Margaret de Castro

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

CONTEXT Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. OBJECTIVE A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. DESIGN AND SETTING The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. PATIENTS OR OTHER PARTICIPANTS 345 probands, 18 family members, and 292 controls were studied. INTERVENTION Reproductive phenotypes throughout reproductive life and before and after therapy were examined. MAIN OUTCOME MEASURE Rare sequence variants in TAC3/TACR3 were detected. RESULTS In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. CONCLUSIONS Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time.

MicroRNAs Differentially Expressed in ACTH-Secreting Pituitary Tumors

CONTEXT MicroRNAs (miRNAs) are small noncoding RNAs, functioning as antisense regulators of gene expression by targeting mRNA and contributing to cancer development and progression. More than 50% of miRNA genes are located in cancer-associated genomic regions or in fragile sites of the genome. OBJECTIVE The aim of the study was to analyze the differential expression of let-7a, miR-15a, miR-16, miR-21, miR-141, miR-143, miR-145, and miR-150 in corticotropinomas and normal pituitary tissue and verify whether their profile of expression correlates with tumor size or remission after treatment. MATERIAL AND METHODS ACTH-secreting pituitary tumor samples were obtained during transphenoidal surgery from patients with Cushing disease and normal pituitary tissues from autopsies. The relative expression of miRNAs was measured by real-time PCR using RNU44 and RNU49 as endogenous controls. Relative quantification of miRNA expression was calculated using the 2(-DeltaDeltaCt) method. RESULTS We found underexpression of miR-145 (2.0-fold; P = 0.04), miR-21 (2.4-fold; P = 0.004), miR-141 (2.6-fold; P = 0.02), let-7a (3.3-fold; P = 0.003), miR-150 (3.8-fold; P = 0.04), miR-15a (4.5-fold; P = 0.03), miR-16 (5.0-fold; P = 0.004), and miR-143 (6.4-fold; P = 0.004) in ACTH-secreting pituitary tumors when compared to normal pituitary tissues. There were no differences between miRNA expression and tumor size as well as miRNA expression and ratio of remission after surgery, except in patients presenting lower miR-141 expression who showed a better chance of remission. CONCLUSION Our results support the possibility that altered miRNA expression profile might be involved in corticotrophic tumorigenesis. However, the lack of knowledge about miRNA target genes postpones full understanding of the biological functions of down-regulated or up-regulated miRNAs in corticotropinomas.

Loss-of-Function Mutations in the Genes Encoding Prokineticin-2 or Prokineticin Receptor-2 Cause Autosomal Recessive Kallmann Syndrome

CONTEXT Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. OBJECTIVE To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. DESIGN We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. RESULTS In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p.G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p.I55fsX56 mutation. In PROKR2, four distinct mutations (p.R80C, p.Y140X, p.L173R, and p.R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p.R80C, p.L173R, and p.R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, no mutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p.Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. CONCLUSION We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Inducible nitric oxide synthase pathway in the central nervous system and vasopressin release during experimental septic shock.

BackgroundSeptic shock is characterized by arteriolar vasodilation and hypotension. We have tested the hypothesis that nitric oxide arising from inducible nitric oxide synthase in the central nervous system is responsible for the deficiency in vasopressin release and consequent hypotension during experimental septic shock. Methods and ResultsSeptic shock was induced in male Wistar rats by intravenous injection of 1.5 mg/kg lipopolysaccharide. After lipopolysaccharide administration, we found a significant decrease in mean arterial pressure with a concomitant increase in heart rate, a significant decrease in diuresis, and a transitory decrease in body temperature. An increase in plasma vasopressin concentrations occurred in these animals and was present for 2 hrs after lipopolysaccharide administration, returning close to basal concentrations thereafter and remaining unchanged for the next 24 hrs. When lipopolysaccharide was combined with central administration of aminoguanidine, an inducible nitric oxide synthase inhibitor, we observed a sustained increase in plasma vasopressin concentration and in the maintenance of blood pressure at 4 and 6 hrs after lipopolysaccharide treatment compared with rats treated with lipopolysaccharide alone. ConclusionThese data indicate that central nitric oxide arising from the inducible nitric oxide synthase pathway plays an important inhibitory role in vasopressin release during experimental septic shock and may be responsible for the hypotension occurring in this vasodilatory shock.

