Stefan Riedl

Disorders of sex development: Insights from targeted gene sequencing of a large international patient cohort

BackgroundDisorders of sex development (DSD) are congenital conditions in which chromosomal, gonadal, or phenotypic sex is atypical. Clinical management of DSD is often difficult and currently only 13% of patients receive an accurate clinical genetic diagnosis. To address this we have developed a massively parallel sequencing targeted DSD gene panel which allows us to sequence all 64 known diagnostic DSD genes and candidate genes simultaneously.ResultsWe analyzed DNA from the largest reported international cohort of patients with DSD (278 patients with 46,XY DSD and 48 with 46,XX DSD). Our targeted gene panel compares favorably with other sequencing platforms. We found a total of 28 diagnostic genes that are implicated in DSD, highlighting the genetic spectrum of this disorder. Sequencing revealed 93 previously unreported DSD gene variants. Overall, we identified a likely genetic diagnosis in 43% of patients with 46,XY DSD. In patients with 46,XY disorders of androgen synthesis and action the genetic diagnosis rate reached 60%. Surprisingly, little difference in diagnostic rate was observed between singletons and trios. In many cases our findings are informative as to the likely cause of the DSD, which will facilitate clinical management.ConclusionsOur massively parallel sequencing targeted DSD gene panel represents an economical means of improving the genetic diagnostic capability for patients affected by DSD. Implementation of this panel in a large cohort of patients has expanded our understanding of the underlying genetic etiology of DSD. The inclusion of research candidate genes also provides an invaluable resource for future identification of novel genes.

Ghrelin affects the hypothalamus-pituitary-thyroid axis in humans by increasing free thyroxine and decreasing TSH in plasma

OBJECTIVE Ghrelin promotes a positive energy balance, e.g. by increasing food intake. Stimulation of the activity of the hypothalamus-pituitary-thyroid (HPT) axis promotes a negative energy balance, e.g. by increasing energy expenditure. We therefore hypothesized that ghrelin suppresses the HPT axis in humans, counteracting its energy-saving effect. DESIGN AND METHODS In this single-blind, randomized, cross-over study, we determined secretion patterns of free triiodothyronine (fT(3)), free thyroxine (fT(4)), TSH, and thyroid-binding globulin (TBG) between 2000 and 0700 h in 20 healthy adults (10 males and 10 females, 25.3+/-2.7 years) receiving 50 microg ghrelin or placebo at 2200, 2300, 0000, and 0100 h. RESULTS FT(4) plasma levels were significantly higher after ghrelin administration than after placebo administration from 0000 h until 0620 h except for the time points at 0100, 0520, and 0600 h. TSH plasma levels were significantly lower from 0200 until the end of the study at 0700 h except for the time points at 0540, 0600, and 0620 h. The relative increase of fT(4) (area under the curve (AUC) 0130-0700 h (ng/dl x min): placebo: 1.31+/-0.03; ghrelin: 1.39+/-0.03; P=0.001) was much weaker than the relative decrease of TSH (AUC 0130-0700 h (mIU/ml x min): placebo: 1.74+/-0.12; ghrelin: 1.32+/-0.12; P=0.007). FT(3) and TBG were not affected. CONCLUSIONS This is the first study to report that ghrelin affects the HPT axis in humans. The early fT(4) increase was possibly induced by direct ghrelin action on the thyroid where ghrelin receptors have been identified. The TSH decrease might have been caused by ghrelin-mediated inhibition at hypothalamic level by feedback inhibition through fT(4), or both.

Stool Polymerase Chain Reaction for Helicobacter pylori Detection and Clarithromycin Susceptibility Testing in Children

BACKGROUND & AIMS This study was undertaken in a pediatric gastroenterology clinic to retrospectively evaluate a real-time polymerase chain reaction (PCR) for the detection and clarithromycin susceptibility testing of Helicobacter pylori using stool specimens. METHODS All consecutive children who underwent a gastroscopy between March 2006 and February 2009 and also having been examined by stool PCR were enrolled. Rapid urease test, histology, and culture were the reference methods for the detection of H pylori and E-test for susceptibility testing, respectively. RESULTS A total of 143 children (mean age, 10.8 y; range, 2.8-17.9; males:females, 1:1.5) were evaluable. Sensitivity, specificity, and test accuracy for the detection of H pylori were 83.8%, 98.4%, and 90.2%, respectively. Sensitivity, specificity, and accuracy for the detection of clarithromycin resistance were 89.2%, 100%, and 94.0%, respectively. CONCLUSIONS Stool PCR was a reliable and useful noninvasive tool for detection and clarithromycin susceptibility testing of H pylori in a pediatric population with a high prevalence of clarithromycin-resistant strains.

