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Congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency is a common inherited disorder of adrenal hormone biosynthesis due to mutations in the 21-hydroxylase gene, CYP21A2. Genotyping for ten of the most frequent mutations was performed in 84 Portuguese CAH patients: 10 salt-wasters, 6 simple-virilizers and 68 non-classical patients. The patients were diagnosed by a level of 17-hydroxyprogesterone above 10 ng/ml either in basal conditions or after an ACTH 0,25 mg IV Test. A variety of genotyping techniques were used to detect these ten mutations. CYP21A2 mutations were detected in 91.7% (77/84) of the patients. The frequency of alleles carrying two or more CYP21A2 mutations (9.5% - 16/168) is higher than in other populations. The most frequent mutations identified in our population were V281L (41.7%) and deletions/conversions involving the promoter region of the CYP21A2 gene (28.3%). A decreased frequency of IVS2-12C/A>G mutation (5.6%) was the most characteristic feature of our population. This study allow the characterization of the mutational spectrum of CAH patients, mainly non-classical CAH, with 21-hydroxylase deficiency from Portugal showing specific genetic features of this population which reveals differences with worldwide countries., (© J. A. Barth Verlag in Georg Thieme Verlag KG Stuttgart · New York.)

Context: Because many women with 21-hydroxylase (21-OH)-deficient nonclassic adrenal hyperplasia (NCAH) carry at least one allele affected by a severe mutation of CYP21, they are at risk for giving birth to infants with classic adrenal hyperplasia (CAH)., Objective: Our objective was to determine the frequency of CAH and NCAH infants born to mothers with 21-OH-deficient NCAH., Design and Setting: We conducted an international multicenter retrospective/prospective study., Patients and Methods: The outcome of 203 pregnancies among 101 women with 21-OH-deficient NCAH was reviewed. The diagnosis of 21-OH-deficient NCAH was established by a basal or post-ACTH-stimulation 17-hydroxyprogesterone level of more than 10 ng/ml (30.3 nmol/liter). When possible, genotype analyses were performed to confirm CAH or NCAH in the offspring., Results: Of the 203 pregnancies, 138 (68%) occurred before the mother's diagnosis of NCAH and 65 (32%) after the diagnosis. Spontaneous miscarriages occurred in 35 of 138 pregnancies (25.4%) before the maternal diagnosis of NCAH, and in only four of 65 pregnancies (6.2%) after the diagnosis (P < 0.002). Four (2.5%; 95% confidence interval, 0.7-6.2%) of the 162 live births were diagnosed with CAH. To date, 24 (14.8%; 95% confidence interval, 9.0-20.6%) children, 13 girls and 11 boys, have been diagnosed with NCAH. The distribution of NCAH children and their mothers varied significantly by ethnicity (P < 0.0001, for both)., Conclusions: The risk of a mother with 21-OH-deficient NCAH for giving birth to a child affected with CAH is 2.5%; at least 14.8% of children born to these mothers have NCAH.

Objective: Our aim was to determine whether the clinical features of 21-hydroxylase-deficient nonclassic adrenal hyperplasia are correlated with either age at symptom onset or age at presentation, or both, and with the degree of adrenocortical abnormality., Study Design: In a multicenter cohort design 220 women with nonclassic adrenal hyperplasia, with a basal or adrenocorticotropic hormone-stimulated 17-hydroxyprogesterone level >30.3 nmol/L, were studied, either prospectively (n = 39) or retrospectively (n = 181). Patients were stratified by age of presentation into 5 groups: (1) <10 years (n = 25), (2) 10 to 19 years (n = 64), (3) 20 to 29 years (n = 83), (4) 30 to 39 years (n = 30), and (5) 40 to 49 years (n = 16). Two patients >50 years old were excluded from the analysis because of age., Results: Ninety-two percent of patients <10 years old had premature pubarche at presentation, whereas clitoromegaly and acne were each present in only 20% of these younger subjects. With only patients > or =10 years old considered, presenting clinical features included hirsutism (59%), oligomenorrhea (54%), acne (33%), infertility (13%), clitoromegaly (10%), alopecia (8%), primary amenorrhea (4%), and premature pubarche (4%). Among the patients >/=10 years old, the prevalence but not the degree of hirsutism increased significantly with age. Basal levels of 17-hydroxyprogesterone in adolescents were significantly higher than the levels found either in children (<10 years old) or women 40 to 49 years old (P <.01 and P <.03, respectively), although no difference was noted in the stimulated 17-hydroxyprogesterone levels between age groups. The adrenocorticotropic hormone-stimulated levels but not the basal levels of 17-hydroxyprogesterone were significantly higher in patients with clitoromegaly than in women without clitoromegaly. Alternatively, there were no differences in either basal or stimulated 17-hydroxyprogesterone levels between patients with and those without hirsutism, acne, or alopecia., Conclusion: In children <10 years old the most common presenting complaint was premature pubarche, whereas hirsutism and oligomenorrhea were more common in older patients. The prevalence of hirsutism increased with age, suggesting the progressive nature of nonclassic adrenal hyperplasia. Furthermore, the adrenocorticotropic hormone-stimulated levels of 17-hydroxyprogesterone were higher in patients with clitoromegaly, which suggests that the degree of adrenocortical dysfunction in nonclassic adrenal hyperplasia determines, at least in part, the clinical presentation.

