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Congenital adrenal hyperplasia owing to 21-hydroxylase deficiency is caused by mutation in the CYP21A2 gene. The frequency and spectrum of CYP21A2 mutations and genotype-phenotype correlations among different populations are variable. Aim of this study was to define mutation frequency and spectrum of CYP21A2 gene mutations in patients with classical 21-hydroxylase deficiency (21OHD) and their family members in Croatia and study genotype-phenotype correlation. Clinical features and mutations of CYP21A2 gene in 93 unrelated 21OHD patients and 193 family members were examined. In this cohort, 66 patients were affected with salt wasting (SW) form, and 27 were affected with simple virilizing (SV) form of the disease. Mutations were identified in both alleles (67% compound heterozygous and 33% homozygous) in 91 of 93 patients. Deletions and conversions were found in 18.8% and point mutations in 79.6% alleles. Mutations in 3 alleles (1.6%) remained unidentified (in one patient we found only one, while in other no mutations were found at all). The most common point mutations were Intron 2 splice mutation IVS2-13 A/C>G (35.5%) and p.R357W (16.7%). Genotypes were categorized into Groups 0, A, B and C according to the extent of enzyme impairment. Genotype-phenotype concordance was 100%, 85% and 75% for Groups 0, A and B, respectively. Since only classical 21OHD patients were studied, Group C comprised solely p.P31L mutation and had 73% patients with SV and 27% with SW phenotype. Intrafamilial phenotypic variability was found in two families. CYP21A2 genetic analysis in 193 family members showed that 126 parents were heterozygous carriers, 3 were newly discovered patients, 2 fathers were not biological parents, and mutations were not detected in 3. Among 59 siblings, 32 were heterozygous carriers, 15 carried normal alleles, and 12 were patients (4 newly diagnosed). Genotype-phenotype divergence observed in this study suggests caution in preconceptional counseling and prenatal diagnosis of CAH. High frequency of p.R357W mutation was found in Croatian patients with classical 21-OHD. Genotyping of family members discovered new patients and thus avoided pitfalls in genetic counseling when the parents were found to be affected., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Patients with Congenital Adrenal Hyperplasia (CAH) owing to 21-hydroxylase deficiency and whose karyotype is 46, XX are usually assigned to the female gender. Reported herein are the long term outcomes in three patients with CAH whose karyotype is 46, XX and who were reared as males. A retrospective review of three CAH patients with a 46, XX karyotype who were reared as males was conducted. Gender assignment, clinical and biochemical data, pre and post-genitoplasty genital examinations were reviewed. Gender identity was tested by an extensive questionnaire. Gender role, sexual preference, marital status and sexual satisfaction were evaluated by interview. The three patients were genotyped for the CYP21A2 gene confirming the diagnosis of CAH. Owing to genital virilization, cultural preferences for male gender and the lack of newborn screening programs the three patients reported herein were assigned to the male gender at birth before the diagnosis of CAH was established. In adulthood the patients remained significantly virilized. Thorough psychosexual assessments in adulthood revealed well established male gender identities compatible with their male gender assignments at birth. In all three patients, gender role and behavior were consistent with male gender identity including sexual intercourse with female partners. The three patients reported herein revealed that male gender assignment to CAH patients with a 46, XX karyotype may have a successful outcome providing there is strong parental support and expert endocrine care. No standard guidelines have been published for the gender assignment of CAH patients with a 46, XX karyotype and genital ambiguity. More studies concerning gender assignment in CAH patients with a 46, XX karyotype reared as males are needed., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Congenital adrenal hyperplasia (CAH) refers to a group of inherited genetic disorders involving deficiencies in enzymes that convert cholesterol to cortisol within the adrenal cortex. There are five key enzymes involved in the production of cortisol. Of these key enzymes, deficiency of 21-hydroxylase is the most commonly defective enzyme leading to CAH representing more than 90% of cases. The low adrenal cortisol levels associated with CAH affects the hypothalamic-pituitary-adrenal negative feedback system leading to increased pituitary adrenocorticotropic hormone (ACTH) production, which overstimulates the adrenal cortex in an attempt to increase cortisol production resulting in a hyperplastic adrenal cortex. The deficiency of enzyme 21-hydroxylase results from mutations or deletions in the CYP21A2 gene found on chromosome 6p. The disorder is transmitted