Clinical, Molecular, Functional, and Structural Characterization of CYP17A1 Mutations in Brazilian Patients with 17-Hydroxylase Deficiency.

17-Hydroxylase-deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The aim of the work was to study clinical, biochemical, and the follow up of 17OHD patients and evaluate the function and structure of CYP17A1 mutations. Brazilian patients (three 46, XX and four 46, XY; 17±1.9 years) with combined 17-hydroxylase/17,20-lyase deficiency were evaluated. CYP17A1 gene was sequenced. Functional analysis was performed transfecting COS7 cells, which were exposed to progesterone or 17α-hydroxypregnolone substrates. Hormones were determined by RIA or LC-MS/MS. Three-dimensional structural modeling was performed by Modeller software. All patients presented prepubertal female external genitalia, primary amenorrhea, hypergonadotrophic hypogonadism, hypokalemic hypertension, decreased cortisol, and increased ACTH and corticosterone levels. Five patients presented previously described mutations: p.W406R/p.W406R, IVS2-2A>C/p.P428L, and p.P428L/p.P428L. Two patients presented the compound heterozygous p.G478S/p.I223Nfs*10 mutations, whose CYP17A1 activity and the three dimensional structural modeling are originally studied in this paper. CYP17A1 activity of p.G478S was 13 and 58% against progesterone and 17-hydroxypregnenolone, respectively. The p.I223Nfs*10 caused a truncated inactive protein. Three-dimensional p.G478S structural modeling showed different internal hydrophobic interaction with W313 and created an additional chain side contact with L476 residue. Due to the rarity of 17OHD, the long term follow up (15.3±3.1 years) of our patients will help endocrinologists on the management of patients with 17OHD. The mutation p.G478S/pI223Nfs*10 led to severe 17OHD and impaired CYP17A1 structure and function. The integration of in silico and in vitro analysis showed how the amino acid changes affected the CYP17A1 activity and contributed to clarify the molecular interactions of CYP17A1.

How do mutations affect the structural characteristics and substrate binding of CYP21A2? An investigation by molecular dynamics simulations.

Congenital adrenal hyperplasia (CAH) is one of the most frequent inborn errors of metabolism, inherited as an autosomal recessive trait. Above 95% of CAH cases are caused by mutations in cytochrome P450 21A2 (CYP21A2). It is a pity that how these mutations affect the structural characteristics and substrate binding of CYP21A2 is still unclear. To this end, molecular dynamics (MD) simulations and binding free energy calculations are performed to investigate the effects of single point mutations (L108R, G292C, G292S, G293D, and T296N) in CYP21A2. The results indicate that mutations could cause the local conformational changes of CYP21A2, affecting the substrate binding by changing the interaction between the protein and heme, changing the charge environment of residues, or introducing steric hindrance. In addition, our work gives a wonderful explanation of the phenomenon that though the substrate binding ability increases, the reaction activity decreases in T296N. The present study provides detailed atomistic insights into the structure-function relationships of CYP21A2, which could contribute to further understanding about 21-hydroxylase deficiency and also provide a theoretical basis for CAH prediction and treatment.

Variability in human drug metabolizing cytochrome P450 CYP2C9, CYP2C19 and CYP3A5 activities caused by genetic variations in cytochrome P450 oxidoreductase.

