A subset of patients with monogenic disorders lacks disease causing mutations in the protein coding region of the corresponding gene. Here we describe a recurrent germline mutation found in two ...unrelated patients with complete androgen insensitivity syndrome (CAIS) generating an upstream open reading frame (uORF) in the 5' untranslated region (5'-UTR) of the androgen receptor (AR) gene. We show in patient derived primary genital skin fibroblasts as well as in cell-based reporter assays that this mutation severely impacts AR function by reducing AR protein levels without affecting AR mRNA levels. Importantly, the newly generated uORF translates into a polypeptide and the expression level of this polypeptide inversely correlates with protein translation from the primary ORF of the AR thereby providing a model for AR-5'UTR mediated translational repression. Our findings not only add a hitherto unrecognized genetic cause to complete androgen insensitivity but also underline the importance of 5'UTR mutations affecting uORFs for the pathogenesis of monogenic disorders in general.
Abstract
Context
Low birthweight (bw) and unfavorable intrauterine conditions have been associated with metabolic sequelae in later life, but little is known about their impact on glucocorticoid ...metabolism.
Objective
We studied monozygotic twins with intratwin bw differences to analyze the long-term impact of bw on glucocorticoid metabolism.
Methods
46 monozygotic twin pairs with bw differences of <1 SDS (concordant; n = 29) and ≥1 SDS (discordant; n = 17) were recruited. At 6.9 years (mean age), saliva samples were collected (at 7 hours, 13 hours, 18 hours and 21 hour) and analyzed with liquid chromatography–tandem mass spectrometry (LC-MS/MS).
Results
We found significant or highly significant intratwin correlations in all twin pairs at 3 of 4 (cortisol), and 4 of 4 (cortisone) time points. Graphic evaluation of the diurnal cortisol patterns for each twin pair showed a distinct alignment in all groups. Analyses of the change of intratwin differences over the day by mixed linear modeling showed no intratwin differences in diurnal patterns. Regression analyses of intratwin differences at 7:00 hours showed a significant influence of catch-up growth, indicating lower cortisol concentrations in smaller twins with more catch-up growth (adj. R2 = 0.159, P = .014, ß = −3.71, F(1,42) = 9.15, f2 = 0.19).
Conclusion
In monozygotic twins with intratwin bw differences, intratwin catch-up growth showed a moderate influence on intratwin differences in morning cortisol concentrations. We observed no differences regarding diurnal patterns. In contrast, in all groups, we found significant intratwin correlations for cortisol and cortisone over the day and a pronounced graphic alignment of cortisol diurnal patterns. We therefore suggest a predominant significance of the genetic background compared with bw differences on cortisol metabolism.
Low birthweight may have adverse sequelae in later life. Therefore, we analyzed behavioral difficulties and salivary glucocorticoid profiles in monozygotic twins with intra-twin birthweight ...differences due to twin-to-twin transfusion syndrome (TTTS).
46 monozygotic TTTS twin pairs with birthweight differences of <1SDS (concordant; n=29) and ≥1SDS (discordant; n=17) were recruited at a mean age of 6.9 years for a prospective longitudinal cohort study. For glucocorticoid analysis, saliva samples were collected (at 7 h, 13 h, 18 h and 21 h) and analyzed with liquid chromatography-tandem mass spectrometry. Parents completed the Strengths and Difficulties Questionnaire.
From the parents’ perspective, the formerly smaller twins had statistically higher scores regarding hyperactivity (mean 4.63 vs 3.48, p=0.003) and emotional problems (mean 2.67 vs 2.02, p=0.042). Less catch-up growth (Δintra-twin height SDS 4 years of age - Δintra-twin birth length SDS) of the smaller twins was associated with higher scores for hyperactivity (Adj. R²=0.261, p<0.001, β=-1.88, F(1.44)=16.86, n=46, f²=0.35), while smaller birthweight (Adj. R²=0.135, p=0.007, β=-0,87, F(1.44)=8.03, n=46, f²=0.16) and birth length (Adj. R²=0.085, p=0.028, β=-0,78, F(1.44)=5.19, n=46, f²=0.09) were associated with higher scores for peer problems. Greater Δintra-twin for cortisol (7 h: rho=0.337, p=0.029; cumulative: rho=0.458; p=0.024) and cortisone (7 h: rho=0.329, p=0.029; 13 h: rho=0.436, p=0.005) correlated with a greater Δintra-twin for conduct problems. In the discordant group, circa 1 SDS in head circumference persisted from birth (mean SDS: smaller twin −1.18, larger twin −0.08, p<0.001) to present (mean SDS: smaller twin −1.16, larger twin −0.14, p<0.001).
Higher cortisol and cortisone concentrations in smaller twins were associated with higher scores for conduct problems. Lower birthweight and absent catch-up growth affected the parents’ perspective on the smaller twins’ behavior. They saw those children as more hyperactive, with more peer problems and emotional problems. Thus, it seems important to introduce regular check-ups where behavioral difficulties can be assessed, and assistance and advice can be given to the families. Due to the persisting smaller head circumference in the smaller discordant twins, this should be measured regularly.
•Correlation between greater intra-twin difference for conduct problems and cortisol•Parents reported more emotional problems and hyperactivity in formerly smaller twin•Absent catch-up growth affected perception of parents on children’s behavior•No head-circumference catch-up in smaller twins with greater birthweight difference•Parents and smaller twins differ in perception of health-related quality of life
Reliable estradiol (E2) reference intervals (RIs) are crucial in pediatric endocrinology.
