A novel mutation of the StAR gene with congenital adrenal hyperplasia and its association with heterochromia iridis: a case report.

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.

Carboxyl-terminal mutations in 3beta-hydroxysteroid dehydrogenase type II cause severe salt-wasting congenital adrenal hyperplasia.

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.

Epigenetic Repression of Androgen Receptor Transcription in Mutation-Negative Androgen Insensitivity Syndrome (AIS Type II).

Context: Inactivating mutations within the AR gene are present in only ~40% of individuals with clinically and hormonally diagnosed androgen insensitivity syndrome (AIS). Previous studies revealed the existence of an AR gene mutation-negative group of patients with AIS who have compromised androgen receptor (AR) function (AIS type II). Objective: To investigate whether AIS type II can be due to epigenetic repression of AR transcription. Design: Quantification of AR mRNA and AR proximal promoter CpG methylation levels in genital skin-derived fibroblasts (GFs) derived from patients with AIS type II and control individuals. Setting: University hospital endocrine research laboratory. Patients: GFs from control individuals (n = 11) and patients with AIS type II (n = 14). Main Outcome Measure(s): Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro. Results: Fifty-seven percent of individuals with AIS type II (n = 8) showed a reduced AR mRNA expression in their GFs. A significant inverse correlation was shown between AR mRNA abundance and methylation at two consecutive CpGs within the proximal AR promoter. Methylation of a 158-bp-long region containing these CpGs was sufficient to severely reduce reporter gene expression. This region was bound by the runt related transcription factor 1 (RUNX1). Ectopic expression of RUNX1 in HEK293T cells was able to inhibit reporter gene expression through this region. Conclusions: Aberrant CpGs methylation within the proximal AR promoter plays an important role in the control of AR gene expression and may result in AIS type II. We suggest that transcriptional modifiers, such as RUNX1, could play roles therein offering new perspectives for understanding androgen-mediated endocrine diseases.

The Aldosterone/Renin Ratio as a Diagnostic Tool for the Diagnosis of Primary Hypoaldosteronism in Newborns and Infants.

BACKGROUND/AIMS: Primary hypoaldosteronism is a rare inborn disorder with life-threatening symptoms in newborns and infants due to an aldosterone synthase defect. Diagnosis is often difficult as the plasma aldosterone concentration (PAC) can remain within the normal range and thus lead to misinterpretation and delayed initiation of life-saving therapy. We aimed to test the eligibility of the PAC/plasma renin concentration (PRC) ratio as a tool for the diagnosis of primary hypoaldosteronism in newborns and infants. Meth ods: Data of 9 patients aged 15 days to 12 months at the time of diagnosis were collected. The diagnosis of primary hypoaldosteronism was based on clinical and laboratory findings over a period of 12 years in 3 different centers in Switzerland. To enable a valid comparison, the values of PAC and PRC were correlated to reference methods. RESULTS: In 6 patients, the PAC/PRC ratio could be determined and showed constantly decreased values <1 (pmol/l)/(mU/l). In 2 patients, renin was noted as plasma renin activity (PRA). PAC/PRA ratios were also clearly decreased. The diagnosis was subsequently genetically confirmed in 8 patients. CONCLUSION: A PAC/PRC ratio <1 pmol/mU and a PAC/PRA ratio <28 (pmol/l)/(ng/ml × h) are reliable tools to identify primary hypoaldosteronism in newborns and infants and help to diagnose this life-threatening disease faster.

In vivo investigations of the effect of short- and long-term recombinant growth hormone treatment on DNA-methylation in humans.

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.

Relationships between 24-hour urinary free cortisol concentrations and metabolic syndrome in obese children.