A broad spectrum of human diseases are caused by mutations in the NADPH cytochrome P450 oxidoreductase (POR). Cytochrome P450 proteins perform several reactions, including the metabolism of steroids, drugs, and other xenobiotics. In 2004 the first human patients with defects in POR were reported, and over 250 variations in POR are known. Information about the effects of POR variants on drug metabolizing enzymes is limited and has not received much attention. By analyzing the POR sequences from genomics databases, we identified potentially disease-causing variations and characterized these by in vitro functional studies using recombinant proteins. Proteins were expressed in bacteria and purified for activity assays. Activities of cytochrome P450 enzymes were tested in vitro using liposomes prepared with lipids into which P450 and P450 reductase proteins were embedded. Here we are reporting the effect of POR variants on drug metabolizing enzymes CYP2C9, CYP2C19, and CYP3A5 which are responsible for the metabolism of many drugs. POR Variants A115V, T142A, A281T, P284L, A287P, and Y607C inhibited activities of all P450 proteins tested. Interestingly, the POR variant Q153R showed a reduction of 20-50% activities with CYP2C9 and CYP2C19 but had a 400% increased activity with CYP3A5. The A287P is most common POR mutation found in patients of European origin, and significantly inhibited drug metabolism activities which has important consequences for monitoring and treatment of patients. In vitro, functional assays using recombinant proteins provide a useful model for establishing the metabolic effect of genetic mutations. Our results indicate that detailed knowledge about POR variants is necessary for correct diagnosis and treatment options for persons with POR deficiency and the role of changes in drug metabolism and toxicology due to variations in POR needs to be addressed.

The Spectrum of Genetic Defects in Congenital Adrenal Hyperplasia in the Population of Cyprus: A Retrospective Analysis.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21-OHD) is caused by mutations in the CYP21A2 gene. The study refers to CAH patients of Greek-Cypriot ancestry between years 2007 and 2018. One hundred and twenty patients with various degrees of CAH were categorized and genotyped. The patients were categorized in 4 mutation groups based on their clinical and biochemical findings. The majority of patients (85.0%) belonged to the non-classic (NC)-CAH form and the disorder was more often diagnosed in females (71.7%). The most severe classic salt-wasting (SW) form was identified in 11 neonates (9.2%). Seven (5.8%) children were also identified with the simple virilizing (SV) form and a median presentation age of 5 years [interquartile range (IQR) 3.2-6.5]. In the 240 nonrelated alleles, the most frequent mutation was p.Val281Leu (60.0%) followed by c.655 A/C>G (IVS2-13A/C>G) (8.8%), p.Pro453Ser (5.8%), DelEx1-3 (4.6%), p.Val304Met (4.6%), and p.Gln318stop (4.2%). Other less frequent mutations including rare deletions were also identified. Following our recent report that the true carrier frequency of CYP21A2 in Greek-Cypriots is 1:10, this study reports that the CAH prevalence is predicted around 1.7 cases per 10 000 people. Therefore, the up-to-date 120 CAH patients identified by our group make only the 6.9% of the ones estimated (approximately 1750) to exist in the Greek Cypriot population. The compiled data from a coherent population such as the Greek-Cypriot could be valuable for the antenatal diagnosis, management and genetic counselling of the existing and prospect families with CAH.

Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia.

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.

[17α-Hydroxylase deficiency with severe hypertension as the initial symptom in a child].

A 14-year-old female (social gender) patient was admitted to the hospital due to severe hypertension for 11 days. The patient had primary amenorrhea. The blood pressure was 146/90 mm Hg. The skin color was slightly black. The development of secondary sexual characteristics was poor. The labia majora could be observed in the vulva. However, the labia minora, clitoris, vagina, and hymen were absent. The levels of renin, cortisol, and sex hormone were low, while the levels of adrenocorticotropic hormone and gonadotropin were high. The levels of blood potassium and aldosterone were both normal. Radiography indicated retardation of bone age. Ultrasound examination revealed that the ovary and uterus were both absent. The patient had bilateral adrenal hyperplasia and cryptorchid testes located in both inguinal canals. The patient had a 46,XY karyotype. Whole genome sequencing revealed two homozygous mutations, c.985T>C and c.987delC, in exon 6 of the CYP17A1 gene of the patient and heterozygous mutations in the same sites of the parents. The patient was diagnosed with congenital adrenal hyperplasia-17α-hydroxylase deficiency. After treatment with hydrocortisone for 2 months, blood pressure returned to normal and the level of adrenocorticotropic hormone was reduced. According to the request of the patient and the parents, hydrocortisone was replaced with estrogen to allow the patient to live as a female. The patient also received surgical excision of cryptorchid testes to prevent gonadal malignancy. It is concluded that in the differential diagnosis of pediatric hypertension, sexual development should be considered and the levels of adrenocorticotropic hormone and cortisol should be evaluated. The rare disease 17α-hydroxylase deficiency should be considered for patients with low-renin hypertension and gonadal dysgenesis.

