A novel function of CYP21A2 in regulating cell migration and invasion via Wnt signaling.

CYP21A2, which is responsible for 21-hydroxylase activity, is prominent to the development of congenital adrenal hyperplasia (CAH). The aim of our current study is to investigate the role of CYP21A2 in the tumor processes. Here, we used HepG2 cell lines and generated CYP21A2 overexpressing vector and siRNA to investigate the effect of CYP21A2 on the tumor development processes, particularly cell migration and invasion; genes expression related to these processes were further examined. Results showed that CYP21A2 over-expressed or silenced had no effects on cell viability as well as the process of cell apoptosis. Further study suggested that CYP21A2 silenced significantly decreased the G0/G1 phase and increased the S phase of the cell cycle. However, no differences were observed when CYP21A2 was overexpressed. Moreover, we found that cell migration and invasion significantly improved with CYP21A2 overexpressed and impaired with silenced CYP21A2. Finally, we examined the expression of genes related to tumor processes and found that the Wnt signaling genes were changed. Taken together, our results demonstrated a novel function of CYP21A2 in the regulation of tumor processes, particularly cell migration and invasion, which this may be mediated by the Wnt signaling pathway.

[The alternative androgen synthesis pathway in humans]. / Der alternative Androgensyntheseweg des Menschen.

During the last year, alternative androgen synthesis pathways have been discovered in humans. This review article highlights these new concepts of androgen synthesis.We performed a selective literature research using PubMed.After the discovery of a new androgen synthesis pathway in marsupials, this new path-way of androgen synthesis could be established in humans during the last year from two independent studies. One of them could demonstrate that two pathways of androgen synthesis are needed for male sexual differentiation in humans; the other study established that the new pathway is an important source of androgen synthesis in congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Additionally, it has been shown that an alternative androgen synthesis pathway that bypasses testosterone drives castration resistant prostate cancer.New and alternative androgen path-ways occur in humans. Importantly, these path-ways remain cryptic for the clinician, because the androgen synthesis circumvents classical intermediates like dehydroepiandrosterone, androstenedione and testosterone.

Phenotypic profiling of parents with cryptic nonclassic congenital adrenal hyperplasia: findings in 145 unrelated families.

OBJECTIVE: To comprehensively phenotype parents identified with nonclassic congenital adrenal hyperplasia (NCCAH) by family genetic studies, termed here as cryptic NCCAH and to define the incidence of cryptic NCCAH in the parents of a large cohort of patients with 21-hydroxylase deficiency. DESIGN: Genotyping was performed on 249 parents of 145 unrelated congenital adrenal hyperplasia (CAH) patients. Parents with two CYP21A2 mutations underwent extensive evaluation. RESULTS: Of the 249 parents, ten (4%; seven females and three males) were identified as having cryptic NCCAH. The majority was of ethnicities previously reported to have a higher incidence of NCCAH. Cosyntropin stimulation performed in eight parents provided biochemical confirmation (17-hydroxyprogesterone range 56-364 nmol/l) and cortisol response was ≤500 nmol/l in three parents (38%). Of the seven women (27-54 years) with cryptic NCCAH, four had prior infertility, two reported irregular menses, two had treatment for hirsutism, one had androgenic alopecia. Men were asymptomatic. All cryptic NCCAH parents reported normal puberty and had normal height. Adrenal hypertrophy and a small adrenal myelolipoma were observed in two parents; testicular adrenal rest tissue was not found. CONCLUSIONS: Parents diagnosed with NCCAH by genetic testing are mostly asymptomatic. Temporary female infertility and suboptimal cortisol response were commonly observed. Ongoing glucocorticoid therapy is not indicated in adults with CAH identified by family genotype studies unless symptomatic, but glucocorticoid stress coverage should be considered in select cases. Parents of a child with CAH have a 1:25 risk of having NCCAH; if the mother of a child with CAH has infertility, evaluation for NCCAH is indicated.

Expression of paralogs of cytochrome P 450 21a1 pseudogene (Cyp21a1-ps) and endogenous retrovirus SC1 (SC1) in the rat ovary during the ovulatory process.

