beta-HSD Deficiency Research Articles

Successful Management of Significant Maternal 3 Beta-Hydroxysteroid Dehydrogenase Deficiency

We report a case of successful management of pregnancy in a patient with significant 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency. A 25-year-old female presented for interfertility evaluation after failure to conceive for 8 months. The patient began full menses each month at the time of ovulation. Her unique clinical presentation led to the diagnosis of a significant 3beta-HSD deficiency. Treatment for infertility with clomiphene citrate, gonadotropins, dexamethasone and human chorionic gonadotropin trigger was unsuccessful. The patient conceived spontaneously while taking continuous combination oral contraceptive pills. Due to her history of instability and full menses in the luteal phase, oral estrogen and progesterone in oil were administered to support the early pregnancy. The results of elevated 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), and ratio of 17 OH-pregnenolone to 17-OHP were consistent with diagnosis of significant 3beta-HSD deficiency. Successful pregnancy was achieved after exogenous hormone supplementation. Fertility in 3beta-HSD deficiency, a rare form of congenital adrenal hyperplasia, has not been extensively studied. Varying phenotype can make the diagnosis challenging. Heightened suspicion in patients with reported bleeding at ovulation or breakthrough bleeding on oral contraceptive pills could aid in the diagnosis. Successful pregnancy in these patients is possible. J Clin Gynecol Obstet. 2021;10(2):51-54 doi: https://doi.org/10.14740/jcgo730

17β-Hydroxysteroid dehydrogenase type 3 deficiency as a result of a homozygous 7 base pair deletion in 17βHSD3 gene

17-beta-Hydroxysteroid dehydrogenase type 3 (17betaHSD-3) converts delta4 androstenedione (A) to testosterone (T) in the testes. This enzyme plays a key role in androgen synthesis and it is essential for normal fetal development of male genitalia. 17betaHSD-3 deficiency is a rare cause of 46,XY disorders of sexual development. Here, we report a 16-year-old 46,XY patient with 17betaHSD-3 deficiency raised as a female and significantly virilized in puberty. A homozygous 7 base pair deletion on exon 10 was determined in HSD17B3 gene (c.777-783del_GATAACC). Our patient had one of the very rare mutations, which was previously unencountered in Turkish patients with 17betaHSD type 3, and she is the second reported case with this deletion.

Pediatric disorders of sex development

The management of disorders of sexual differentiation (DSD) involves a multidisciplinary approach. The main aim of analysis was to study the phenotype-karyotype correlation in North Indian children with DSD. The records of pediatric DSD were retrieved and characteristics noted. Of total of 58 children, 43 (74.1%) and 10 (17.2%) were raised as males and females respectively. The mean age at presentation was 31.3+/-9 months. The karyotype was 46XY in 45 (77.6%) and 46XX in 12 (20.7%). CAH was commonest cause of DSD (36.2%), followed by gonadal dysgenesis. Of the 15 patients of 46 XY CAH, there were 5 with 17-alpha hydroxylase deficiency, 2 with 3-beta HSD deficiency and one case of lipoid adrenal hyperplasia. There was an excess of genetic males, possibly due to prevalent socio-cultural factors and gender bias favoring males. There is a need to improve the diagnostic facilities and incorporate a team approach in management of DSD.

Oral Cholic Acid for Hereditary Defects of Primary Bile Acid Synthesis: A Safe and Effective Long-term Therapy

Oral bile acid replacement has been shown to be an effective therapy in primary bile acid synthesis defects, but to date there have been no reports of the long-term effects of this therapy. The aim of the study was to evaluate the long-term effectiveness and safety of cholic acid (CA) therapy. Fifteen patients with either 3beta-hydroxy-Delta(5)-C(27)-steroid oxidoreductase (3beta-HSD) (n = 13) or Delta(4)-3-oxosteroid 5beta-reductase (Delta(4)-3-oxo-R) (n = 2) deficiency confirmed by mass spectrometry and gene sequencing received oral CA and were followed up prospectively. CA therapy was started at a median age of 3.9 years (range, 0.3-13.1 years). The median follow-up with treatment was 12.4 years (range, 5.6-15 years). The mean daily dose of CA was initially 13 mg/kg and was 6 mg/kg at last evaluation. During CA therapy, physical examination findings, laboratory test results, and findings on sonography normalized. Mass spectrometry analysis of urine showed that excretion of the atypical metabolites was reduced by 500-fold and 30-fold in 3beta-HSD and Delta(4)-3-oxo-R deficiency, respectively, and total urinary bile acid excretion decreased dramatically. Liver biopsies performed in 14 patients after at least 5 years of CA therapy showed marked improvement, especially in patients with the 3beta-HSD deficiency. CA was well tolerated with all children developing normally, including 2 women having 4 normal pregnancies during treatment. Oral CA therapy is a safe and effective long-term treatment of the most common primary bile acid synthesis defects.