Improving the diagnosis of 11ß-hydroxylase deficiency using home-made MLPA probes: identification of a novel chimeric CYP11B2/CYP11B1 gene in a Sicilian patient.

PURPOSE: 11ß-Hydroxylase deficiency (11OHD) represents the second most common cause of congenital adrenal hyperplasia. It is caused by mutations in the CYP11B1 gene localized about 40 kb from the CYP11B2 gene with which it shares a homology of 95 %. The asymmetric recombination of these two genes is involved both in 11OHD and in glucocorticoid-remediable aldosteronism (GRA). Our objective was to set up an easy and rapid method to detect these hybrid genes and other kinds of deletions, to improve the molecular diagnosis of 11OHD. METHODS: A set of 8 specific probes for both the CYP11B1 and the CYP11B2 genes to be used for multiplex ligation-dependent probe amplification (MLPA) analysis was designed to detect rearrangements of these genes. RESULTS: The method developed was tested on 15 healthy controls and was proved to be specific and reliable; it led us to identify a novel chimeric CYP11B2/CYP11B1 gene in one patient that carried the known A306V mutation on the other allele. Specific amplification and sequencing of the hybrid gene confirmed the breakpoint localization in the second intron. CONCLUSIONS: The MLPA kit developed enables the detection of deletions, duplications or chimeric genes and represents an optimal supplement to DNA sequence analysis in patients with 11OHD. In addition, it can also be used to show the presence of the opposite chimaera associated with GRA.

Combined 17α-hydroxylase/17,20-lyase deficiency with short stature: case study.

BACKGROUNDS: 17α-hydroxylase/17,20-lyase deficiency (17-OHD) is an uncommon form of congenital adrenal hyperplasia. Most patients are tall owing to delayed closure of epiphyses as a result of deficiency of sex hormones. METHODS: We present a 17-OHD case with unusual short stature and reviewed related literature. RESULTS: A 17-year-old female patient presented with primary amenorrhea, hypertension, hypokalemia and hypergonadotropic hypogonadism (HH). Sequencing of the CYP17A1 gene identified a homozygous c.985_987delTACinsAA in exon 6 that confirmed the diagnosis of 17-OHD. However, her height (148 cm, height standard deviation score [HSDS] -2.28) was unusually low compared with that of other 17-OHD patients. Levels of growth hormone (GH) and insulin-like growth factor (IGF)-1 were normal, and the GH provocation test excluded the possibility of GH deficiency. She underwent glucocorticoid and sex-hormone replacement therapy, reaching a final height of 152 cm (HSDS -1.59). These data suggest that tall stature is not a requisite characteristic of 17-OHD. Further studies are needed to clarify the effects of sex hormone on linear bone growth (LBG) in 17-OHD patients.

Congenital Adrenal Hyperplasia with Non-functional Mutations in Both Alleles in a Clinically Unaffected Infant.

BACKGROUND: Results in neonatal screening programs aiming at detection of congenital adrenal hyperplasia (CAH) can only report elevated levels of 17-hydroxy-progesterone (17-OHP), without being able to differentiate presence or absence of salt loss. AIM: To predict presence or absence of salt loss in newborn infants with CAH. METHODS: The first specimen of suspected CAH in samples sent from People's Democratic Republic of Laos (Lao PDR) was investigated for known mutations in CAH associated with salt loss. RESULTS: Molecular genetic diagnosis revealed mutations associated with loss of function in both alleles; however, the infant was clinically unaffected even without any corticosteroid substitution therapy. CONCLUSIONS: Although molecular genetic methods can theoretically predict loss of function in CAH, our infant was clinically unaffected even without therapy at 6 years of age. We speculate that in CAH, remaining enzyme activity can be sufficiently high, despite the presence of loss of function mutations, which do not affect infants clinically.