Salivary cortisol as a tool for physiological studies and diagnostic strategies

Salivary cortisol is an index of plasma free cortisol and is obtained by a noninvasive procedure. We have been using salivary cortisol as a tool for physiological and diagnostic studies, among them the emergence of circadian rhythm in preterm and term infants. The salivary cortisol circadian rhythm in term and premature infants was established between 8 and 12 postnatal weeks. In the preterm infants the emergence of circadian rhythm was parallel to the onset of sleep rhythm. We also studied the use of salivary cortisol for screening for Cushings syndrome (CS) in control and obese outpatients based on circadian rhythm and the overnight 1 mg dexamethasone (DEX) suppression test. Salivary cortisol was suppressed to less than 100 ng/dl after 1 mg DEX in control and obese patients. A single salivary cortisol measurement at 23:00 h and again after 1 mg DEX above the 90th percentile of the obese group values had sensitivity and specificity of 93 and 93% (23:00 h), and 91 and 94% (after DEX), respectively. The sensitivity improved to 100% when we combined both parameters. We also studied 11 CS children and 21 age-matched primary obese children for whom salivary cortisol sensitivity and specificity were 100/95% (23:00 h), and 100/95% (1 mg DEX), respectively. Similar to adults, sensitivity and specificity of 100% were obtained by combining 23:00 h and 1 mg DEX. The measurement of salivary cortisol is a useful tool for physiological studies and for the diagnosis of CS in children and adults on an outpatient basis.

AIP expression in sporadic somatotropinomas is a predictor of the response to octreotide LAR therapy independent of SSTR2 expression.

mutation and/or a history of medical treatment with SRL before surgery. Biochemical assessment was performed 3 months after surgery by oral glucose tolerance test (OGTT) and evaluations of IGF1 levels. Patients were considered non-cured based on nonsuppressible GH levels on OGTT and plasma IGF1 levels higher than those of age-matched normal subjects. Medical therapy with OCT-LAR was started at a dose of 20 mg every 4 weeks, and the dose was increased to 30 mg in patients with non-controlled disease after 3 months of therapy. The efficacy of medical therapy was evaluated during the last patient visit. Patients were considered to have non-controlled disease if they had plasma IGF1 levels greater than those of age-matched normal subjects or basal GH levels O1.0 ng/ml after at least 6 months of treatment with OCT-LAR at a dosage of 30 mg. The AIP and SSTR2 expressions were analyzed through immunohistochemistry in paraffin-embedded

Central Precocious Puberty that appears to be sporadic caused by Paternally inherited mutations in the imprinted GENE makorin ring finger 3

CONTEXT Loss-of-function mutations in makorin ring finger 3 (MKRN3), an imprinted gene located on the long arm of chromosome 15, have been recognized recently as a cause of familial central precocious puberty (CPP) in humans. MKRN3 has a potential inhibitory effect on GnRH secretion. OBJECTIVES The objective of the study was to investigate potential MKRN3 sequence variations as well as copy number and methylation abnormalities of the 15q11 locus in patients with apparently sporadic CPP. SETTING AND PARTICIPANTS We studied 215 unrelated children (207 girls and eight boys) from three university medical centers with a diagnosis of CPP. All but two of these patients (213 cases) reported no family history of premature sexual development. First-degree relatives of patients with identified MKRN3 variants were included for genetic analysis. MAIN OUTCOME MEASURES All 215 CPP patients were screened for MKRN3 mutations by automatic sequencing. Multiplex ligation-dependent probe amplification was performed in a partially overlapping cohort of 52 patients. RESULTS We identified five novel heterozygous mutations in MKRN3 in eight unrelated girls with CPP. Four were frame shift mutations predicted to encode truncated proteins and one was a missense mutation, which was suggested to be deleterious by in silico analysis. All patients with MKRN3 mutations had classical features of CPP with a median age of onset at 6 years. Copy number and methylation abnormalities at the 15q11 locus were not detected in the patients tested for these abnormalities. Segregation analysis was possible in five of the eight girls with MKRN3 mutations; in all cases, the mutation was inherited on the paternal allele. CONCLUSIONS We have identified novel inherited MKRN3 defects in children with apparently sporadic CPP, supporting a fundamental role of this peptide in the suppression of the reproductive axis.