This study assesses the relatively high incidence of the expression of paralogs of several pseudogenes within the cascade of expression of functional genes in the rat ovary in response to an ovulation-stimulating dose of gonadotropin. Immature Wistar rats were primed with 10 IU equine chorionic gonadotropin subcutaneously, and 48 h later the 12-h ovulatory process was initiated by 10 IU hCG subcutaneously. Ovarian RNA was extracted at 0, 2, 4, 8, 12, and 24 h after injecting the animals with hCG. The RNA extracts were used for RT-PCR differential display to detect gene expression in the ovarian tissue. Sequence analyses of differentially expressed cDNAs revealed that approximately 27% (i.e. 22/82 clones) of the transcripts were fragments of paralogs of known pseudogenes. Out of the 22 clones reported here, 12 have high sequence similarity to the cytochrome P450 pseudogene Cyp21a1-ps, and 5 have high sequence similarity to both the Cyp21a1-ps and the aldo-keto reductase gene Akr1c6. The remaining five clones were paralogs of the endogenous retrovirus SC1 that has heavily infested the rat genome. Northern analyses reveal that peak expression of all the 22 paralogs occurs at 4-8 h into the ovulatory process. In situ hybridization shows that expression of these pseudogenes is primarily in the granulosa layer of ovulatory follicles. In summary, the results reveal that ovarian expression of Cyp21a1-ps- and SC1-like pseudogenes occurs concurrently with the ovulatory process.

Inhibition of CYP21A2 enzyme activity caused by novel missense mutations identified in Brazilian and Scandinavian patients.

BACKGROUND: Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles. OBJECTIVE: Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed. DESIGN: The study included 10 Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone. RESULTS: Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P, and p.R408C mutants classified them as classical congenital adrenal hyperplasia mutations, whereas the p.H62L showed an activity within the range of nonclassical mutations. Apparent kinetic constants for p.H62L confirmed the nonclassical classification as the substrate binding capacity was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination because it caused a significant reduction in the enzymatic activity. CONCLUSIONS: We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, and p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R, that might not be the case, and the p.H62L is definitely a nonclassical mutation.

The role of 21-hydroxylase in the pathogenesis of adrenal masses: review of the literature and focus on our own experience.

An exaggerated response of 17- hydroxyprogesterone (17-OHP) to exogenous ACTH stimulation has been found in 30 to 70% of patients with incidentally discovered adrenal tumors, supporting the concept that congenital 21- hydroxylase deficiency may be a predisposing factor for adrenocortical tumorigenesis. Decreased expression of 21-hydroxylase gene has been observed in sporadic non-functioning adrenocortical adenomas and adrenocortical carcinomas, in agreement with the reduced steroidogenic activity found in these types of tumors. Screening studies for the presence of mutations in CYP21A2 gene, encoding 21-hydroxylase, in patients with sporadic adrenocortical tumors yielded discordant results. Overall, a higher frequency of germline 21-hydroxylase mutation carriers has been found among patients with adrenal tumors, including incidentalomas, than in the general population. However, the presence of mutations did not correlate with endocrine test results and tumor mass features, suggesting that 21-hydroxylase deficiency does not represent a relevant mechanism in adrenal tumorigenesis. Mechanisms leading to reduced 21-hydroxylase expression and activity are still unknown.

Prenatal diagnosis and treatment of congenital adrenal hyperplasia owing to 21-hydroxylase deficiency.

Classical forms of congenital adrenal hyperplasia are caused by a severe deficiency of 21-hydroxylase, an enzyme involved in steroid biosynthesis, which triggers excessive androgen production before birth. Affected females experience virilization both physically and psychologically. Prenatal diagnosis and treatment of congenital adrenal hyperplasia has been implemented for more than 20 years. In utero gene-specific diagnosis is now feasible for fetal cell samples derived from chorionic villi or amniotic cells in culture, and this gene-specific diagnosis guides the treatment of the affected female fetus. Appropriate dexamethasone administration to the at-risk pregnant mother is effective in reducing genital virilization in the fetus, and thus avoids unnecessary genitoplasty in affected females. Current data from large human studies show the benefit and safety of prenatal treatment. Long-term follow-up of the safety of prenatal treatment is currently underway. This practice is a rare example of effective prenatal treatment to prevent a malformation caused by an inborn error of metabolism.