CONTEXT: Clinical features of Metabolic Syndrome (MetS) and Cushing's Syndrome are similar, suggesting a pathogenetic role of hypothalamus-pituitary-adrenal axis in MetS. OBJECTIVE: The aim of the study was to determine whether MetS diagnosis and specific clusters of MetS components (waist circumference, dyslipidemia, hypertension, and impaired glucose metabolism) are associated with serum cortisol (SC) or 24-h urinary free cortisol (UFC) levels. DESIGN AND SETTING: We conducted cross-sectional analyses of data from our obesity cohort. We studied 264 obese children (age, 11.0 ± 2.8 years; male, 48%; BMI, 28.2 ± 5.4 kg/m(2)). We examined UFC, SC, homeostasis model assessment (HOMA), and features of MetS (waist circumference, blood pressure, fasting lipids, and glucose). RESULTS: Slightly increased UFC concentrations were measured in 30.7% of the children. Obese children with MetS had significantly (P = .003) higher UFC levels compared with obese children without MetS. Girls demonstrated significantly higher UFC concentrations compared with boys independent of pubertal stage. UFC and SC levels were significantly related to features of MetS, but the associations were stronger for UFC. In multivariate analyses adjusted for age, sex, and body mass index, none of the features of MetS but HOMA index was correlated with UFC, whereas SC demonstrated no significant association to any parameter of MetS or HOMA. CONCLUSIONS: Our findings support the hypothesis that changes in the hypothalamus-pituitary-adrenal axis are related to MetS in obesity. UFC seems to be a suitable marker for this relationship. Norm values for UFC adapted to obese children may help to avoid unnecessary dexamethasone suppression tests.

17α-hydroxylase deficiency diagnosed in early infancy caused by a novel mutation of the CYP17A1 gene.

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.

Implementation of a liquid chromatography tandem mass spectrometry assay for eight adrenal C-21 steroids and pediatric reference data.

BACKGROUND: Sensitive and accurate determination of steroids is essential for diagnosing congenital and acquired adrenal diseases. Since plasma concentrations change during childhood, age-specific reference ranges are the prerequisite for clinical interpretation. The objectives of this study were to develop a sensitive and reliable method for simultaneous detection and quantification of progesterone, 17-hydroxyprogesterone, deoxycorticosterone (DOC), 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol (F) and cortisone (E) by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and to establish age- and sex-specific reference ranges from birth to adulthood. METHODS: All eight steroids were measured simultaneously in 0.1 ml plasma by UPLC-MS/MS. Samples of 905 children were measured and grouped in five age groups. RESULTS: The assay was linear up to 70 ng/ml (700 ng/ml for F; r(2) > 0.992). The limit of detection ranged between 0.01 ng/ml for DOC and 0.07 ng/ml for E. Correlations with radioimmunoassays yielded a coefficient of determination between 0.82 and 0.99. Reference data are reported as a function of age and sex. CONCLUSIONS: The UPLC-MS/MS method presented here for the simultaneous detection of eight C-21 adrenal hormones together with the detailed reference ranges for children provides a valuable methodology for assessing adrenal steroids in clinical routine and research.

Steroid hormone profiles in prepubertal obese children before and after weight loss.

CONTEXT: Little information is available on the steroid hormone profiles in obese children and their changes after weight loss. OBJECTIVE: We compared liquid chromatography-tandem mass spectrometry of serum steroid hormone profiles between obese and normal-weight children and studied the differential effects of weight loss on these hormones. DESIGN: This study was a cross-sectional comparison between obese and normal-weight children and a longitudinal 1-year follow-up study during lifestyle intervention in obese children. SETTING: The setting of the study was primary care. PATIENTS: Forty obese prepubertal (mean age 8.5 ± 2.1 years, 48% female, mean body mass index 24.8 ± 3.5 kg/m(2)) and 40 normal-weight children matched for gender, age, and pubertal stage. INTERVENTION: The study consisted of an outpatient 1-year intervention program based on exercise, behavior, and nutrition therapy. MAIN OUTCOMES MEASURES: Progesterone, 17-hydroxyprogesterone, 11-deoxycorticosterone, corticosterone, aldosterone, 11-deoxycortisol, cortisol, cortisone, dehydroepiandrosterone sulfate (DHEAS), androstenedione, T, dihydrotestosterone, insulin resistance index of the homeostasis model assessment, and blood pressure were measured. RESULTS: Prepubertal obese children showed significantly increased androgens (DHEAS, androstenedione, T), mineralocorticoid precursor corticosterone, and glucocorticoids (11-deoxycortisol, cortisol, cortisone) compared with normal-weight children. In contrast to 20 obese children without weight loss, the 20 obese children with substantial weight loss demonstrated a significant decrease of cortisol, cortisone, and corticosterone. Androstenedione and T decreased but DHEAS remained elevated. Changes of the homeostasis model assessment correlated significantly positively with changes of cortisol (r = 0.38) and cortisone (r = 0.43) in partial regression analyses adjusted to changes of weight status. CONCLUSIONS: In obese prepubertal children, the increased androgens, mineralocorticoid precursors, and glucocorticoids were responsive to weight loss in contrast to DHEAS, suggesting that DHEAS does not seem to be regulated by changes in body mass index.