as an autosomal recessive pattern and specific mutations may be correlated to enzymatic compromise of varying degrees, leading to the clinical manifestation of 21-hydroxylase deficiency (21-OHD) CAH., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Apparent mineralocorticoid excess (AME) is a genetic disorder causing severe hypertension, hypokalemia, and hyporeninemic hypoaldosteronism owing to deficient 11 beta-hydroxysteroid dehydrogenase type-2 (11βHSD2) enzyme activity. The 11βHSD2 enzyme confers mineralocorticoid receptor specificity for aldosterone by converting cortisol to its inactive metabolite, cortisone and inactivating the cortisol-mineralocorticoid receptor complex. The 20year follow-up of a consanguineous Iranian family with three sibs affected with AME shows the successes and pitfalls of medical therapy with spironolactone. The three sibs, (female, male, female) were diagnosed at the ages of 14, 11, and 4 years, respectively. At diagnosis, hypertensive retinopathy and left ventricular hypertrophy were present in the eldest female and retinopathy was noted in the male sib. Spironolactone treatment resulted in decreased blood pressure and rise in serum potassium levels. The older female, age 36, developed reduced left ventricular function with mitral and tricuspid regurgitation and renal failure after her second pregnancy. She was treated with renal transplantation resulting in cure of AME with decreased blood pressure and weaning from antihypertensives. Her younger sibs, age 34 and 26, do not have end organ damage. Early and vigilant treatment improves morbidity in patients with AME. Mineralocorticoid receptor antagonists normalize blood pressure, correct hypokalemia and reduce hypertensive end-organ damage in patients with AME. Low dose dexamethasone can be considered, though the response may be variable. Future directions of therapy include selective mineralocorticoid antagonists., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

A 14-year-old Native American girl from the Iroquois Nation was referred as a potential patient with the syndrome of Apparent Mineralocorticoid Excess. Instead, her evaluation revealed resistance to glucocorticoids, mineralocorticoids, and androgens. She lacked Cushingoid features in spite of significantly high cortisol levels. Menstruation was regular and there was no clinical evidence of masculinization despite high serum androgen levels in the male range. The patient's sister had similar clinical features. Partial resistance to exogenous glucocorticoid and mineralocorticoid administration was well demonstrated in both patients. It is proposed that these patients represent the first cases of partial resistance to multiple steroids, possibly owing to a coactivator defect.

The diagnostic term congenital adrenal hyperplasia (CAH) applies to a family of inherited disorders of steroidogenesis caused by an abnormality in one of the five enzymatic steps necessary in the conversion of cholesterol to cortisol. The enzyme defects are translated as autosomal recessive traits, with the enzyme deficient in more than 90% of CAH cases being 21-hydroxylase. In the classical forms of CAH (simple virilizing and salt wasting), owing to 21-hydroxylase deficiency (21-OHD), androgen excess causes external genital ambiguity in newborn females and progressive postnatal virilization in males and females. Non-classical 21-OHD (NC21OHD) refers to the condition in which partial deficiencies of 21-hydroxylation produce less extreme hyperandrogenemia and milder symptoms. Females do not demonstrate genital ambiguity at birth. The gene for adrenal 21-hydroxylase, CYP21, is located on chromosome 6p in the area of HLA genes. Specific mutations may be correlated with a given degree of enzymatic compromise and the clinical form of 21-OHD. NC21OHD patients are predicted to have mild mutations on both alleles or one severe and one mild mutation of the 21-OH locus (compound heterozygote). In most cases the mutation groups represent one diagnosis (e.g., Del/Del with SW CAH), however we have found several non-correlations of genotype to phenotype. Non-classical and classical patients were found within the same mutation group. Phenotypic variability within each mutation group has important implications for prenatal diagnosis and treatment. Prenatal treatment of 21-OHD with dexamethasone has been utilized for a decade. An algorithm has been developed for prenatal diagnosis and treatment, which, when followed closely, has been safe for both the mother and the fetus, and has been effective in preventing ambiguous genitalia in the affected female newborn. This is an instance of an inborn metabolic error successfully treated prenatally. Since 1986, prenatal diagnosis and treatment of congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21-OHD) has been carried out in 403 pregnancies in The New York Hospital Cornell Medical Center. In 280, diagnoses were made by amniocentesis, while 123 were diagnosed using chorionic villus sampling. Of the 403 pregnancies