Congenital adrenal hyperplasia caused by compound heterozygosity of two novel CYP11B1 gene variants.

BACKGROUND: Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by pathogenic variants in seven genes involved in the cortisol and aldosterone biosynthetic pathway. The second most common cause, 11ß-hydroxylase deficiency (11ßOHD), is attributed to pathogenic variants in the CYP11B1 gene encoding for the enzyme 11ß-hydroxylase (11ßOH). CASE PRESENTATION: A 13-year-old girl was referred to the pediatric endocrinologist due to a syncopal episode. She is the third child of non-consanguineous parents. She presented with premature adrenarche at the age of 6 years and menarche at the age of 12 years. On physical examination, her height was 154.5 cm and weight 50 kg, while she presented with acne, hirsutism, clitoromegaly, and normal blood pressure. Laboratory investigation revealed increased androgen levels and poor cortisol response to the ACTH stimulation test. From the family history, the mother was diagnosed with CAH at the age of 10 years and was under treatment with methylprednisolone. Previous molecular investigation of the CYP21A2 gene was negative. Due to the increased androstenedione levels in the index patient, the suspicion of 11ßOH was raised, and she was investigated for 11-deoxycortisol, 11-deoxycorticosterone, and CYP11B1 gene pathogenic variants. The patient and her mother were found to be compound heterozygous for two novel variants of the CYP11B1 gene. CONCLUSION: We present a case of CAH due to compound heterozygosity of two novel pathogenic variants of the CYP11B1 gene, emphasizing the importance of molecular investigation in order to confirm clinical diagnosis and allow proper genetic counseling of the family.

The spectrum of clinical, hormonal and molecular findings in 280 individuals with nonclassical congenital adrenal hyperplasia caused by mutations of the CYP21A2 gene.

BACKGROUND: Nonclassical congenital adrenal hyperplasia (NC-CAH) is caused by mutations of the CYP21A2 gene. The clinical manifestations and hormonal derangements of NC-CAH are quite variable. OBJECTIVES: (i) To define the phenotype and its relation to genotype according to gender and age and (ii) to evaluate the validity of currently applied hormonal criteria for establishing the diagnosis of NC-CAH. PATIENTS AND METHODS: The clinical, hormonal and molecular data of 280 subjects (235 female) with NC-CAH and a median age of 17·6 years were analysed. CYP21A2 genotyping was performed in all subjects. RESULTS: The majority of females aged less than 8 years presented with premature pubarche (88·3%), while those older than 8 presented with a polycystic ovary-like phenotype (63·2%). A total of 7·7% of the females and 51·1% of the males were asymptomatic at the time of diagnosis. In the total group, 50·4% of the subjects were compound heterozygotes for one classical (C) and one nonclassical (NC) mutation, while 46% of the alleles studied carried the p.V281L mutation. Basal 17OHP values were below 6 nm (2 ng/ml) in 2·1% of the subjects with NC-CAH, but none had peak 17OHP values post-ACTH lower than 30 nm (10 ng/ml). CONCLUSIONS: NC-CAH has a variable phenotype depending on the age, gender and the presence of a classical mutation. A peak cut-off value of 17OHP post-ACTH lower than 30 nm excludes the diagnosis of NC-CAH, whereas basal 17OHP <6 nm may represent a false-negative result. A significant number of patients harboured a classical mutation, a finding which requires genotyping of the partner for genetic counselling.

Routine method for the simultaneous quantification of 17alpha-hydroxyprogesterone, testosterone, dehydroepiandrosterone, androstenedione, cortisol, and pregnenolone in human serum of neonates using gas chromatography-mass spectrometry.

Steroid determination by immunoassays results in significant interferences and inaccurate results. This study describes the development and validation of a new gas chromatographic-mass spectrometric method for the simultaneous quantification of 17alpha-hydroxyprogesterone (17alphaOHP), testosterone (T), dehydroepiandrosterone (DHEA), androstenedione (Delta4-A), cortisol (F) and pregnenolone (Preg) in serum of neonates. Steroids were extracted and purified from 0.5 mL serum using diethyl ether and Extrelut mini NT1 column. The extracts were derivatized with N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA)/trimethylsilyl iodide (TMSI)/dithioerythritol (DTE) and the resulting trimethylsilyl derivatives were quantified by gas chromatography-selected ion monitoring-mass spectrometry (GC-SIM-MS). The detection limit for all steroids was lower than 0.1 ng/mL. The limit of quantification was 0.1 ng/mL for all steroids except cortisol which was at 0.25 ng/mL. d3-Testosterone and methyltestosterone served as internal standards. Precision for all compounds at the concentrations of 0.5, 1, 5 and 10 ng/mL (n = 10) in fortified steroid-free serum samples ranged from 0.8% to 16.6%. Accuracy was calculated at the concentrations of 0.5, 1, 5 and 10 ng/mL and ranged from -9.2% to 10.6% (n = 10). Linear calibration equations were obtained for all five steroids (0.125-31.25 ng/mL) and for cortisol (0.125-200 ng/mL). Relative recoveries at concentrations 1.0 and 12.5 ng/mL ranged from 70.5% to 97.5%. Absolute recoveries at the same concentrations ranged from 73.2% to 96.6%. Reference intervals were estimated for infants aged from 9 to 40 days. The proposed steroid profile is suitable for routine analysis and provides meaningful data for samples within normal range as well as those with elevated levels.

