone is similar to that of progesterone in the same cell-culture system; however, there are two exceptions. The 21-hydroxylation of 16 alpha-hydroxyprogesterone occurs at a rate similar to that of its 11 beta-hydroxylation, whereas the 21-hydroxylation of progesterone is faster than its 11 beta-hydroxylation. The ratio of 11 beta- to 18-hydroxylation of 16 alpha-hydroxyprogesterone is about 3, whereas the ratio of 11 beta- to 18-hydroxylation of progesterone, 20 alpha-dihydroxyprogesterone and DOC is between 1./ and 2. It is most likely the rate of 18-hydroxylation which is decreased by the hydroxyl group at C-16. The use of adrenal cell cultures is a practical, simple method for the preparation of a variety of 16 alpha-hydroxylated steroids from a single substrate. Its adaptation to the production of important amounts of 16 alpha-hydroxylated corticosteroids will permit the study of their biological activity.

Homologous sequences in steroidogenic enzymes, steroid receptors and a steroid binding protein suggest a consensus steroid-binding sequence.

The amino acid sequences of two steroidogenic enzymes, P450c17 (steroid 17 alpha-hydroxylase/17,20 lyse) and P450c21 (steroid 21-hydroxylase), are only 28.9% identical. However, these proteins share a region of 21 amino acids bearing 17 identical residues, which we previously suggested may represent the steroid binding site. We assembled a sequence database of known steroid-binding proteins and searched this with the sequence of this 21 amino acid region. The steroidogenic enzymes, P450c17, P450c21, P450scc (the cholesterol side-chain cleavage enzyme), and P450c11 (steroid 11 beta/18-hydroxylase) share a subregion of 17 amino acids having at least 15 identical residues. Related sequences were identified in a computerized search of the available sequences of steroid hormone receptors and binding proteins. These sequences were invariably found within larger domains previously associated with steroid binding. From these we propose a more general consensus sequence of LPLLL +/- 000KDRE0LKRL +/- PV, where +/- refers to any charged amino acid, and 0 refers to an uncharged amino acid. This consensus sequence predicts 147 or 187 total amino acids in 11 human proteins examined (78.6%). An equivalent degree of sequence identity, 178 of 221 amino acids (80.5%) was found among 13 animal homologs of these human proteins. The ability of this consensus sequence to predict 325 of 408 amino acids (79.7%) strongly suggests this sequence is necessary, if not sufficient, for a steroid binding site in many proteins. Lecithin-cholesterol acetyl transferase, cholesterol ester transfer protein, and steroid sulfatase did not have sequences similar to our consensus sequence.

[Histological and molecular study of fetal human adrenal cortex (12-36 wk)]. / Etude histologique et moléculaire de la corticosurrénale foetale humaine (12e-36e SD). La corticosurrénale foetale humaine (12e-36e SD).

Histological and functional characteristics of the fetal human adrenals was studied in 119 normal fetuses aged 12 to 36 weeks development (WD). Immunocytochemical detection of steroidogenesis enzyme (3beta-HSD and P450 c21) and evaluation of cell proliferation using two nuclear markers (Ki-67 and PCNA) were performed in 70 of them. The human fetal adrenal cortex is composed of two morphologically distinct zones: the definitive peripheral zone and the fetal inner zone. From the 12th WD, we observed expression of an adherence protein (NCAM) and two steroidogenesis enzymes (3beta-HSD and P450 c21) in the definitive zone cells, attesting to the capacity of these cells to synthesize mineralocorticoids and/or cortisol. In the fetal zone, only P450 c21 immunoreactivity was detected. From the 14th WD, a transitional zone between the definitive zone and the fetal zone was identified by immunocytochemistry, with expression of 3b-HSD from the 21st WD. Only cells of the definitive zone proliferated from the 12th to 25th WD. The indexes of proliferation of PCNA and Ki-67, 40% and 25% respectively, decreased gradually and were lower than 1% at the 25th WD.

Cytochrome P450 2D catalyze steroid 21-hydroxylation in the brain.

