Structural aspects of the p.P222Q homozygous mutation of HSD3B2 gene in a patient with congenital adrenal hyperplasia.

Type II 3ß-hydroxysteroid dehydrogenase/Δ(5)-Δ(4)-isomerase (3ß-HSD2), encoded by the HSD3B2 gene, is a key enzyme involved in the biosynthesis of all the classes of steroid hormones. Deleterious mutations in the HSD3B2 gene cause the classical deficiency of 3ß-HSD2, which is a rare autosomal recessive disease that leads to congenital adrenal hyperplasia (CAH). CAH is the most frequent cause of ambiguous genitalia and adrenal insufficiency in newborn infants with variable degrees of salt losing. Here we report the molecular and structural analysis of the HSD3B2 gene in a 46,XY child, who was born from consanguineous parents, and presented with ambiguous genitalia and salt losing. The patient carries a homozygous nucleotide c.665C>A change in exon 4 that putatively substitutes the proline at codon 222 for glutamine. Molecular homology modeling of normal and mutant 3ß-HSD2 enzymes emphasizes codon 222 as an important residue for the folding pattern of the enzyme and validates a suitable model for analysis of new mutations.

Refining hormonal diagnosis of type II 3beta-hydroxysteroid dehydrogenase deficiency in patients with premature pubarche and hirsutism based on HSD3B2 genotyping.

Congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3betaHSD), a rare autosomal recessive disorder that affects both sexes, has a heterogeneous clinical presentation ranging from the severe salt-wasting to the non-salt-wasting forms and results from mutations in the HSD3B2 gene. The hormonal criteria for diagnosing the mild variant of 3betaHSD deficiency have been controversial because the initial studies were not based on genetic evidence. We investigated the relationship between the hormonal phenotype and HSD3B2 genotype in 22 patients with clinical and/or biochemical features suggestive of 3betaHSD2 deficiency, including nine female children with premature pubarche, 12 hirsute females, and one boy with salt-wasting and ambiguous genitalia. Serum 17-hydroxypregnenolone (Delta5-17P), cortisol (F), 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione levels were determined by RIA and were compared with Tanner pubic hair stage-matched control groups. The genomic DNA was extracted, and the entire HSD3B2 gene was amplified by PCR followed by automatic sequencing. Besides two different mutations previously observed in three patients (T259M and G129R/P222Q mutations), we observed the P222Q mutation in the male patient with salt-wasting form of 3betaHSD2 deficiency. Basal and ACTH-stimulated Delta5-17P levels (nanomoles per liter) ranged from 4-41 (-0.2 to 14 sd) and 36-97 (3.5-15.5 sd), respectively, in patients without mutation in HSD3B2 and from 69-153 (25-57 sd) and 201-351 (36-65 sd), respectively, in patients with mutation in HSD3B2. Basal and ACTH-stimulated Delta5-17P to F ratios ranged from 11-159 (0.5-25 sd) and 42-122 (2.4-11.3 sd), respectively, in patients without mutation in HSD3B2 and from 181-1700 (29-282 sd) and 487-1523 (52-167 sd), respectively, in patients with mutation in HSD3B2. The hormone findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3betaHSD2 deficiency in children with premature pubarche: ACTH-stimulated Delta5-17P and Delta5-17P to F ratios at or greater than 201 and 487 nmol/liter, respectively, equivalent to or greater than 36 and 52 sd above matched control mean. Basal and ACTH-stimulated Delta5-17P and Delta5-17P to F ratios in all genotype-proven patients in childhood were unequivocally higher than the levels of either genotype-normal patients. All the other parameters overlapped between the patients with and without mutations in the HSD3B2 gene. In conclusion, genotyping more patients in the present study, we confirm that patients with mutations in the HSD3B2 gene have extremely elevated basal and ACTH-stimulated Delta5-17P levels and Delta5-17P to F ratios. Therefore, these data refine the hormonal criteria proposed to predict more accurately 3betaHSD2 deficiency.

[Rare forms of female pseudohermaphroditism: when to investigate?]. / Causas raras de pseudo-hermafroditismo feminino: quando suspeitar?