17β-Hydroxysteroid dehydrogenase-3 deficiency: From pregnancy to adolescence

Aim of this study is to report on basal clinical phenotype and follow up after diagnosis, of patients with 17beta-hydroxysteroid-dehydrogenase type 3 (17beta-HSD3) deficiency in Italy. Pediatric Endocrine Departments, University Hospitals. The cases of 5 Italian subjects affected by 17beta-HSD3 deficiency are presented in this study. Laboratory and genetic assessment. Gonadectomy and female sex assignment (4 patients) or GnRH analog therapy to regress puberty and gender identity disorder (1 patient). Presentation lasted from pregnancy (pre-natal diagnosis of a 46,XY fetus with female external genitalia) to infancy (inguinal hernia containing testes/clitoromegaly) and adolescence (virilisation). All subjects but one (subject 1, Central-Northern Italy) were from small areas of Southern Italy. Endocrine data (baseline and/or stimulated testosterone/ Delta4-androstenedione ratio) were informative. Two girls were homozygous for 17beta-HSD3 gene mutations (G289S/G289S; R80W/R80W), while the others were compound heterozygous (IVS325+4 A>T/A203V; L212Q/M235V; R80W/A235E). Four patients were confirmed as females and were well-adjusted with assigned sex; gender identity disorder improved during treatment with GnRH analog in the last subject. 17betaHSD3 deficiency may present from pregnancy to puberty for different clinical issues. Albeit testosterone/Delta4-androstenedione ratio represents the most accurate endocrine marker to diagnose the disorder, hCGstimulation is mandatory in pre-puberty. Molecular analysis of 17beta-HSD3 gene should be performed to confirm the diagnosis. Temporary GnRH analog treatment may regress gender identity disorder and provide time to confirm or change the birth sex assignment. Female individuals seems to be compliant with their sex, providing that virilisation does not occur. In Italy, the disorder seems to be more prevalent in the Southern regions and shows genetic heterogeneity.

Carboxyl-Terminal Mutations in 3β-Hydroxysteroid Dehydrogenase Type II Cause Severe Salt-Wasting Congenital Adrenal Hyperplasia

3beta-Hydroxysteroid dehydrogenase (3beta-HSD) deficiency is a rare cause of congenital adrenal hyperplasia caused by inactivating mutations in the HSD3B2 gene. Most mutations are located within domains regarded crucial for enzyme function. The function of the C terminus of the 3beta-HSD protein is not known. We studied the functional consequences of three novel C-terminal mutations in the 3beta-HSD protein (p.P341L, p.R335X and p.W355X), detected in unrelated 46,XY neonates with classical 3beta-HSD type II deficiency showing different degrees of under-virilization. In vitro expression of the two truncated mutant proteins yielded absent conversion of pregnenolone and dehydroepiandrosterone (DHEA), whereas the missense mutation p.P341L showed a residual DHEA conversion of 6% of wild-type activity. Additional analysis of p.P341L, including three-dimensional protein modeling, revealed that the mutant's inactivity predominantly originates from a putative structural alteration of the 3beta-HSD protein and is further aggravated by increased protein degradation. The stop mutations cause truncated proteins missing the final G-helix that abolishes enzymatic activity irrespective of an augmented protein degradation. Genital appearance did not correlate with the mutants' residual in vitro activity. Three novel C-terminal mutants of the HSD3B2 gene are responsible for classical 3beta-HSD deficiency. The C terminus is essential for the enzymatic activity. However, more studies are needed to clarify the exact function of this part of the protein. Our results indicate that the genital phenotype in 3beta-HSD deficiency cannot be predicted from in vitro 3beta-HSD function alone.

Clinical, endocrine, and molecular findings in 17β -hydroxysteroid dehydrogenase type 3 deficiency