The effects of restraint stress on nociceptive responses induced by formalin injected in rat's TMJ

It has been reported that stress can alter nociception from superficial tissues, such as skin and subcutaneous region. However, the influence of stress on an experimental deep nociception model is not understood. In this study, the temporomandibular joint (TMJ) formalin test was used to evaluate the effects of acute and chronic restraint stress on nociceptive responses in rats. Animals were initially submitted to one session of acute restraint stress (1 h) or exposed to chronic stress (40 days-1 h/day). Then, animals were killed immediately to collect blood for hormonal determinations by radioimmunoassay, or submitted to the TMJ formalin test to evaluate nociception. Rats submitted to acute restraint presented a performance similar to unstressed controls in the TMJ formalin test, whereas chronically stressed rats showed an increase in nociceptive responses. After 40 days of restraint, morphine was injected i.p. (1, 5 mg/kg or saline). The stressed rats displayed decreased morphine effects on nociception compared to unstressed controls. These findings suggest that repeated stress can produce hyperalgesia, which is, at least in part, due to alterations in the activity of opioid systems. This model may help elucidate the underlying neural mechanisms that mediate the effects of repeated stress on orofacial pain.

A dose–response study of salivary cortisol after dexamethasone suppression test in Cushing's disease and its potential use in the differential diagnosis of Cushing's syndrome

objective  A dose–response study with different doses of dexamethasone (dex) to assess the corticotrophic resistance in Cushings disease (CD) using salivary cortisol as an end point has not yet been evaluated. We also reported our experience with salivary cortisol compared to plasma cortisol determination during dex suppression test (DST) and after ovine corticotrophin release hormone (oCRH) test in the differential diagnosis of Cushings syndrome (CS).

Late remote ischemic preconditioning in children undergoing cardiopulmonary bypass: a randomized controlled trial.

OBJECTIVE Cardiopulmonary bypass is associated with ischemia-reperfusion injury to multiple organs. We aimed to evaluate whether remote ischemic preconditioning performed the day before surgery for congenital heart disease with cardiopulmonary bypass attenuates the postoperative inflammatory response and myocardial dysfunction. METHODS This was a prospective, randomized, single-blind, controlled trial. Children allocated to remote ischemic preconditioning underwent 4 periods of 5 minutes of lower limb ischemia by a blood pressure cuff intercalated with 5 minutes of reperfusion. Blood samples were collected 4, 12, 24, and 48 hours after cardiopulmonary bypass to evaluate nuclear factor kappa B activation in leukocytes by quantification of mRNA of I kappa B alpha by real-time quantitative polymerase chain reaction and for interleukin-8 and 10 plasma concentration measurements by enzyme-linked immunosorbent assay. Myocardial dysfunction was assessed by N-terminal pro-B-type natriuretic peptide and cardiac troponin I plasma concentrations, measured by chemiluminescence, and clinical parameters of low cardiac output syndrome. RESULTS Twelve children were allocated to remote ischemic preconditioning, and 10 children were allocated to the control group. Demographic data and Risk Adjustment for Congenital Heart Surgery 1 classification were comparable in both groups. Remote ischemic preconditioning group had lower postoperative values of N-terminal pro-B-type natriuretic peptide, but cardiac troponin I levels were not significantly different between groups. Interleukin-8 and 10 concentrations and I kappa B alpha gene expression were similar in both groups. Postoperative morbidity was similar in both groups; there were no postoperative deaths in either group. CONCLUSIONS Late remote ischemic preconditioning did not provide clinically relevant cardioprotection to children undergoing cardiopulmonary bypass.