[Genotypes and phenotypes in Uygur children with 21-hydroxylase deficiency in Xinjiang, China].

OBJECTIVE: To investigate gene mutations and the relationship between genotypes and clinical phenotypes in Uygur children with 21-hydroxylase deficiency (21-OHD) in Xinjiang, China. METHODS: A total of 20 Uygur children with 21-OHD who visited the hospital between October 2013 and October 2014 were enrolled. Full-length direct sequencing and multiplex ligation-dependent probe amplification (MLPA) were used to detect the mutations of CYP21A2 gene, which encoded 21-hydroxylase. According to the type of mutation, the patients with 21-OHD were divided into different groups to analyze the consistency between predicted clinical phenotypes and actual clinical phenotypes. RESULTS: A total of 9 mutation types were found in the 20 patients, and 8 of them were identified as pathogenic mutations, i.e., Del, conv, I2g, I172N, Cluster E6, 8-bp del, V281L, and R356W. The other mutation is the new mutation occurring in intron 5 (c.648+37A>G), which had not been reported, and its pathological significance remains unknown. Most clinical phenotypes predicted by mutation types had a higher coincidence rate with actual clinical phenotypes (above 67%), and the clinical phenotypes predicted by P30L and V281L had a lower coincidence rate with actual clinical phenotypes (below 33%). CONCLUSIONS: The genotype of 21-OHD has a good correlation with phenotype, and the clinical phenotype can be predicted by detecting the patient′s genotype. The new mutation (c.648+37A>G) may be related to the pathogenesis of 21-OHD.

A rare CYP 21 mutation (p.E431K) induced deactivation of CYP 21A2 and resulted in congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the CYP21A2 gene. The residual enzyme activity is strongly associated with the phenotype. We describe a rare case of CAH with a rare CYP21A2 mutation. The patient was a one-year-old Japanese boy. At 16 days old, he was referred to our hospital because of elevated serum 17-OH-progesterone (17-OHP) levels in neonatal screening. The compound heterozygous mutations (IVS2-13 A/C>G, and p.E431K) in CYP21A2 were identified at 2 months old, and we diagnosed non-classical CAH, since he did not have significant physical signs (pigmentation and salt-wasting). However, his body weight decreased, and his serum 17-OHP level (99.5 ng/mL) was elevated at 3 months old. Steroid replacement therapy was started at 3 months old. Our patient's clinical course resembled simple virilizing (SV) CAH, but classification was difficult because the patient showed increased renin activity indicating an aldosterone deficiency, and late onset of symptoms. While the IVS 2-13 A/C>G mutation is common in the classical form of CAH, p.E431K is a rare point mutation. Functional analysis revealed that the residual enzyme activity of p.E431L was 5.08±2.55% for 17-OHP and 4.12±2.37% for progesterone, which is consistent with SV CAH. p.E431 is localized in the L-helix near the heme-binding site. The mutation might interfere with heme binding, leading to deactivation of CYP21A2. This report showed that CYP21A2 p.E431 has an important effect on enzyme activity.

Reduced activity of 11ß-hydroxylase accounts for elevated 17α-hydroxyprogesterone in preterms.