The 17beta-hydroxysteroid dehydrogenases (17betaHSD) gene family comprises different enzymes involved in the biosynthesis of active steroid hormones. The 17betaHSD type 3 (17betaHSD3) isoenzyme catalyzes the reductive conversion of the inactive C19-steroid, Delta4-androstenedione (Delta4- A), into the biologically active androgen, testosterone (T), in the Leydig cells of the testis. It is encoded by the 17beta-hydroxysteroid dehydrogenase type 3 (HSD17B3) gene, which maps to chromosome 9q22. Mutations in the HSD17B3 gene are associated with a rare form of 46,XY disorder of sex development referred to as 17betaHSD3 deficiency (or as 17-ketosteroid reductase deficiency), due to impaired testicular conversion of Delta4-A into T. 46,XY patients with 17betaHSD3 deficiency are usually classified as female at birth, raised as such, but develop secondary male features at puberty. Diagnosis, and consequently early treatment, is difficult because clinical signs from birth until puberty may be mild or absent. Biochemical diagnosis of 17betaHSD3 deficiency requires measurement of serum T/Delta4-A ratio after hCG stimulation test in pre-pubertal subjects, while baseline values seem to be informative in early infancy and adolescence. However, low basal T/Delta4-A ratio is not specific for 17betaHSD3 deficiency, being sometimes also found in patients with other defects in T synthesis or with Leydig cells hypoplasia. Mutational analysis of the 17HSDB3 gene is useful in confirming the clinical diagnosis of 17betaHSD3 deficiency. This review describes clinical findings, diagnosis, and molecular basis of this rare disease.

17β-hydroxysteroid dehydrogenase 3 deficiency in a male pseudohermaphrodite

To present the clinical, biochemical, and genetic features of a male pseudohermaphrodite whose condition was caused by 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) deficiency. Case report. Gynecology practice in a university teaching hospital. A 15-year-old black American male pseudohermaphrodite with 17beta-HSD3 deficiency. Laboratory evaluation, genetic mutation analysis, bilateral gonadectomy, and hormone replacement. Endocrinologic evaluation and genetic analysis. A diagnosis of 17beta-HSD3 deficiency made on the basis of hormone evaluation was confirmed through genetic mutation analysis of the HSD17B3 gene. Female phenotype was attained after gonadectomy, passive vaginal dilatation, and hormone therapy. Deficiency of 17beta-HSD3 was diagnosed in this patient on the basis of endocrinologic evaluation and was confirmed with genetic mutation analysis. The patient was able to retain her female sexual identity after surgical and medical treatment.

Phenotypic variability in 17β‐hydroxysteroid dehydrogenase‐3 deficiency and diagnostic pitfalls

17beta-hydroxysteroid dehydrogenase type 3 isoenzyme (17beta-HSD3) is required to produce testosterone for male sex differentiation. Mutations in the HSD17B3 gene cause 17betaHSD3 deficiency and result in XY sex reversal of varying degree. We report the phenotypes of 14 subjects with 17betaHSD3 deficiency in relation to sex of rearing, androgen production, and HSD17B3 mutations. Cases were identified through the Cambridge Disorders of Sex Development Database where detailed clinical information was recorded, results of hCG stimulation tests were available, and HSD17B3 mutation was identified. Fourteen subjects from seven pedigrees (four consanguineous) had the following seven mutations: A56T, N130S, E215D, S232L, C268Y, V205E, and a novel mutation M197K. XY sex reversal was classified as complete in 10 infants at birth. Inguinal masses suggestive of androgen insensitivity syndrome (AIS) occurred in five infants. Contrasexual virilization reminiscent of 5alpha-reductase deficiency occurred in four subjects at puberty. The median (range) testosterone : androstenedione (T/A) ratio after a short hCG stimulation test was 0.32 (0.12-3.4). The S232L mutation identified in three affected family members caused isolated, severe hypospadias in one member who was raised male; virilization occurred despite in vitro studies showing an inactive mutant enzyme. Ratios of T/A in this pedigree were more than 0.8. XY sex reversal is sufficiently variable in 17betaHSD3 deficiency to cause problems in accurate diagnosis, particularly in distinguishing it from AIS. It should be considered in undervirilized male infants with normal Wolffian duct structures, absent Müllerian ducts, and normal adrenal steroid biosynthesis; or when an assigned female subject virilizes at puberty. Elevated hCG-stimulated T/A ratio may occur, and sex of rearing may not be concordant within affected families with the same HSD17B3 mutation. The T/A ratio, mutation analysis and functional analysis of the mutant enzyme taken in isolation, respectively, may not conclusively establish a diagnosis of 17betaHSD3 deficiency in undervirilized male subjects; the reasons for these discrepancies remain unknown.