"White coat hypertension" in adolescents: increased values of urinary cortisol and endothelin.

OBJECTIVE: To investigate whether "white coat hypertension" (WCH) in adolescents is an innocent phenomenon or is associated with early changes of the vascular system and/or increased stress response, reflected in the urinary endothelin and cortisol values, respectively. STUDY DESIGN: The study group included 36 subjects, 14 with WCH (8 males and 6 females) aged 12.9 +/- 3 years and 22 normotensive control subjects (12 males and 10 females) aged 13 +/- 3.5 years. WCH was defined as systolic and/or diastolic blood pressure (BP) > or =95th percentile for age, sex, and height and with reported normal BP measurements at home. Urinary endothelin (UET1), urinary free cortisol (UFC), and plasma renin levels were determined by radioimmunoassay; and urinary albumin levels were determined by nephelometry. For statistical analysis, the Mann Whitney U test, Spearman correlation coefficient, and multivariate analysis of variance/multivariate analysis of covariance were used, as applicable. RESULTS: The 24-hour values of UET1 and UFC were greater in male subjects with WCH than in male control subjects (P =.02), whereas no such difference was found in female subjects. The difference in UFC values in male subjects was accounted for by the day values. In subjects with WCH, and not in control subjects, a positive correlation of UET1 to UFC (r = 0.59, P =.027), diastolic BP (r = 0.55, P =.04), and mean BP (r = 0.65, P =.012) was detected. CONCLUSIONS: Our data indicate that WCH in adolescence may not be an innocent phenomenon and may represent a prelude to permanent idiopathic hypertension of adulthood.

High incidence of molecular defects of the CYP21 gene in patients with premature adrenarche.

On the basis of hormonal studies, the incidence of defective steroidogenesis in children with premature adrenarche (PA) in the various reports ranges from 0-54%. Molecular studies have not been reported to date. The aim of the present study was to search for defects in the CYP21 gene in children with PA and to detect possible correlations of the molecular defect to pertinent hormonal and clinical data. In 48 children with PA (40 females and 8 males) and without signs of virilization, a Synachten test and molecular studies were carried out. DNA analysis was performed using the Southern blot technique and allele-specific PCR. Synachten (0.25 mg) was given i.v., and 17-hydroxyprogesterone and cortisol were determined at 0 and 60 min. At baseline, delta4-androstenedione, dehydroepiandrosterone sulfate, and 11-deoxycortisol were also determined. Bone age was evaluated using the Greulich and Pyle atlas. Abnormal genotype was detected in 45.8% of the studied subjects; 8.3% were homozygotes, with genotypes concordant with the nonclassical phenotype of 21 hydroxylase deficiency, and 37.5% were heterozygotes for 9 different molecular defects of the CYP21 gene. The children with no detectable molecular defect were designated normal. The 60 min post-Synachten values in homozygotes (17.9 +/- 7.1 ng/mL) and heterozygotes (7.1 +/- 3.6 ng/mL) were significantly higher than that in normal subjects (3.3 +/- 1.5 ng/mL), but with significant overlapping of values. The mean difference between bone age and chronological age differed in the three groups with overlapping values. The basal delta4-androstenedione level was lower in the normal subjects (0.65 +/- 0.3 ng/mL) than in those with abnormal genotype (1.1 +/- 0.8 ng/mL). The data indicate that the incidence of molecular defects in PA is quite high. The CYP21 heterozygocity is clinically expressed in some subjects prepubertally. In a significant number of cases the genotype cannot be predicted by the age of onset of PA, the mean difference between bone age and chronological age, or the results of a Synachten test. Follow-up of these children through puberty is imperative and may reveal the clinical significance of the molecular defect, namely more hypertrichosis, intense acne, early puberty, possible abnormal menses, and/or fertility problems in the affected.

Urinary endothelin in adolescents and young adults with insulin-dependent diabetes mellitus: relation to urinary albumin, blood pressure, and other factors.

