Phase 3 Trial of Crinecerfont in Pediatric Congenital Adrenal Hyperplasia.

BACKGROUND: Children with classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency require treatment with glucocorticoids, usually at supraphysiologic doses, to address cortisol insufficiency and reduce excess adrenal androgens. However, such treatment confers a predisposition to glucocorticoid-related complications. In 2-week phase 2 trials, patients with CAH who received crinecerfont, a new oral corticotropin-releasing factor type 1 receptor antagonist, had decreases in androstenedione levels. METHODS: In this phase 3, multinational, randomized trial, we assigned pediatric participants with CAH, in a 2:1 ratio, to receive crinecerfont or placebo for 28 weeks. A stable glucocorticoid dose was maintained for 4 weeks, and the dose was then adjusted to a target of 8.0 to 10.0 mg per square meter of body-surface area per day (hydrocortisone dose equivalents), provided that the androstenedione level was controlled (≤120% of the baseline level or within the reference range). The primary efficacy end point was the change in the androstenedione level from baseline to week 4. A key secondary end point was the percent change in the glucocorticoid dose from baseline to week 28 while androstenedione control was maintained. RESULTS: A total of 103 participants underwent randomization, of whom 69 were assigned to crinecerfont and 34 to placebo; 100 (97%) remained in the trial at 28 weeks. At baseline, the mean glucocorticoid dose was 16.4 mg per square meter per day, and the mean androstenedione level was 431 ng per deciliter (15.0 nmol/liter). At week 4, androstenedione was substantially reduced in the crinecerfont group (-197 ng per deciliter [-6.9 nmol/liter]) but increased in the placebo group (71 ng per deciliter [2.5 nmol/liter]) (least-squares mean difference [LSMD], -268 ng per deciliter [-9.3 nmol/liter]; P<0.001); the observed mean androstenedione value, obtained before the morning glucocorticoid dose, was 208 ng per deciliter (7.3 nmol/liter) in the crinecerfont group, as compared with 545 ng per deciliter (19.0 nmol/liter) in the placebo group. At week 28, the mean glucocorticoid dose had decreased (while androstenedione control was maintained) by 18.0% with crinecerfont but increased by 5.6% with placebo (LSMD, -23.5 percentage points; P<0.001). Headache, pyrexia, and vomiting were the most common adverse events. CONCLUSIONS: In this phase 3 trial, crinecerfont was superior to placebo in reducing elevated androstenedione levels in pediatric participants with CAH and was also associated with a decrease in the glucocorticoid dose from supraphysiologic to physiologic levels while androstenedione control was maintained. (Funded by Neurocrine Biosciences; CAHtalyst Pediatric ClinicalTrials.gov number, NCT04806451.).

Phase 3 Trial of Crinecerfont in Adult Congenital Adrenal Hyperplasia.

BACKGROUND: Adrenal insufficiency in patients with classic 21-hydroxylase deficiency congenital adrenal hyperplasia (CAH) is treated with glucocorticoid replacement therapy. Control of adrenal-derived androgen excess usually requires supraphysiologic glucocorticoid dosing, which predisposes patients to glucocorticoid-related complications. Crinecerfont, an oral corticotropin-releasing factor type 1 receptor antagonist, lowered androstenedione levels in phase 2 trials involving patients with CAH. METHODS: In this phase 3 trial, we randomly assigned adults with CAH in a 2:1 ratio to receive crinecerfont or placebo for 24 weeks. Glucocorticoid treatment was maintained at a stable level for 4 weeks to evaluate androstenedione values, followed by glucocorticoid dose reduction and optimization over 20 weeks to achieve the lowest glucocorticoid dose that maintained androstenedione control (≤120% of the baseline value or within the reference range). The primary efficacy end point was the percent change in the daily glucocorticoid dose from baseline to week 24 with maintenance of androstenedione control. RESULTS: All 182 patients who underwent randomization (122 to the crinecerfont group and 60 to the placebo group) were included in the 24-week analysis, with imputation of missing values; 176 patients (97%) remained in the trial at week 24. The mean glucocorticoid dose at baseline was 17.6 mg per square meter of body-surface area per day of hydrocortisone equivalents; the mean androstenedione level was elevated at 620 ng per deciliter. At week 24, the change in the glucocorticoid dose (with androstenedione control) was -27.3% in the crinecerfont group and -10.3% in the placebo group (least-squares mean difference, -17.0 percentage points; P<0.001). A physiologic glucocorticoid dose (with androstenedione control) was reported in 63% of the patients in the crinecerfont group and in 18% in the placebo group (P<0.001). At week 4, androstenedione levels decreased with crinecerfont (-299 ng per deciliter) but increased with placebo (45.5 ng per deciliter) (least-squares mean difference, -345 ng per deciliter; P<0.001). Fatigue and headache were the most common adverse events in the two trial groups. CONCLUSIONS: Among patients with CAH, the use of crinecerfont resulted in a greater decrease from baseline in the mean daily glucocorticoid dose, including a reduction to the physiologic range, than placebo following evaluation of adrenal androgen levels. (Funded by Neurocrine Biosciences; CAHtalyst ClinicalTrials.gov number, NCT04490915.).