Molecular Biology of the 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase Gene Family

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. In humans, expression of the type I isoenzyme accounts for the 3beta-HSD activity found in placenta and peripheral tissues, whereas the type II 3beta-HSD isoenzyme is predominantly expressed in the adrenal gland, ovary, and testis, and its deficiency is responsible for a rare form of congenital adrenal hyperplasia. Phylogeny analyses of the 3beta-HSD gene family strongly suggest that the need for different 3beta-HSD genes occurred very late in mammals, with subsequent evolution in a similar manner in other lineages. Therefore, to a large extent, the 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. Recent studies indicate that HSD3B2 gene regulation involves the orphan nuclear receptors steroidogenic factor-1 and dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1). Other findings suggest a potential regulatory role for STAT5 and STAT6 in transcriptional activation of HSD3B2 promoter. It was shown that epidermal growth factor (EGF) requires intact STAT5; on the other hand IL-4 induces HSD3B1 gene expression, along with IL-13, through STAT 6 activation. However, evidence suggests that multiple signal transduction pathways are involved in IL-4 mediated HSD3B1 gene expression. Indeed, a better understanding of the transcriptional factors responsible for the fine control of 3beta-HSD gene expression may provide insight into mechanisms involved in the functional cooperation between STATs and nuclear receptors as well as their potential interaction with other signaling transduction pathways such as GATA proteins. Finally, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily. Furthermore, several recent studies using type I and type II purified enzymes have elegantly further characterized structure-function relationships responsible for kinetic differences and coenzyme specificity.

Molecular study of the 3 beta-hydroxysteroid dehydrogenase gene type II in patients with hypospadias.

To determine whether some patients with idiopathic hypospadias have HSD3B2 mutations, we genotyped this locus in 90 patients with hypospadias (age, 6.0 +/- 0.4 yr) and 101 healthy fertile male controls. We measured basal plasma renin activity and performed an ACTH test for determination of 17-OH-pregnenolone, 17-OH-progesterone, cortisol, dehydroepiandrosterone sulfate, and androstenedione and an human chorionic gonadotropin test for determination of androstenedione, testosterone, and dihydrotestosterone. We did not observe a clear steroidogenic pattern suggestive of 3 beta-HSD deficiency in any patient. DNA was extracted from peripheral lymphocytes; and exons 1, 2, 3, and 4 were amplified by PCR and analyzed by denaturing gradient gel electrophoresis. An abnormal electrophoretic migration pattern of exon 4 was observed in five patients. Two patients had missense heterozygous mutations (S213T and S284R). In another three patients, we observed heterozygous nucleotide variants in exon 4 that did not produce a change in amino acids (A238, T259, T320). In vitro enzymatic activity was diminished by 40% and 32% in the S213T and S284R heterozygous mutations, respectively. One control exhibited a heterozygous mutation in exon 3 (V78I), which did not alter in vitro enzyme activity. In addition, we observed possible polymorphisms in intron 1 in four patients and one control. We conclude that subtle molecular abnormalities in the HSD3B2 gene may be observed in some patients with apparent idiopathic hypospadias but that this finding is uncommon.

The prevalence of late onset congenital adrenal hyperplasia in hirsute women from Central Anatolia.

Late onset congenital adrenal hyperplasia (LO CAH) can be seen in association with polycystic ovary syndrome (PCOS) or idiopathic hirsutism (IH). The study aimed to find out the prevalence of LO CAH in Central Anatolia among hirsute women. Sixty-three patients with hirsutism were evaluated to determine the frequency of LO CAH by comparing them with their age and body mass index matched 28 healthy controls. Of those 63 hirsute women, 43 were diagnosed as PCOS, and 20 were diagnosed as IH. Following basal hormonal evaluation, all subjects underwent ACTH stimulation test and ACTH stimulated 17-hydroxyprogesterone (17-OH P), 11-desoxycortisol (11-DOC), cortisol (F), and dehydroepiandrosterone sulfate (DHEA-S) levels were determined in all subjects. ACTH stimulated 17-OH P, 11-DOC, and DHEA-S levels did not differ between groups. However, stimulated F levels were found to be higher in hirsute women (p<0.001). Six out of 63 (9.52%) patients with hirsutism met the criterion for 21 hydroxylase deficiency. We found no subject presumed to have 11-beta hydroxylase deficiency, but one subject in control group (3.57%) and two patients among PCOS subjects (4.65%) had exaggerated DHEA-S response which was suggestive of mild 3-beta hydroxysteroid dehydrogenase deficiency. In conclusion, the most frequent form of LO CAH seems to be due to 21 OH deficiency among women with PCOS and IH in Central Anatolia. Mild 3-beta HSD deficiency may also be an underlying cause for hirsutism and it may be seen without any clinical presentation. Adrenal hyperactivity is likely to be the main reason of hyperandrogenemia in women with hirsutism.

Newly proposed hormonal criteria via genotypic proof for type II 3beta-hydroxysteroid dehydrogenase deficiency.

