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CONTEXT: KISS1R mutations have been reported in few patients with normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). OBJECTIVE: To describe in detail nCHH patients with biallelic KISS1R mutations belonging to 2 unrelated families, and to functionally characterize a novel KISS1R mutation. RESULTS: An original mutant, p.Tyr313His, was found in the homozygous state in 3 affected kindred (2 females and 1 male) from a consanguineous Portuguese family. This mutation, located in the seventh transmembrane domain, affects a highly conserved amino acid, perturbs the conformation of the transmembrane segment, and impairs MAP kinase signaling and intracellular calcium release. In the second family, a French Caucasian male patient with nCHH was found to carry two recurrent mutations in the compound heterozygous state (p.Leu102Pro/Stop399Arg). In this man, pulsatile GnRH (Gonadotropin Releasing Hormone) administration restored pulsatile LH (Luteinizing Hormone) secretion and testicular hormone secretion. Later, long-term combined gonadotropin therapy induced spermatogenesis, enabling 3 successive pregnancies that resulted in 2 miscarriages and the birth of a healthy boy. CONCLUSION: We show that a novel loss-of-function mutation (p.Tyr313His) in the KISS1R gene can cause familial nCHH, revealing the crucial role of this amino acid in KISS1R function. The observed restoration of gonadotropin secretion by exogenous GnRH administration further supports, in humans, the hypothalamic origin of the gonadotropin deficiency in this genetic form of nCHH.

Normosmic congenital hypogonadotropic hypogonadism (nCHH) is a rare reproductive disease leading to lack of puberty and infertility. Loss-of-function mutations of GNRH1 gene are a very rare cause of autosomal recessive nCHH. R31C GNRH1 is the only missense mutation that affects the conserved GnRH decapeptide sequence. This mutation was identified in a CpG islet in nine nCHH subjects from four unrelated families, giving evidence for a putative "hot spot". Interestingly, all the nCHH patients carry this mutation in heterozygosis that strikingly contrasts with the recessive inheritance associated with frame shift and non-sense mutations. Therefore, after exclusion of a second genetic event, a comprehensive functional characterization of the mutant R31C GnRH was undertaken. Using different cellular models, we clearly demonstrate a dramatic reduction of the mutant decapeptide capacity to bind GnRH-receptor, to activate MAPK pathway and to trigger inositol phosphate accumulation and intracellular calcium mobilization. In addition it is less able than wild type to induce lh-beta transcription and LH secretion in gonadotrope cells. Finally, the absence of a negative dominance in vitro offers a unique opportunity to discuss the complex in vivo patho-physiology of this form of nCHH.

Progesterone receptor isoforms (PRA and PRB) are expressed at equal levels in normal mammary cells. However, alteration in PRA/PRB expression is often observed in aggressive breast cancer suggesting differential contribution of PR isoforms in carcinogenesis. The mechanisms underlying such processes remain to be established mainly due to paucity of appropriate cellular models. To investigate the role of PR isoforms and the impact of imbalanced PRA/PRB ratio in transcriptional regulation, we have generated an original human breast cancer cell line conditionally expressing PRA and/or PRB in dose-dependence of non-steroid inducers. We first focused on PR-dependent transcriptional regulation of the paracrine growth factor gene amphiregulin (AREG) playing important role in cancer. Interestingly, unliganded PRA increases AREG expression, independently of estrogen receptor, yet inhibitable by antiprogestins. We show that functional outcome of epidermal growth factor (EGF) on such regulation is highly dependent on PRA/PRB ratio. Using this valuable model, genome-wide transcriptomic studies allowed us to determine the differential effects of PRA and PRB as a function of hormonal status. We identified a large number of novel PR-regulated genes notably implicated in breast cancer and metastasis and demonstrated that imbalanced PRA/PRB ratio strongly impact their expression predicting poor outcome in breast cancer. In sum, our unique cell-based system strongly suggests that PRA/PRB ratio is a critical determinant of PR target gene selectivity and responses to hormonal/growth factor stimuli. These findings provide molecular support for the aggressive phenotype of breast cancers with impaired expression of PRA or PRB.