Severe adrenal insufficiency in six neonates with normal newborn screening for CAH.

BACKGROUND: Newborn screening (NBS) reduces the risk of mortality in congenital adrenal hyperplasia (CAH), mainly due to the salt-wasting form of 21-hydroxylase deficiency. There is limited knowledge regarding the results of NBS in non-CAH primary adrenal insufficiency (non-CAH PAI). PATIENTS AND METHODS: Clinical and NBS for CAH data of neonates who were diagnosed with non-CAH PAI between January and December 2022 were examined. RESULTS: Patients (n = 6, 4 females) were presented with severe hyperpigmentation (n = 6), hypoglycemia (n = 4), hyponatremia (n = 3), hyperkalemia (n = 1), respiratory distress syndrome (n = 1) between 3rd hour to 2 months of life. All had normal NBS results. The median first-tier 17-hydroxyprogesterone (17OHP) concentration in NBS for CAH was 0.14 ng/mL (range; 0.05-0.85). Molecular studies revealed biallelic mutations in the MC2R (n = 4; 3 homozygous, 1 compound heterozygous), MRAP (n = 1) and STAR (n = 1) genes. Glucocorticoid with or without mineralocorticoid replacement was initiated once the diagnosis of non-CAH PAI was established. CONCLUSION: Neonates with non-CAH PAI have always normal NBS due to persistently low 17OHP, even when these newborn infants are severely symptomatic for adrenal insufficiency. Clinicians should be alert for signs of adrenal insufficiency in neonates, even if the patient has a 'normal' screening for CAH, so as not to delay diagnosis and treatment. This fact should be kept in mind particularly in countries where these conditions are more common than elsewhere.

Development and evaluation of a candidate reference measurement procedure for detecting 17α-hydroxyprogesterone in dried blood spots using isotope dilution liquid chromatography tandem mass spectrometry.

17α-Hydroxyprogesterone (17α-OHP) quantification in dried blood spots (DBS) is essential for newborn screening for congenital adrenal hyperplasia (CAH), which is challenging due to its low physiological concentration. The high false-positive rates of immunoassays necessitate the development of more accurate methods. Liquid chromatography tandem mass spectrometry (LC-MS/MS) offers increased specificity and sensitivity, yet standardized procedures for 17α-OHP measurement are required for clinical application. A candidate reference measurement procedure (cRMP) using isotope dilution LC-MS/MS was developed for 17α-OHP quantification in DBS. By utilizing stable isotope-labeled D8-17α-OHP as an internal standard, the cRMP was optimized, covering sample preparation, calibration, and LC-MS/MS analysis. The method performance was validated across several parameters, including precision, accuracy, specificity, detection limits, and matrix effects. Clinical applicability was further assessed through the establishment of reference intervals for healthy newborns. The developed cRMP exhibited a linear range of 1.00 to 80.00 ng/mL for 17α-OHP, with detection and quantification limits of 0.14 ng/mL and 0.52 ng/mL, respectively. Inter- and intraday precision demonstrated coefficients of variation within 1.27 to 5.69%. The recovery rates and matrix effects were well within acceptable limits, ensuring method reliability. Clinical application showed distinct reference intervals for healthy newborns that were unaffected by sex but influenced by weight and gestational age. This method significantly enhances CAH diagnostic accuracy in newborns, providing a valuable tool for clinical laboratories and improving newborn screening program standardization and traceability.

Targeted long-read sequencing identifies missing pathogenic variant in unsolved 11ß-hydroxylase deficiency.

