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Summary Background GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome is caused by aberrant expression of the GIP receptor in adrenal lesions. The bilateral nature of this disease suggests germline genetic predisposition. We aimed to identify the genetic driver event responsible for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Methods We conducted a multicentre, retrospective, cohort study at endocrine hospitals and university hospitals in France, Canada, Italy, Greece, Belgium, and the Netherlands. We collected blood and adrenal samples from patients who had undergone unilateral or bilateral adrenalectomy for GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome. Adrenal samples from patients with primary bilateral macronodular adrenal hyperplasia who had undergone an adrenalectomy for overt or mild Cushing's syndrome without evidence of food-dependent cortisol production and those with GIP-dependent unilateral adrenocortical adenomas were used as control groups. We performed whole genome, whole exome, and targeted next generation sequencing, and copy number analyses of blood and adrenal DNA from patients with familial or sporadic disease. We performed RNA sequencing on adrenal samples and functional analyses of the identified genetic defect in the human adrenocortical cell line H295R. Findings 17 patients with GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome were studied. The median age of patients was 43·3 (95% CI 38·8–47·8) years and most patients (15 [88%]) were women. We identified germline heterozygous pathogenic or most likely pathogenic variants in the KDM1A gene in all 17 patients. We also identified a recurrent deletion in the short p arm of chromosome 1 harboring the KDM1A locus in adrenal lesions of these patients. None of the 29 patients in the control groups had KDM1A germline or somatic alterations. Concomitant genetic inactivation of both KDM1A alleles resulted in loss of KDM1A expression in adrenal lesions. Global gene expression analysis showed GIP receptor upregulation with a log2 fold change of 7·99 (95% CI 7·34–8·66; p=4·4 × 10−125), and differential regulation of several other G protein-coupled receptors in GIP-dependent primary bilateral macronodular hyperplasia samples compared with control samples. In vitro pharmacological inhibition and inactivation of KDM1A by CRISPR-Cas9 genome editing resulted in an increase of GIP receptor transcripts and protein in human adrenocortical H295R cells. Interpretation We propose that GIP-dependent primary bilateral macronodular adrenal hyperplasia with Cushing's syndrome results from a two-hit inactivation of KDM1A, consistent with the tumour suppressor gene model of tumorigenesis. Genetic testing and counselling should be offered to these patients and their relatives. Funding Agence Nationale de la Recherche, Fondation du Grand defi Pierre Lavoie, and the French National Cancer Institute.

International audience; Androgen receptor gene (AR) mutations are responsible for androgen insensitivity syndrome (AIS) presenting with a clinical phenotype that ranges from gynaecomastia and/ or infertility in mild AIS (MAIS) to complete testicular feminisation in complete AIS. We report a novel AR gene mutation in two unrelated adult patients with MAIS and we studied its functional impact using 3D modelling. Patient 1, referred for infertility, presented with gynaecomastia, mild hypospadias and bilateral testicular hypotrophy contrasting with high testosterone levels, an elevated FSH, an elevated androgen sensitivity index (ASI) and oligoasthenoteratospermia. In vitro fertilisation and intracytoplasmic sperm injection resulted in a successful twin pregnancy. Patient 2 referred for a decrease in athletic performance had surgically treated gynaecomastia, oligoasthenospermia, high testosterone levels and an elevated ASI. Despite his impaired spermogram, he fathered two children without assisted reproductive technology. AR gene sequencing in the two patients revealed a common novel missense mutation, Ala699Thr, in exon 4 within the ligand-binding domain. 3D modelling studies showed that this mutation may impact dimer stability upon ligand binding or may affect allosteric changes upon dimerisation. This study illustrates the value of structural analysis for the functional study of mutations and expands the database of AR gene mutations.