The novel mutation p.Trp147Arg of the steroidogenic acute regulatory protein causes classic lipoid congenital adrenal hyperplasia with adrenal insufficiency and 46,XY disorder of sex development.

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.

Five novel mutations in the SCNN1A gene causing autosomal recessive pseudohypoaldosteronism type 1.

BACKGROUND: Pseudohypoaldosteronism type 1 (PHA1) is a monogenic disease caused by mutations in the genes encoding the human mineralocorticoid receptor (MR) or the α (SCNN1A), ß (SCNN1B) or γ (SCNN1G) subunit of the epithelial Na(+) channel (ENaC). While autosomal dominant mutation of the MR cause renal PHA1, autosomal recessive mutations of the ENaC lead to systemic PHA1. In the latter, affected children suffer from neonatal onset of multi-organ salt loss and often exhibit cystic fibrosis-like pulmonary symptoms. OBJECTIVE: We searched for underlying mutations in seven unrelated children with systemic PHA1, all offsprings of healthy consanguineous parents. METHODS AND RESULTS: Amplification of the SCNN1A gene and sequencing of all 13 coding exons unraveled mutations in all of our patients. We found five novel homozygous mutations (c.587_588insC in two patients, c.1342_1343insTACA, c.742delG, c.189C>A, c.1361-2A>G) and one known mutation (c.1474C>T) leading to truncation of the αENaC protein. All parents were asymptomatic heterozygous carriers of the respective mutations, confirming the autosomal recessive mode of inheritance. Five out of seven patients exhibited pulmonary symptoms in the neonatal period. CONCLUSION: The α subunit is essential for ENaC function and mutations truncating the pore-forming part of the protein leading to systemic PHA1. Based on current knowledge, the pulmonary phenotype cannot be satisfactorily predicted.

Transcriptional response of peripheral blood mononuclear cells to recombinant human growth hormone in a routine four-days IGF-I generation test.

BACKGROUND: There are very few laboratory markers which reflect the biological sensitivity of children to recombinant human growth hormone (rhGH) treatment. Genome-wide transcriptional changes in peripheral blood mononuclear cells (PBMC) have been widely used as functional readout for different pharmacological stimuli. OBJECTIVE: To characterize transcriptional changes in PBMC induced by rhGH during a routine short-term IGF-I generation test (IGFGT) in children with growth disorders. MATERIALS AND METHODS: Blood was obtained for IGF-I determination and RNA-preparation from PBMC of 12 children before and after 4days treatment with 30µgrhGH/kg body weight/day s.c. Transcriptional changes were assessed by cDNA-microarrays in the first six children. Selected genes were validated in all 12 cases by RT-qPCR. RESULTS: Serum IGF-I rose in all patients except one (p<0.0001), confirming biological response to rhGH. Unsupervised microarray data analysis in the first six children revealed 313 transcripts with abundant transcriptional changes but considerable inter-individual variability of response patterns. Many patients showed a large cluster of up-regulated genes, including EGR1, EGR2, FOS and to a lesser extent STAT2 and 5b. Exemplarily, EGR1, EGR2 and FOS data were independently reproduced by RT-qPCR. Gene ontology analysis revealed that pathways involved in cell proliferation and immune functions were significantly over represented. CONCLUSION: The IGFGT is a suitable method for measuring reproducible and biologically conclusive transcriptional changes in PBMC. As our unsupervised data analysis strategy exposed a considerable inter-individual variability of response profiles a search for molecules of diagnostic and even prognostic value needs to be based on large long-term studies.