BACKGROUND: 11ß-hydroxylase deficiency (11ß-OHD), caused by homozygosity or compound heterozygosity CYP11B1 variants, is the second most common cause of congenital adrenal hyperplasia (CAH). Due to the high degree of sequence identity between CYP11B1 and CYP11B2, chimeric genes, and complex structural variants (SVs), the conventional approach to gene testing for 11ß-OHD is facing challenges. The study aimed to clarify the underlying genetic causes of two siblings of a Chinese family with 11ß-OHD. METHODS: Peripheral blood samples and clinical information were collected from subjects and their family members. Sex steroid concentrations were measured using LC-MS/MS. Long-range PCR-based next-generation sequencing (NGS), PCR assay and target long-read sequencing were used to detect the pathogenic variants. RESULTS: Early onset hypertension, increased serum levels of adrenocorticotropin (ACTH), progesterone, testosterone, and decreased cortisol and potassium were detected in both affected siblings. Long-range PCR-based NGS identified a heterozygous missense variant (NM_000497.4:c.281 C > T, p.P94> L) in CYP11B1 gene in the two siblings. PCR detected no chimeric CYP11B2/CYP11B1 gene. We finally identified a second pathogenic variant in CYP11B1 gene via target long-read sequencing (T-LRS). This novel variant was a deletion-insertion variant and located chr8:143957269-143,957,579 (hg19) with the insertion of 'ACAG' (NM_000497.4:c.954 + 78_980delinsACAG), which was in trans with CYP11B1: c.281 C > T. CONCLUSIONS: Our study suggests that the integrated long-range PCR-based NGS and T-LRS seem to be the most reliable and accurate method for 11ß-OHD genetic diagnosis and carrier sequencing.

Clinical characteristics and treatment during preconception and perinatal period of infertile women with non-classical 21-hydroxylase deficiency.

OBJECTIVE: A single-center observational study to determine the clinical characteristics and therapeutic dose adjustments in women of reproductive age with infertility and non-classical 21-hydroxylase deficiency (NC-21OHD). DESIGN: A retrospective analysis of 20 women of reproductive age who were diagnosed with NC-21OHD during an infertility evaluation at Shengjing Hospital of China Medical University from January 2013 to May 2024 was performed. The clinical manifestations, auxiliary examinations, adjustment of glucocorticoid (GC) treatment during preconception and perinatal period, and pregnancy outcomes were analyzed. RESULTS: 14 of 16 patients (87.5%) had inappropriately elevated progesterone levels during the follicular phase. The average levels of 17α-hydroxyprogesterone, testosterone, androstenedione, and dehydroepiandrosterone sulfate in the follicular phase were also significantly increased. All 20 infertile patients received GC treatment before preparing for pregnancy. During the follow-up, six of 20 patients had seven conceptions. three patients had spontaneous abortions in the first trimester and four patients delivered babies (4/20). Three patients had a GC dose that was maintained throughout pregnancy and one had an increase in the GC dose starting in the second trimester. Of the remaining 16 patients, seven are still trying to conceive and nine had discontinued treatment. CONCLUSIONS: An abnormal increase in the follicular phase progesterone level is the most common serologic marker for NC-21OHD among infertile women. Ovulation can be restored after GC treatment, but the proportion of successful conceptions remains low. The dose of GCs in most pregnant women remained unchanged throughout pregnancy.

Genotypic spectrum of 21-hydroxylase deficiency in an endogamous population.

BACKGROUND: Congenital adrenal hyperplasia (CAH) due to autosomal recessive 21-hydroxylase deficiency (21-OHD) is caused by defects in the CYP21 (CYP21A2) gene. Several mutations have been identified in the CYP21 (CYP21A2) gene of patients with 21-OHD. We aimed at determining the frequency of these mutations among a group of Egyptian patients and studying the genotype-phenotype correlation. METHODS: Forty-seven patients with CAH due to 21-OHD from 42 different families diagnosed by clinical and hormonal evaluation and classified accordingly into salt wasting (SW) and simple virilizing (SV) phenotypes were enrolled. Their ages ranged between 1.78 and 18.99 years. Molecular analysis of the CYP21 (CYP21A2) gene was performed for the detection of eleven common mutations: P30L, I2 splice (I2 G), Del 8 bp E3 (G110del8nt), I172N, cluster E6 (I236N, V237E, M239K), V281L, L307 frameshift (F306 + T), Q318X, R356W, P453S, R483P by polymerase chain reaction (PCR) and reverse hybridization. RESULTS: Disease-causing mutations were identified in 47 patients, 55.31% of them were compound heterozygous. The most frequent mutations were I2 splice (25.43%), followed by cluster E6 (16.66%) and P30L (15.78%). Two point mutations (P453S, R483P) were not identified in any patient. In the SW patients, genotypes were more compatible with their phenotypes. CONCLUSION: Molecular characterization should be considered along with clinical and biochemical diagnosis of CAH since it could confirm the diagnosis, outline the treatment strategy and morbidity, and ensure proper genetic counseling.