STUDY QUESTION: Does the phenotype of women with normosmic congenital hypogonadotrophic hypogonadism (nCHH) and pituitary resistance to GnRH caused by biallelic mutations in the GnRH receptor (GNRHR) (nCHH/bi-GNRHR) differ from that of women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Women with nCHH/bi-GNRHR have variable pubertal development but nearly all have primary amenorrhea and an exaggerated LH response to GnRH stimulation, similar to that seen in women with PCOS. WHAT IS KNOWN ALREADY: Women with nCHH/bi-GNRHR are very rare and their phenotype at diagnosis is not always adequately documented. The results of gonadotrophin stimulation by acute GnRH challenge test and ovarian features have not been directly compared between these patients and women with PCOS. STUDY DESIGN, SIZE, DURATION: We describe the phenotypic spectrum at nCHH/bi-GNRHR diagnosis in a series of 12 women. Their reproductive characteristics and acute responses to GnRH were compared to those of 70 women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients and controls (healthy female volunteers aged over 18 years) were enrolled in a single French referral centre. Evaluation included clinical and hormonal studies, pelvic ultrasonography and GnRH challenge test. We also functionally characterized two missense GNRHR mutations found in two new consanguineous families. MAIN RESULTS AND THE ROLE OF CHANCE: Breast development was highly variable at nCHH/bi-GNRHR diagnosis, but only one patient had undeveloped breasts. Primary amenorrhea was present in all but two cases. In untreated nCHH/bi-GNRHR patients, uterine height (UH) correlated (P = 0.01) with the circulating estradiol level and was shorter than in 23 nulliparous post-pubertal age-matched controls (P < 0.0001) and than in 15 teenagers with PCOS under 20-years-old (P < 0.0001) in which PCOS was revealed by primary amenorrhea or primary-secondary amenorrhea. Unexpectedly, the stimulated LH peak response in nCHH/bi-GNRHR patients was variable, and often normal or exaggerated. Interestingly, the LH peak response was similar to that seen in the PCOS patients, but the latter women had significantly larger mean ovarian volume (P < 0.001) and uterine length (P < 0.001) and higher mean estradiol (P < 0.001), anti-Müllerian hormone (AMH) (P = 0.02) and inhibin-B (P < 0.001) levels. In the two new consaguineous families, the affected nCHH/bi-GNRHR women carried the T269M or Y290F GNRHR missense mutation in the homozygous state. In vitro analysis of GnRHR showed complete or partial loss-of-function of the T269M and Y290F mutants compared to their wildtype counterpart. LIMITATIONS, REASONS FOR CAUTION: The number of nCHH/bi-GNRHR patients reported here is small. As this disorder is very rare, an international study would be necessary to recruit a larger cohort and consolidate the phenotypic spectrum observed here. WIDER IMPLICATIONS OF THE FINDINGS: In teenagers and young women with primary amenorrhea, significant breast and uterine development does not rule out CHH caused by biallelic GNRHR mutations. In rare patients with PCOS presenting with primary amenorrhea and a mild phenotype, the similar exaggerated pituitary LH responses to GnRH in PCOS and nCHH/bi-GNRHR patients could lead to diagnostic errors. This challenge test should therefore not be recommended. As indicated by consensus and guidelines, careful analysis of clinical presentation and measurements of testosterone circulating levels remain the basis of PCOS diagnosis. Also, analysis of ovarian volume, UH and of inhibin-B, AMH, estradiol and androgen circulating levels could help to distinguish between mild PCOS and nCHH/bi-GNRHR. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the French National Research Agency (ANR) grant ANR-09-GENO-017 KALGENOPATH, France; and by the Italian Ministry of Education, University and Research (MIUR) grant PRIN 2012227FLF_004, Italy. The authors declare no conflict of interest.

