Very few reference intervals for salivary steroids in children have been established to date (1 ). Even the manufacturers of salivary steroid assays do not provide sufficient reference data for their products. This lack of information is surprising because the measurement of salivary steroids has been accepted as being noninvasive and stress-free (2, 3 ). In particular, psychiatric and neuroendocrinologic experiments are frequently designed with saliva as the medium of choice for steroid analysis (4 -6 ).A large variety of stressors can rapidly affect the adrenal cortex, causing increased adrenal steroid concentrations. For example, hypoglycemia (7 ) or physical exercise (8 ) are potent physiologic stressors, whereas fear (9 ), feelings of inferiority (10, 11 ), or experiences at school (12, 13 ) can affect the adrenal cortex activity as psychologic stressors. The taking of blood can also influence adrenal steroid concentrations in children; saliva collection, however, is almost stress-free (14 ). The use of saliva for steroid analysis in children is therefore an excellent alternative to blood.The aim of our study was to establish age-dependent reference values for salivary cortisol, 17␣-hydroxyprogesterone (17OHP), and progesterone in a large cohort of healthy children. The availability of such reference intervals will improve the applicability of saliva analysis as a diagnostic tool in pediatric endocrinology.We collected 252 saliva profiles from healthy children and adolescents (125 boys; age range, 4 days to 15 years; 127 girls; age range, 6 days to 13 years) with normal body length/height and weight. None of the girls had developed a regular menstrual cycle. The parents of the children gave informed consent.Saliva was collected either with the Salivette ® , using polyester swabs (Sarstedt), from children Ͼ1 year of age or with modified medical pacifiers (Bü ttner-Frank; see Fig. 1 in the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem. org/content/vol49/issue10/) from infants Ͻ1 year. The teats of the pacifiers were perforated, and strips of filter paper, as used for blood-spot collection, were inserted into the teats. The absorbent material had no effect on the steroid analysis in the RIAs, in contrast to the cotton Salivette, particularly for 17OHP (15 ) and progesterone (16 ).Samples were taken three times a day (0700, 1300, and 1900) at home. After sample collection, the saliva was immediately frozen and sent to the laboratory in a cooler. We tried to exclude any potential stressful events; thus the samples were taken only on so-called "quiet" days at home, e. g., days with no school-related stress or sports, or on weekends. All of the children participating in the study lived in an intact familial environment.We used adapted commercial RIAs (DSL), as described in detail elsewhere (17,18 ), that require 50 L of saliva per tube. The sensitivities and intra-and interassay CVs were 0.2 nmol/L (6% and 9.6%) for cortisol, 9 pmol/L (6% and 8.6%) for 17OHP, ...

PurposeShort stature is a common condition of great concern to patients and their families. Mostly genetic in origin, the underlying cause often remains elusive due to clinical and genetic heterogeneity.MethodsWe systematically phenotyped 565 patients where common nongenetic causes of short stature were excluded, selected 200 representative patients for whole-exome sequencing, and analyzed the identified variants for pathogenicity and the affected genes regarding their functional relevance for growth.ResultsBy standard targeted diagnostic and phenotype assessment, we identified a known disease cause in only 13.6% of the 565 patients. Whole-exome sequencing in 200 patients identified additional mutations in known short-stature genes in 16.5% of these patients who manifested only part of the symptomatology. In 15.5% of the 200 patients our findings were of significant clinical relevance. Heterozygous carriers of recessive skeletal dysplasia alleles represented 3.5% of the cases.ConclusionA combined approach of systematic phenotyping, targeted genetic testing, and whole-exome sequencing allows the identification of the underlying cause of short stature in at least 33% of cases, enabling physicians to improve diagnosis, treatment, and genetic counseling. Exome sequencing significantly increases the diagnostic yield and consequently care in patients with short stature.

Short stature is a common pediatric disorder affecting 3% of the population. However, the clinical variability and genetic heterogeneity prevents the identification of the underlying cause in about 80% of the patients. Recently, heterozygous mutations in the ACAN gene coding for the proteoglycan aggrecan, a main component of the cartilage matrix, were associated with idiopathic short stature. To ascertain the prevalence of ACAN mutations and broaden the phenotypic spectrum in patients with idiopathic short stature we performed sequence analyses in 428 families. We identified heterozygous nonsense mutations in four and potentially disease-causing missense variants in two families (1.4%). These patients presented with a mean of −3.2 SDS and some suggestive clinical characteristics. The results suggest heterozygous mutations in ACAN as a common cause of isolated as well as inherited idiopathic short stature.

Calcilytics might offer medical treatment for patients with autosomal dominant hypocalcemia caused by calcilytic-sensitive CaSR mutants.