Clinical applications of genetic analysis and liquid chromatography tandem-mass spectrometry in rare types of congenital adrenal hyperplasia.

BACKGROUND: Our study aims to summarize the clinical characteristics of rare types of congenital adrenal hyperplasia (CAH) other than 21-hydroxylase deficiency (21-OHD), and to explore the clinical applications of genetic analysis and liquid chromatography tandem-mass spectrometry (LC-MS/MS) in rare CAH. METHODS: We retrospectively analysed the clinical data of 5 rare cases of CAH admitted to our hospital and summarized their clinical manifestations, auxiliary examinations, diagnosis and mutational spectrum. RESULTS: After gene sequencing, complex heterozygous variants were detected in all patients (2 cases were lipoid congenital adrenal hyperplasia (LCAH), 11ß-hydroxylase deficiency (11ß-OHD), 3ß-hydroxysteroid dehydrogenase deficiency (3ß-HSD deficiency) and P450 oxidoreductase deficiency (PORD) each accounted for 1 case), which were consistent with their clinical manifestations. Among them, 4 novel variants were detected, including c.650 + 2 T > A of the StAR gene, c.1145 T > C (p. L382P) of the CYP11B1 gene, c.1622C > T (p. A541V) and c.1804C > T (p. Q602 *) of the POR gene. The LC-MS/MS results for steroid hormones in patients were also consistent with their genetic variants: 2 patients with LCAH showed a decrease in all steroid hormones; 11ß-OHD patient showed a significant increase in 11-deoxycortisol and 11-deoxycorticosterone; patient with 3ß-HSD deficiency showed a significant increase in DHEA; and PORD patient was mainly characterized by elevated 17OHP, progesterone and impaired synthesis of androgen levels. CONCLUSIONS: The clinical manifestations and classification of CAH are complicated, and there are cases of missed diagnosis or misdiagnosis. It's necessary to combine the analysis of clinical manifestations and auxiliary examinations for diagnosis; if necessary, LC-MS/MS analysis of steroid hormones or gene sequencing is recommended for confirming diagnosis and typing.

[Clinical characteristics of 21 infertile women with non-classic 21-hydroxylase deficiency].

Objective: To investigate the clinical features of infertile women with non-classic 21-hydroxylase deficiency (21-OHD). Methods: The study enrolled 21 infertile women with non-classic 21-OHD in Sun Yat-sen Memorial Hospital of Sun Yat-sen University from January 2009 to December 2018. The clinical presentation, endocrine hormone, glucolipid metabolism and treatment outcome were retrospectively analyzed. The diagnosis of non-classic 21-OHD was comprehensively based on clinical and hormonal characteristics. Results: Among 21 cases, the age was (29.9±2.9) years, the mean age at menarche was (13.6±2.0) years, body mass index was (22.1±2.9) kg/m2, and 38% (8/21) had oligomenorrhea. Hirsutism was diagnosed in 3 cases (14%, 3/21). Clitoromegaly was seen in 14% (3/21) and polycystic ovarian morphology was found in 33% (7/21) of the patients. The mean serum level of basal progesterone was (11.3±21.0) nmol/L, with 48% (10/21) having high basal progesterone level; after therapy by glucocorticoid, the level of progesterone was (1.9±2.0) nmol/L. Serum 17-hydroxyprogesterone concentration was (66.4±123.6) nmol/L; after therapy by glucocorticoid, it was (2.4±1.8) nmol/L. In the study increased testosterone, androstenedione and dehydroepiandrosterone sulfate were present in 62% (13/21), 52% (11/21) and 43% (9/21), respectively; and 52% (11/21) of patients manifested androgen excess and basal progesterone elevation; androgen levels decreased after therapy by glucocorticoid. The pregnancy rate was 76% (16/21). Out of 19 pregnancies, 6/19 ended in spontaneous miscarriages. Conclusions: Infertile women with non-classic 21-OHD are characterized by hyperandrogenism and basal progesterone elevation, whereas gonad axis disorder is not apparent. After no response to conventional therapy, adult infertile women with non-classic 21-OHD could achieve a desirable pregnancy outcome with proper treatment of glucocorticoid.