Purpose To describe abnormalities in choroidal and retinal vasculature associated with Val30Met familial transthyretin amyloidosis (V30M-FTA) using fluorescein and indocyanine green (ICG) angiography. Design Prospective, cross-sectional study. Methods This study was conducted at the French National Reference Center for FTA. We included 18 consecutive genetically confirmed V30M-FTA patients (36 eyes) who underwent complete neurologic examination, including staging with polyneuropathy disability (PND) score, and complete ophthalmic evaluation, including staging of intraocular amyloid deposits and fluorescein and ICG angiograms (ICG-A). The grading of choroidal and retinal angiopathy, and their association with neurologic functional impairment, were the main outcome measures. Results Eleven men and 7 women, mean age 61.6 ± 12.1 years, were included. Retinal amyloid angiopathy (RAA) was detected in 24 eyes (92%) of 13 patients, with microaneurysms, retinal hemorrhages, and retinal ischemia of variable extent. Three patients (5 eyes) had neovascular glaucoma and 2 (2 eyes) had preretinal neovascularization. ICG-A indicated choroidal amyloid angiopathy (CAA) in all patients, with 3 distinct patterns—diffuse (9/18 patients), focal (5/18 patients), or punctiform (4/18 patients)—based on the extent of late hypercyanescence along the choroidal arteries. PND scores were significantly higher in patients with diffuse CAA (firework pattern) compared to those with limited CAA (focal and punctiform patterns) (2.89 vs 1.78, P = .045). Conclusion RAA is a frequent and severe complication of V30M-FTA that may lead to anterior and posterior segment neovascularization. CAA was detected in all patients, with a late hypercyanescent delineation of the choroidal arterial vasculature, which was more extensive with increased disease severity.

CONTEXT: Although endogenous estradiol, generally considered as the female hormone, has been little investigated in men, it could play a role in men's health, mortality in particular. The influence of estrogen receptors (ER) genetic polymorphisms on this relationship has never been studied. DESIGN AND PARTICIPANTS: The Three-City cohort study included (1999-2001) 3650 men >/=65 years who were followed for mortality over 12 years. At baseline, total estradiol (tE2) was measured and ER genotyped in a random subsample of 472 men without hormonal treatment. Free estradiol (fE2) was estimated using Vermeulen and Sodergard algorithms. MAIN OUTCOME: Mortality data were obtained from death certificates. We used inverse probability weighted Cox models to examine the association of estradiol with all-cause and cause-specific mortality and its interaction with ER genetic polymorphisms. RESULTS: 183 men died over the follow-up (cardiovascular disease (CVD), n=44; cancer, n=57; other causes, n=82). After adjustment, there was a quadratic relationship of all-cause mortality with tE2 and fE2 (p-quadratic=0.04 and 0.05, respectively), with higher mortality for the top and bottom tertiles compared to the middle one. These associations were stronger for CVD mortality (p-quadratic=0.01 and 0.02 for tE2 and fE2, respectively) and disappeared for cancer mortality. There was no evidence of an interaction of estradiol with any ER polymorphisms on all-cause mortality. CONCLUSION: In elderly men, we showed a nonlinear association of tE2 and fE2 with all-cause mortality. These quadratic relationships were stronger for CVD mortality and did not exist for cancer mortality. ER genetic polymorphisms did not modify this association. This article is protected by copyright. All rights reserved.

International audience; OBJECTIVES: Previous studies have reported controversial findings regarding the association of testosterone with mortality in older men. This heterogeneity might be partially explained by comorbidities and the presence of metabolic syndrome, as well as differential associations according to causes of death.METHODS: We used data from a random subsample of the Three-City study, in which hormone levels were measured in 338 men ≥65 years without metabolic syndrome who were followed-up for 12 years. Vital status was determined for all participants from different sources. We used inverse-probability-weighted Cox regression to estimate the hazard ratios (HRs) of cause-specific mortality and 95% confidence intervals (CIs).RESULTS: Over the follow-up period, 130 men died (30 from cardiovascular disease, 45 from cancer, 55 from other causes). The association of testosterone with mortality showed significant heterogeneity across causes of death (p=0.027 and p=0.022 for total and bioavailable testosterone, respectively). Higher testosterone levels were associated with increased cardiovascular mortality (HR for 1-standard deviation increase, 1.86; 95% CI, 1.28 to 2.71 and 1.50; 95% CI, 1.04 to 2.17 for total and bioavailable testosterone, respectively). By contrast, there were no significant associations of testosterone with mortality from cancer and other causes.CONCLUSIONS: Our data suggest that the association of testosterone with mortality in men without metabolic syndrome might be differential according to the cause of death. These findings may partially explain the heterogeneity across studies on the relationship between testosterone levels and mortality.