The congenital adrenal hyperplasia is the commonest cause of ambiguity of the external genitalia at birth, due to classic forms of 21-hydroxylase and 11beta-hydroxylase deficiencies. 3beta-hydroxysteroid dehydrogenase (3betaHSD) is a rare disorder that affects both sexes and female patients may have ambiguous genitalia. Familial glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hypercortisolism, but with manifestations of androgen and mineralocorticoid excess, caused by glucocorticoid receptor gene mutation, and rarely can lead to female pseudohermaphroditism. Placental aromatase deficiency is a rare disease characterized by a masculinized female fetus and a virilized mother, which should be considered in the absence of fetal adrenal hyperplasia and maternal androgen-secreting tumours. Finally, mutations of P450 oxidoreductase causes disordered steroidogenesis with ambiguous genitalia. The investigation of abnormal sexual development requires an initial karyotype analysis and serum 17OH progesterone, 11 deoxycortisol, 17 pregnenolone, and androgen measurements to assess the diagnosis of different forms of congenital adrenal hyperplasia.

[Abnormalities of adrenal steroidogenesis in Chilean boys with micropenis]. / Anomalías de la esteroidogénesis suprarrenal en niños chilenos con micropene.

BACKGROUND: Mutations in type II 3 beta hydroxysteroid Dehydrogenase (3 beta HSD) are found in male children with severe undervirilized genitalia. Mild undervirilization (isolated micropenis or with distal hypospadia) can be associated with a partial deficit in 3 beta HSD activity. AIM: To assess the frequency of abnormal adrenal response to ACTH, suggesting a deficit in adrenal enzymatic activity, in children with mild undervirilization. PATIENTS AND METHODS: We studied 26 male children with micropenis, aged one to eight years. Children with evidences of puberal development or in treatment with drugs that affect steroidal metabolism were excluded from the study. Serum levels of androstenedione (A), dehydroepiandrosterone (DHEA), progesterone (P), 17 hydroxyprogesterone (17 P) and the ratios DHEA/A, P/17 P, 17 P/DHEA were measured after an adrenal stimulation with 0.25 mg/m2 intramuscular ACTH. RESULTS: Two children had DHEA y DHEA/A values suggesting a defective 3 beta HSD activity. Other two children had high levels of 17 P, suggesting a deficiency of cytochrome p450c21. A CYP 21 gene mutation was found in one of the later children. CONCLUSIONS: A low proportion of children with micropenis have a deficient 3 beta HSD activity.

[Polycystic ovary syndrome of extra-ovarian origin. Review]. / Síndrome de ovarios poliquísticos de origen extraovárico. Revisión.

An established fact in the polycystic ovarian syndrome (POS) is an abnormal ovarian steroidogenesis. Though this suggest an intrinsic ovarian defect, the syndrome could also be influenced by factors outside the ovaries. Although of unknown etiology, the POS is one of the most frequent endocrine disorders in the gynecologic practice. The disorder is characterized by ultrasound findings of enlarged polycystic ovaries, hyperandrogenism, menstrual disorders, obesity and including the appearance of infertility. There are a series of mechanisms involved in the extraovarian androgen increase in patients with POS. Among these mechanisms are implicated those of central and peripheral origin, genetic factors and adrenocortical dysfunction. In the same way, the alterations produced could imply genetic, molecular biological, biochemical, physiological and endocrinological factors. Sometimes all these factors could interact at the same time. The high serum androgen level could stop the pituitary gonadotropin production, either as a direct mechanism or as a result of its peripheral conversion. The increased androgens also explain the manifestations of clinical acne, hirsutism, and the detention in follicular ovarian maturation. All these manifestations are related with the menstrual disorders, anovulation, and infertility that these patients develop. The characteristics of the extraovarian POS include the 17-hydroxyprogesterone elevation in response to the ACTH test and the dexamethasone suppression of adrenal androgens. It is possible to improve the ovarian function in some patients with POS. This could be achieved with clomiphene citrate associated with glucocorticoids to induce ovulation.

Evaluation of the tubular and interstitial functions of the testis in 46,XY patients with ambiguous genitalia.