Does fludrocortisone treatment cause hypomagnesemia in children with primary adrenal insufficiency?

Background/aim: Aldosterone is a mineralocorticoid that secreted from adrenal glands and a known factor to increase magnesium excretion by direct and indirect effects on renal tubular cells. Although the frequency of hypomagnesemia was found to be approximately 5% in adult studies, there is no study in the literature investigating the frequency of hypomagnesemia in children by using fludrocortisone, which has a mineralocorticoid activity. Materials and methods: A multi-center retrospective study was conducted, including children who were under fludrocortisone treatment for primary adrenal insufficiency and applied to participant pediatric endocrinology outpatient clinics. Results: Forty-three patients (58.1% male, 41.9% prepubertal) included in the study, whose median age was 9.18 (0.61-19) years, and the most common diagnosis among the patients was a salt-wasting form of congenital adrenal hyperplasia (67.4%). Mean serum magnesium level was 2.05 (±0.13) mg/dL, and hypomagnesemia was not observed in any of the patients treated with fludrocortisone. None of the patients had increased urinary excretion of magnesium. Conclusion: Unlike the studies performed in adults, we could not find any evidence of magnesium wasting effect of fludrocortisone treatment with normal or even high doses in children and adolescents.

Sensitive Monogenic Noninvasive Prenatal Diagnosis by Targeted Haplotyping.

During pregnancy, cell-free DNA (cfDNA) in maternal blood encompasses a small percentage of cell-free fetal DNA (cffDNA), an easily accessible source for determination of fetal disease status in risk families through non-invasive procedures. In case of monogenic heritable disease, background maternal cfDNA prohibits direct observation of the maternally inherited allele. Non-invasive prenatal diagnostics (NIPD) of monogenic diseases therefore relies on parental haplotyping and statistical assessment of inherited alleles from cffDNA, techniques currently unavailable for routine clinical practice. Here, we present monogenic NIPD (MG-NIPD), which requires a blood sample from both parents, for targeted locus amplification (TLA)-based phasing of heterozygous variants selectively at a gene of interest. Capture probes-based targeted sequencing of cfDNA from the pregnant mother and a tailored statistical analysis enables predicting fetal gene inheritance. MG-NIPD was validated for 18 pregnancies, focusing on CFTR, CYP21A2, and HBB. In all cases we could predict the inherited alleles with >98% confidence, even at relatively early stages (8 weeks) of pregnancy. This prediction and the accuracy of parental haplotyping was confirmed by sequencing of fetal material obtained by parallel invasive procedures. MG-NIPD is a robust method that requires standard instrumentation and can be implemented in any clinic to provide families carrying a severe monogenic disease with a prenatal diagnostic test based on a simple blood draw.

Digit ratio (2D:4D) and congenital adrenal hyperplasia (CAH): Systematic literature review and meta-analysis.

The ratio of length between the second and fourth fingers (2D:4D) is commonly used as an indicator of prenatal sex hormone exposure. Several approaches have been used to try to validate the measure, including examining 2D:4D in people with congenital adrenal hyperplasia (CAH), a suite of conditions characterised by elevated adrenal androgen production secondary to defective steroidogenesis. We present a systematic review and meta-analysis that examines the relationship between these two variables. Twelve articles relating to nine CAH cohorts were identified, and 2D:4D comparisons have been made between cases and controls in eight of these cohorts. Altogether, at least one 2D:4D variable has been compared between n = 251 females with CAH and n = 358 unaffected females, and between n = 108 males with CAH and n = 204 unaffected males. A previous meta-analysis (Hönekopp and Watson, 2010) reported lower right hand (R2D:4D) and left hand (L2D:4D) digit ratios in patients with CAH relative to sex-matched controls. Our meta-analysis showed the same pattern, with medium effect sizes for R2D:4D and small effect sizes for L2D:4D. Differences of small magnitude were also observed for M2D:4D, and no significant effects were observed for D[R-L]. Notably, the only effects that remained statistically significant when stratified by sex were R2D:4D in males and L2D:4D in females, and the average effect size had reduced by 46.70% since the meta-analysis of Hönekopp and Watson (2010). We also found that individual comparisons in this literature were considerably underpowered, and that patterns of sexual dimorphism in 2D:4D were similar in CAH samples as in typically developing populations. Findings are discussed in relation to the prenatal androgen hypothesis as well as alternative explanations.