Objective To evaluate the prevalence of FMR1 premutations and X chromosome cytogenetic abnormalities in a large cohort of Tunisian women with premature ovarian failure (POF). Patients and methods The cohort consisted of 127 Tunisian women with POF referred by endocrinologists and gynecologists for genetic investigation in the context of idiopathic POF and altered hormonal profiles. Clinical information concerning the reproductive function in the family, previous hormonal measurements and/or possible fertility treatment were collected. Karyotype, FISH analyses, FMR1 and FMR2 testing were performed for all patients. Results Fifteen patients (11.81%) presented structural or numerical X chromosomal abnormalities. Moreover, we detected in 12 patients (10.71%) a high level of X mosaicism. Analysis of FMR1 gene in the 100 patients without X chromosomal abnormalities showed that five percent of the patients carried a FMR1 premutation allele. On the other hand, the FMR2 screening did not reveal any deletion. Conclusion Our study confirms the major role of X chromosome abnormalities in POF and highlights the importance of karyotype analyses and FMR1 screening. These investigations provide valuable information for diagnosis and genetic counseling for these women who still have a 5% chance of spontaneous conception.

We report the prenatal detection of a de novo unbalanced complex chromosomal rearrangement (CCR), in a fetus with growth delay and bilateral cataracts. Standard karyotype and FISH analyses on amniotic fluid revealed a complex de novo translocation, resulting in a 46,XY,t(1;12;14)(q42;q14;q32) karyotype. CGH-array showed a significant deletion of 387 kb at 12q14.3, at a distance of only 200–700 kb from the breakpoint at 12q14, which encompassed the HMGA2 gene and occurred de novo . Although 12q14 microdeletions are associated with growth delay in several reports in the literature, we present here the smallest deletion prenatally detected, and we detail the clinical description of the fetus. The correlation between cataracts and this complex genotype is puzzling. Among the genes disrupted by the breakpoint in 12q14, GRIP1 has been associated with abnormal eye development in mice, including lens degeneration. Interestingly, HMGA2 is expressed in the mouse's developing lens, and its expression is decreased in lens of elderly humans, correlated with the severity of lens opacity. In this report, we refine the link between HMGA2 loss of function and growth delay during prenatal development. We also discuss the correlation between cataracts and genotype in this unbalanced CCR case of unexpected complexity.

Accurate typing of amyloidosis is still a major issue for pathologists and clinicians. Besides clinical data and immunohistochemistry, the histologic distribution of amyloid could represent a useful tool to prevent typing errors, such as the misdiagnosis of hereditary and senile amyloidosis as light chain-related amyloidosis (AL). Minor salivary gland biopsy (MSGB) is a widely performed procedure for amyloidosis diagnosis and typing. In the largest clinicopathologic series of amyloid-containing MSGB specimens to date, we investigated for the first time whether amyloidosis subtypes can be distinguished according to their pattern of salivary amyloid deposition. The histologic distribution and semiquantification of amyloid within salivary tissue were thoroughly reassessed for each case using Congo red-fluorescence. Clinical data were retrospectively collected. The cohort included 92 patients with amyloid-containing minor salivary gland biopsies. The type of amyloidosis was AL in 51 patients (55.4%), non-V30M mutant ATTR in 10 (10.9%), V30M mutant ATTR in 8 (8.7%), serum amyloid A-derived amyloidosis (AA) in 6 (6.5%), wild-type ATTR in 4 (4.3%), gelsolin in 3 (3.3%), and unclassified in 10 (10.9%). Amyloid was more abundant in AL and AA compared with ATTR amyloidosis, because of more extensive basement membranes and vascular deposits. Conversely, non-V30M mutant ATTR and wt-ATTR were strongly associated with peculiar amyloid nodules located in close contact with salivary excretory ducts, with a specificity of 91.7%. In conclusion, our study suggests for the first time that MSGB, in addition to its high sensitivity for amyloidosis diagnosis, is a simple and effective tool for the recognition of ATTR amyloidosis.