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

Autoantibodies to cytochrome P450 enzymes P450scc, P450c17, and P450c21 in autoimmune polyglandular disease types I and II and in isolated Addison's disease.

Patients with idiopathic Addison's disease have autoantibodies reacting with adrenal cortex. If Addison's disease is associated with other endocrine immune diseases like autoimmune polyglandular diseases (APD) type I and type II, antibodies may recognize all steroid-producing cells. We showed previously that one antigen recognized by APD-I sera is the cytochrome P450c17 hydroxylase. We have now looked for antibodies to P450c17 and to two other key enzymes in the steroid biosynthetic pathway, the P450scc and P450c21, in a series of patients with isolated Addison's disease (8 patients) or with APD-I or APD-II (50 and 9 patients, respectively). The result of antienzyme antibodies were further correlated with the immunofluorescence pattern against adrenal gland, testis, ovary, and placenta, and with the clinical findings presented. In APD-I patients with Addison's disease and in APD-II patients, antibodies to at least one of the P450 enzymes were frequently found (positive findings in 81% and 78%, respectively). Such antibodies were less frequent in APD-I patients without Addison's disease (21%) and in the isolated Addison cases (25%). In APD-I, antibodies recognized as frequently P450c17 and P450scc, specific for all steroid-producing cells as the adrenal specific enzyme P450c21. In contrast, patients with APD-II or with the isolated Addison's disease reacted almost exclusively with P450c21. Immunofluorescence studies showed good correlation with the known fact that the zona glomerulosa of the adrenal cortex is devoid of the P450c17, that the Leydig cells of the testis and the theca interna cells of the ovary express P450c17 and P450scc, and that the placental trophoblasts express only P450scc. The presence of antibodies to P450scc or to at least one of the tested P450 enzymes correlated significantly to gonadal failure in the females but not in the males.

Gestational age-dependent changes in the levels of mRNAs encoding cortisol biosynthetic enzymes and IGF-II in the adrenal gland of fetal sheep during prolonged hypoxemia.

Hypoxemia represents a major stress for the fetus, and is associated with alterations and adaptations in cardiovascular, metabolic and endocrine responses, which in turn may affect tissue growth and differentiation. To determine the effects of hypoxemia on fetal adrenal activity and growth, we subjected sheep fetuses at days 126-130 and 134-136 (term 145 days) to reduced PaO2 by reducing the maternal fraction of oxygen for 48 h (mean reduction of 6.8 mmHg), without change in arterial pH or PaCO2. This stimulus resulted in similar increases in the plasma immunoreactiveACTH response at both ages. Among adrenal steroids, plasma cortisol (C21Delta4) rose in both groups of animals, but plasma androstenedione (C19Delta4) declined marginally, resulting in a pronounced increase in the cortisol:androstenedione ratio in the plasma that was greater and more sustained in the older fetuses. In the younger fetuses, after 48 h of hypoxemia, there were no significant changes in mRNAs encoding steroidogenic enzymes in the fetal adrenal gland. However, in the older fetuses, hypoxemia resulted in significantly increased levels of mRNAs encoding P450scc, P450C21 and 3beta-hydroxysteroid dehydrogenase, but not for P450C17, in the fetal adrenal gland. Levels of IGF-II mRNA in the fetal adrenal gland fell in both groups of fetuses, and this response was greater at the later gestational age. We conclude that sustained hypoxemia is a potent stimulus which activates adrenal steroidogenesis in the late gestation fetal sheep. The resultant increase in cortisol synthesis is associated with decreased expression of adrenal IGF-II mRNA. We speculate that this relationship might influence patterns of fetal organ growth and differentiative function in response to fetal stress such as hypoxemia.

Immunohistochemical localization of cytochrome P-450C21 in human adrenal cortex and its relation to endocrine function.