To define the hormonal criteria via genotypic proof for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) deficiency in the adrenals and gonads, we investigated the type II 3beta-HSD genotype in 55 patients with clinical and/or hormonal presentation suggesting compromised adrenal with or without gonadal 3beta-HSD activity. Fourteen patients (11 males and 3 females) had ambiguous genitalia with or without salt wasting and with or without premature pubarche. One female neonate had salt wasting only. Twenty-five children (4 males and 21 females) had premature pubarche only. Fifteen adolescent and adult females had hirsutism with or without menstrual disorder. The type II 3beta-HSD gene, including the promoter region up to -1053 base, all exons I, II, III, IV, and exon and intron boundaries, was sequenced in all subjects. Eight patients had a proven or predictably deleterious mutation in both alleles of the type II 3beta-HSD gene, and 47 patients had no apparent mutation in the gene. ACTH-stimulated (1 h post iv bolus of 250 microg Cortrosyn) serum 17-hydroxypregnenolone (Delta5-17P) levels and basal and ACTH-stimulated ratios of Delta5-17P to cortisol (F) in the genotypic proven patients were unequivocally higher than those of age-matched or pubic hair stage matched genotype-normal patients or control subjects (n = 7-30 for each group). All other baseline and ACTH-stimulated hormone parameters, including dehydroepiandrosterone (DHEA) levels, ratios of Delta5-17P to 17-OHP and DHEA to androstenedione in the genotype-proven patients, overlapped with the genotype-normal patients or control subjects. The hormonal findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3beta-HSD deficiency congenital adrenal hyperplasia (numeric and graphic reference standards from infancy to adulthood are provided): ACTH-stimulated Delta5-17P levels in 1) neonatal infants with ambiguous genitalia at or greater than 378 nmol/liter equivalent to or greater than 5.3 SD above the control mean level [95 +/- 53 (SD) nmol/liter]; 2) Tanner I children with ambiguous genitalia at or greater than 165 nmol/liter equivalent to or greater than 35 SD above the control mean level [12 +/- 4.3 (SD) nmol/liter]; 3) children with premature pubarche at or greater than 294 nmol/liter equivalent to or greater than 54 SD above Tanner II pubic hair stage matched control mean level [17 +/- 5 (SD) nmol/liter]; and 4) adults with at or greater than 289 nmol/liter equivalent to or greater than 21 SD above the normal mean level [25 +/- 12 (SD) nmol/liter]. ACTH-stimulated ratio of Delta5-17P to F in 1) neonatal infants at or greater than 434 equivalent to or greater than 6.4 SD above the control mean ratio [88 +/- 54 (SD)]; 2) Tanner I children at or greater than 216 equivalent to or greater than 23 SD above the control mean ratio [12 +/- 9 (SD)]; 3) children with premature pubarche at or greater than 363 equivalent to or greater than 38 SD above the control mean ratio [20 +/- 9 (SD)]; and 4) adults at or greater than 4010 equivalent to or greater than 221 SD above the normal mean ratio [29 +/- 18 (SD)]. Conversely, the hormonal data in the genotype-normal patients suggest the following hormonal criteria are not consistent with 3beta-HSD deficiency congenital adrenal hyperplasia: ACTH-stimulated Delta5-17P levels in children with premature pubarche up to 72 nmol/liter equivalent to up to 11 SD above the control mean level, and in hirsute females up to 150 nmol/liter equivalent to up to 12 SD above the normal female mean level [28 +/- 10 (SD) nmol/liter]; and ACTH-stimulated Delta5-17P to F ratio in children with premature pubarche up to 67 equivalent to up to 5 SD above the control mean ratio, and in hirsute females up to 151 equivalent to up to 10 SD above the normal mean ratio [32 +/- 12 (SD)]. These findings help define newly proposed hormonal criteria to accurately predict inherited 3beta-HSD deficiency.

A novel nonstop mutation in the stop codon and a novel missense mutation in the type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing, respectively, nonclassic and classic 3beta-HSD deficiency congenital adrenal hyperplasia.