Previous studies have shown controversial results about the role of testosterone in all-cause mortality in elderly men. We hypothesized that metabolic syndrome (MetS) could partly explain this discrepancy. We therefore examined the association of all-cause mortality with total and bioavailable testosterone, taking into account the MetS. We used data from the Three-City Cohort (3C) study with 12-year follow-up. The 3C study included 3650 men aged >65 years in three French cities. Hormone was measured in a random subsample of 444 men, and MetS was determined as stated by the International Diabetes Federation criteria. We used inverse-probability–weighted Cox regression to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs). Of 444 men included in the analysis, 106 (23.9%) had MetS at baseline, and 166 died over the follow-up. There was a significant interaction between testosterone level and MetS for all-cause mortality (P = 0.002 and P = 0.008 for total and bioavailable testosterone, respectively). Among men with MetS, a decrease in one standard deviation of testosterone was associated with higher mortality risk [HR 1.78 (95% CI 1.13 to 2.78) and HR 1.83 (95% CI 1.17 to 2.86) for total and bioavailable testosterone, respectively]. By contrast, there was no association of testosterone with mortality risk among men without MetS. Our results suggest that MetS modifies the association between testosterone and mortality in older men. If confirmed, these findings could contribute to improve risk stratification and better manage the health of older men., We evaluated the association of testosterone with mortality in men and found metabolic syndrome (MetS) was an effect modifier of this relationship. Low testosterone increased mortality among men with MetS.

Background Recently discovered mutations of NR3C1 gene, encoding for the GR, in patients with glucocorticoid resistance and bilateral adrenal incidentalomas prompted us to investigate whether GR mutations might be associated with adrenal hyperplasia. Objective The multicenter French Clinical Research Program (Muta-GR) was set up to determine the prevalence of GR mutations and polymorphisms in patients harboring bilateral adrenal incidentalomas associated with hypertension and/or biological hypercortisolism without clinical Cushing’s signs. Results One hundred patients were included in whom NR3C1 sequencing revealed five original heterozygous GR mutations that impaired GR signaling in vitro. Mutated patients presented with mild glucocorticoid resistance defined as elevated urinary free cortisol (1.7 ± 0.7 vs 0.9 ± 0.8 upper limit of normal range, P = 0.006), incomplete 1 mg dexamethasone suppression test without suppressed 8-AM adrenocorticotrophin levels (30.9 ± 31.2 vs 16.2 ± 17.5 pg/mL) compared to the non-mutated patients. Potassium and aldosterone levels were lower in mutated patients (3.6 ± 0.2 vs 4.1 ± 0.5 mmol/L, P = 0.01, and 17.3 ± 9.9 vs 98.6 ± 115.4 pg/mL, P = 0.0011, respectively) without elevated renin levels, consistent with pseudohypermineralocorticism. Ex vivo characterization of mutated patients’ fibroblasts demonstrated GR haploinsufficiency as revealed by below-normal glucocorticoid induction of FKBP5 gene expression. There was no association between GR polymorphisms and adrenal hyperplasia in this cohort, except an over-representation of BclI polymorphism. Conclusion The 5% prevalence of heterozygous NR3C1 mutations discovered in our series is higher than initially thought and encourages GR mutation screening in patients with adrenal incidentalomas to unambiguously differentiate from Cushing’s states and to optimize personalized follow-up.

Congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) are rare, related diseases that prevent normal pubertal development and cause infertility in affected men and women. However, the infertility carries a good prognosis as increasing numbers of patients with CHH/KS are now able to have children through medically assisted procreation. These are genetic diseases that can be transmitted to patients’ offspring. Importantly, patients and their families should be informed of this risk and given genetic counseling. CHH and KS are phenotypically and genetically heterogeneous diseases in which the risk of transmission largely depends on the gene(s) responsible(s). Inheritance may be classically Mendelian yet more complex; oligogenic modes of transmission have also been described. The prevalence of oligogenicity has risen dramatically since the advent of massively parallel next-generation sequencing (NGS) in which tens, hundreds or thousands of genes are sequenced at the same time. NGS is medically and economically more efficient and more rapid than traditional Sanger sequencing and is increasingly being used in medical practice. Thus, it seems plausible that oligogenic forms of CHH/KS will be increasingly identified making genetic counseling even more complex. In this context, the main challenge will be to differentiate true oligogenism from situations when several rare variants that do not have a clear phenotypic effect are identified by chance. This review aims to summarize the genetics of CHH/KS and to discuss the challenges of oligogenic transmission and also its role in incomplete penetrance and variable expressivity in a perspective of genetic counseling.

Pituitary adenomas are benign intracranial neoplasms that can result in morbidity owing to local invasion and/or excessive or deficient hormone production. The prevalence of symptomatic pituitary adenomas is approximately 1:1,000 in the general population. The vast majority of these tumours occur sporadically and are not part of syndromic disorders. However, germline mutations in genes known to predispose individuals to familial pituitary adenomas are found in a few patients with sporadic pituitary adenomas. Mutations in AIP (encoding aryl-hydrocarbon receptor-interacting protein) are the most frequently observed germline mutations. The prevalence of these mutations in patients with sporadic pituitary adenomas is ∼4%, but can increase to 8-20% in young adults with macroadenomas or gigantism, and also in children. Germline mutations in MEN1 (encoding menin) result in multiple endocrine neoplasia type 1 and are found in very young patients with isolated sporadic pituitary adenomas, which highlights the importance of the chromosome 11q13 locus in pituitary tumorigenesis. In this Review, we describe the clinical features of patients with sporadic pituitary adenomas that are associated with AIP or MEN1 mutations, and discuss the molecular mechanisms that might be involved in pituitary adenoma tumorigenesis. We also discuss genetic screening of patients with sporadic pituitary adenomas and investigations of relatives of these patients who also have the same genetic mutations.

Alagille syndrome (ALGS) is a complex, multisystem disease associated with mutations in the JAG1 gene. In the liver, ALGS is characterized by paucity of intrahepatic bile ducts. Gene dosage analysis performed on a large, central regenerative nodule with preserved interlobular bile ducts of 2 unrelated ALGS patients, and on surrounding cirrhotic and ductopenic liver parenchyma, showed in both cases complete JAG1 heterozygous deletion in the regenerative nodule and the ductopenic liver, with no differences in gene dosage. Thus, JAG1 mosaicism and differential haploinsufficiency do not explain the presence of bile ducts in centrally located regenerative nodules.