evaluated, 84 babies were affected with classical 21-OHD. Of these, 52 were females, 36 of whom were treated prenatally with dexamethasone. Dexamethasone administered at or before 10 weeks of gestation (23 affected female fetuses) was effective in reducing virilization. Thirteen cases had affected female sibs (Prader stages 1-4); 6 of these fetuses were born with entirely normal female genitalia, while 6 were significantly less virilized (Prader stages 1-2) than their sibs, and one was Prader stage 3. Eight newborns had male sibs: 4 were born with normal genitalia, 3 were Prader stages 1-2, and 3 were born Prader stages 3-4. No significant or enduring side effects were noted in either the mothers or the fetuses, indicating that dexamethasone treatment is safe. Prenatally treated newborns did not differ in weight, length, or head circumference from untreated, unaffected newborns. Based on our experience, proper prenatal diagnosis and treatment of 21-OHD is effective in significantly reducing or eliminating virilization in the newborn female. This spares the affected female the consequences of genital ambiguity of genital surgery, sex misassignment, and gender confusion.

The enzyme 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) catalyses an essential step in the biosynthesis of all classes of steroid hormones. Classical 3 beta-HSD deficiency is responsible for CAHII, a severe form of congenital adrenal hyperplasia (CAH) that impairs steroidogenesis in both the adrenals and gonads. Newborns affected by 3 beta-HSD deficiency exhibit signs and symptoms of adrenal insufficiency of varying degrees associated with pseudohermaphroditism in males, whereas females exhibit normal sexual differentiation or mild virilization. Elevated ratios of 5-ene-to 4-ene-steroids appear as the best biological parameter for the diagnosis of 3 beta-HSD deficiency. The nonclassical form has been suggested to be related to an allelic variant of the classical form of 3 beta-HSD as described for steroid 21-hydroxylase deficiency. To elucidate the molecular basis of the classical form of 3 beta-HSD deficiency, we have analysed the structure of the highly homologous type I and II 3 beta-HSD genes in 12 male pseudohermaphrodite 3 beta-HSD deficient patients as well as in four female patients. The 14 different point mutations characterized were all detected in the type II 3 beta-HSD gene, which is the gene predominantly expressed in the adrenals and gonads, while no mutation was detected in the type I 3 beta-HSD gene predominantly expressed in the placenta and peripheral tissues. The finding of a normal type I 3 beta-HSD gene provides the explanation for the intact peripheral intracrine steroidogenesis in these patients and increased androgen manifestations at puberty. The influence of the detected mutations on enzymatic activity was assessed by in vitro expression analysis of mutant enzymes generated by site-directed mutagenesis in COS-1 cells. The mutant type II 3 beta-HSD enzymes carrying mutations detected in patients affected by the salt-losing form exhibit no detectable activity in intact transfected cells, whereas those with mutations found in nonsalt-loser index cases have some residual activity ranging from approximately 1-10% compared to the wild-type enzyme. Although in general, our findings provide a molecular explanation for the enzymatic heterogeneity ranging from the severe salt-losing form to the clinically inapparent salt-wasting form of the disease, we have observed that the mutant L108W or P186L enzymes found in a compound heterozygote male presenting the salt-wasting form of the disease, has some residual activity (approximately 1%) similar to that observed for the mutant N100S enzyme detected in a homozygous male patient suffering from a nonsalt-losing form of this disorder.(ABSTRACT TRUNCATED AT 400 WORDS)

Congenital adrenal hyperplasia (CAH) results from an enzymatic block at any stage in the synthesis of cortisol. All enzyme defects causing CAH are autosomal recessive traits. It is a relatively common disease, occurring in 1 in 5000 to 1 in 15,000 births in most populations. Since the isolation of the gene responsible for steroid 21-hydroxylase deficiency (involved in about 90% of the cases of CAH) in 1984, knowledge of the specific mutations that cause the different forms of CAH has grown rapidly. Defects in the encoding gene have been confirmed as the basis of endocrine disease in the case of all but one of the adrenal steroidogenic enzymes. Analysis of DNA obtained by chorionic villus sampling in early pregnancy permits prenatal diagnosis and treatment of 21-hydroxylase deficiency CAH. The correlation between the clinical expression of endocrine disease and the mutations of the primary structural gene is not absolute. Clinicians cannot accurately predict the course of the disease or make therapeutic decisions based on the genotype alone. We will review the various forms of clinical presentation of 21-hydroxylase CAH, its etiology, diagnosis, molecular genetics, and treatment.