We investigated two novel point mutations in the human type II 3beta-hydroxysteroid dehydrogenase (3beta-HSD) gene causing a mild and a severe form of 3beta-HSD deficiency congenital adrenal hyperplasia. The first is a nonstop mutation in the normal stop codon 373 of the gene in exon IV [TGA (Stop) --> TGC (Cys) = Stop373C) identified from one allele of a female child with premature pubarche whose second allele had an E142K mutation. The Stop373C mutation predictably results in an open reading frame and a mutant-type (MT) II 3beta-HSD protein containing 467 amino acid residues, compared with the 372 amino acid residues of wild-type (WT) protein. The second is a homozygous missense mutation in codon 222 [CCA (Pro) --> ACT (Thr) = P222T] in the gene identified from a female neonate with salt-wasting disorder. The pcDNA vectors containing the constructs of WT II 3beta-HSD cDNA, WT cDNA with the open reading frame (WT cDNA(+)), MT Stop373C with the open reading frame (Stop373C(+)) and MT P222T cDNA were transfected in COS-I and 293T cells and expressed a similar amount of 3beta-HSD mRNA. The enzyme activity in intact cells using pregnenolone and dehydroepiandrosterone as substrate in the medium (1 micromol/liter) was identical between the WT cDNA and the WT cDNA(+), but was decreased to 27% of the WT enzymes at 6 h by MT Stop373C(+) enzyme, and was undetectable by P222T enzyme. In the homogenates of the cells, both MT Stop373C(+) and P222T enzyme activities and enzymes were undetectable despite clear detection of WT enzyme activities and WT enzymes. LH response to an LHRH analog stimulation in the pubertal female with the Stop373C/E142K genotypes and in a pubertal female with compound 273/318 frameshift genotypes were comparable to and higher than control females, respectively. In conclusion, a structurally lengthy MT II 3beta-HSD enzyme due to a nonstop mutation was relatively detrimental in intact cells causing the nonclassic phenotype of 3beta-HSD deficiency. A missense P222T mutation was seriously detrimental, causing the classic phenotype of 3beta-HSD deficiency. The undetectable Stop373C and P222T enzymes on Western blottings, together with the respective in vivo and in vitro data, suggest that a relative instability of Stop373C enzyme and a profound instability of the P222T enzyme are likely the detrimental molecular mechanisms. The increased LH in the female with the frameshift genotype and the appropriate LH response in the female with the nonstop genotype correlated with predictably severe and mild ovarian type II 3beta-HSD deficiency, respectively.

Congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerase deficiency.

The 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)isomerase (3beta-HSD) isoenzymes are responsible for the oxidation and isomerization of Delta(5)-3beta-hydroxysteroid precursors into Delta(4)-ketosteroids, thus catalyzing an essential step in the formation of all classes of active steroid hormones. The 3beta-HSD gene family should have evolved to facilitate differential patterns of tissue- and cell-specific expression and regulation involving multiple signal transduction pathways, which are activated by several growth factors, steroids, and cytokines. In humans, there are two 3beta-HSD isoenzymes, which were chronologically designated type I and II encoded by HSD3B1 and HSD3B2 gene, respectively. HSD3B1 gene encodes the almost exclusive 3beta-HSD isoenzyme expressed in the placenta and peripheral tissues, whereas HSD3B2 gene encodes the predominant 3beta-HSD isoenzyme expressed in the adrenal gland, ovary, and testis and its deficiency is responsible for a rare form of congenital adrenal hyperplasia causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. Although an elevated ratio of Delta(5)-Delta(4)-steroids was considered to be the best biological parameter for the diagnosis of this autosomal recessive disorder, the most accurate criteria now appears to be the plasma levels of 17-OH-pregnenolone greater than 100 nmol/L following ACTH stimulation. To date a total of 34 mutations (including 5 frameshift, 4 nonsense, 1 in-frame deletion, 1 splicing, and 23 missense mutations) have been identified in the HSD3B2 gene in 56 individuals from 44 families suffering from classical 3beta-HSD deficiency. In almost all the cases, the functional characterization of HSD3B2 mutations has provided a molecular explanation for the heterogeneous clinical presentation of this disorder. Indeed these experiments confirm that no functional 3betaHSD type II isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form, whereas the non-salt-losing form results from specific missense mutation(s) in the HSD3B2 gene, which causes an incomplete loss of enzymatic activity thus leaving sufficient enzymatic activity to prevent salt wasting. Moreover, various mutations appear to have a drastic effect upon stability of the protein, therefore providing molecular evidence of a new mechanism involved in classical 3beta-HSD deficiency. Thus, the elucidation of the molecular basis of 3beta-HSD deficiency has highlighted the fact that mutations in the HSD3B2 gene can result in a wide spectrum of molecular repercussions, which are associated with the different phenotypic manifestations of classical 3beta-HSD deficiency and also provide valuable information concerning the structure-function relationships of the 3beta-HSD superfamily.

CONGENITAL ADRENAL HYPERPLASIA OWING TO 3β-HYDROXYSTEROID DEHYDROGENASE DEFICIENCY

Recent advances in the genetics of the family of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) enzymes have helped in the understanding of the molecular basis and hormonal phenotype of bona fide 3 beta-HSD deficiency congenital adrenal hyperplasia (CAH). This article revisits the clinical spectra of 3 beta-HSD deficiency disorders, pathophysiology of 3 beta-HSD deficiency CAH, and updates genotype findings and diagnostic hormonal criteria for bona fide classic and nonclassic 3 beta-HSD deficiency CAH. The delta-5 steroid abnormality for the nonclassic 3 beta-HSD deficiency CAH, proven by genotype study, is substantially greater than the hormonal criteria for the disorder published before the advent of molecular information on the gene encoding adrenals and gonads in humans. In hyperandrogenic children and women, the pathogenic mechanism of a subtle abnormality in adrenal 3 beta-HSD activity, determined by modestly elevated ACTH stimulated delta-5 steroid levels, which led to the diagnosis of mild nonclassic 3 beta-HSD deficiency in the past, is outside of the type II 3 beta-HSD gene which encodes adrenals and gonads in humans and remains to be further explored.