The calcium-sensing receptor (CASR) is the main calcium sensor in the maintenance of calcium metabolism. Mutations of the CASR, the G protein alpha 11 (GNA11) and the adaptor-related protein complex 2 sigma 1 subunit (AP2S1) genes can shift the set point for calcium sensing causing hyper-or hypo-calcemic disorders. Therapeutic concepts for these rare diseases range from general therapies of hyper-and hypo-calcemic conditions to more pathophysiology oriented approaches such as parathyroid hormone (PTH) substitution and allosteric CASR modulators. Cinacalcet is a calcimimetic that enhances receptor function and has gained approval for the treatment of hyperparathyroidism. Calcilytics in turn attenuate CASR activity and are currently under investigation for the treatment of various diseases. We conducted a literature search for reports about treatment of patients harboring inactivating or activating CASR, GNA11 or AP2S1 mutants and about in vitro effects of allosteric CASR modulators on mutated CASR. The therapeutic concepts for patients with familial hypocalciuric hypercalcemia (FHH), neonatal hyperparathyroidism (NHPT), neonatal severe hyperparathyroidism (NSHPT) and autosomal dominant hypocalcemia (ADH) are reviewed. FHH is usually benign, but symptomatic patients benefit from cinacalcet. In NSHPT patients pamidronate effectively lowers serum calcium, but most patients require parathyroidectomy. In some patients cinacalcet can obviate the need for surgery, particularly in heterozygous NHPT. Symptomatic ADH patients respond to vitamin D and calcium supplementation but this may increase calciuria and renal complications. PTH treatment can reduce relative hypercalciuria. None of the currently available therapies for ADH, however, prevent tissue calcifications and complications, which may become possible with calcilytics that correct the underlying pathophysiologic defect.

Objective: To evaluate adrenal crises after the start of treatment up to the age of 6 years in children with classic congenital adrenal hyperplasia (CAH). Design: Analysis of data extracted from a population-based prospective long-term follow-up study of children detected in neonatal screening. Methods: Data of 102 Bavarian children with classic CAH due to 21-hydroxylase deficiency were analyzed, using parental questionnaires and medical reports. Parent-reported hospital admissions of children diagnosed with acute health impairment were included in the analysis if salt loss (hyponatremia) or hypoglycemia was documented in the discharge summary. Results: A total of 74 children (72.5%) had no report of hospital admissions with salt loss or hypoglycemia during the observational period. However, in 27.5% of the children, 22 salt-wasting crises (seven of these also with low blood glucose) and 16 hypoglycemic episodes without salt loss were reported. Furthermore, the cumulative incidence for seizures was elevated; 13 children experienced seizures during hyponatremia or hypoglycemia. Most adrenal crises were triggered by infections, often with inappropriate emergency management, but in 11 cases hypoglycemia occurred unexpectedly, without evidence of severe illness and without any management errors. Frequency of adrenal crises was 6.5 per 100 patient years (95% CI: 4.6-8.8). Conclusions: Crisis prevention remains a permanent challenge for families and physicians caring for children with classic CAH. Expert care and compliance with emergency recommendations are crucial. Further research on the interactions among glucocorticoid deficiency, adrenomedullary dysfunction, and glucose metabolism is necessary for the prevention of hypoglycemia, especially in young CAH patients.

The IGF1 generation test (IGFGT) is often used during the assessment of suspected GH insensitivity (GHI). We report the results of a survey undertaken in 2010 to determine the use of IGFGT amongst members of the European Society for Paediatric Endocrinology to evaluate suspected GHI. The literature surrounding the usefulness and limitations of IGFGT are reviewed, and recommendations provided for its use. Of 112 paediatric endocrinologists from 30 countries who responded to the survey, 91 (81%) reported that they had used the IGFGT in the previous 2 years; O10 IGFGT protocols were used. The IGFGT impacted treatment decisions for 97% of the respondents and was a prerequisite for recombinant human IGF1 treatment for 45% of respondents. From a literature review, sensitivity of the IGFGT was evaluated as 77-91% in molecularly proven cases of GHI; specificity was %97%, depending on the protocol. The positive predictive value of the IGFGT is likely to be low, as the frequency of normality is predictably higher than that of abnormality in GH signalling. Given the limitations of the IGFGT in the most severe cases of GHI syndrome (GHIS), the ability of the IGFGT to detect less severe GHIS is doubtful. In a pragmatic approach, the IGFGT may not be useful for the diagnosis of GHIS.

Objective: GH deficiency is diagnosed in children if serum GH fails to rise above a predefined cutoff value in response to at least two stimuli. Diagnostic decisions based on this testing are highly variable between centers and depend on the GH assays used. Considering the large spectrum of commercially available GH assays, we wanted to evaluate the agreement between assays, and to test whether assayrelated variability of diagnostic decisions could be reduced by reassessment of peak GH concentrations in a reference center. Design: We reanalysed 699 peak GH serum samples obtained after GH testing of 382 children and adolescents from 19 centers using three reference assays and compared these results with those obtained with the local assays. A subgroup of 132 patients tested with the combination of insulin hypoglycemia test and arginine test was evaluated for changes in the assignment to the diagnostic group of GH deficiency. Results: The mean difference between methods ranged from 5.4 to 10.3 mU/l, slopes of the regression lines from 1.28 to 1.65. Significant non-linearity was detected in five of six assay comparisons, indicating that most assay results cannot be interconverted by the use of a factor. Overall agreement between reference and local assays was only moderate. Significant changes in diagnostic assignment occurred when different assays were used on the same patient (P , 0.0001 -P , 0.0023). Based on GH remeasurement by one reference assay, 36 of 132 patients were categorized differently, with 35 patients changing into the GH-deficient group. Similar findings were obtained with the other reference assays. Conclusions: To decrease variability in GH testing related to assays and cutoff values, we recommend nationwide reassessment of GH peak sera in reference centers. Decisions to treat GH deficiency should incorporate the reference center results.European Journal of Endocrinology 150 291-297