Investigation of the origin of sexual ambiguity is complex. Although testicular function has traditionally been assessed only by examining the steroidogenic capacity of Leydig cells and spermatogenesis, it has recently been shown that the measurement of serum anti-Müllerian hormone (AMH) as a marker of Sertoli cell function may also help clinicians. The aim of this study was to evaluate both Leydig and Sertoli cell functions in 46,XY patients with intersex states in order to establish biochemical patterns that would help to reach an etiologic diagnosis. We measured serum androgens, AMH and gonadotropins in 24 patients with sexual ambiguity and XY karyotype: 8 with gonadal dysgenesis (GD), 3 with 3beta-hydroxysteroid dehydrogenase deficiency (3betaHSD), 5 with androgen insensitivity syndrome (AIS), 4 with 5alpha-reductase 2 (SRD5A2) deficiency, and 4 were of unknown origin or idiopathic. Our results showed that while testosterone was low and gonadotropins elevated in patients with either GD or 3betaHSD, AMH was low in the former and high in the latter. Serum AMH and gonadotropins were normal or high in patients with 3betaHSD or AIS, but these could be distinguished by testosterone levels. Serum testosterone and gonadotropins were normal or high in AIS and SRD5A2 deficiency patients; however, while AMH was elevated in AIS, it was not the case in SRD5A2 deficiency patients, indicating that testosterone is sufficient to inhibit AMH within the testis. In idiopathic cases gonadotropins and testosterone were normal, and AMH was normal or low. We conclude that the combined measurement of androgens, AMH and gonadotropins helps to establish the diagnosis in intersex patients.

Mutations in the type II 3beta-hydroxysteroid dehydrogenase (HSD3B2) gene can cause premature pubarche in girls.

OBJECTIVE: Most previous studies have failed to demonstrate any mutations in the type II 3beta hydroxysteroid dehydrogenase (HSD3B2) gene in patients satisfying the hormonal criteria of nonclassic 3beta-hydroxysteroid dehydrogenase deficiency, suggesting that a mutant 3beta-hydroxysteroid dehydrogenase protein is not the cause of this disorder. We screened the HSD3B2 gene for mutations in girls with premature pubarche and a hormonal diagnosis of 3beta-hydroxysteroid dehydrogenase deficiency. DESIGN: From 30 girls with premature pubarche, we selected 9 whose ACTH-stimulated 17-hydroxypregnenolone levels were elevated (> or =6 SD) and screened the HSD3B2 gene for mutations. MEASUREMENTS: All patients were submitted to a standard ACTH stimulation test. Serum steroids were measured and compared to the mean level of pubertal stage matched control subjects. The four exons and exon-intron boundaries of the HSD3B2 gene were amplified by polymerase chain reaction and screened for mutations by denaturing gradient gel electrophoresis. The fragments with abnormal migration on denaturing gradient gel electrophoresis were directly sequenced. RESULTS: A homozygous T259M mutation was identified in one girl and a new compound heterozygous G129R/P222H mutation was identified in two sisters. The highest ACTH-stimulated 17-hydroxypregnenolone levels, 147, 339 and 351 nmol/l, were found in those patients with mutations in the HSD3B2 gene. In the patients without mutations, ACTH-stimulated 17-hydroxypregnenolone ranged from 48 to 111 nmol/l. ACTH-stimulated dehydroepiandrosterone levels had an overlap among the girls with and without mutations and the normal controls. CONCLUSIONS: Premature pubarche can be caused by mutations in the type II 3beta hydroxysteroid dehydrogenase gene.

Aspectos genéticos e dúvidas diagnósticas da deficiência da 3 beta-hidroxiesteróide desidrogenase: atualizaçäo / Genetic and diagnostic aspects on 3 beta-hidroxisteroid dehidrogenase defficiency: an update

Nos últimos 10 anos, muito tem-se publicado sobre a diminuiçao da atividade da 3 beta-hidroxiesteróide desidrogenase (3 beta-HSD) por ocasiao da infância, menarca ou mesmo após, determinando por um lado hiperplasia supra-renal congênita e na área ginecológica síndrome dos ovários policísticos devido a instalaçao de um meio androgênico coadjuvante do desvio da funçao ovulatória. Tida como a deficiência enzimática mais freqüente na forma de início tardio das hiperplasias supra-renais, seu diagnóstico era feito através apenas de estudos hormonais em que se comparavam os precursores delta 5 aos delta 4 da esteroidogênese. Tais dados, aceitos como universais, começaram a ser questionados a partir das descobertas recentes da localizaçao dos genes que codificam a atividade enzimática da 3 beta-HSD. Esta revisao procura mostrar os avanços na conduçao diagnóstica desta patologia a partir das últimas publicaçoes sobre o tema. Questiona-se por fim a existência da forma tardia desta enzimopatia.