Congenital adrenal hyperplasia caused by 21-hydroxylase deficiency is a common autosomal recessive disorder resulting from mutations in the 21-hydroxylase (CYP21) gene. To develop a strategy to screen for the most commonly occurring CYP21 mutations in Brazil, we performed molecular genotype analysis on 73 children with CAH representing 71 unrelated families. The techniques used for CYP21 molecular genotype analysis were: restriction fragment length polymorphism, single-strand conformational polymorphism, allele-specific oligonucleotide hybridization, allele-specific polymerase chain reaction amplification, and heteroduplex analyses. Mutations were identified on all but eight affected alleles. The intron 2 splicing mutation was the most frequently identified mutation. Screening for the most common mutations detected at least one mutation on 132/142 (93%) alleles. Multiple CYP21 mutations were detected on 16.2% of alleles. The high frequency of multiple mutations on a single allele emphasizes the importance of thorough and accurate molecular genotype analysis of the complex CYP21 locus.

Objective: To determine the role of heterozygosity for mutations in the 21-hydroxylase gene (CYP21) in the pathogenesis of hyperandrogenism., Design: Controlled clinical study., Setting: Tertiary care institutional hospital., Patient(s): Forty hirsute women and 13 healthy control women., Intervention(s): The source of androgen excess was determined by the changes in serum testosterone levels in response to a single 3.75-mg i.m. dose of triptorelin., Main Outcome Measure(s): CYP21 molecular genetic analysis and serum 17-hydroxyprogesterone levels., Result(s): Eight patients and one control were heterozygous carriers of CYP21 mutations. Two patients with adrenal hyperandrogenism and one patient with ovarian hyperandrogenism, who carried the V281L mutation had an increased ACTH-stimulated 17-hydroxyprogesterone level (>4.1 ng/mL) that persisted during gonadal suppression. Another patient with adrenal hyperandrogenism carried the V281L mutation, and her ACTH-stimulated 17-hydroxyprogesterone level was elevated only during gonadal suppression. Four patients (three with idiopathic hirsutism, one with ovarian hyperandrogenism) and one control were carriers of CYP21 mutations typically associated with classic congenital adrenal hyperplasia but had normal basal and ACTH-stimulated 17-hydroxyprogesterone levels. Nine patients without CYP21 mutations had increased ACTH-stimulated 17-hydroxyprogesterone levels; these decreased to normal in six of the patients during gonadal suppression., Conclusion(s): The response of serum 17-hydroxyprogesterone to ACTH does not predict CYP21 carrier status. No clear concordance was found between the CYP21 genotype and the functional origin of androgen excess.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal recessive disorder (MIM# 201910) due to mutations in the 21-hydroxylase (CYP21) gene (GDB Accession # M12792). Using our protocol for single strand conformational polymorphism (SSCP) analysis, we have identified two mutations not known to exist in the 21-hydroxylase pseudogene (CYP21P). One mutation involving codon 169, TGC to AC appears to be novel. The 46,XX patient carried the codon 169 mutation on her paternal allele and a large gene deletion/conversion event on her maternal allele. This patient had been referred in the immediate neonatal period for the evaluation of genital ambiguity and had developed hyponatremia and hyperkalemia. The second patient presented with premature pubic hair. She carried R356Q on her maternal allele and V281L on her paternal allele.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. To ascertain carrier status, adrenocorticotropin (ACTH) stimulation tests are often used. To determine the sensitivity of ACTH stimulation to detect heterozygotes and to correlate stimulated 17-hydroxyprogesterone responses with molecular genotype, we compared molecular genetic analysis of the 21-hydroxylase (CYP21) gene with 17-hydroxyprogesterone responses at 30 min in 51 individuals. Molecular genotype analysis and ACTH stimulation tests were performed in healthy volunteers (n = 20) and relatives of patients with congenital adrenal hyperplasia (n = 31). Polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, allele-specific oligonucleotide hybridization (ASOH) analysis, and restriction fragment length polymorphism (RFLP) analysis were utilized to screen for 14 CYP21 mutations which account for >90% of the mutations associated with 21-hydroxylase deficiency. Molecular genotype analysis classified 28 individuals as heterozygotic carriers and 23 individuals as normal for all mutations tested. As a group, the heterozygotes had significantly greater stimulated 17-hydroxyprogesterone responses at 10 and 30 min (P < 0.0005). However, on an individual basis, 14/28 (50%) genotyped heterozygotic carriers had stimulated 17-hydroxyprogesterone concentrations, 17-hydroxyprogesterone/cortisol ratios, and 17-hydroxyprogesterone incremental elevations indistinguishable from the genotyped normal individuals. Thus, a normal 17-hydroxyprogesterone response to ACTH stimulation testing does not exclude carrier status for 21-hydroxylase deficiency. Molecular genotype analysis is a more reliable method to determine 21-hydroxylase heterozygotes.