Endothelin (ET) is a potent vasoconstrictive peptide that may play a role in vascular pathology in general and diabetic nephropathy in particular. The aim of this study was to investigate (1) alterations of urinary ET1 (UET1) in adolescents and young adults with insulin-dependent diabetes mellitus (IDDM) and (2) the relation of UET1 to other indices of diabetic nephropathy and to risk factors of diabetic angiopathy in general. In 130 IDDM subjects aged 15.2+/-4.9 years with a diabetes duration of 7.3+/-5.1 years, UET1 by radioimmunoassay, urinary albumin by nephelometry, plasma renin by immunoradiometric assay, hemoglobin A1c (HbA1c) by high-performance liquid chromatography, and routine biochemistry analyses were determined. Forty-eight controls, healthy siblings of the diabetics of comparable age, were similarly studied. Total 24-hour UET1 excretion was higher in diabetics than in controls (10,866+/-7,270 and 6,598+/-3,294 pg/24 h, respectively, P=.000). This difference was also noted if male and female diabetics were separately compared with controls. In diabetics with normoalbuminuria (<20 microg/min), total 24-hour UET1 excretion was also higher than in controls (P=.002). In diabetics but not in controls, 24-hour UET1 values were higher in males than in females (P=.018). In IDDM subjects, UET1 showed a linear relationship with age (P=.002), urinary albumin (P=.000), serum creatinine (P=.001), systolic blood pressure (P=.038), triglycerides (P=.003), and HbA1c (P=.041). Multiple regression analysis demonstrated that the variables interacting independently with UET1 were urinary albumin (P=.003) and serum creatinine (P=.038). UET1 is elevated early (in adolescence) in IDDM subjects, and it is positively correlated with the degree of albuminuria. These data suggest that the amount of UET1 possibly reflects the severity of diabetic renovascular damage. It may thus be speculated that UET1 could be used as another index of diabetic nephropathy or its progress.

Carotid atherosclerosis in adolescents and young adults with IDDM. Relation to urinary endothelin, albumin, free cortisol, and other factors.

OBJECTIVE: To investigate 1) alterations of carotid intimal-plus-medial thickness (IMT) in subjects with IDDM and 2) the relation of IMT to indexes of diabetic angiopathy and to risk factors of atherosclerosis. RESEARCH DESIGN AND METHODS: IMT was assessed by ultrasound B-mode imaging in 39 subjects with IDDM (23 male, 16 female young adults aged 17.5 +/- 5.2 years, diabetes duration 8.8 +/- 5.9) and in 22 control subjects (healthy siblings of the IDDM subjects) of comparable age. Urinary endothelin (UET1) and urinary free cortisol (UFC) were determined by radioammunoassay (RIA), urinary albumin by nephelometry, HbA1c by high-performance liquid chromatography (HPLC), and plasma renin by immunoradiometric assay (IRMA). RESULTS: The IMT values were greater in IDDM subjects than in control subjects (0.49 +/- 0.1 mm, 0.44 +/- 0.09 mm, respectively; P = 0.048) and greater in IDDM male subjects than in control male subjects (0.52 +/- 0.09 and 0.44 +/- 0.06 mm, respectively; P = 0.015), with no difference between IDDM and control female subjects. The IMT values were greater in diabetic male subjects than in female subjects (0.52 +/- 0.09 and 0.45 +/- 0.1 mm, respectively; P = 0.017). In IDDM subjects, but not in control subjects, there was a positive correlation of IMT to urinary albumin (P = 0.008), systolic blood pressure (P = 0.023), UET1 (P = 0.016), UFC (P = 0.002), and BMI (P = 0.021). Multiple regression analysis demonstrated that in IDDM subjects the variable that interacts independently with IMT was the BMI (P = 0.001). CONCLUSIONS: IMT, an index of atherosclerosis (macroangiopathy), is increased in IDDM subjects quite early (already in adolescence), and it is positively related to urinary albumin, UET1, blood pressure, and UFC.

Urinary free cortisol and its nyctohemeral variation in adolescents and young adults with IDDM: relation to endothelin 1 and indices of diabetic angiopathy.

The existence of a hyperactive hypothalamic pituitary adrenal (HPA) axis in diabetics and its relevance to diabetic complications has been controversial. In this study we determined the 24 hour urinary excretion of free cortisol (UFC), its nyctohemeral variation and its relation to indices of diabetic angiopathy. In 130 subjects with IDDM, aged 15.2+/-4.8 years and diabetes duration 7.3+/-5 years, and in 48 controls of comparable age, UFC, urinary endothelin (UET1), urinary albumin, HbA1c, and plasma renin were determined. The total 24-hour UFC excretion was greater in diabetics than in controls (p=0.002) and also greater in diabetic males than in females (p=0.006), while no sex difference was detected in the controls. Day UFC excretion was greater than night UFC excretion (p<0.001) in all subjects. UFC correlated to carotid intimal plus medial thickness (r=0.48, p=0.002), urinary albumin (r=0.50, p<0.001), UET1 (r=0.56, p<0.001), diastolic, systolic and mean blood pressure (r=0.27, p=0.003; r=0.41, p<0.001; r=0.34, p<0.001), BMI (r=0.50, p<0.001), serum creatinine (r=0.35, p<0.001), total cholesterol (r= -0.19, p=0.036), HDL (r= -0.22, p=0.038), LDL (r= -0.23, p=0.032), age (r=0.61, p<0.001) and diabetes duration (r=0.37, p<0.001). These results suggest that hyperactivity of the HPA axis or of the adrenals, due to chronic stress, hypoglycemic episodes or other factors, possibly contributes to the establishment or progression of diabetic micro- or macroangiopathy.