Cytochrome P-450C21 was successfully demonstrated in the human adrenal glands by a peroxidase-antiperoxidase method. All three cortical layers were stained in the normal adrenal glands, particularly the zonae glomerulosa and reticularis. Well-stained and faintly stained cells were intermingled in the zona fasciculata, suggestive of intrazonal variations. The immunoreactivity was particularly intense at the site of ACTH action, i.e., cells in micronodules and cells around myelolipomatous lesions in adrenocortical hyperplasia of Cushing's disease and sites of regeneration in the normal adrenal glands. In adrenocortical adenomas with Cushing's syndrome and primary aldosteronism, cells with large nuclei were generally stained well. In the adrenocortical tissue adjacent to a functioning adenoma, the immunoreactivity was observed only in the zona glomerulosa, especially in cases of primary aldosteronism. This finding is consistent with morphologic observations.

A localization study of the cytochrome P-450(21)-linked monooxygenase system in adult rat brain.

Immunohistochemical studies were performed to investigate the localization of the cytochrome P-450(21)-linked monooxygenase system in rat brain using a specific antibody against bovine adrenal cytochrome P-450(21) (P-450XXIA1), which was purified electrophoretically as a single protein band and with a heme content of 18.0 nmol/mg protein from adrenocortical microsomes. The cytochrome P-450(21) was found to be mainly localized in the tractus reticulothalamicus and other ascending fibers in adult rat brains. This finding indicated that deoxycorticosterone or its derivatives could be implicated in the regulation of consciousness and the induction of an anesthetic effect.

Dual subcellular localization of the 3 beta-hydroxysteroid dehydrogenase isomerase: characterization of the mitochondrial enzyme in the bovine adrenal cortex.

The enzyme 3 beta-hydroxysteroid dehydrogenase isomerase (3 beta-HSD/I) is an essential step in the biosynthesis of steroid such as progesterone, mineralo- and gluco-corticoids, estrogens and androgens in steroidogenic tissues. It is considered to be mainly localized in microsomes; however, 3 beta-HSD/I activity has also been described to be associated with mitochondrial preparations. In this study, we examined the subcellular distribution of 3 beta-HSD/I in bovine adrenocortical tissue and we characterized the catalytic properties of the enzyme present in the various cell compartments. About 30% of the total 3 beta-HSD/I activity was found to remain tightly associated with the purified mitochondrial pellet. The 3 beta-HSD/I and 3-ketoreductase activities were found in microsomes as well as in mitochondria. The 3 beta-HSD/I associated with the mitochondrial fraction did not require addition of exogenous NAD+. When the pyridine nucleotide was reduced following addition of substrates of the tricarboxylic acids cycle, the mitochondrial 3 beta-HSD/I activity decreased, suggesting that the enzyme utilizes NAD+ available from the matrix space. By contrast, the microsomal enzyme was inactive in the absence of exogenous NAD+. Submitochondrial fractionation disclosed that 3 beta-HSD/I was associated (i) with the inner membrane and (ii) with a particulate fraction sedimenting in a density gradient between inner and outer membranes. This fraction was characterized as contact sites between the two membranes. 3 beta-HSD/I specific activity was much higher in this fraction than in the inner mitochondrial membrane. Altogether, these observations suggest that these mitochondrial intermembrane contact sites may represent a special organization of functional significance, facilitating both the access of cholesterol to the inner membrane where cytochrome P-450scc is located and the rapid transformation of its product, pregnenolone, to progesterone, through 3 beta-HSD/I activity.

Monitoring of menstrual cycles, ovulation, and adrenal suppression by saliva sampling in female patients with 21-hydroxylase deficiency.