Steroid 17-Hydroxyprogesterone in Hair Is a Potential Long-Term Biomarker of Androgen Control in Congenital Adrenal Hyperplasia due to 21-Hydroxylase Deficiency.

INTRODUCTION: To evaluate scalp hair steroid concentrations as a monitoring tool for androgen control and metabolic outcomes in adults with congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. METHODS: 17-hydroxyprogesterone (17-OHP), androstenedione, testosterone, cortisol, cortisone, progesterone, prednisolone, and prednisone concentrations were measured in scalp hair by means of LC-MS/MS in 27 women and 15 men with CAH and controls (37 women, 42 men). RESULTS: In CAH men and women, 17-OHP levels in hair showed a significant positive correlation with corresponding levels in serum (ρ = 0.654; p = 0.01; ρ = 0.553, p = 0.003 respectively), while total testosterone levels were only significantly correlated in CAH men (ρ = 0.543; p = 0.036). Androstenedione levels did not show a significant correlation. Receiver-operating characteristic (ROC) curve analysis indicated that a cutoff value of 21.7 pg/mg for 17-OHP in hair provided a sensitivity of 100% and a specificity of 88.9% for identifying men with elevated serum androstenedione. Hair 17-OHP in women showed a poorer performance in terms of identifying those with elevated androstenedione serum levels. However, when applying a cutoff value of 5.5 for the free androgen index as a marker of significant hyperandrogenism in CAH women, 17-OHP >27.6 pg/mg in hair provided a sensitivity of 100% and a specificity of 95.8% (AUC 0.986, 95% CI 0.945-1.000; p < 0.001). Neither hair cortisol nor markers of adrenal androgen control in hair showed significant associations with cardiometabolic outcome or bone health. CONCLUSION: This study shows that scalp hair 17-OHP concentrations may be a promising noninvasive long-term parameter for treatment monitoring in adult patients with CAH.

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.

Serum Fetuin-A and Insulin Levels in Classic Congenital Adrenal Hyperplasia.

Androgens play a pivotal role in non-reproductive organs such as the kidney, heart, liver, and pancreas. As androgen receptors are expressed in pancreatic and liver cells, excess testosterone can result in hypersecretion of insulin and fetuin-A, a protein produced in the liver. The expression of fetuin-A, a natural inhibitor of tyrosine kinase activity in muscle and liver, leads to insulin resistance. In addition, insulin and fetuin-A levels are thought to be affected by drugs such as glucocorticoids (GCs) and fludrocortisone. However, whether fetuin-A and insulin levels are affected by androgens and GCs in patients with classic congenital adrenal hyperplasia (CAH) is unknown. This cross-sectional study included 56 CAH patients and 70 controls. Analyses were stratified by sex and prepubertal/pubertal status to control for potential changes in serum metabolic/inflammatory markers associated with the production of sex steroids. Fasting blood glucose, insulin, triglyceride, total cholesterol, high density lipoprotein-cholesterol, aspartate aminotransferase, alanine aminotransferase, fetuin-A, and high-sensitivity C-reactive protein (hs-CRP) levels were measured in blood samples. In addition, 17α-hydroxyprogesterone, androstenedione, total testosterone, free testosterone, and dehydroepiandrosterone sulfate levels were measured before medication was administered. Insulin and fetuin-A levels were significantly higher in CAH patients than in controls. The unfavourably high levels of these substances exhibited a positive correlation with total and free testosterone. Regression analysis revealed that fetuin-A and free testosterone were the only independent predictors of the insulin level, while insulin and free testosterone levels significantly predicted the fetuin-A level (R2=42.7% and 59.8%). Differences were also observed in triglyceride and hs-CRP levels between the pubertal and prepubertal groups. We conclude that serum fetuin-A and insulin levels may be associated with androgens in CAH patients.

Steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency in a population of PCOS with suspicious levels of 17OH-progesterone.

