Clemens Kamrath

A novel entity of clinically isolated adrenal insufficiency caused by a partially inactivating mutation of the gene encoding for P450 side chain cleavage enzyme (CYP11A1).

CONTEXT Cytochrome P450 side-chain cleavage enzyme (CYP11A1) facilitates the first and rate-limiting step of steroidogenesis. Only nine patients with CYP11A1 deficiency have been described. All patients presented with adrenal insufficiency (AI) and disorder of sex development in 46,XY individuals. OBJECTIVE Our objective was to define the pathogenic consequences of a novel CYP11A1 mutation (p.R451W) found in two brothers with isolated adrenal insufficiency. PATIENTS The two brothers (46,XY) presented with AI and normal male genital development. The older boy first presented with signs and symptoms suggestive of AI at the age of 2.8 yr but was only diagnosed at the age of 4.1 yr during an adrenal crisis. The younger brother was diagnosed with AI at the age of 2.5 yr while being clinically asymptomatic. Both boys had entirely normal appearance of their external genitalia. RESULTS The novel p.R451W mutation and five published missense CYP11A1 mutations were characterized employing two in vitro approaches using the natural substrate cholesterol and the intermediate 22R-hydroxycholesterol, respectively. Pregnenolone generation was measured by highly specific liquid chromatography tandem mass spectrometry. p.R451W had 30% of wild-type activity consistent with the clinical phenotype in our patients. Two previously published mutations (p.L222P and p.A359V) had 2- to 3-fold higher in vitro activities than originally reported, correlating better with the associated phenotypes. CONCLUSIONS We provide the first evidence that partial CYP11A1 deficiency has to be considered as a differential diagnosis in clinically isolated adrenal insufficiency. Our assays demonstrate a tighter genotype-phenotype correlation in CYP11A1 deficiency than previous in vitro studies.

Steroid 17α-Hydroxylase Deficiency: Functional Characterization of Four Mutations (A174E, V178D, R440C, L465P) in the CYP17A1 Gene

CONTEXT Steroid 17alpha-hydroxylase (CYP17A1, alias P450c17) deficiency (17OHD) is a rare form of congenital adrenal hyperplasia. The CYP17A1 enzyme catalyzes two distinct reactions, 17alpha-hydroxylase and 17,20-lyase activities. OBJECTIVE The aim of the study was to analyze the structural and functional consequences of three novel (A174E, V178D, and L465P) and one previously reported (R440C) CYP17A1 mutation found in three patients clinically and biochemically presenting with 17OHD. PATIENTS AND METHODS Two patients suffering from 46,XY disordered sex development presented at ages 5.5 and 8.8 yr, respectively, with tall stature and hypertension. Mutation analysis revealed compound heterozygous CYP17A1 mutations (A174E/K388X; V178D/R440C). The third patient (46,XX) presented with primary amenorrhea and hypertension at age 15 yr. She was homozygous for the novel L465P mutation. Functional studies employing a yeast microsomal expression system compared wild-type and mutant CYP17A1 both with regard to 17alpha-hydroxylase and 17,20-lyase activity. Mutants were examined in a computational three-dimensional model of the CYP17A1 protein. RESULTS The activity assays showed that all three mutants retain only 0-7% of both 17alpha-hydroxylase and 17,20-lyase activity relative to CYP17A1 wild-type activity, corresponding to the in vivo situation. Enzyme kinetic studies proved the impairment of both reactions, respectively. Computer-based three-dimensional model analysis of CYP17A1 using CYP2B4 as template showed that three of the mutations had no direct effect on the active center, whereas one affects the heme coordination. CONCLUSION The functional studies revealed that the described missense mutations result in severe 17OHD. Our data are important to predict the phenotypic expressions and provide important information for patient management and genetic counseling.

Diagnosis of 21-hydroxylase deficiency by urinary metabolite ratios using gas chromatography-mass spectrometry analysis: Reference values for neonates and infants.