We aimed to investigate the impact of endogenous estradiol (E2) on dementia and to evaluate the contribution of vascular risk factors and inflammatory and blood coagulation markers to this association.Using data from a French population-based prospective study (the Three-City Study) including 5,644 postmenopausal women aged 65 years or older, we investigated the association of endogenous total-E2 and bioavailable-E2 and total-testosterone with the 4-year incidence of all-cause dementia. We further focused on the role of dementia and cardiovascular risk factors as well as inflammation (C-reactive protein, fibrinogen) and hypercoagulability (fibrin d-dimers, thrombin generation) in these associations. We used a case-cohort design consisting of a random subcohort of 562 women not using hormone therapy and 132 incident dementia cases.Adjusted Cox proportional hazards models showed a J-shaped relationship between total-E2 and risk of dementia (p = 0.001). Total-E2 values in the lower and upper quartiles were associated with an increased dementia risk (adjusted hazard ratio [HR] [95% confidence interval] = 2.2 [1.1-4.5] and HR = 2.4 [1.2-5.2], respectively). Importantly, the risk associated with higher E2 levels was dramatically increased in women with diabetes compared with nondiabetic women (adjusted HR associated with the upper E2 quartile = 14.2 [1.60-123] and HR = 3.4 [0.1-147], respectively, p interaction0.05). Similar results were found for bioavailable-E2. Adjustment for inflammatory and blood coagulation markers did not modify our results. No significant association was found for total-testosterone.High E2 level is an independent predictor of incident dementia, particularly in postmenopausal women with diabetes.

International audience; GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Germline mutations of the AIP (aryl-hydrocarbon receptor interacting protein) gene are associated with a predisposition to pituitary adenomas. Such mutations are found in about half of patients with familial acromegaly, but penetrance is incomplete.We studied the prevalence of germline AIP mutations in a large cohort of patients with apparently sporadic pituitary adenomas.A total of 443 patients with pituitary adenomas of all histotypes, who had no familial history of pituitary adenomas or multiple endocrine neoplasia and who were examined at Bicêtre University Hospital, a tertiary referral center, between 2007 and 2010, were enrolled in this prospective study.The entire coding sequence of the AIP gene was screened for germline mutations. A subgroup of patients were screened for large deletions or duplications of the AIP and MEN1 genes by multiplex ligation-dependent probe amplification.AIP mutations were detected in 16 (3.6%) of the 443 patients, comprising six of 148 patients with acromegaly (4.1%), six of 132 patients with prolactinomas (4.5%), one of 113 patients with nonfunctioning adenomas (0.9%), three of 44 patients with corticotropic adenomas (6.8%), and none of the six patients with thyrotropic adenomas. This is the first report of an AIP mutation leading to a truncated protein in a patient with Cushing's disease. Patients with AIP mutation were younger at diagnosis (24.1 vs. 42.8 yr) and had predominantly macroadenoma (12 of 16). No mutations were found in patients diagnosed after age 40 yr, whereas the prevalence before this age was 7.2% (16 of 222). Studies of seven of the AIP-mutated patients' families showed that one asymptomatic parent carried the same mutation in each case.This large prospective cohort study confirms the very low prevalence of germline AIP mutations in patients with apparently sporadic pituitary adenomas. We propose to limit AIP testing to patients diagnosed before age 40 yr with apparently sporadic large pituitary adenomas, especially GH- or PRL-secreting adenomas.

background: Kallmann syndrome (KS) is a genetic disorder associating pubertal failure with congenitally absent or impaired sense of smell. KS is related to defective neuronal development affecting both the migration of olfactory nerve endings and GnRH neurons. The discovery of several genetic mutations responsible for KS led to the identification of signaling pathways involved in these processes, but the mutations so far identified account for only 30% of cases of KS. Here, we attempted to identify new genes responsible for KS by using a pan-genomic approach. methods: From a cohort of 120 KS patients, we selected 48 propositi with no mutations in known KS genes. They were analyzed by comparative genomic hybridization array, using Agilent 105K oligonucleotide chips with a mean resolution of 50 kb. results: One propositus was found to have a heterozygous deletion of 213 kb at locus 7q21.11, confirmed by real-time qPCR, deleting 11 of the 17 SEMA3A exons. This deletion cosegregated in the propositus’ family with the KS phenotype, that was transmitted in autosomal dominant fashion and was not associated with other neurological or non-neurological clinical disorders. SEMA3A codes for semaphorin 3A, a protein that interacts with neuropilins. Mice lacking semaphorin 3A expression have been showed to have a Kallmann-like phenotype. conclusions: SEMA3A is therefore a new gene whose loss-of-function is involved in KS. These findings validate the specific role of semaphorin 3A in the development of the olfactory system and in neuronal control of puberty in humans.