Congenital hypogonadotropic hypogonadism (CHH) is characterized by low gonadotropins and failure to progress normally through puberty. Mutations in the gene encoding the GnRH receptor (GNRHR1) result in CHH when present as compound heterozygous or homozygous inactivating mutations. This study identifies and characterizes the properties of two novel GNRHR1 mutations in a family in which three brothers display normosmic CHH while their sister was unaffected. Molecular analysis in the proband and the affected brothers revealed two novel non-synonymous missense GNRHR1 mutations, present in a compound heterozygous state, whereas their unaffected parents possessed only one inactivating mutation, demonstrating the autosomal recessive transmission in this kindred and excluding X-linked inheritance equivocally suggested by the initial pedigree analysis. The first mutation at c.845 C>G introduces an Arg substitution for the conserved Pro 282 in transmembrane domain (TMD) 6. The Pro282Arg mutant is unable to bind radiolabeled GnRH analogue. As this conserved residue is important in receptor conformation, it is likely that the mutation perturbs the binding pocket and affects trafficking to the cell surface. The second mutation at c.968 A>G introduces a Cys substitution for Tyr 323 in the functionally crucial N/DPxxY motif in TMD 7. The Tyr323Cys mutant has an increased GnRH binding affinity but reduced receptor expression at the plasma membrane and impaired G protein-coupling. Inositol phosphate accumulation assays demonstrated absent and impaired Gα(q/11) signal transduction by Pro282Arg and Tyr323Cys mutants, respectively. Pretreatment with the membrane permeant GnRHR antagonist NBI-42902, which rescues cell surface expression of many GNRHR1 mutants, significantly increased the levels of radioligand binding and intracellular signaling of the Tyr323Cys mutant but not Pro282Arg. Immunocytochemistry confirmed that both mutants are present on the cell membrane albeit at low levels. Together these molecular deficiencies of the two novel GNRHR1 mutations lead to the CHH phenotype when present as a compound heterozygote.

ContextTAC3/TACR3 mutations have been reported in normosmic congenital hypogonadotropic hypogonadism (nCHH) (OMIM #146110). In the absence of animal models, studies of human neuroendocrine phenotypes associated with neurokinin B and NK3R receptor dysfunction can help to decipher the pathophysiology of this signaling pathway.ObjectiveTo evaluate the prevalence of TAC3/TACR3 mutations, characterize novel TACR3 mutations and to analyze neuroendocrine profiles in nCHH caused by deleterious TAC3/TACR3 biallelic mutations.ResultsFrom a cohort of 352 CHH, we selected 173 nCHH patients and identified nine patients carrying TAC3 or TACR3 variants (5.2%). We describe here 7 of these TACR3 variants (1 frameshift and 2 nonsense deleterious mutations and 4 missense variants) found in 5 subjects. Modeling and functional studies of the latter demonstrated the deleterious consequence of one missense mutation (Tyr267Asn) probably caused by the misfolding of the mutated NK3R protein. We found a statistically significant (pConclusionThe gonadotropin axis dysfunction associated with nCHH due to TAC3/TACR3 mutations is related to a low GnRH pulsatile frequency leading to a low frequency of alpha-subunit pulses and to an elevated FSH/LH ratio. This ratio might be useful for pre-screening nCHH patients for TAC3/TACR3 mutations.

Primary glucocorticoid resistance (OMIM 138040) is a rare hereditary disease that causes a generalized partial insensitivity to glucocorticoid action, due to genetic alterations of the glucocorticoid receptor (GR). Investigation of adrenal incidentalomas led to the discovery of a family (eight affected individuals spanning three generations), prone to cortisol resistance, bilateral adrenal hyperplasia, arterial hypertension and hypokalemia. This phenotype exacerbated over time, cosegregates with the first heterozygous nonsense mutation p.R469[R,X] reported to date for the GR, replacing an arginine (CGA) by a stop (TGA) at amino-acid 469 in the second zinc finger of the DNA-binding domain of the receptor. In vitro, this mutation leads to a truncated 50-kDa GR lacking hormone and DNA binding capacity, devoid of hormone-dependent nuclear translocation and transactivation properties. In the proband's fibroblasts, we provided evidence for the lack of expression of the defective allele in vivo. The absence of detectable mutated GR mRNA was accompanied by a 50% reduction in wild type GR transcript and protein. This reduced GR expression leads to a significantly below-normal induction of glucocorticoid-induced target genes, FKBP5 in fibroblasts. We demonstrated that the molecular mechanisms of glucocorticoid signaling dysfunction involved GR haploinsufficiency due to the selective degradation of the mutated GR transcript through a nonsense-mediated mRNA Decay that was experimentally validated on emetine-treated propositus' fibroblasts. GR haploinsufficiency leads to hypertension due to illicit occupation of renal mineralocorticoid receptor by elevated cortisol rather than to increased mineralocorticoid production reported in primary glucocorticoid resistance. Indeed, apparent mineralocorticoid excess was demonstrated by a decrease in urinary tetrahydrocortisone-tetrahydrocortisol ratio in affected patients, revealing reduced glucocorticoid degradation by renal activity of the 11β-hydroxysteroid dehydrogenase type 2, a GR regulated gene. We propose thus that GR haploinsufficiency compromises glucocorticoid sensitivity and may represent a novel genetic cause of subclinical hypercortisolism, incidentally revealed bilateral adrenal hyperplasia and mineralocorticoid-independent hypertension.