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Background: We report a novel mutation within the StAR gene, causing congenital adrenal hyperplasia, with the so far unreported association with heterochromia iridis., Case Presentation: In a now 15-year-old girl (born at 41 + 6 weeks of gestation) adrenal failure was diagnosed in the neonatal period based on the clinical picture with spontaneous hypoglycaemia, hyponatremia and an extremely elevated concentration of ACTH (3381 pmol/l; ref. level 1,1-10,1 pmol/l), elevated renin (836 ng/l; ref. level 5-308 ng/l), and a decreased concentration of aldosterone (410 pmol/l; ref. level 886-3540 pmol/l). In addition to hyperpigmented skin the patient exhibited sectorial heterochromia iridis. Sequence analysis of the steroidogenic acute regulatory protein (StAR) gene showed a novel homozygous mutation (c.652G > A (p.Ala218Thr), which was predicted in-silico to be possibly damaging. Under daily steroid substitution her electrolyte levels are balanced while she became obese. Puberty occurred spontaneously., Conclusion: A novel mutation in the StAR gene was identified in a patient with severe adrenal hypoplasia and sectorial heterochromia iridis. We discuss a causal relationship between these two rare phenotypes, i.e. whether very high levels of ACTH and alpha-MSH during early development might have disturbed early differentiation and distribution of uveal melanocytes. If confirmed in additional cases, discolorization of the iris might be considered as an additional phenotypical feature in the differential diagnosis of congenital adrenal insufficiency.

We present a boy with a genetically proven congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency. While massively elevated 17-hydroxyprogesterone (17-OHP) concentrations after birth led to the diagnosis, 17-OHP concentrations became immeasurable starting with the second year of life even though the dose of hydrocortisone was continuously decreased to ∼7 mg/m2/day. Furthermore, 17-OHP levels were immeasurable during the ACTH test and after withdrawing hydrocortisone medication. In contrast, ACTH levels increased after cessation of hydrocortisone treatment suggesting complete primary adrenal cortex failure. We discuss this case based on the differential diagnosis of complete adrenal cortex failure including other genetic causes in addition to CAH, prednisolone treatment, autoimmune adrenalitis, adrenoleukodystrophy, CMV infection, and adrenal hemorrhage infarction. The most likely disease in our boy is autoimmune adrenalitis, which is difficult to prove years after the onset of the disease. Treatment of CAH had masked the classical symptoms of complete adrenal cortex insufficiency leading to delayed diagnosis in this case., (© 2018 S. Karger AG, Basel.)

Background/aims: The high complexity of pediatric reference ranges across age, sex, and units impairs clinical application and comparability of steroid hormone data, e.g., in congenital adrenal hyperplasia (CAH). We developed a multiples-of-median (MoM) normalization tool to overcome this major drawback in pediatric endocrinology., Methods: Liquid chromatography tandem mass spectrometry data comprising 10 steroid hormones representing 905 controls (555 males, 350 females, 0 to > 16 years) from 2 previous datasets were MoM transformed across age and sex. Twenty-three genetically proven CAH patients were included (21-hydroxylase deficiency [21OHD], n = 19; 11β-hydroxylase deficiency [11OHD], n = 4). MoM cutoffs for single steroids predicting 21OHD and 11OHD were computed and validated through new, independent patients (21OHD, n = 8; adrenal cortical carcinoma, n = 6; obesity, n = 40)., Results: 21OHD and 11OHD patients showed disease-typical, easily recognizable MoM patterns independent of age, sex, and concentration units. Two single-steroid cutoffs indicated 21OHD: 3.87 MoM for 17-hydroxyprogesterone (100% sensitivity and 98.83% specificity) and 12.28 MoM for 21-deoxycortisol (94.74% sensitivity and 100% specificity). A cutoff of 13.18 MoM for 11-deoxycortisol indicated 11OHD (100% sensitivity and 100% specificity)., Conclusions: Age- and sex-independent MoMs are straightforward for a clinically relevant display of multi-steroid patterns. In addition, defined single-steroid MoMs can serve alone as predictors of 21OHD and 11OHD. Finally, MoM transformation offers substantial enhancement of routine and scientific steroid hormone data exchange due to improved comparability., (© 2018 S. Karger AG, Basel.)