3beta-hydroxysteroid dehydrogenase/delta5-->4-isomerase activity associated with the human 17beta-hydroxysteroid dehydrogenase type 2 isoform.

The type 2 isoform of human 17beta-hydroxysteroid dehydrogenase (17betaHSD2) efficiently catalyzes the oxidative metabolism of androgens and estrogens, and it is expressed in a large series of human peripheral tissues. To obtain a better understanding of the regulation of local steroid biosynthesis and metabolism in human tissues, we have established a dual steroidogenic activity of the 17betaHSD2 enzyme after transfection of human 17betaHSD2-transfected human embryonic kidney (293) cells. After transient transfection, the metabolism of testosterone, pregnenolone, and dehydroepiandrosterone (DHEA) in intact transfected 293 cells was evaluated by TLC-based radiometric assays. 17betaHSD2-transfected cells converted 91% of testosterone (1 micromol/L) into androstenedione in a 2-h incubation period. In addition, pregnenolone (1 micromol/L) was converted to progesterone (18.5%), whereas DHEA (1 micromol/L) was metabolized to androstenedione (8.3% conversion) in a 15-h incubation period. The kinetics of the 3beta-hydroxysteroid dehydrogenase (3betaHSD) and 17betaHSD2 activities using cell homogenate protein of stably transfected 293 cells indicated that the catalytic efficiency (apparent catalytic efficiency = maximum velocity/Km) of this 3betaHSD activity is approximately 2000-fold (pregnenolone as substrate) or 3000-fold (DHEA as substrate) weaker than that of 17betaHSD2 activity. It is noteworthy, however, that the apparent catalytic efficiency of the HSD3B2 gene product is only approximately 50-fold higher than that of the 3betaHSD aspect of the 17betaHSD2 gene product. Pregnenolone or DHEA effectively inhibited 17betaHSD2 activity in a noncompetitive fashion. Furthermore, the potent 5alpha-reductase/3betaHSD inhibitor, 17beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5alpha-androstane-3-one , inhibited neither 3betaHSD nor 17betaHSD2 activities. We conclude that human 17betaHSD2 enzyme exhibits 3betaHSD activity. Notwithstanding that this 3betaHSD activity is reduced compared to 17betaHSD oxidative activity, it may account for at least some of the reports of 3betaHSD activity found in human peripheral tissues that express notable amounts of the 17betaHSD2 isozyme as well as in individuals with severe classic 3betaHSD deficiency.

New insight into the molecular basis of 3beta-hydroxysteroid dehydrogenase deficiency: identification of eight mutations in the HSD3B2 gene eleven patients from seven new families and comparison of the functional properties of twenty-five mutant enzymes.

Classical 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3betaHSD) deficiency is a form of congenital adrenal hyperplasia that impairs steroidogenesis in both the adrenals and gonads resulting from mutations in the HSD3B2 gene and causing various degrees of salt-wasting in both sexes and incomplete masculinization of the external genitalia in genetic males. To identify the molecular lesion(s) in the HSD3B2 gene in the 11 patients from the seven new families suffering from classical 3betaHSD deficiency, the complete nucleotide sequence of the whole coding region and exon-intron splicing boundaries of this gene was determined by direct sequencing. Five of these families were referred to Morel's molecular diagnostics laboratory in France, whereas the two other families were investigated by Peter's group in Germany. Functional characterization studies were performed by Simard's group in Canada. Following transient expression in 293 cells of each of the mutant recombinant proteins generated by site-directed mutagenesis, the effect of the 25 mutations on enzyme activity was assessed by incubating intact cells in culture with 10 nM [14C]-DHEA as substrate. The stability of the mutant proteins has been investigated using a combination of Northern and Western blot analyses, as well as an in vitro transcription/translation assay using rabbit reticulocyte lysates. The present report describes the identification of 8 mutations, in seven new families with individuals suffering from classical 3betaHSD deficiency, thus increasing the number of known HSD3B2 mutations involved in this autosomal recessive disorder to 31 (1 splicing, 1 in-frame deletion, 3 nonsense, 4 frameshift and 22 missense mutations). In addition to the mutations reported here in these new families, we have also investigated for the first time the functional significance of previously reported missense mutations and or sequence variants namely, A82T, A167V, L173R, L205P, S213G and K216E, P222H, T259M, and T259R, which have not previously been functionally characterized. Furthermore, their effects have been compared with those of the 10 previously reported mutant enzymes to provide a more consistent and comprehensive study. The present results are in accordance with the prediction that no functional 3betaHSD type 2 isoenzyme is expressed in the adrenals and gonads of the patients suffering from a severe salt-wasting form of CAH due to classical 3betaHSD deficiency. Whereas the nonsalt-losing form also results from missense mutation(s) in the HSD3B2 gene, which cause an incomplete loss in enzyme activity, thus leaving sufficient enzymatic activity to prevent salt wasting. The functional data described in the present study concerning the sequence variants A167V, S213G, K216E and L236S, which were detected with premature pubarche or hyperandrogenic adolescent girls suspected to be affected from nonclassical 3betaHSD deficiency, coupled with the previous studies reporting that no mutations were found in both HSD3B1 and/or HSD3B2 genes in such patients strongly support the conclusion that this disorder does not result from a mutant 3betaHSD isoenzyme. The present study provides biochemical evidence supporting the involvement of a new molecular mechanism in classical 3betaHSD deficiency involving protein instability and further illustrates the complexity of the genotype-phenotype relationships of this disease, in addition to providing further valuable information concerning the structure-function relationships of the 3betaHSD superfamily.