Critérios diagnósticos das formas näo-clássicas de hiperplasia adrenal congênita / Diagnostic criteria of non-classic forms of congenital adrenal hyperplasia

A hiperplasia adrenal congênita (HAC) resulta de deficiência herdada em uma das várias enzimas necessárias para a síntese do cortisol. Deficiência na produçao de cortisol é comum a todas as formas de HAC, em decorrência da deficiência enzimática. Devido à existência de um mecanismo de feedback negativo entre a hipófise anterior e a supra-renal, ocorre aumento da secreçao de ACTH. Dependendo da severidade da deficicência enzimática, a concentraçao sérica de cortisol pode ou nao ser normalizada; porém sempre acompanhado de elevaçao dos esteróides precursores, resultando, nas formas clássicas de HAC, de alteraçoes bioquímicas e fenotípicas. O grau do bloqueio determina qual precursor estará elevado, enquanto que a severidade da deficiência enzimática determinará a extensao do comprometimento clínico. A forma nao-clássica da HAC diferencia-se da forma clássica pelo fato de os indivíduos afetados manifestarem sinais e sintomas de excesso androgênico tardiamente, ma infância ou adolescência, através de um espectro clínico variável, como pubarca precoce, acne, hirsutismo, distúrbio menstrual e infertilidade, podendo até mesmo nao manifestar sintomas, na chamada forma críptica da doença. O diagnóstico da forma nao-clássica de HAC, em geral, nao pode ser feito levando-se em consideraçao apenas a concentraçao basal dos hormônios esteróides, uma vez que estes podem se encontrar normais ou em concentraçao similar à das demais síndromes hiperandrogênicas. A dosagem dos hormônios esteróides após estímulo com ACTH sintético é o método que se impoe para o diagnóstico. Entretanto, os critérios diagnósticos ainda nao se encontram estabelecidos.

Hyperandrogenism due to 3 beta-hydroxysteroid dehydrogenase deficiency with accessory adrenocortical tissue: a hormonal and metabolic evaluation.

1. Adrenal ectopic tissue has been detected in the paragonadal region of normal women. In patients with congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency, the manifestation of hyperplasia of paragonadal accessory adrenal tissue has been usually reported to occur in males. Probably, this is the first report of a female with 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) deficiency with ectopic adrenal tissue in ovaries. However, the occurrence of hyperplasia of adrenal rests among women with classical congenital adrenal hyperplasia may not be rare, especially among patients with a late diagnosis. 2. We report a woman with 3 beta-HSD deficiency whose definitive diagnosis was made late at 41 years of age immediately before surgery for the removal of a uterine myoma. During surgery, exploration of the abdominal cavity revealed the presence of bilateral accessory adrenal tissue in the ovaries and in the para-aortic region. The patient had extremely high levels of ACTH (137 pmol/l), DHEA (901.0 nmol/l), DHEA-S (55.9 mumol/l), androstenedione (70.2 nmol/l), testosterone (23.0 nmol/l) and 17 alpha-hydroxypregnenolone (234.4 nmol/l) suggesting 3 beta-HSD deficiency. 3. In view of these elevated androgen levels, with an absolute predominance of DHEA and DHEA-S, we evaluated the effect of this hormonal profile on carbohydrate tolerance and insulin response to glucose ingestion. 4. The patient presented normal glucose tolerance but her insulin response was lower than that of 14 normal women (area under the curve, 3 beta-HSD = 17,680 vs 50,034 pmol/l for the control group over a period of 3 h after glucose ingestion).(ABSTRACT TRUNCATED AT 250 WORDS)

[3 beta-hydroxysteroid dehydrogenase defect: frequency of presentation in a sample of Chilean hirsute women]. / Defecto de 3 beta hidroxiesteroide deshidrogenasa: frecuencia de presentación en una muestra de mujeres chilenas hirsutas.

The defect of 3 beta hydroxysteroid dehydrogenase (3 beta HSD) is frequent among hirsute women and clearly dependent on the ethnic composition of the studied population. Our aim was to study the frequency of 3 beta HSD deficit in a group of Chilean hirsute women. Basal and post ACTH concentrations of cortisol, 17 hydroxyprogesterone and 17 hydroxypregnenolone were measured by RIA in 40 hirsute post puberal women and in 15 normal age matched female volunteers. Criteria for considering a 3 beta HDS deficit were 17 hydroxypregnenolone values and 17 hydroxypregnenolone/17 hydroxyprogesterone and 17 hydroxypregnenolone/cortisol ratios after ACTH stimulation over the 95% confidence intervals of normal women. Basal dehydroepiandrosterone sulphate and testosterone levels were also measured in hirsute women. All samples were obtained during the follicular phase of the menstrual cycle. ACTH stimulated hormone values and ratios were diagnostic for 3 beta HDS deficit in 7.5% of hirsute women. Basal testosterone was over 80 ng/dl in 47.5% and dehydroepiandrosterone sulphate over 3.9 micrograms/ml in 52.5% of these women. There was no correlation between dehydroepiandrosterone or testosterone values and ACTH stimulated hormone values. It is concluded that 3 beta HSD is frequent in hirsute women and that its diagnosis requires the determination of ACTH stimulated 17 hydroxypregnenolone values and 17 hydroxypregnenolone/17 hydroxyprogesterone ratio.