Congenital adrenal hyperplasia (CAH), resulting from mutations in CYP11B1 , a gene encoding 11β-hydroxylase, represents a rare autosomal recessive Mendelian disorder of aberrant sex steroid production. Unlike CAH caused by 21-hydroxylase deficiency, the disease is far more common in the Middle East and North Africa, where consanguinity is common often resulting in identical mutations. Clinically, affected female newborns are profoundly virilized (Prader score of 4/5), and both genders display significantly advanced bone ages and are oftentimes hypertensive. We find that 11-deoxycortisol, not frequently measured, is the most robust biochemical marker for diagnosing 11β-hydroxylase deficiency. Finally, computational modeling of 25 missense mutations of CYP11B1 revealed that specific modifications in the heme-binding (R374W and R448C) or substrate-binding (W116C) site of 11β-hydroxylase, or alterations in its stability (L299P and G267S), may predict severe disease. Thus, we report clinical, genetic, hormonal, and structural effects of CYP11B1 gene mutations in the largest international cohort of 108 patients with steroid 11β-hydroxylase deficiency CAH.

Objective: Heterozygosity in 21-hydroxylase deficiency (21OHD) has been associated with hyperandrogenemic symptoms in children and adults. Moreover, the carrier status is mandatory for genetic counseling. We aimed at defining a hormonal parameter for carrier detection by mass spectrometry., Design: Eleven basal and ACTH-stimulated steroid hormones of heterozygous carriers of CYP21A2 mutations and control individuals were compared., Method: Hormones were determined in plasma samples by liquid chromatography tandem mass spectrometry (LC-MS/MS) in 58 carriers (35 males, 23 females, age range 6-78 years) and 44 random controls (25 males, 19 females, age range 8-58 years)., Results: Heterozygotes could be identified best applying the 17-hydroxyprogesterone+21-deoxycortisol/cortisol×1000 ((17OHP+21S)/F×1000) equation 30  min after ACTH injection. An optimal cut-off value of 8.4 provided 89% sensitivity and specificity. Considering this data and a published frequency of heterozygotes of 1/50 to 1/61, the positive predictive value (PPV) of this cut-off is 12%. Of note, the negative predictive value (NPV) excluding heterozygosity in a given patient is 99.8%., Conclusion: Considering only marginal biochemical effects anticipated from heterozygosity, the stimulated ((17OHP+21S)/F×1000) identifies and excludes heterozygotes remarkably well. Nevertheless, LC-MS/MS cannot replace genetic testing, since sensitivity and specificity did not reach 100%. However, due to the considerably high NPV of the optimal cut-off and to a specificity of even 100% applying a cut-off higher than 14.7, hormonal assessment of heterozygosity can be of significant aid in conditions with limited access to genetic testing, as in some health care systems. The ((17OHP+21S)/F×1000) equation can guide diagnostic considerations in the differential diagnosis of hyperandrogenism., (© 2015 European Society of Endocrinology.)

Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine disease. Steroid 11β-hydroxylase deficiency (11β-OHD) is the second most common form of CAH. The aim of the study was to study the functional consequences of three novel and one previously described CYP11B1 gene mutations (p.(Arg143Trp), p.(Ala306Val), p.(Glu310Lys) and p.(Arg332Gln)) detected in patients suffering from classical and non-classical 11β-OHD. Functional analyses were performed by using a HEK293 cell in vitro expression system comparing wild type (WT) with mutant 11β-hydroxylase activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein. Two mutations (p.(Ala306Val) and p.(Glu310Lys)) detected in patients with classical 11β-OHD showed a nearly complete loss of 11β-hydroxylase activity. The mutations p.(Arg143Trp) and p.(Arg332Gln) detected in patients with non-classical 11β-OHD showed a partial functional impairment with approximately 8% and 6% of WT activity, respectively. Functional mutation analysis allows the classification of novel CYP11B1 mutations as causes of classical and non-classical 11β-OHD. The detection of patients with non-classical phenotypes underscores the importance to screen patients with a phenotype comparable to non-classical 21-hydroxylase deficiency for mutations in the CYP11B1 gene in case of a negative analysis of the CYP21A2 gene. As CYP11B1 mutations are most often individual for a family, the in vitro analysis of novel mutations is essential for clinical and genetic counselling.