One major issue of newborn screening programs for 21-hydroxylase deficiency (21OHD) is the high rate of false-positive results, especially in preterm neonates. Urinary steroid metabolite analysis using gas chromatography-mass spectrometry (GC-MS) is suitable as a confirmatory diagnostic tool. The objective of this study was to analyze retrospectively diagnostic metabolite ratios in neonates and infants with and without 21OHD using GC-MS with emphasis on glucocorticoid metabolism, and to develop reference values for the steroid metabolite ratios for the diagnosis of 21OHD. We retrospectively analyzed urinary steroid hormone metabolites determined by GC-MS of 95 untreated neonates and infants with 21OHD (1-148 days), and 261 neonates and infants (100 preterms) without 21OHD (0-217 days). Metabolites of 17α-hydroxyprogesterone showed specificities below 98%, whereas the 21-deoxycortisol metabolite pregnanetriolone clearly separated 21OHD from non-21OHD subjects. The best diagnostic ratio for 21OHD was pregnanetriolone to 6α-hydroxy-tetrahydrocortisone. The lowest value of this ratio in the 21OHD group (0.47) was at least eight times higher than the highest values in the non-21OHD group (0.055). We have given appropriate reference values for steroid metabolite ratios in the largest 21OHD cohort so far described. Consideration of glucocorticoid metabolism, especially the use of typical neonatal 6α-hydroxylates metabolites, leads to improvement of diagnostic metabolite ratios.

The urinary steroidome of treated children with classic 21-hydroxylase deficiency.

Monitoring treatment of children with classic congenital adrenal hyperplasia (CAH) is difficult and biochemical targets are not well defined. We retrospectively analysed 576 daily urinary steroid hormone metabolite profiles determined by gas chromatography-mass spectrometry of 150 children aged 3.0-17.9 years with classic 21-hydroxylase deficiency (21-OHD) on hydrocortisone and fludrocortisone treatment. Daily urinary excretion of glucocorticoid-, 17α-hydroxyprogesterone (17-OHP)-, and androgen metabolites as well as growth and weight gain are presented. Children with classic CAH exhibited increased height velocity during prepubertal age, which was then followed by diminished growth velocity during pubertal age until final height was reached. Final height was clearly below the population mean. 11β-Hydroxyandrosterone was the dominant urinary adrenal-derived androgen metabolite in CAH children. Adrenarche is blunted in children with CAH under hydrocortisone treatment and androgen metabolites except 11β-hydroxyandrosterone were suppressed. Cortisol metabolite excretion reflected supraphysiological hydrocortisone treatment dosage, which resulted in higher body-mass-indices in children with CAH. Reference values of daily urinary steroid metabolite excretions of treated children with CAH allow the clinician to adequately classify the individual patient regarding the androgen-, 17-OHP-, and glucocorticoid status in the context of the underlying disorder. Additionally, urinary 21-OHD-specific reference ranges will be important for research studies in children with CAH.

Diagnosis of Glucocorticoid-Remediable Aldosteronism in Hypertensive Children

Objective: Glucocorticoid-remediable aldosteronism (GRA) is caused by the presence of a chimeric gene originating from an unequal cross-over between the CYP11B1 and CYP11B2 genes. Aldosterone suppression by dexamethasone and high 18-hydroxycortisol (18-OHF) levels have been used to differentiate GRA from the other forms of primary aldosteronism. Methods: A dexamethasone suppression test including serum 18-OHF determination and the measurement of urinary excretion of aldosterone, its metabolites and 18-OHF were performed in 3 children of a family with primary aldosteronism. Polymerase chain reactions were performed to identify the chimeric gene. Results: The chimeric gene was identified in 2 children, their mother and grandmother. The affected children had an aldosterone-to-plasma renin activity ratio >30, elevated serum 18-OHF concentration and increased urinary excretion of aldosterone, its metabolites, and 18-OHF. Post-dexamethasone concentrations of serum aldosterone and 18-OHF concentrations were suppressed. Conclusion: Although very rare, the possible diagnosis of GRA should be considered in all children or young adults with low-renin hypertension. Since genetic testing is more specific than biochemical testing, a definitive diagnosis can only be obtained by identification of the CYP11B1/CYP11B2 chimeric gene.