This study aims to develop a sensitive ultra-performance liquid chromatographic tandem mass spectrometry ...(UPLC-MS/MS) method for E2 in serum, to establish graphically represented RI percentiles and annual RIs for both sexes, and to perform a systematic literature comparison.
First, a UPLC-MS/MS method for E2 was developed. Second, graphically represented RI percentiles and annual RIs covering 0-18 years were computed (cohort of healthy children 1181 girls and 543 boys). Subsequently, RIs were compared with published data by systematic searches.
Lower limit of quantification was 11 pmol/L, indicating high sensitivity. Estradiol first peaked during mini-puberty in both sexes (girls up to 192 pmol/L; boys up to 225 pmol/L). As could be expected, girls showed higher pubertal E2 (up to 638 pmol/L). However, boys' RIs (up to 259 pmol/L) overlapped considerably. We found 4 studies in the literature that also used LC-MS/MS to determine E2 and published RIs for the complete pediatric age range. Reference intervals varied considerably. Pre-pubertal and pubertal phases were present in all studies. Higher E2 during the time of mini-puberty in both sexes was documented in 3 studies including ours.
Variability of RIs for E2 between studies illustrates the importance of laboratory-specific RIs despite using a LC-MS/MS reference method. In boys, the striking E2 peak during mini-puberty as well as high pubertal E2 without phenotypic estrogenization in regular male puberty indicates that the role of E2 in children and, especially in boys, requires better functional understanding.
Abstract
The 2006 Chicago consensus statement of management of disorders/difference of sex development (DSD) has achieved advantages in clinical care and diagnosis for patients and families affect by ...DSD. This article provides a brief overview of contexts of care for physicians, and points out specific challenges in clinical practice that have arisen from the transformations of the sex/gender system in recent years. We focus on the impact of diagnosis and laboratory measurements. Both laboratory measurements and hormonal therapies still depend on the binary system. One problem is the lack of reference intervals for the different forms of DSD, which means that diversity is often neglected. In the following, we will give a brief insight into this complex topic.
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).
To investigate whether AIS type II can be due to epigenetic repression of AR transcription.
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.
University hospital endocrine research laboratory.
GFs from control individuals (n = 11) and patients with AIS type II (n = 14).
Measurement of AR mRNA and AR promoter CpG methylation as well as activity of AR proximal promoter in vitro.
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.
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.
CYP17A1 is a cytochrome P450 enzyme with 17-alpha-hydroxylase and C17,20-lyase activities. CYP17A1 genetic variants are associated with coronary artery disease, myocardial infarction and visceral and ...subcutaneous fat distribution; however, the underlying pathological mechanisms remain unknown. We aimed to investigate the function of CYP17A1 and its impact on atherosclerosis in mice. At 4-6 months, CYP17A1-deficient mice were viable, with a KO:Het:WT ratio approximating the expected Mendelian ratio of 1:2:1. All Cyp17a1 knockout (KO) mice were phenotypically female; however, 58% were Y chromosome-positive, resembling the phenotype of human CYP17A1 deficiency, leading to 46,XY differences/disorders of sex development (DSD). Both male and female homozygous KO mice were infertile, due to abnormal genital organs. Plasma steroid analyses revealed a complete lack of testosterone in XY-KO mice and marked accumulation of progesterone in XX-KO mice. Elevated corticosterone levels were observed in both XY and XX KO mice. In addition, Cyp17a1 heterozygous mice were also backcrossed onto an Apoe KO atherogenic background and fed a western-type diet (WTD) to study the effects of CYP17A1 on atherosclerosis. Cyp17a1 x Apoe double KO XY mice developed more atherosclerotic lesions than Apoe KO male controls, regardless of diet (standard or WTD). Increased atherosclerosis in CYP17A1 XY KO mice lacking testosterone was associated with altered lipid profiles. In mice, CYP17A1 deficiency interferes with sex differentiation. Our data also demonstrate its key role in lipidomic profile, and as a risk factor in the pathogenesis of atherosclerosis.
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.
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.
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.
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.
Purpose
To study differences in metabolic outcomes between testosterone and estradiol replacement in probands with complete androgen insensitivity syndrome (CAIS).
Methods
In this multicentre, ...double-blind, randomized crossover trial, 26 women with CAIS were included of whom 17 completed the study. After a two-months run in phase with estradiol, probands either received transdermal estradiol followed by crossover to transdermal testosterone or vice versa. After six months, differences in lipids, fasting glucose, insulin, hematocrit, liver parameters and blood pressure between the treatment phases were investigated.
Results
Linear mixed models adjusted for period and sequence did not reveal major group differences according to treatment for the investigated outcomes. In each treatment group, there were however significant uniform changes in BMI and cholesterol. BMI increased significantly, following six months of estradiol ( + 2.7%;
p
= 0.036) as well as testosterone treatment ( + 2.8%;
p
= 0.036). There was also a significant increase in total ( + 10.4%;
p
= 0.001) and LDL-cholesterol ( + 29.2%;
p
= 0.049) and a decrease in HDL-cholesterol (−15.8%;
p
< 0.001) following six months of estradiol as well as six months of testosterone treatment (total cholesterol: + 14.6%;
p
= 0.008; LDL-cholesterol: + 39.1%;
p
= 0.005, HDL-cholesterol: −15.8%;
p
= 0.004). Other parameters remained unchanged.
Conclusion
Transdermal estradiol as well as testosterone treatment in women with CAIS results in worsening in lipid profiles. Given the relatively small sample size, subtle group differences in other metabolic parameters may have remained undetected.
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/m2). 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.
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