OBJECTIVE: We aimed at defining the most effective routine immunoassay- or liquid chromatography-tandem mass spectrometry (LC-MS/MS)-determined steroid biomarkers for identifying non-classic adrenal hyperplasia due to 21-hydroxylase deficiency (21-NCAH) in a PCOS-like population before genotyping. METHODS: Seventy PCOS-like patients in reproductive age with immunoassay-determined follicular 17OH-progesterone (17OHP) ≥ 2.00 ng/mL underwent CYP21A2 gene analysis and 1-24ACTH test. Serum steroids were measured by immunoassays at baseline and 60 min after ACTH stimulation; basal steroid profile was measured by LC-MS/MS. RESULTS: Genotyping revealed 23 21-NCAH, 15 single allele heterozygous CYP21A2 mutations (21-HTZ) and 32 PCOS patients displaying similar clinical and metabolic features. Immunoassays revealed higher baseline 17OHP and testosterone, and after ACTH stimulation, higher 17OHP (17OHP60) and lower cortisol, whereas LC-MS/MS revealed higher 17OHP (17OHPLC-MS/MS), progesterone and 21-deoxycortisol and lower corticosterone in 21-NCAH compared with both 21-HTZ and PCOS patients. Steroid thresholds best discriminating 21-NCAH from 21-HTZ and PCOS were estimated, and their diagnostic accuracy in identifying 21-NCAH from PCOS was established by ROC analysis. The highest accuracy was observed for 21-deoxycortisol ≥ 0.087 ng/mL, showing 100% sensitivity, while the combination of 17OHPLC-MS/MS ≥ 1.79 ng/mL and corticosterone ≤ 8.76 ng/mL, as well as the combination of ACTH-stimulated 17OHP ≥ 6.77 ng/mL and cortisol ≤ 240 ng/mL by immunoassay, showed 100% specificity. CONCLUSIONS: LC-MS/MS measurement of basal follicular 21-deoxycortisol, 17OHP and corticosterone seems the most convenient method for diagnosing 21-NCAH in a population of PCOS with a positive first level screening, providing high accuracy and reducing the need for ACTH stimulation test.

The role and importance of auxiliary tests in differential diagnosis in patients with mildly high basal 17-OH-progesterone levels in the evaluation of hirsutism

Background/aim: In the differential diagnosis of hirsutism, early follicular basal 17-OH-progesterone levels sometimes overlap with the diagnosis of late onset congenital adrenal hyperplasia (LOCAH) and other causes of hyperandrogenism. This study aims to investigate the role of some common tests and clinical findings in differential diagnosis in such cases. Materials and methods: One hundred seventy-five female patients with hirsutism and mildly high initial 17-OH-progesterone levels (2-10 ng/mL) were included in the study. The cases were divided into three groups according to their diagnosis: LOCAH (n = 16, mean age = 26.1 ± 6.9), polycystic ovary syndrome (PCOS) (n = 122, mean age = 23.9 ± 5.1), and intracranial hypertension (IH) (n = 37, mean age = 25.2 ± 7.3). Clinical signs and symptoms, such as menstrual irregularity and hirsutism score, and hormone levels including total testosterone and dehydroepiandrosterone sulfate (DHEAS), were compared between the groups. Results: There was no difference between the groups with PCOS, LOCAH, and IH for total testosterone level results (P = 0.461). The DHEAS level was higher in the PCOS group than in the LOCAH group (449.6 ± 151.14 vs. 360.31 ± 152.40, P = 0.044). While there was no difference between the PCOS and LOCAH groups in terms of menstrual irregularity (P = 0.316), the hirsutism score for IH was significantly lower than those of PCOS and LOCAH (9.2 vs. 12.2 and 11.1, respectively; P < 0.001). Basal 17-OH-progesterone levels were higher in the LOCAH group than in the other groups (P = 0.016). Conclusion: While DHEAS level was lower in LOCAH than in PCOS, it was not different from that in IH. While the severity of hirsutism was higher in LOCAH than in IH, it was not different from that in PCOS. Menstrual irregularity was similar between PCOS and LOCAH. According to these results, although the auxiliary tests and clinical findings for the diagnosis of LOCAH contribute to the clinical interpretation, they are not superior to the 17-OH-progesterone level for diagnosis.

Impaired systolic and diastolic left ventricular function in children and adolescents with congenital adrenal hyperplasia receiving corticosteroid therapy.