OBJECTIVE: To investigate the correlation between menstrual cycles, ovulation, and adrenal suppression in congenital adrenal hyperplasia. DESIGN: Prospective observational study. SETTING: An academic outpatient clinic. PATIENT(S): Five females with salt-wasting 21-hydroxylase deficiency, aged 15.5 to 22.9 years; one had amenorrhea, one had irregular bleeding, and three had regular bleeding. INTERVENTION(S): Daily morning saliva sampling for 40 to 280 days. MAIN OUTCOME MEASURE(S): Salivary levels of progesterone (P), 17-hydroxyprogesterone (17-OHP), and androstenedione. RESULT(S): In the amenorrheic patient, the elevated P and 17-OHP levels decreased when the glucocorticoid dose was increased, and subsequently menarche occurred. The androstenedione levels were normal. The correlations between P and 17-OHP levels before and after menarche suggest that adrenal progesterone had prevented menarche. The patient with irregular bleeding showed slightly elevated androstenedione levels and increased levels of 17-OHP and P in an irregular pattern, without correlation in time with vaginal bleeding. Three patients with regular cycles showed a biphasic pattern of P levels, indicating ovulation. CONCLUSION(S): These longitudinal data support the hypothesis that menstrual cycling in females with 21-hydroxylase deficiency can be prevented or disturbed by elevated progesterone levels of adrenal origin, in the absence of androgen excess. Increasing glucocorticoid dose could suppress adrenal progesterone production, resulting in menarche.

Analysis of CYP21 coding polymorphisms in three ethnic populations: further evidence of nonamplifying CYP21 alleles among whites.

BACKGROUND: Adrenal steroid 21-hydroxylase is essential for the synthesis of both mineralocorticoids and glucocorticoids. The gene for this enzyme, CYP21, contains several frequent coding polymorphisms. Because of its essential function in steroid synthesis, polymorphisms in this enzyme might influence a variety of disease processes. However, before disease-association studies are performed, it is important to understand the frequency of these polymorphisms among normal individuals. METHODS: Using polymerase chain reaction (PCR) with restriction enzyme digestion or size length polymorphism analysis, we measured the frequencies of the +Leu(10), Arg102Lys, and Ser268Thr polymorphisms in CYP21 in healthy whites, blacks, and Indian Americans. The subjects were all young female college students participating in a study of relative risks for cardiovascular disease in these populations. RESULTS: The frequency of each polymorphism among whites, blacks, and Indian Americans were as follows: +Leu(10), 0.55, 0.96, 0.75; Arg102, 0.63, 0.97, 0.82; and Ser268, 0.92, 0.68, 0.79, respectively. With the exception of the frequencies of the Ser268Thr polymorphism among blacks and Indian Americans, there were significantly different frequencies of each polymorphism among all groups (P<.05). Among whites, the distribution of genotypes for the +Leu(10) and Arg102Lys polymorphisms deviated significantly from expected Hardy-Weinberg values because of an excess of homozygotes. CONCLUSIONS: Among the ethnic groups, there are statistically significant differences in the frequencies of these common coding polymorphisms in CYP21 that need to be considered in disease-association studies. Deviation from Hardy-Weinberg distributions might be explained by allelic dropout during PCR, a phenomenon previously reported at this locus.

The new world primates as animal models of glucocorticoid resistance.

Many New World primate species have greatly increased plasma cortisol concentrations, decreased plasma cortisol binding globulin capacity and affinity, marked resistance of the hypothalamic-pituitary-adrenal axis to suppression by dexamethasone, and no biological evidence of glucocorticoid excess. These primates also have high levels of circulating progesterone, estrogen, mineralocorticoid, androgen and vitamin D. The glucocorticoid target tissues that have been examined (circulating mononuclear lymphocytes and cultured skin fibroblasts) have normal concentrations of glucocorticoid receptors with decreased affinity for dexamethasone. Transformation of B-lymphocytes with the Epstein-Barr virus leads to glucocorticoid receptor induction that is less than that observed with cells from Old World primates. The receptor in these cells has a low affinity for dexamethasone. The low affinity leads to an increased loss of specific bound ligand during thermal activation. Meroreceptor generation is normal. The molecular weight of the receptor, determined by SDS-PAGE, is similar to that of Old World primates (approximately 92,000) and the activation pattern per se, examined in vitro by heating cytosol and performing phosphocellulose chromatography, appears similar to that of human controls. The ratios of nuclear to cytosolic hormone-receptor-complexes and of cytosolic activated to unactivated receptor complexes in intact cells are similar to Old World primates. Results from mixing studies do not support the hypothesis that a binding inhibitor(s) or a deficient cytosolic positive modifier(s) of binding underlies the findings in these primates. The New World primates, unlike men with the syndrome of primary cortisol resistance, have compensated for their condition with intra-adrenal and mineralocorticoid receptor adaptations. Thus, unlike Old World primates, cortisol in New World primates has only weak sodium-retaining potency because the aldosterone receptor has a low affinity for cortisol. The common element that would explain the apparent resistance to six steroid hormones in New World primates remains unknown.