Premature adrenarche and functional adolescent androgen excess are common disorders which may evolve into polycystic ovary syndrome (PCOS). In all three disorders, ACTH-stimulated 17-hydroxyprogesterone concentrations are often somewhat elevated. To determine the role of 21-hydroxylase (CYP21) gene mutations in these disorders, we performed molecular genotype analysis on 48 children and adolescents referred for evaluation of hyperandrogenic findings and diagnosed as having premature adrenarche or functional androgen excess. For comparison, DNA samples from 80 healthy adults were genotyped. Seventeen of the 48 hyperandrogenic patients were found to be heterozygotic carriers of CYP21 mutations. The frequency of heterozygosity was significantly greater among symptomatic patients (35%) than among the healthy controls (6%), P < 0.001. Seven mutation-positive patients (50%) and only one mutation-negative patient had ACTH-stimulated 17-hydroxyprogesterone concentrations typical for heterozygotic carriers of 21-hydroxylase deficiency, 400-1000 ng/dl. The significant difference in heterozygote frequency between symptomatic patients and healthy controls suggests that heterozygosity for 21-hydroxylase deficiency may be associated with premature adrenarche and functional adolescent hyperandrogenism. Longitudinal studies are necessary to determine if heterozygosity for 21-hydroxylase deficiency predicts risk for PCOS.

Blood samples for plasma steroid hormone determinations and molecular genotype analysis of the 21-hydroxylase gene (CYP21) were obtained from 15 infants identified through a voluntary newborn screening program. Mutations were identified on both CYP21 alleles in 12 (80%) of 15 infants; all had confirmatory plasma 17-hydroxyprogesterone concentrations > 3500 ng/dl. No patient was found to carry mutations associated with late-onset 21-hydroxylase deficiency. Newborn screening hastened diagnosis in eight infants.

21-Hydroxylase deficiency is one of the most common inherited disorders, with carrier frequencies of approximately 10% in all world populations studied to date. The high prevalence of the mutant gene is probably due to a flanking pseudogene serving as a reservoir for mutations. Despite the potential for a high rate of de novo mutations, a founder effect for specific gene conversions is observed in most populations. We hypothesized that there was a survival advantage to 21-hydroxylase heterozygotes, and here we report endocrinological and molecular investigations to test this hypothesis. We defined 28 carriers and 22 mutation-negative controls by molecular genotyping and determined ACTH-stimulated adrenal hormone responses. We found significantly elevated cortisol responses in the carriers compared to controls (30 min cortisol levels: normal, 24.2 +/- 4.6 micrograms/dL; carrier, 28.1 +/- 4.2 micrograms/dL; P < 0.005). Cortisol has a crucial role in maintaining homeostasis, influencing differentiation, suppressing inflammation, and effecting cross-talk among the immune, nervous, and endocrine systems. The brisk cortisol response we have documented in carriers of 21-hydroxylase may enable a rapid return to homeostasis in response to infectious, inflammatory, or other environmental stresses and may protect from inappropriate immune responses, such as autoimmune diseases.

One mutation frequently identified in 21-hydroxylase deficiency is the intron 2 splicing mutation, in which the normal polymorphic C or A at nucleotide 655 has been converted to G. Using allele-specific oligonucleotide hybridization, single strand conformational polymorphism analysis, and heteroduplex analyses, we identified 38 individuals from 21 different families who had 2 deleterious mutations. All were homozygous or compound heterozygotes for the splicing mutation. Comparison of the phenotypic features with the molecular genotypes shows phenotypic heterogeneity extending from classical salt-losing 21-hydroxylase deficiency to asymptomatic. Single strand conformational polymorphism analysis followed by DNA sequence analysis revealed numerous sequence variations in intron 2, most commonly at nucleotides 601 and 683. Transient transfection experiments show that the 3'-portion of intron 2 is sufficient to transfer the effect of the 655c/a-->g mutation to a chimeric heterologous gene. Clinical correlations and initial transfection studies suggest that sequence variations at nucleotides 601 and 683 do not correlate with clinical severity or substantially affect splicing. In summary, a single nucleotide change, 655c/a-->g, alters the splice acceptor site at the intron 2/exon 3 boundary. The molecular basis of the phenotypic heterogeneity associated with the mutation remains to be elucidated.

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a common autosomal-recessive disorder. During our routine genotyping of affected individuals and their relatives using allele-specific oligonucleotide hybridization and single-strand conformational polymorphism analysis, we identified two families each segregating three mutations. In both families, a mutation known to be associated with 21-hydroxylase deficiency was identified in healthy individuals but was not detected in the propositus. The propositus in family 1 was shown to be a homozygous carrier for G at nucleotide 655, which alters the splice acceptor site at exon 3. The propositus in family 2 carried the same splicing mutation on the maternal allele and a gene deletion/conversion on the paternal allele. In both families, other clinically unaffected relatives carried the Q318X mutation in exon 8. If molecular diagnostic studies had been limited to the mutation carried by the propositi, relatives would have been misinformed regarding their status as carriers or mildly affected individuals. The findings in these two families emphasize the high frequency of alleles causing 21-hydroxylase deficiency in the population.