Auxological, Ophthalmological, Neurological and MRI Findings in 25 Austrian Patients with Septo-Optic Dysplasia (SOD)

Septo-optic dysplasia (SOD) comprises ophthalmological, endocrinological and neurological disorders resulting from varying degrees of midline malformation of the forebrain like visual impairment by optic nerve hypoplasia, endocrine deficits due to hypothalamic and/or pituitary anomalies, and psychomental retardation by associated cortical malformation. MRI shows aplasia/hypoplasia of the septum pellucidum and corpus callosum as a radiological hallmark. For etiology, genetic defects (Hesx1/HESX1 gene) as well as vascular disruption during embryonic brain development are discussed. Aim: To perform detailed analysis of morphological findings and clinical symptoms and to improve care of SOD patients by interdisciplinary management. Patients: We investigated 25 patients with a mean age of 5.1 years at diagnosis. Results: Pituitary insufficiency was present in 11/25 patients, multiple deficits in 6 of them. Bilateral optic nerve hypoplasia was found in 70% of patients, unilateral in 20%. Mild or moderate neurological disorders were observed in the majority of patients (14/20), EEG was usually normal (12/19). Analysis of MRI films revealed very heterogeneous morphological anomalies, ranging from isolated agenesis of the septum pellucidum to multiple malformations, involving the cortex. Malrotation of the hippocampal structures was a common finding. Conclusion: We conclude that only interdisciplinary management of SOD patients can ameliorate the exact diagnosis and outcome, depending on early visual or developmental support as well as early diagnosis and substitution of potentially life-threatening endocrine deficits.

Long-term treatment in children with hypopituitarism: pubertal development and final height.

Auxological data, pubertal development and final height were analyzed in 25 patients with growth hormone (GH) deficiency who were treated only with pituitary GH in 3 doses/week. 15 patients had a spontaneous onset of puberty and in 10 puberty was induced. The duration of therapy was 7.5 years, the dosage was about 11 U/m2/week and was not changed during puberty. 80% of the patients with induced puberty, but only 33% of the children with spontaneous puberty reached final heights within the 2 SD range. Final height was correlated with height at the start of treatment only in patients with spontaneous puberty, which shows the necessity of early treatment initiation in this group. The lack of this correlation in patients with induced puberty might be attributed to gonadotropin deficiency. Height at the onset of puberty was related to final height only in the group with gonadotropin deficiency. A prediction model for calculation of the first year height velocity which was derived from present treatment procedures showed a too favorable prediction for our patients. The reasons for the insufficient final results may be due to impure GH preparations, suboptimal dosage, low frequency of injections and late onset of therapy.

Death in two female Prader-Willi syndrome patients during the early phase of growth hormone treatment

UNLABELLED Reports on sudden death in Prader-Willi syndrome (PWS) patients after the start of growth hormone (GH) treatment have been published recently. We observed a 4.7-y-old girl who showed a continuous increase in pulmonary artery pressure and died of cardiorespiratory failure 7 wk after GH therapy had been initiated, and a 9.3-y-old girl with additional trisomy 21 who died during a minor respiratory infection 6 mo after GH had been started. Both patients were overweight (weight for height 127% and 224%, respectively). GH-induced fluid retention may have occurred in the younger girl. In contrast to the reported cases, our PWS patients were female. CONCLUSION Our cases illustrate the difficulty of differentiation between possible GH side effects and the natural course of disease, in particular with respect to obesity-related comorbidity and mortality.

Posterior pituitary ectopy in children with idiopathic growth hormone deficiency.

AIMS To evaluate the underlying pathogenesis in children with pituitary hormone deficiency by means of high resolution MRI of the brain. PATIENTS/METHODS Thirty-seven children with short stature and isolated GH deficiency (IGHD, n = 17) or multiple pituitary hormone deficiency (MPHD, n = 20) were subjected to an MRI of the brain at the age of 1.0-17.3 years. The anatomic condition of the hypothalamo-pituitary area was analyzed and the height of the pituitary gland was measured and compared to the data of age-matched healthy subjects. RESULTS Seventy percent of the patients had a characteristic anomaly: the adenohypophysis was hypoplastic, the infundibulum was absent and the posterior pituitary lobe was ectopic at the bottom of the median eminence. The height of the anterior pituitary was significantly reduced in these patients (1.9 +/- 0.1 mm; mean +/- SD) when compared to age-matched healthy controls (4.1 +/- 0.8 mm, p<0.001) or hypopituitary patients with a normal MRI (4.3 +/- 0.8 mm). MPHD was found in 62% of patients with the pituitary anomaly whereas only 27% of children with a normal MRI had MPHD (p<0.05). CONCLUSIONS The pathogenesis of the pituitary anomaly is unknown; a disorder during embryonal development or perinatal events have been discussed as causal factors. MRI should have a prominent position in the work-up of hypopituitary children. When an anatomical malformation is visualized by MRI, the diagnostic terminology should be adapted accordingly.

