GH-Releasing Hormone Receptor Gene: A Novel Splice-Disrupting Mutation and Study of Founder Effects

Background: Mutations in GH-releasing hormone receptor gene (GHRHR) are emerging as the most common cause of autosomal recessive isolated GH deficiency (IGHD). Objective: To search for GHRHR mutations in patients with familial or sporadic IGHD and to investigate founder effects in recurring mutations. Methods: The coding region of GHRHR was entirely amplified and sequenced from DNA of 18 patients with IGHD (16 unrelated) with topic posterior pituitary lobe on MRI. Haplotypes containing promoter SNPs and microsatellites flanking GHRHR were analyzed in patients with c.57+1G>A (IVS1+1G>A) mutation of our previously published kindred and also a Brazilian patient and 2 previously reported Japanese sisters with c. 1146G>A (p.E382E) mutation. Results: A novel homozygous intronic GHRHR c.752-1G>A (IVS7-1G>A) mutation, predicting loss of the constitutive splice acceptor site, was identified in two siblings with IGHD. A compound heterozygous c.[57+1G>A];[1146G>A] and a heterozygous c.527C>T (p.A176V) were found in two sporadic cases. Haplotype analysis provided evidence for a founder effect for the c.57+1G>A mutation and independent recurrence for the c.1146G>A mutation. Conclusion: We report a novel splice-disrupting mutation in GHRHR in 2 siblings and provide evidence that all c.57+1G>A (IVS1+1G>A) mutant chromosomes have the same haplotype ancestor, indicating the occurrence of a founder effect in Brazilian patients with IGHD. Copyright (C) 2012 S. Karger AG, Basel

The consequences of growth hormone-releasing hormone receptor haploinsufficiency for bone quality and insulin resistance

Objective Growth hormone (GH)/insulin-like growth factor (IGF) axis and insulin are key determinants of bone remodelling. Homozygous mutations in the GH-releasing hormone receptor (GHRHR) gene (GHRHR) are a frequent cause of genetic isolated GH deficiency (IGHD). Heterozygosity for GHRHR mutation causes changes in body composition and possibly an increase in insulin sensitivity, but its effects on bone quality are still unknown. The objective of this study was to assess the bone quality and metabolism and its correlation with insulin sensitivity in subjects heterozygous for a null mutation in the GHRHR. Patients and methods A cross-sectional study was performed on 76 normal subjects (68.4% females) (N/N) and 64 individuals (64.1% females) heterozygous for a mutation in the GHRHR (MUT/N). Anthropometric features, quantitative ultrasound (QUS) of the heel, bone markers [osteocalcin (OC) and CrossLaps], IGF-I, glucose and insulin were measured, and homeostasis model assessment of insulin resistance (HOMAIR) was calculated. Results There were no differences in age or height between the two groups, but weight (P = 0.007) and BMI (P = 0.001) were lower in MUT/N. There were no differences in serum levels of IGF-I, glucose, T-score or absolute values of stiffness and OC, but insulin (P = 0.01), HOMAIR (P = 0.01) and CrossLaps (P = 0.01) were lower in MUT/N. There was no correlation between OC and glucose, OC and HOMAIR in the 140 individuals as a whole or in the separate MUT/N or N/N groups. Conclusions This study suggests that one allele mutation in the GHRHR gene has a greater impact on energy metabolism than on bone quality.

Assessment of letrozole and tamoxifen alone and in sequence for postmenopausal women with steroid hormone receptor-positive breast cancer: the BIG 1-98 randomised clinical trial at 8·1 years median follow-up

Postmenopausal women with hormone receptor-positive early breast cancer have persistent, long-term risk of breast-cancer recurrence and death. Therefore, trials assessing endocrine therapies for this patient population need extended follow-up. We present an update of efficacy outcomes in the Breast International Group (BIG) 1-98 study at 8·1 years median follow-up.