The balance of cortisol–cortisone interconversion is shifted towards cortisol in neonates with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

BACKGROUND Patients with congenital adrenal hyperplasia due to 21-hydroxylase deficiency (21OHD) have an impaired cortisol synthesis, but it is unknown whether the metabolism of glucocorticoids differs between neonates and infants with and without 21OHD. OBJECTIVE The objective of this study was to compare the glucocorticoid metabolism between neonates and infants with and without 21OHD. METHODS We analyzed 14 urinary glucocorticoid metabolites, 7 metabolites each of cortisol and cortisone, by gas chromatography-mass spectrometry of 89 untreated 21OHD neonates and infants and 161 neonates and infants without 21OHD. RESULTS Neonates with 21OHD exhibit elevated relative amounts of cortisol metabolites in total glucocorticoid metabolism and an increased ratio of cortisol to cortisone metabolites (p<0.0001). This reflects a shift toward cortisol in the relative balance of the interconversion between cortisol and cortisone. The ratio of cortisol to cortisone metabolites correlated significantly with low urinary glucocorticoid concentrations (p<0.03), with low 21-hydroxylase activity (p<0.001) and high urinary sodium and chloride concentrations (p<0.05) in neonates with 21OHD. CONCLUSIONS Our results demonstrate substantial changes in the relative cortisone to cortisol interconversion in neonates with 21OHD. The shift of glucocorticoid metabolism toward active cortisol in neonates with 21OHD seems to be related to the severity of 21OHD and adrenal dysfunction. Our data provide new insights into the regulation of glucocorticoid homeostasis in 21OHD.

Androgen excess is due to elevated 11-oxygenated androgens in treated children with congenital adrenal hyperplasia

Adrenal androgen excess is the hallmark of classic congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. Recently, 11-oxygenated C19 steroids, a class of highly active adrenal-derived androgens, have been described in patients with CAH. The aim of our study was to elucidate the significance of 11-oxygenated androgens in children with CAH. We retrospectively analysed 190 daily urinary excretion rates of glucocorticoid-, 17α-hydroxyprogesterone (17OHP)-, and androgen metabolites determined by gas chromatography-mass spectrometry of 99 children aged 3.0-10.9 years with classic CAH on hydrocortisone and fludrocortisone treatment. Daily urinary steroid metabolite excretions were transformed into z-scores using references of healthy children. Androgen metabolite z-scores were separately calculated for androsterone (AN), the major urinary metabolite of androstenedione (A4), testosterone and 5α-dihydrotestosterone, for urinary metabolites of dehydroepiandrosterone (DHEA), and for 11β-hydroxyandrosterone (11OHAN), the major urinary metabolite of adrenal-derived 11-oxygenated androgens. Multivariate regression analysis was applied to analyse the precursors of 11OHAN synthesis. 11OHAN, cortisol-, and 17OHP metabolite z-scores were elevated in treated children with CAH, whereas AN- and DHEA metabolite z-scores were normalized or suppressed. Multivariate regression analysis revealed that 11OHAN excretion was strongest associated with 21-deoxycortisol (β = 0.379; P =.0006), followed by A4 (β = 0.280; P = .0008)) and 17OHP (β = 0.243; P = .04) metabolite excretion. Androgen excess in treated children with CAH is solely due to elevated 11-oxygenated androgens that derive in addition to the known conversion from A4 also by direct conversion from 21-deoxycortisol. 11-Oxygenated androgens may represent better biomarkers of adrenal androgen status and treatment response than conventional androgens.