A Homozygous L299P Mutation in the CYP11B1 Gene Leads to Complete Virilization in 46,XX Individuals with 11-Beta-Hydroxylase Deficiency

Background/Aim: 11-β-hydroxylase deficiency (11βOHD) is caused by CYP11B1 gene defects and leads to congenital adrenal hyperplasia associated with hypertension. Recently, a novel L299P mutation has been described in a compound heterozygous male individual. We observed two 46,XX siblings with a homozygous L299P mutation and investigated the functional properties of this CYP11B1 variant. Patients: The index patient from a consanguineous Turkish family showed complete external virilization and was diagnosed incidentally at the age of 19 months during hospital admission for severe combined bacterial (urosepsis) and viral (CMV and EBV) infection. The younger sibling was diagnosed at the age of 5 months. Their genital phenotype was identical and both demonstrated borderline elevated blood pressure. Results: Biochemical findings revealed 11βOHD. A homozygous L299P mutation of the CYP11B1 gene was detected. In vitro expression studies performed in HCT116 cells showed a markedly decreased CYP11B1 activity in the L299P mutant (1.6 ± 0.8%) for the conversion of 11-deoxycortisol to cortisol. Conclusions: Our study provides clear data on the functional properties and clinical phenotype in 46,XX individuals homozygous for this point mutation. Adrenal insufficiency might have contributed to the severe infectious disease that was present in the index patient at diagnosis.

Effects of growth hormone (GH) and insulin-like growth factor-I therapy in patients with gene defects in the GH axis

We report on four patients (3 F) who were diagnosed as having either a 6.7 kb GH1 gene deletion, a GH1 signalling peptide mutation, or a GH receptor mutation, with particular regard to treatment modalities (GH, rhIGF-I) and final height. Patients with GH1 gene defects developed anti-GH antibodies (GH-Ab) following GH treatment. Surprisingly, growth response to GH was unrestricted in one girl, who reached a final height within her target height range, whereas her cousin with the identical genetic defect responded far less favourably. Variability in the growth inhibiting potency of GH-Ab may therefore depend on genetic disposition, specific epitopes, or induction of immunological tolerance. Growth response during rhIGF-I treatment carried out in three of the patients was moderate, but pubertal development and bone age acceleration occurred in the two patients treated at pubertal age. GH resistance, either caused by GH-Ab or GH receptor mutations, is still difficult to treat and results in a heterogeneous outcome.

Reverse-hybridization assay for rapid detection of common CYP21A2 mutations in dried blood spots from newborns with elevated 17-OH progesterone

BACKGROUND Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder most commonly caused by defects in the CYP21A2 gene. Neonatal CAH-screening based on 17-hydroxyprogesterone (17-OHP) measurements prevents life-threatening salt wasting conditions in newborns, but results in a considerable false-positive rate. Therefore, efficient second tier tests are required. METHODS We developed a reverse-hybridization test strip-based assay (CAH StripAssay) covering the most prevalent CYP21A2 point mutations/small insertions/deletions occurring in Middle European populations. Assay specificity was validated using plasmid clones, and wild-type and mutant reference DNAs. Its practicability was evaluated in 271 samples from patients with CAH, suspected CAH, and dried blood spots from screening-positive newborns. RESULTS All eleven point mutations and 51% of large deletions/conversions could be unambiguously identified when compared to reference methods (DNA sequencing, MLPA). After exclusion of rare mutations (6.4%) not covered by the StripAssay, the overall detection rate was 85%. Undetected heterozygous deletions/conversions caused a lack of information, but did not result in an incorrect prediction of phenotypes. CONCLUSIONS Our novel CAH StripAssay proved to be a fast (7h) and reliable method for detection of common CYP21A2 mutations. Implemented as a second-tier test in CAH newborn screening, it has the potential to significantly reduce recall rates.