AimThe present study aimed to evaluate systolic and diastolic myocardial function in children and adolescents with congenital adrenal hyperplasia. METHODS: The study included 44 children with the diagnosis of classic congenital adrenal hyperplasia and 39 healthy children whose age, pubertal status, and gender were similar to those of the patient group. Anthropometric parameters and 17-hydroxyprogesterone levels were measured, and bone age was calculated. The average daily hydrocortisone dose was calculated over the last 1-year file records. Hyperandrogenic state was defined according to bone age SD score (⩾2) and 17-hydroxyprogesterone levels (>10 ng/ml). Echocardiographic examinations were assessed by conventional two-dimensional Doppler echocardiography and tissue Doppler imaging. RESULTS: Patients had higher morphological parameters, such as left ventricular end-systolic diameter, interventricular septal thickness at end diastole, left ventricular posterior wall thickness at end diastole, left ventricular mass and index, than the control group (p<0.05). On pulsed-wave and tissue Doppler echocardiography, significant subclinical alterations were observed in systolic (isovolumic contraction time), diastolic (isovolumic relaxation time), and global left ventricular functional (myocardial performance index) parameters in the congenital adrenal hyperplasia group compared to the control group (p<0.05). In partial correlation analyses, after controlling the effect of hyperandrogenism, the mean hydrocortisone dosage was positively correlated with isovolumic relaxation time in congenital adrenal hyperplasia group (p<0.05). CONCLUSION: This study demonstrated that the patients with congenital adrenal hyperplasia are at risk for left ventricular hypertrophy, systolic and diastolic myocardial subclinical alterations. Overtreatment may be responsible for the increased risk of myocardial dysfunction in patients with congenital adrenal hyperplasia.

[Clinical features and genetic characteristics of 33 patients with simple virilizing form of 21-hydroxylase deficiency].

Objective: To analyze the clinical features and genotypes of adult patients with simple virilizing form of 21-hydroxylase deficiency (SV 21-OHD). Methods: This is a retrospective study including 33 patients with SV 21-OHD from January 2015 to March 2018 in the Ninth People's Hospital of Shanghai Jiao Tong University School of Medicine. Results: The diagnostic age of the patients was (26.3±6.5) years old. All patients presented with signs of masculinization, such as short stature (100%), clitoromegaly/microphallus (89.65%, 26/29), undeveloped breasts (82.76%, 24/29), deep voice (55.17%,16/29) and primary amenorrhea (89.65%, 26/29). The serum levels of 17-hydroxyprogesterone (17-OHP), androstenedione (AD) and testosterone were significantly elevated in 90.9%, 93.9% and 91.2% of the patients, respectively. Thirteen types of mutations were identified in CYP21A2 from these patients. Among them, I173N accounted for 40% and I2 G accounted for 18.33%. Four patients were found with multiple mutations in CYP21A2. Conclusions: Short stature, clitoromegaly/microphallus and primary amenorrhea are the most common clinical features in adult patients with SV 21-OHD. Serum levels of 17-OHP and AD are important indices for the diagnosis and monitoring of the patients. I173N and I2 G are the two most prevalent mutations in patients of the present study. Limitation of clinical recognition and delay in treatment contribute to the short stature of the SV 21-OHD patients.

Long-term use of continuous subcutaneous hydrocortisone infusion therapy in patients with congenital adrenal hyperplasia.

BACKGROUND: In a phase 2 short-term (6 months) study of patients with congenital adrenal hyperplasia (CAH), continuous subcutaneous hydrocortisone infusion (CSHI) was found to be a safe, effective and well-tolerated method of replacing cortisol with improved disease and patient-related outcomes. OBJECTIVE: To evaluate the safety and efficacy of long-term CSHI. DESIGN: Single-centre, open-label, phase 2 extension study. PATIENTS: Five adults with classic CAH. MEASUREMENTS: Biomarkers of disease control, metabolic indices and health-related quality-of-life (HRQoL) estimates. RESULTS: Six of eight patients chose to continue on long-term CSHI therapy. Compared to baseline, eighteen months of CSHI resulted in decreased (P = 0.043) 0700-hour ACTH, 17-hydroxyprogesterone, androstenedione and progesterone; increased whole-body lean mass (P = 0.024); and improved HRQoL, especially symptoms of adrenal insufficiency (P = 0.003). Findings at six and eighteen months did not differ, and improvements achieved in androgen control, lean body mass and HRQoL after 6 months of CSHI were maintained at eighteen months. The hydrocortisone dose appeared to decrease with time [6 vs 18 months: 38.3 ± 8.8 vs 33.6 ± 12.2 mg/day (P = 0.062)], especially in women receiving oral contraceptives. Reduction of testicular adrenal rest and adrenal size observed at 6 months remained stable. In one patient, an adrenal adenoma continually decreased over time. Subjective improvement in hirsutism was reported. CONCLUSIONS: Long-term use of CSHI is a safe and well-tolerated treatment option in a select set of adults with classic CAH. Improvements observed short term in disease control and subjective health status continued long term.