[Polycystic ovary syndrome as expression of 3-beta-hydroxysteroid dehydrogenase deficiency]. / Síndrome de ovario poliquístico como expresión de déficit de la enzima 3 beta hidroxiesteroide deshidrogenasa.

Late-onset adrenal enzymatic deficiencies are a common cause of hirsutism. They can produce a PCO syndrome-like clinical course, underscoring the multi-causality of this condition. We report a 18 year old woman with secondary amenorrhea and hirsutism, whose serum levels of testosterone and DHEA-S were 175 ng/dl and 7.3 micrograms/ml and the LH/FSH ratio was 5.9. Dexamethasone decreased testosterone and DHEA-S levels, while the ACTH stimulation test increased the 17 hydroxypregnenolone serum concentration and the 17 hydroxypregnenolone/17 hydroxyprogesterone ratio to values considered diagnostic of 3 beta hydroxysteroid dehydrogenase deficiency.

Síndrome de ovario poliquístico como expresión de déficit de la enzima 3ß hidroxiesteroide deshidrogenasa / Polycystic ovary syndrome as expression of 3-beta-hydroxysteroid dehydrogenase deficit

Los defectos enzimáticos de expresión tardía de la esteroidogénesis suprarrenal están entre las causas más comunes de hirsutismo. Esta forma de hiperandrogenismo puede asumir las características clínicas de un síndrome de ovario poliquístico, lo que viene a confirmar la multicausalidad de esta última condición. Se comunica el caso de una mujer de 18 años con amenorrea secundaria e hirsutismo, cuya concentración sérica de testosterona y de DHEA-S eran 175 ng/dl y 7,3 *g/ml respectivamente y la relación LH/FSH de 5,9. La administración de dexametasona produjo una marcada reducción en la concentración de testosterona y DHEA-S, en tanto que el estímulo con ACTH se asoció a un aumento de 17 hidroxipregnenolona y de la relación 17 hidroxipregnenolona/17 hidroxiprogesterona en el rango descrito para el déficit de 3ß hidroxiesteroide deshidrogenasa

Male pseudohermaphroditism due to nonsalt-losing 3 beta-hydroxysteroid dehydrogenase deficiency: gender role change and absence of gynecomastia at puberty.

Adrenal and gonadal functions were evaluated on two adult cousins with male pseudohermaphroditism due to congenital 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta-HSD) without clinical salt-losing. Both patients had been reared as females since birth. Case 1 presented at age 17 with perineal hypospadias virilization without gynecomastia and a female to male gender role change at puberty. Case 2 had previously undergone bilateral orchidectomy in childhood and presented "primary amenorrhea", absence of virilization and a female gender role at the age of 24. In the basal state, as well as after ACTH and hCG stimulation, 3 beta-hydroxy-5-ene-steroid levels were disproportionately elevated, resulting in abnormal 3 beta-hydroxy-5-ene: 3-oxi-4-ene steroids ratios. Normal basal serum cortisol with inadequate cortisol response to ACTH was observed in both patients. Elevated basal plasma renin activity (PRA) and normal basal serum aldosterone (ALDO) were present in both subjects. After ACTH stimulation serum ALDO rose adequately in Case 1 but subnormally in Case 2. Salt restriction resulted in an increase in serum ALDO and no salt loss in Case 1 whereas in Case 2 the substantial rise in PRA and serum ALDO were unable to prevent slight urinary sodium loss. Case 1 had normal basal serum testosterone with subnormal response to hCG stimulation. Incubation of testicular tissue in vitro with [3H]DHEA resulted in large Androstenediol production but diminished testosterone conversion confirming the 3 beta-HSD deficiency in the testes. We conclude that (1) absence of gynecomastia and a female to male gender role change may be observed in the male pubertal presentation of nonsalt-losing 3 beta-HSD deficiency and (2) the different functional behavior of zona glomerulosa in our patients suggests the presence of variable degrees of 3 beta-HSD deficiency in the zona glomerulosa of the nonsalt-losing form.