Growth hormone receptor polymorphism and growth hormone therapy response in children: a bayesian meta-analysis

Recombinant human growth hormone (rhGH) therapy is used in the long-term treatment of children with growth disorders, but there is considerable treatment response variability. The exon 3-deleted growth hormone receptor polymorphism (GHR(d3)) may account for some of this variability. The authors performed a systematic review (to April 2011), including investigator-only data, to quantify the effects of the GHR(fl-d3) and GHR(d3-d3) genotypes on rhGH therapy response and used a recently established Bayesian inheritance model-free approach to meta-analyze the data. The primary outcome was the 1-year change-in-height standard-deviation score for the 2 genotypes. Eighteen data sets from 12 studies (1,527 children) were included. After several prior assumptions were tested, the most appropriate inheritance model was codominant (posterior probability = 0.93). Compared with noncarriers, carriers had median differences in 1-year change-in-height standard-deviation score of 0.09 (95% credible interval (CrI): 0.01, 0.17) for GHR(fl-d3) and of 0.14 (95% CrI: 0.02, 0.26) for GHR(d3-d3). However, the between-study standard deviation of 0.18 (95% CrI: 0.10, 0.33) was considerable. The authors tested by meta-regression for potential modifiers and found no substantial influence. They conclude that 1) the GHR(d3) polymorphism inheritance is codominant, contrasting with previous reports; 2) GHR(d3) genotypes account for modest increases in rhGH effects in children; and 3) considerable unexplained variability in responsiveness remains.

Interpreting Breast International Group (BIG) 1-98: a randomized, double-blind, phase III trial comparing letrozole and tamoxifen as adjuvant endocrine therapy for postmenopausal women with hormone receptor-positive, early breast cancer

The Breast International Group (BIG) 1-98 study is a four-arm trial comparing 5 years of monotherapy with tamoxifen or with letrozole or with sequences of 2 years of one followed by 3 years of the other for postmenopausal women with endocrine-responsive early invasive breast cancer. From 1998 to 2003, BIG -98 enrolled 8,010 women. The enhanced design f the trial enabled two complementary analyses of efficacy and safety. Collection of tumor specimens further enabled treatment comparisons based on tumor biology. Reports of BIG 1-98 should be interpreted in relation to each individual patient as she weighs the costs and benefits of available treatments.

Re-evaluating adjuvant breast cancer trials: assessing hormone receptor status by immunohistochemical versus extraction assays

BACKGROUND: Tumor levels of steroid hormone receptors, a factor used to select adjuvant treatment for early-stage breast cancer, are currently determined with immunohistochemical assays. These assays have a discordance of 10%-30% with previously used extraction assays. We assessed the concordance and predictive value of hormone receptor status as determined by immunohistochemical and extraction assays on specimens from International Breast Cancer Study Group Trials VIII and IX. These trials predominantly used extraction assays and compared adjuvant chemoendocrine therapy with endocrine therapy alone among pre- and postmenopausal patients with lymph node-negative breast cancer. Trial conclusions were that combination therapy provided a benefit to pre- and postmenopausal patients with estrogen receptor (ER)-negative tumors but not to ER-positive postmenopausal patients. ER-positive premenopausal patients required further study. METHODS: Tumor specimens from 571 premenopausal and 976 postmenopausal patients on which extraction assays had determined ER and progesterone receptor (PgR) levels before randomization from October 1, 1988, through October 1, 1999, were re-evaluated with an immunohistochemical assay in a central pathology laboratory. The endpoint was disease-free survival. Hazard ratios of recurrence or death for treatment comparisons were estimated with Cox proportional hazards regression models, and discriminatory ability was evaluated with the c index. All statistical tests were two-sided. RESULTS: Concordance of hormone receptor status determined by both assays ranged from 74% (kappa = 0.48) for PgR among postmenopausal patients to 88% (kappa = 0.66) for ER in postmenopausal patients. Hazard ratio estimates were similar for the association between disease-free survival and ER status (among all patients) or PgR status (among postmenopausal patients) as determined by the two methods. However, among premenopausal patients treated with endocrine therapy alone, the discriminatory ability of PgR status as determined by immunohistochemical assay was statistically significantly better (c index = 0.60 versus 0.51; P = .003) than that determined by extraction assay, and so immunohistochemically determined PgR status could predict disease-free survival. CONCLUSIONS: Trial conclusions in which ER status (for all patients) or PgR status (for postmenopausal patients) was determined by immunohistochemical assay supported those determined by extraction assays. However, among premenopausal patients, trial conclusions drawn from PgR status differed--immunohistochemically determined PgR status could predict response to endocrine therapy, unlike that determined by the extraction assay.