Altered 24-hour blood pressure profiles in children and adolescents with classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

OBJECTIVE: Children and adolescents with classical congenital adrenal hyperplasia have been shown to be at risk for obesity associated with higher insulin and leptin levels. Because these factors are also known to cause hypertension, the aim of this study was to analyze 24-h blood pressure profiles and their relation to different clinical and laboratory parameters. DESIGN: Fifty-five subjects, aged between 5.3 and 19.0 yr, were enrolled in a prospective, cross-sectional study. All patients had genetically proven 21-hydroxylase deficiency and underwent ambulatory 24-h blood pressure monitoring during a period off school/work. RESULTS (MEDIAN, RANGE): The median body mass index of the cohort was significantly elevated [1.09 sd score (SDS), -2.45 to 3.77]. Daytime and nighttime systolic blood pressures were also significantly elevated (0.67 SDS, -1.5-4.1; 0.63 SDS, -0.91 to 3.3), whereas daytime diastolic blood pressure was significantly lowered (-0.81 SDS, -2.6 to 3.2) and normal during the night (0.11 SDS, -2.0 to 2.0). Overall, there was a normal nocturnal drop of systolic (12.8%, 2.1-22.8) but not diastolic blood pressure (17.2%, 0.90-25.8). The different parameters of systolic and diastolic blood pressures were significantly correlated with body mass index and skinfold thickness (r(s) = 0.271-0.486). There was no correlation with equivalent hydrocortisone and fludrocortisone dosage and laboratory parameters except for serum leptin and insulin. CONCLUSIONS: Our data show altered 24-h blood pressure profiles with elevated systolic levels correlated with the degree of overweight and obesity, whereas normal-weight patients tended to diastolic hypotension.

Blood pressure in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

BACKGROUND: In patients with congenital adrenal hyperplasia (CAH) recording of blood pressure (BP) must be included in monitoring treatment to detect hypertension. AIM: To investigate the BP patterns in patients with CAH. METHODS: Twenty-three children and adolescents (age 6-17 years) and 11 adult patients (age 18-26 years) were studied (21 females, 13 males; 28 salt-wasting patients). In the whole group BP in the outpatient clinic was compared with BP under hospitalisation and in 11 of the children and adolescents also with 24-hour ambulatory blood pressure monitoring (ABPM). RESULTS: BP in the ward in children and adolescents but not in adults was significantly higher than BP in the outpatient clinic, where BP was in the upper normal range. There was also a significant difference between BP in the outpatient clinic and the lower ABPM in the 11 patients tested. Atrial natriuretic peptide (ANP) in blood serum showed normal values. CONCLUSIONS: BP measured in outpatients in a relaxed and calm atmosphere meets the requirements for monitoring of treatment. Measurement of BP on the ward leads to falsely high results. ABPM is not necessary. Estimation of ANP provides no additional information.

[Congenital adrenal hyperplasia due to 21-hydroxylase deficiency: final height in 27 patients with the classical form]. / Hiperplasia congênita das supra-renais por deficiência da 21-hidroxilase: altura final de 27 pacientes com a forma clássica.

OBJECTIVE: To determine final height (FH) in congenital adrenal hyperplasia (CAH) patients and investigate conditions allowing better height outcome. METHODOLOGY: 13 salt-wasting (SW) and 14 simple virilizing (SV) patients were studied. FH and target height (TH) were transformed into standard deviation score (z). Data were analyzed according to sex, clinical form, age at treatment onset and length of treatment before attaining FH. RESULTS: zFH (n = 27) was -1.57 +/- 1.01. FH (-1.50 +/- 1.03) was below TH (-0.78 +/- 0.84) (n = 25, p < 0.001). FH has not differed according to sex, clinical form and age at therapy onset although there was a trend towards better FH in SW patients and in early treated cases; there was significant difference (p = 0.018) between patients treated for less than 5 years (-2.49 +/- 1.03) and those accompanied longer than 10 years (-1.21 +/- 0.88) before attaining FH. CONCLUSIONS: There was a FH impairment and adult height improvement seems to depend mainly on early diagnosis and treatment.

Functional characterization of two novel point mutations in the CYP21 gene causing simple virilizing forms of congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

Congenital adrenal hyperplasia is a group of autosomal recessive disorders most often caused by deficiency of steroid 21-hydroxylase due to mutations in the CYP21 gene. We studied the functional and structural consequences of two novel missense mutations in the CYP21 gene, detected in two simple virilizing congenital adrenal hyperplasia patients. Both the male and female patient were compound heterozygous for the novel I77T and A434V point mutations, respectively. The in vitro expression analysis in COS-7 cells revealed a reduced 21-hydroxylase activity in the I77T mutant of 3 +/- 2% (sd) for the conversion of 17-hydroxyprogesterone to 11-deoxycortisol and of 5 +/- 3% for the conversion of progesterone to 11-deoxycorticosterone. The A434V mutant had a residual enzyme activity of 14 +/- 2% for 17-hydroxyprogesterone and 12 +/- 6% for progesterone. Substrate affinity was similar in the mutants as in the CYP21 wild-type protein, whereas reaction velocity was markedly decreased in both mutants. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. We hypothesize that the I77T mutation markedly decreases the reaction product release and/or substrate entrance to the enzyme's active site, whereas the A434V mutant reduces both the catalytic capacity and reaction velocity. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 21-hydroxylase deficiency and also provides new insights into cytochrome P450 structure-function relationships.