Congenital adrenal hyperplasia: experience at Calcutta.

Eight patients (7 females, 1 male) with congenital adrenal hyperplasia (CAH), were seen over a 24-month period beginning from March 1988. Seven patients had 21 hydroxylase (21-OH) deficiency while one had 11 beta hydroxylase deficiency. Of the 7 patients with 21-OH deficiency, 3 were of the salt losing (SL-CAH), and 4 were of the non-salt losing (NSL-CAH) type. The patients with NSL-CAH were diagnosed by their elevated 17-hydroxyprogesterone (17-OHP) levels. The 3 cases with SL-CAH were diagnosed on the basis of ambiguous external genitalia, typical electrolyte picture, normal female internal genitalia, sex chromatin and response to steroids. In one patient post-ACTH 17 OHP was alter measured. All 3 patients with SL-CAH were assigned the male sex. Sex reassignment was advised for two children; one accepted the advice and the child is doing well; one family did not accept sex reassignment and the child died. One patient died due to non-availability of fludrocortisone. Six patients are under follow-up. All are doing well except one patient with NSL-CAH who started treatment late. We conclude that a high index of suspicion, early diagnosis and meticulous patient education are the key features of successful management of CAH in India.

Characterization of pathogenic mutations in 21-hydroxylase gene of Pakistani patients with congenital adrenal hyperplasia and their family members--a preliminary report.

OBJECTIVE: To characterize specific mutations within the 21-hydroxylase gene (CYP21-B) using ARMS-PCR assay in patients with congenital adrenal hyperplasia (CAH) and to compare it with that reported in other populations. SUBJECTS AND METHODS: Five families, having an index case with CAH diagnosed on the basis of clinical and biochemical findings volunteered to give blood samples for analysis. A strategy, based on ARMS-PCR (Amplified Refractory Mutation System) was employed for the detection of mutations in 21-hydroxylase gene. The products of ARMS-PCR were resolved on agarose gels and the PCR products were visualized over ultra violet illumination. RESULTS: Twenty-six specimens were analyzed for common point mutations in the 21-hydroxlase genes at the nucleotide positions 659, 1004 and 1688. Seven samples belonged to index cases with CAH. Of these 7, the assigned sex was male in 5 and female in 2 cases. However, genotypic sex was 3 males and 4 females. The mean age was 8 months in 5 cases while the median 17-OH Progesterone levels was 273.2 ng/ml. Vomiting, precocious puberty and ambiguous genitalia were the presenting features in 2, 1 and 4 cases respectively. Analysis for mutation at 659, 100 and 1688 was performed on 7 index cases and the family members of 5 index cases. The mutation analysis for the family members of index case 6 and 7 was not performed due to non-availability of their blood specimens. Index case No. 1, 4 and 7 showed homozygosity for splice mutations at nucleotide position 659, intron 2 with a sequence change of A to G, while the index case No. 2 and 6 showed heterozygosity for the same mutation. No mutation was found at 659, 1004 or 1688 in index case No. 3 and 4 at the analyzed nucleotide position. Nineteen family members of Case Nos. 1-5 were also analyzed for the same mutations. (Family No. 1, 2, 3, 4 and 5 included 3, 2, 7, 4 and 5 members respectively). These included 8 males and 11 females. All were asymptomatic. Both the parents of index case 1 and 4 were heterozygous at 659 while the father of index case No. 2 was heterozygous at 659 and mother was normal. CONCLUSION: Our results demonstrated the A to G transition at nucleotide 659 causing aberrant splicing, reported for some other populations as the most commonly identified point mutations. All cases were appropriately assigned to paternal or maternal chromosomes.