Predictors of early relapse in postmenopausal women with hormone receptor-positive breast cancer in the BIG 1-98 trial

BACKGROUND: Aromatase inhibitors are considered standard adjuvant endocrine treatment of postmenopausal women with hormone receptor-positive breast cancer, but it remains uncertain whether aromatase inhibitors should be given upfront or sequentially with tamoxifen. Awaiting results from ongoing randomized trials, we examined prognostic factors of an early relapse among patients in the BIG 1-98 trial to aid in treatment choices. PATIENTS AND METHODS: Analyses included all 7707 eligible patients treated on BIG 1-98. The median follow-up was 2 years, and the primary end point was breast cancer relapse. Cox proportional hazards regression was used to identify prognostic factors. RESULTS: Two hundred and eighty-five patients (3.7%) had an early relapse (3.1% on letrozole, 4.4% on tamoxifen). Predictive factors for early relapse were node positivity (P < 0.001), absence of both receptors being positive (P < 0.001), high tumor grade (P < 0.001), HER-2 overexpression/amplification (P < 0.001), large tumor size (P = 0.001), treatment with tamoxifen (P = 0.002), and vascular invasion (P = 0.02). There were no significant interactions between treatment and the covariates, though letrozole appeared to provide a greater than average reduction in the risk of early relapse in patients with many involved lymph nodes, large tumors, and vascular invasion present. CONCLUSION: Upfront letrozole resulted in significantly fewer early relapses than tamoxifen, even after adjusting for significant prognostic factors.

Growth hormone regulates growth hormone receptor gene transcription in primary human thyroid cells

In this study the regulation of GH-receptor gene (GHR/GHBP) transcription by different concentrations of GH (0, 12.5, 25, 50, 150, 500 ng/ml) with and without variable TSH concentrations (0.5, 2, 20 mU/l) in primary human thyroid cells cultured in serum-free hormonally-defined medium was studied. The incubation time was 6 h and GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification at hourly intervals. Correlating with the GH-concentrations added a constant and significant increase of GHR/GHBP gene transcription was found. After the addition of 12.5 ng/ml GH, GHR/GHBP mRNA concentration remained constant over the incubation period of 6 h but in comparison with the experiments where no GH was added there was a significant change of GHR/GHBP mRNA expression. Following the addition of 25 ng/ml GH a slight but further increase of GHR/GHBP transcription products was seen which increased even more in the experiments where higher GH concentrations were used. These data focusing on GHR/GHBP gene transcription derived from cDNA synthesis and quantitative PCR amplification were confirmed by run-on experiments. Furthermore, cycloheximide did not affect these changes supporting the notion that GH stimulates GHR/GHBP gene transcription directly. In a second set of experiments, in combination with variable TSH levels, identical GH concentrations were used and no difference in either GHR/GHBP mRNA levels or in transcription rate (run-on experiments) could be found. In conclusion, we report data showing that primary thyroid cells express functional GH-receptors in which GH has a direct and dose dependent effect on the GHR/GHBP gene transcription. Furthermore, TSH does not a have a major impact on GHR/GHBP gene regulation.

Regulation of human growth hormone receptor gene transcription by triiodothyronine (T3)

In this study the hypothesis that triiodothyronine (T3) and growth hormone (GH) may have some direct or indirect effect on the regulation of GH-receptor/GH-binding protein (GHR/GHBP) gene transcription was tested. Different concentrations of T3 (0, 0.5, 2, 10 nmol/l) and GH (0, 10, 150 ng/ml) were added to human hepatoma (HuH7) cells cultured in serum-free hormonally-defined medium for 0, 1 and 2 h. Thereafter GHR/GHBP mRNA expression was quantitatively assessed by using PCR amplification. GH at a concentration of 10 ng/ml resulted in a significant increase of GHR/GHBP gene expression whereas a supraphysiological concentration of GH (150 ng/ml) caused a significant decrease of GHR/GHBP mRNA levels. The simultaneous addition of 0.5 nmol/l T3 to the variable concentrations of GH did not modify GHR/GHBP mRNA levels whereas the addition of 2 nmol/l up-regulated GHR/GHBP gene expression already after 1 h, an increase which was even more marked when 10 nmol/l of T3 was added. Interestingly, there was a positive correlation between the increase of GHR/GHBP mRNA levels and the T3 concentration used (r: 0.8). In addition, nuclear run-on experiments and GHBP determinations were performed which confirmed the changes in GHR/GHBP mRNA levels. Cycloheximide (10 microg/ml) did not alter transcription rate following GH addition but blocked GHR/GHBP gene transcription in T3 treated cells indicating that up-regulation of GHR/GHBP gene transcription caused by T3 requires new protein synthesis and is, therefore, dependent on indirect mechanisms. In conclusion, we present data showing that T3 on its own has a stimulatory effect on GHR/GHBP gene transcription which is indirect and additive to the GH-induced changes.