OBJECTIVE: To characterize the urinary steroid metabolome of neonates and infants born either at term or preterm. STUDY DESIGN: We retrospectively analyzed urinary steroid hormone metabolites determined by gas chromatography-mass spectrometry of 78 neonates and infants born at term and 83 neonates and infants born preterm (median 34 weeks of gestational age). The subjects' 11ß-hydroxylase and 21-hydroxylase activities were assessed on the basis of urinary metabolite substrate-to-product ratios. RESULTS: Preterm neonates and infants had elevated urinary concentrations of 17α-hydroxyprogesterone (17OHP) metabolites (P<.001) but lower urinary concentrations of the 21-deoxycortisol metabolite pregnanetriolone (PTO) (P<.01). One reason was lower 11ß-hydroxylase activity in preterms. We could demonstrate a correlation between low 11ß-hydroxylase activity and high urinary concentrations of 17OHP metabolites (r=0.51, P<.001) but low urinary concentrations of the 21-deoxycortisol metabolite PTO (r=-0.24, P=.03) in preterms. CONCLUSIONS: Low 11ß-hydroxylase activity may explain increased 17OHP but decreased 21-deoxycortisol metabolite excretion in preterms. Our analysis clarifies, first, why preterms have higher 17OHP levels and thus higher rates of false-positive screening results for congenital adrenal hyperplasia than do term infants, and, second, why 21-deoxycortisol or its urinary metabolite PTO is more specific than 17OHP for the diagnosis of 21-hydroxylase deficiency.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

CYP17A1 gene mutations and hypertension variations found in 46, XY females with combined 17α-hydroxylase/17, 20-lyase deficiency.

The aim of this study was to analyze the structural consequences of the mutations in CYP17A1 gene and their relationship with the variations of clinical manifestations in three patients who presented with complete or partial combined 17α-hydroxylase/17,20-lyase deficiency (17OHD). DNA sequences of the coding exons and intron/exon boundaries of the CYP17A1 gene were analyzed for mutations. In silico analysis with computational three-dimensional model of human P450c17 and multiple alignments analysis were performed to evaluate the spatial conformational changes by missense mutations. Five mutations p.S117fs (c.351_352delCT), p.H373L (c.1184 A>T), p.Y329fs (c.985_987delTACinsAA), p.A82D (c.245 C>A) and p.L209P (c.626 T>C) were identified in three patients, respectively. The novel mutation p.S117fs (c.351_352delCT) has not been reported previously. In silico analysis explained the conformational changes by the described mutations, which resulted in different severe 17OHD. Our studies also suggest that molecular data accompanying with in silico analysis of the CYP17A1 gene are much helpful for the diagnosis, management and genetic counseling of 17OHD.

[Establishment of an allele-specific PCR method for direct screening of CYP21A2 gene mutation].

OBJECTIVE: To establish an allele-specific PCR method for detect screening of CYP21A2 gene mutation. METHODS: Allele-specific PCR primers and analogy primers were designed based on the sequence alignment of CYP21A2 and CYP21AP genes. Genomic DNA was extracted from blood specimens of 4 patients with 21-hydroxylase deficiency and 5 healthy controls and respectively amplified with allele-specific PCR primers and analogy primers and sequenced. RESULTS: Mutations of CYP21A2 including IVS2-13A/C>G, Arg356Trp and Arg149Pro were found with the established method in all of the 4 patients but not in the healthy controls. When detected with the analogy primers set, IVS2-13A/C>G and Arg356Trp were observed in both patients and healthy controls. CONCLUSION: The allele-specific PCR-based method is a simple, effective and reliable method for the detection of CYP21A2 gene mutation.

[Analysis of CYP21A2 gene mutations in two families with 21-hydroxylase deficiency].