Background: Congenital adrenal hyperplasia (CAH) is one of the most common autosomal recessive inherited endocrine diseases. Steroid 11β-hydroxylase (P450c11) deficiency (11OHD) is the second most common form of CAH., Aim: The aim of the study was to study the functional consequences of three novel CYP11B1 gene mutations (p.His125Thrfs*8, p.Leu463_Leu464dup and p.Ser150Leu) detected in patients suffering from 11OHD and to correlate this data with the clinical phenotype., Methods: Functional analyses were done by using a HEK293 cell in vitro expression system comparing WT with mutant P450c11 activity. Mutant proteins were examined in silico to study their effect on the three-dimensional structure of the protein., Results: Two mutations (p.His125Thrfs*8 and p.Leu463_Leu464dup) detected in patients with classic 11OHD showed a complete loss of P450c11 activity. The mutation (p.Ser150Leu) detected in a patient with non-classic 11OHD showed partial functional impairment with 19% of WT activity., Conclusion: Functional mutation analysis enables the correlation of novel CYP11B1 mutations to the classic and non-classic 11OHD phenotype respectively. Mutations causing a non-classic phenotype show typically partial impairment due to reduced maximum reaction velocity comparable with non-classic mutations in 21-hydroxylase deficiency. The increasing number of mutations associated with non-classic 11OHD illustrate that this disease should be considered as diagnosis in patients with otherwise unexplained hyperandrogenism.

Background: Mutations of the CYP17A1 gene cause 17α-hydroxylase deficiency (17OHD) resulting in 46,XY disorder of sex development, hypertension, hypokalemia and absent pubertal development. It is a rare, autosomal recessive form of congenital adrenal hyperplasia (CAH)., Patient: We report on a neonate with prenatally determined 46,XY karyotype. At 20 weeks of gestation, lack of development of male external genitalia was noticed. A phenotypically female child was born at 41 weeks of gestation., Results: Postnatal ultrasound revealed testes in both labia majora, an absence of uterus and normal adrenal glands. Steroid hormone analysis in serum revealed low basal levels of cortisol, testosterone and androstenedione in the presence of massively elevated corticosterone at the age of 2 weeks. The urinary steroid profile from spot urine showed excessive excretion of 17-desoxysteroids, decreased glucocorticoid metabolites and absent C19 steroids, thus proving 17OHD. Molecular analysis identified a novel mutation of the CYP17A1 gene: c.896T>A (p.I299N) in exon 5. Substitution with hydrocortisone was started. The child is raised as a girl and is developing well so far., Conclusion: Herein, we report the unusually early diagnosis of a newborn with the rare CAH form of 17OHD allowing an early start of treatment., (© 2014 S. Karger AG, Basel.)

Background: The steroidogenic acute regulatory protein (StAR) is essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause lipoid congenital adrenal hyperplasia., Objective and Methods: To identify causative mutations in a patient presenting with adrenal failure during early infancy. The objective was to study the functional and structural consequences of the novel StAR mutation p.Trp147Arg in a Turkish patient detected in compound heterozygosity with the p.Glu169Lys mutation., Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.Trp147Arg mutant. The previously described p.Glu169Lys mutant led to significantly lower cholesterol conversion than wild-type StAR protein. As derived from three-dimensional protein modeling, the residue W147 is stabilizing the C-terminal helix in a closed conformation hereby acting as gatekeeper of the ligand cavity of StAR., Conclusions: The novel mutation p.Trp147Arg causes primary adrenal insufficiency and complete sex reversal in the 46,XY patient. Clinical disease, in vitro studies and three-dimensional protein modeling of the mutation p.Trp147Arg underscore the relevance of this highly conserved residue for StAR protein function., (Copyright © 2013 S. Karger AG, Basel.)