Behavioral and physical masculinization are related to genotype in girls with congenital adrenal hyperplasia.

Girls with congenital adrenal hyperplasia (CAH) exhibit behavioral masculinization. There is controversy about the roles of pre- and postnatal androgens, social factors, and chronic illness in its etiology. To assess the effect of chronic illness, we compared behavioral masculinity in 24 CAH girls and 25 diabetic girls aged 3-12 yr from Manchester using two sensitive questionnaires, and an overall masculinity score M (high = masculine) was derived. To assess the contributions of pre- and postnatal androgens, the CAH subjects were categorized into genotype groups (G) according to the reported severity of loss of CYP21 function: G1 (n = 10, null mutations), G2 (n = 9, intron 2G), G3 (n = 3, I172N), and G4 (n = 2, unknown loss of function). In CAH girls, relationships between G, Prader degree of genital masculinization at birth, bone age advance, and M were assessed. CAH girls were less feminine and more masculine than diabetic girls (P < 0.001), who were not significantly different from U.S. controls. Among the CAH girls, those in G1 and 2 were more genitally masculinized than those in G3 and 4 (P < 0.009) and had higher M (P < 0.025). M was negatively correlated with advanced bone age (r = -0.5; P = 0.02). CAH girls, but not diabetic girls, demonstrated behavioral masculinization. Both physical and behavioral masculinization were related to each other and to genotype, indicating that behavioral masculinization is a consequence of prenatal androgen exposure.

Unilateral adrenal tumor, erectile dysfunction and infertility in a patient with 21-hydroxylase deficiency: effects of glucocorticoid treatment and surgery.

In untreated congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHDS) the presence of adrenal and testicular tumors had been described; however little is known about the effect of the enzymatic defect on fertility in males. We studied a male adult patient affected by 21OHDS for infertility, after a long period of discontinuation of glucocorticoid therapy and then during resumption of treatment and 8 months after monoadrenalectomy. The initial spermatic count revealed azoospermia and testicular needle aspiration showed a cytological picture consistent with prepuberty. The morphofunctional study revealed a right adrenal mass with reduced uptake at radioscan. Treatment was resumed with onset of impotency, which improved after reduction of the dose of glucocorticoids. The patient was monoadrenalectomised and his spermatic count increased. The patient shows that corticosteroid therapy in 21OHDS should be continued lifelong to avoid adrenal hyperplasia with possible areas of autonomy and to allow regular fertility. Impotence during treatment is probably due to a decrease of excessive adrenal androgens while testicular androgen production is still suppressed.

Structure and expression of the CYP21 (P450c21, steroid 21-hydroxylase) gene with respect to its deficiency.

Steroid 21-hydroxylase (P450c21) deficiency is the major cause of a common genetic disease, congenital adrenal hyperplasia, with the symptoms of virilization due to steroid imbalance. We have devised a fast diagnostic method to detect common mutations in the c21B gene by a two-step gene amplification procedure coupled to restriction digestion. This procedure does not require isotopes and is suitable for routine use in a hospital setting. In addition, we have developed a procedure for the production of active P450c21 in E. coli. We tested many different vector and bacterial strain combinations to find out the best condition for P450c21 expression. The bacteria harboring the P450c21 expression plasmid were grown in a rich media supplemented with trace metals, heme biosynthesis precursor delta-levulinic acid, and induced with IPTG at 20 degrees C for 48 h. We found that low growth temperature and long induction time were important for abundant synthesis of P450c21 in E. coli.

Autoimmune Addison's disease--evidence for a role of steroid 21-hydroxylase autoantibodies in adrenal insufficiency.

Autoantibodies to steroid 21-hydroxylase (21-OH) are characteristic of adult onset Addison's disease and we have investigated the effects of these autoantibodies on recombinant human 21-OH enzyme activity. Antibody preparations from 11/11 Addison sera inhibited the ability of 21-OH to convert progesterone to deoxycorticosterone with 8 IgGs showing almost complete inhibition, 2 partial inhibition and 1 weak inhibition. Control IgGs from patients with autoimmune thyroid disease and normal blood donors had little or no effect on 21-OH activity. Our results suggest that 21-OH autoantibodies have the potential to contribute to adrenal failure in Addison's disease by inhibiting the 21-OH enzyme.