OBJECTIVE: To analyze CYP21A2 gene mutation in two families with 21-hydroxylase deficiency (21-OHD) and to explore the correlation between genotype and clinical phenotype. METHODS: Two patients with 21-OHD and their families were investigated. CYP21A2 gene mutation was analyzed by PCR and direct sequencing. RESULTS: The probands from family 1 and 2 have been respectively diagnosed with simple virilizing and non-classical 21-OHD. Both showed increased baseline serum 17hydroxyprogesterone, testosterone and adrenocorticotropic hormone (ACTH), but had no evidence of salt loss. Computer tomography revealed bilateral adrenal hyperplasia in both patients. After 1 year treatment, both had conceived successfully. DNA sequencing revealed that the proband of family 1 had compound heterozygous mutations for IVS2 13 A>G and Ile172Asn. Her father was heterozygous for Ile172Asn, whilst her mother and brother were heterozygous for IVS213A/C>G. In family 2, the proband was heterozygous for Arg341Trp and Gln318X. Her father, sister and nephew were heterozygous for Arg341Trp, whilst her mother was heterozygous for Gln318X. her brother and niece were non-affected. Carriers of single heterozygous mutations in both families had no clinical sign. CONCLUSION: In both families, the disease has been caused by compound heterozygous mutations, for which there has been a good genotype-phenotype agreement. Screening of CYP21A2 gene can facilitate both diagnosis and genetic counseling.

Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants.

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Šresolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.

What is the best approach to the teenage patient presenting with nonclassical congenital adrenal hyperplasia: should we always treat with glucocorticoids?

Patients with Congenital adrenal hyperplasia due to partial deficiency in the enzyme 21-hydroxylase can present in childhood or adolescence with signs of adrenal androgen excess. Strategies to reduce the impact of androgen excess in females include cosmetic measures as well as antiandrogens and agents such as the combined oral contraceptive pill. Glucocorticoid may not be appropriate straightaway but can be introduced if other measures are ineffective or when pregnancy is planned.

Androgen synthesis in patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency.

A hallmark of severe congenital adrenal hyperplasia due to 21-hydroxylase deficiency is pre- and postnatal virilization. The most characteristic biochemical abnormality is the elevation of 17α-hydroxyprogesterone, which is metabolized to the most potent androgen receptor agonist dihydrotestosterone. 17α-Hydroxyprogesterone can be metabolized to dihydrotestosterone via 4-androstenedione through the classical Δ4-pathway or via 17α-hydroxypregnenolone and dehydroepiandrosterone through the classical Δ5-pathway, as well as through an alternative route, called the 'backdoor pathway', that bypasses dehydroepiandrosterone, 4-androstenedione, and testosterone as intermediates. This review article will summarize recent advances in the understanding of the activities of androgen synthesis pathways in patients with 21-hydroxylase deficiency obtained by urinary steroid metabolomics based on gas chromatography-mass spectrometry. Compared with healthy controls, the relative activities of the backdoor and Δ4-pathways increase in patients with congenital adrenal hyperplasia during neonatal age and infancy, whereas the activity of the Δ5-pathway remains unchanged. Thereafter, the activity of the Δ5-pathway dominates, whereas a decreasing 5α-reductase activity leads to a diminished role of the backdoor pathway for androgenic steroid production. Beside the backdoor pathway, the Δ4-pathway seems to be responsible for increased androgen generation in patients with 21-hydroxylase deficiency before the onset of adrenarche, whereas the Δ5-pathway might contribute to the increased androgen formation in those patients only after the onset of adrenarche.

Congenital adrenal hyperplasia due to point mutations in the type II 3 beta-hydroxysteroid dehydrogenase gene.

Classical 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase (3 beta-HSD) deficiency is an autosomal recessive form of congenital adrenal hyperplasia characterized by a severe impairment of steroid biosynthesis in both the adrenals and the gonads. We describe the nucleotide sequence of the two highly homologous genes encoding 3 beta-HSD isoenzymes in three classic 3 beta-HSD deficient patients belonging to two apparently unrelated pedigrees. No mutation was detected in the type I 3 beta-HSD gene, which is mainly expressed in the placenta and peripheral tissues. Both nonsense and frameshift mutations, however, were found in the type II 3 beta-HSD gene, which is the predominant 3 beta-HSD gene expressed in the adrenals and gonads, thus providing the first elucidation of the molecular basis of this disorder.