Congenital adrenal hyperplasia in pregnancy

The congenital adrenal hyperplasias (CAH) are a group of inherited enzymatic defects of adrenal steroid biosynthesis. Deficiencies of each enzyme required in the steroid biosynthesis pathway are known, and these deficiencies are all inherited as autosomal recessive disorders. During pregnancy, maternal and fetal problems are confined to women who have 21-hydroxylase deficiency (P450c21 deficiency), 11-hydroxylase deficiency (P450c11 deficiency), and 3 beta-hydroxysteroid dehydrogenase deficiency (3 beta HSD deficiency), because other adrenal enzyme deficiencies are not compatible with fertility. The interposition of the placenta on the hypothalamic-pituitary-adrenal axis and other endocrine changes during pregnancy impact considerably on the clinical evaluation of the congenital adrenal hyperplasias. Successful management of CAH in pregnancy requires a firm knowledge of normal adrenal anatomic and endocrine changes that occur during gestation. Women with severe forms of CAH have decreased fertility rates because of oligo-ovulation, and successful conception requires a combination of good therapeutic compliance, careful endocrine monitoring, and often ovulation induction. From a fetal and neonatal standpoint, accurate prenatal diagnosis of 21-hydroxylase deficiency and 11-hydroxylase deficiency is now possible, which allows for prenatal treatment in an attempt to minimize clinical problems in the neonates. Prevention of masculinization of affected female fetuses by corticosteroid suppression has been attempted in both 21-hydroxylase deficiency and 11beta-hydroxylase deficiency CAH, with variable degrees of success. This review provides an overview of the congenital adrenal hyperplasias and their management during pregnancy.

Natural potent androgens: Lessons from human genetic models

Male pseudohermaphroditism due to 17 beta-hydroxysteroid dehydrogenase-3 (17 beta-HSD-3) deficiency and 5 alpha-reductase-2 (5 alpha-RD-2) deficiency provides natural human genetic models to elucidate androgen actions. To date, five 17 beta-HSD isozymes have been cloned that catalyse the oxidoreduction of androstenedione and testosterone and dihydrotestosterone (DHT), oestrone and oestradiol. Mutations in the isozyme 17 beta-HSD-3 gene are responsible for male pseudohermaphroditism due to 17 beta-HSD deficiency. The type 3 isozyme preferentially catalyses the reduction of androstenedione to testosterone and is primarily expressed in the testes. Fourteen mutations in the 17 beta-HSD-3 gene have been identified from different ethnic groups. Affected males with the 17 beta-HSD-3 gene defect have normal wolffian structures but ambiguous external genitalia at birth. Many are raised as girls but virilize at the time of puberty and adopt a male gender role. Some develop gynaecomastia at puberty, which appears to be related to the testosterone/oestradiol ratio. Two 5 alpha-reductase (5 alpha-RD) isozymes, types 1 and 2, have been identified, which convert testosterone to the more potent androgen DHT. Mutations in the 5 alpha-RD-2 gene cause male pseudohermaphroditism, and 31 mutations in the 5 alpha-RD-2 gene have been reported from various ethnic groups. Such individuals also have normal wolffian structure but ambiguous external genitalia at birth and are raised as girls. Virilization occurs at puberty, often with a gender role change. The prostate remains infantile and facial hair is decreased. Balding has not been reported.