General intelligence and cognitive profile in women with congenital adrenal hyperplasia (CAH).

To investigate possible effects of prenatal androgen exposure on cognitive functions, general intelligence and cognitive profile were studied in 22 women, 17-34 years old, with prenatal virilization due to congenital adrenal hyperplasia (CAH) (21-hydroxylase deficiency) and 22 healthy controls matched for region and date of birth. The tests were selected to measure abilities where gender differences repeatedly had been observed or that had earlier shown differences between CAH subjects and controls. The following cognitive functions were tested: Verbal, Visuo-Spatial, Visuo-Motor, Arithmetical, Logical Inductive abilities, and Field Dependence. Contrary to earlier reports, the mean general intelligence level of the CAH (22) group was significantly lower than the controls' and the earlier observed inferiority for calculation abilities in female CAH subjects was not supported by the results of the present study. A possible verbal disadvantage with significantly inferior results was noted for the two verbal tests for the CAH (22) group. For the 13 CAH/control pairs with equal general intelligence levels, the discrepancy between the inferior verbal vs. visual, arithmetic, and logical category scores was significantly larger for the CAH group than the controls. These results may suggest that CAH women develop a more masculine cognitive pattern under the influence of increased prenatal androgen exposure.

Stimulation by voluntary exercise of adrenal glucocorticoid secretion in mature female hamsters.

The possibility that habitual voluntary running induces a chronic change in adrenal glucocorticoid synthesis and secretion was examined in freely running mature female hamsters, in whom this behavior accelerates growth, reduces body fat levels, and elevates core temperature. Hamsters were free to run on horizontal discs or in vertical wheels between 32 and 80 days, in 14L:10D or in 10L:14D photoperiods, and at the end of this period, corticosterone and cortisol steroidogenesis and serial plasma corticosterone concentrations during day and night were used as measures of the chronic stimulation of adrenal cortical activity. Habitual voluntary running significantly increased steroidogenesis of both glucocorticoids and plasma corticosterone concentrations and alone accounted for all the variance in enhanced synthesis and secretion of corticosterone. Acute exercise and/or the nocturnal phase of circadian period enhanced the chronic stimulatory effects of exercise on cortisol. Despite its voluntary and apparently stress-free nature, running induces chronic increases in basal glucocorticoid secretion in mature female hamsters. Putative oversecretion of corticotropin releasing factor in freely running hamsters could account for increased steroidogenesis, acceleration of growth, reduced body fat levels, and core temperature elevation.

Primary pigmented nodular adrenocortical disease (PPNAD): immunohistochemical and in situ hybridization analysis of steroidogenic enzymes in eight cases.

Primary pigmented nodular adrenocortical disease (PPNAD) is a rare but an interesting adrenocortical disorder associated with ACTH-independent hypercortisolism. We have studied eight cases of the adrenals with PPNAD by immunohistochemistry of all steroidogenic enzymes involved in cortisol biosynthesis (P-45scc, 3 beta-HSD, P-450c21, P-45017 alpha, and P-45011 beta) and also by performing in situ hybridization of P-45017 alpha in seven cases in order to localize the sites of specific steroidogenesis in this unique disorder. Immunoreactivity of all the enzymes examined was intense in almost all of the cells in adrenocortical nodules, especially the cells with abundant eosinophilic cytoplasm in all the cases examined. The internodular cortex, which demonstrated atrophy in five cases, normal appearance in two cases and hyperplasia in one case, was negative for the enzymes with an exception of 3 beta-HSD. Hybridization signals of P-45017 alpha were condensed over the nodules in in situ hybridization study, suggestive of an increased production of the enzyme itself in cortical cells of the nodules. These results may be consistent with autonomous cortisol production by the nodular cells and indicate that almost all of the cells in the nodules produce cortisol, which can also explain the presence of hypercortisolism despite small sizes of adrenals in PPNAD. Immunoreactivity of steroidogenic enzymes is observed in a small cluster of cortical cells with abundant eosinophilic cytoplasm located at the zona reticularis but not in adjacent non-nodular cortex, which may support an abnormal development of the zona reticularis as a possible pathogenesis of this disorder.