Rat liver 3 alpha-hydroxysteroid dehydrogenase cDNA was previously cloned by us. In this study, we used the rat cDNA as the probe to screen a human liver lambda gt11 cDNA library. A total of four different cDNAs were identified and sequenced. The sequence of one of the cDNAs is identical to that of the human chlordecone reductase cDNA except that our clone contains a much longer 5'-coding sequence than previously reported. The other three cDNAs display high degrees of sequence homology to those of both rat 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase. Because 3 alpha-hydroxysteroid dehydrogenase and human chlordecone reductase belong to the aldo-keto reductase superfamily, we named these human clones HAKRa to HAKRd. Northern blot analysis showed that the liver expresses the highest levels of all four clones. Expression of all four clones was also detected in the brain, kidney, lung, and testis, whereas the placenta expressed only the messenger RNA for HAKRb. Genomic blot analysis using HAKRb as the probe detected multiple DNA fragments hybridized to the probe and a high degree of restriction fragment length polymorphism, suggesting the complexity of this supergene family.

The most common enzymatic defect of steroid synthesis is adrenal steroid 21-hydroxylase deficiency. Inhibited formation of cortisol causes increased pituitary release of ACTH, driving the adrenal cortex to overproduce androgens, whose synthesis does not involve the 21-hydroxylase enzyme. This hormonal setting is established in the embryonic period and affects development of genetic females, misdirecting differentiation of the external genitalia toward male type. At birth, the genitalia are visibly ambiguous (enlarged clitoris, fused labia) or in some cases even male in appearance (phallus with urethral opening, rugated scrotal sac), leading to wrong sex assignment. Adrenal steroid 21-hydroxylase deficiency is the most common basis of female pseudohermaphroditism. These females, however, have normal fertility and potential for gestation (gonads are functional and the internal duct-derived structures are well-formed), thus the sex of rearing should always be female. Management is by life-long hormonal (glucocorticoid) replacement, with surgical correction of the genital ambiguity. Prenatal diagnosis of 21-hydroxylase deficiency, first possible by steroid assay of the amniotic fluid, has utilized HLA typing for identification of loci (antigens B and DR) in close linkage with the 21-hydroxylase gene, and now increasingly relies on DNA analysis for linked HLA or C4 genes or for mutant 21-hydroxylase alleles directly by molecular genetic techniques. The most recent clinical advance is a program of combined prenatal diagnosis with karyotyping and suppression of fetal androgen production in genetic females by steroid administration to the mother. This is the first instance of an inborn metabolic error to be prenatally treated. A series of 85 managed pregnancies is reported on, including accuracy of diagnosis, response of the mother to steroid treatment, and outcome for treated and untreated male and female fetuses (of 77 born by 6/91). Prenatal diagnosis by current techniques is accurate. Normal growth and development patterns postnatally suggest that dexamethasone treatment is safe.