Steroid 11β hydroxylase deficiency (11β-OHD) (OMIM # 202010) is the second most common form of congenital adrenal hyperplasia (CAH), accounting for 5-8% of all cases. It is an autosomal recessive enzyme defect impairing the biosynthesis of cortisol. The CYP11B1 gene encoding this enzyme is located on chromosome 8q22, approximately 40kb from the highly homologous CYP11B2 gene encoding for the aldosterone synthase. Virilization and hypertension are the main clinical characteristics of this disease. In Tunisia, the incidence of 11β-OHD appears higher due to a high rate of consanguinity (17.5% of congenital adrenal hyperplasia). The identical presentation of genital ambiguity (females) and pseudo-precocious puberty (males) can lead to misdiagnosis with 21 hydroxylase deficiency. The clinical hallmark of 11β hydroxylase deficiency is variable, and biochemical identification of elevated precursor metabolites is not usually available. In order to clarify the underlying mechanism causing 11β-OHD, we performed the molecular genetic analysis of the CYP11B1 gene in a female patient diagnosed as classical 11β-OHD. The nucleotide sequence of the patient's CYP11B1 revealed two novel mutations in exon 4: a missense mutation that converts codon AGT (serine) to ATT (isoleucine) (c.650G>T; p.S217I) combined with an insertion of a thymine at the c.652-653 position (c.652_653insT). This insertion leads to a reading frame shift, multiple incorrect codons, and a premature stop in codon 258, that drastically affects normal protein function leading to a severe phenotype with ambiguous genitalia of congenital adrenal hyperplasia due to 11β hydroxylase deficiency., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Background/aim: X-linked adrenal hypoplasia congenita (AHC) is a rare disorder characterized by primary adrenal insufficiency and hypogonadic hypogonadism. It is caused by deletions or point mutations of the NR0B1 gene, on Xp21. AHC can be associated with glycerol kinase deficiency, Duchenne muscular dystrophy and mental retardation (MR), as part of a contiguous gene deletion syndrome. A synthetic probe set for multiplex ligation-dependent probe amplification analysis was developed to confirm and characterize NR0B1 deletions in patients with AHC and to correlate their genotypes with their divergent phenotypes., Results: In 2 patients, isolated AHC was confirmed, while a patient at risk for metabolic crisis was revealed as the deletion extends to the GK gene. A deletion extending to IL1RAPL1 was confirmed in both patients showing MR. Thus, a good genotype-phenotype correlation was confirmed., Conclusions: Multiplex ligation-dependent probe amplification analysis is a valuable tool to detect NR0B1 and contiguous gene deletions in patients with AHC. It is especially helpful for IL1RAPL1 deletion detection as no clinical markers for MR are available. Furthermore, multiplex ligation-dependent probe amplification has the advantage to identify female carriers that, depending on the deletion extension, have a high risk of giving birth to children with MR, AHC, glycerol kinase deficiency and Duchenne muscular dystrophy., (Copyright © 2012 S. Karger AG, Basel.)

Context: In 21-hydroxylase (CYP21A2) deficiency (21OHD), the level of in vitro enzymatic function allows for classification of mutation groups (null, A, B, C) and prediction of disease severity. However, genital virilization in affected females correlates only weakly with CYP21A2 mutation groups, suggesting the influence of genetic modifiers., Objective: The objective of the study was to investigate the influence of the polymorphic CAG and GGn repeats of the androgen receptor (AR) gene on the degree of genital virilization in 21OHD females., Design and Patients: Design of the study was the determination of CYP21A2 genotype, degree of genital virilization (Prader stage), and X-weighted biallelic mean of AR CAG and GGn repeat length in 205 females with 21OHD., Outcome Measurements: Correlation of AR CAG and GGn repeat lengths with Prader stages using nested stepwise logistic regression analysis was measured., Results: CYP21A2 mutation groups null and A showed significantly higher levels of genital virilization than groups B and C (P < 0.01). However, Prader stages varied considerably within mutation groups: null, Prader I-V (median IV); A, Prader I-V (median IV); B, Prader I-V (median III); C, 0-III (median I). Mean GGn repeat length of patients was not significantly associated with Prader stages, classified as low (0-I), intermediate (II-III), or severe (IV-V) (odds ratio per repeat: 0.98, 95% confidence interval 0.71-1.35). In contrast, patients with Prader 0-I showed a trend toward longer CAG repeats without reaching statistical significance (P = 0.07, odds ratio per repeat: 0.82, 95% confidence interval 0.65-1.02)., Conclusion: Neither CAG nor GGn repeat lengths are statistically significant modifiers of genital virilization in females with 21OHD.

Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia., Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient)., Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the alpha4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein., Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient's phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

Background: Steroid 11-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Herein, we describe two novel CYP11B1 mutations (g659&#95;660dupTG, p.M92X; g.4817G>A, p.R453Q) found in a patient diagnosed with classic 11OHD, after presenting with borderline elevated 17-hydroxyprogesterone concentrations in CAH newborn screening., Methods: A novel CYP11B1 variant (p.R453Q) identified in a patient with classic 11OHD was characterized employing a COS7 cell assay and a computational three-dimensional CYP11B1 model., Results: The in vitro expression analysis revealed an almost complete loss of function of p.R453Q. This finding was consistent with the clinical presentation of classic 11OHD as the patient was compound heterozygous for p.R453Q and a nonsense mutation (p.M92X) on the other allele. Inserting the p.R453Q mutation into our CYP11B1 model provided two potential explanations for the almost complete loss of enzyme activity. Firstly, the heme coordination is most likely disturbed. A second possibility could be an altered interaction with the redox partner adrenodoxin., Conclusion: Results indicate that both novel mutations are disease-causing mutations. Proving the pathogenic effect of a missense sequence variation is of particular importance for clinical genetic counseling as this provides essential information on the prediction of recurrence risk and disease severity., (Copyright 2009 S. Karger AG, Basel.)

Background: Congenital adrenal hyperplasia is caused by insufficient adrenal steroid biosynthesis due to impaired steroidogenic enzymes. The majority of patients suffer from deficiency of 21-hydroxylase (CYP21) coded by the CYP21A2 gene., Objective: Our objective was to study the functional and structural consequences of the novel CYP21A2 missense mutation c.364A > C (K121Q) detected in a female patient with nonclassical 21-hydroxylase deficiency. The patient was compound heterozygous for the novel K121Q mutation and the mild P453S mutation., Results: In vitro expression analysis of the mutant K121Q enzyme in transiently transfected COS-7 cells revealed reduced CYP21 activity of 14.0 +/- 5% for the conversion of 17-hydroxyprogesterone and 19.5 +/- 4% for the conversion of progesterone. K121 is located on helix C in the CYP21 protein, which is part of the heme coordinating system. In addition, helix C is involved in the interaction with the electron-providing enzyme P450 oxidoreductase. Protein modeling revealed that the substitution of glutamine for the basic amino acid lysine introduces an electrostatic change on the surface of CYP21 and may additionally change heme coordination. We hypothesize that the electron flux between P450 oxidoreductase and CYP21 is impaired and, moreover, that substrate affinity is altered due to heme dislocation with K121Q., Conclusion: Both the interaction of P450 oxidoreductase and CYP21 as well as heme coordination are likely to be disturbed due to the K121Q mutation. Our data exemplify how the combination of in vitro expression and structural protein analysis provide novel insights into molecular mechanisms of reduced CYP21 activity, eventually explaining the patient's phenotype.

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

Context: Congenital adrenal hyperplasia is a group of autosomal recessive inherited disorders of steroidogenesis. The deficiency of steroid 11-hydroxylase (CYP11B1) resulting from mutations in the CYP11B1 gene is the second most frequent cause., Objective: We studied the functional and structural consequences of two CYP11B1 missense mutations, which were detected in a 1.8-yr-old boy with acne and precocious pseudopuberty, to prove their clinical relevance and study their impact on CYP11B1 function., Results: The in vitro expression studies in COS-7 cells revealed an almost complete absence of CYP11B1 activity for the P94L mutant to 0.05% for the conversion of 11-deoxycortisol to cortisol. The A368D mutant severely reduced the CYP11B1 enzymatic activity to 1.17%. Intracellular localization studies by immunofluorescence revealed that the mutants were correctly localized. Introducing these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the effects measured in vitro. We hypothesize that the A368D mutation interferes with structures important for substrate specificity and heme iron binding, thus explaining its major functional impact. However, according to structural analysis, we would expect only a minor effect of the P94L mutant on 11-hydroxylase activity, which contrasts with the observed major effect of this mutation both in vitro and in vivo., Conclusion: Analyzing the in vitro enzyme function is a complementary procedure to genotyping and a valuable tool for understanding the clinical phenotype of 11-hydroxylase deficiency. This is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides valuable insights in cytochrome P450 structural-functional relationships, although one must be aware of the limitations of in silico-based methods.