Correspondence of Gonadotropin-Releasing Hormone and Luteinizing Hormone Secretion during Suckling in Postpartum Cows

The hypothalamus in the lower part of the brain contains neurons that produce a small peptide, gonadotropin- releasing hormone (GnRH, LHRH), that regulates luteinizing hormone (LH) secretion by the anterior pituitary gland. Important functions of LH include induction of ovulation in preovulatory follicles during estrus and the luteinization of granulosa cells lining those collapsed follicles to form corpora lutea that produce progesterone during the luteal phase of the estrous cycle or during pregnancy. The production of progesterone by the corpus luteum conveys a negative feed-back action at the central nervous system (CNS) for further episodic secretion of GnRH and in turn, LH secretion. Gonadal removal (i.e., ovariectomy) allows a greater amount of LH secretion to occur during a prolonged period. The objectives of this study were to characterize the pattern of GnRH secretion in the cerebrospinal fluid (CSF) of the bovine third ventricle region of the hypothalamus, determine its correspondence with the tonic and surge release of LH in ovariectomized cows, and examine the dynamics of GnRH pulse release activity in response to known modulators of LH release (suckling, neuropeptide-Y [NPY]). In ovariectomized cows, both tonic release patterns and estradiol-induced surges of GnRH and LH were highly correlated. A 500-microgram dose of NPY caused an immediate cessation of LH pulses and decreased plasma concentrations of LH for at least 4 hours. This corresponded with a decrease in both GnRH pulse amplitude and frequency. In anestrous cows, GnRH pulse frequency did not change before and 48 to 54 hours after weaning on day 18 postpartum, but GnRH concentration and amplitudes of GnRH pulses increased in association with weaning and heightened secretion of LH. It is clear that high-frequency, highamplitude pulses of LH are accompanied by similar patterns of GnRH in CSF of adult cattle. Yet strong inhibitors of LH pulsatility, putatively acting at the level of the central nervous system (i.e., suckling) or at both the central nervous system and pituitary (NPY) levels, produced periods of discordance between GnRH and LH pulses.

ALTERATIONS IN HYPOTHALAMIC CONTENT OF LUTEINIZING HORMONE RELEASING HORMONE AND PITUITARY RESPONSIVENESS ASSOCIATED WITH TESTICULAR REGRESSION INDUCED BY PINEAL RELATED TREATMENTS IN THE GOLDEN HAMSTER

The adult male golden hamster, when exposed to blinding (BL), short photoperiod (SP), or daily melatonin injections (MEL) demonstrates dramatic reproductive collapse. This collapse can be blocked by removal of the pineal gland prior to treatment. Reproductive collapse is characterized by a dramatic decrease in both testicular weight and serum gonadotropin titers. The present study was designed to examine the interactions of the hypothalamus and pituitary gland during testicular regression, and to specifically compare and contrast changes caused by the three commonly employed methods of inducing testicular regression (BL,SP,MEL). Hypothalamic LHRH content was altered by all three treatments. There was an initial increase in content of LHRH that occurred concomitantly with the decreased serum gonadotropin titers, followed by a precipitous decline in LHRH content which reflected the rapid increases in both serum LH and FSH which occur during spontaneous testicular recrudescence. In vitro pituitary responsiveness was altered by all three treatments: there was a decline in basal and maximally stimulatable release of both LH and FSH which paralleled the fall of serum gonadotropins. During recrudescence both basal and maximal release dramatically increased in a manner comparable to serum hormone levels. While all three treatments were equally effective in their ability to induce changes at all levels of the endocrine system, there were important temporal differences in the effects of the various treatments. Melatonin injections induced the most rapid changes in endocrine parameters, followed by exposure to short photoperiod. Blinding required the most time to induce the same changes. This study has demonstrated that pineal-mediated testicular regression is a process which involves dynamic changes in multiply-dependent endocrine relationships, and proper evaluation of these changes must be performed with specific temporal events in mind. ^