Small supernumerary marker chromosomes (sSMC) are structurally abnormal chromosomes that cannot be unambiguously identified by conventional banding cytogenetics. This study describes four patients with sSMC in relation with infertility. Patient 1 had primary infertility. His brother, fertile, carried the same sSMC (patient 2). Patient 3 presented polycystic ovary syndrome and patient 4 primary ovarian insufficiency. Cytogenetic studies, array comparative genomic hybridization (CGH) and sperm analyses were compared with cases previously reported. sSMC corresponded to the 15q11.2 region (patients 1 and 2), the centromeric chromosome 15 region (patient 3) and the 21p11.2 region (patient 4). Array CGH showed 3.6-Mb gain for patients 1 and 2 and 0.266-Mb gain for patient 4. Sperm fluorescent in-situ hybridization analyses found ratios of 0.37 and 0.30 of sperm nuclei with sSMC(15) for patients 1 and 2, respectively (P < 0.001). An increase of sperm nuclei with disomy X, Y and 18 was noted for patient 1 compared with control and patient 2 (P < 0.001). Among the genes mapped in the unbalanced chromosomal regions, POTE B and BAGE are related to the testis and ovary, respectively. The implication of sSMC in infertility could be due to duplication, but also to mechanical effects perturbing meiosis.

Serpentine fibula-polycystic kidney syndrome (SFPKS) is a rare disorder characterized by the association of craniofacial anomalies, radiological findings (wormian bones, elongated and bowed fibulae), polycystic kidneys, and normal intelligence. SFPKS shares many similarities with Hajdu-Cheney syndrome (HCS). We and others recently showed that truncating mutations in the last exon of NOTCH2 cause HCS. Here, we identify by Sanger sequencing two different heterozygous truncating mutations in the last exon of NOTCH2 in two unrelated patients with SFPKS. In one family, we show that the mutation occurred de novo. These findings demonstrate that SFPKS and HCS are both conditions caused by NOTCH2 mutations.

Gonadotropin-releasing hormone (GnRH) is a crucial neurohormone regulating puberty and reproduction in non primate mammals, monkeys and humans. This neuropeptide is released in synchronized pulses from the nerve endings of several hundred GnRH neurons into the hypophyseal portal system to stimulate the biosynthesis and secretion of LH and FSH from pituitary gonadotrophs. These two dimeric glycoprotein hormones in turn travel through the circulation to the gonads, stimulating the synthesis and secretion of male and female sex steroid hormones and gametogenesis in both genders. Congenital hypogonadotropic hypogonadism (CHH) is a clinical syndrome that results from a decrease in gonadotropin secretion and is characterized by a complete or partial lack of pubertal development. CHH is caused mainly by defective GnRH production or release by the hypothalamus or by defective GnRH-receptor function in the pituitary. The syndromes of GnRH deficiency, either with anosmia or hyposmia, termed Kallmann Syndrome (KS), or with a normal sense of smell, called normosmic non syndromic CHH, (nCHH) are important disease models that have revealed respectively much about the developmental and functional abnormalities that can befall the GnRH neurons. For more than three decades, GNRH1 was an obvious candidate gene for nCHH in human beings. Indeed, in the natural hpg mouse model discovered in 1977, the hypogonadotropic hypogonadal phenotype, secondary to a hypothalamic GnRH deficiency, was caused by a homozygous deletion within the ortholog Gnrh1 gene. In 2009, homozygous inactivating mutations in the GNRH1 gene causing hypothalamic nCHH patients were at last reported in one female and two young male subjects, validating definitively the pivotal role of GnRH in human pubertal development and reproduction.

Background GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Methods We performed molecular analyses on 14 adrenal samples obtained from 12 patients with overt GIP-dependent Cushing's syndrome and from one patient with GIP-dependent aldosteronism. Results Nascent RNA FISH showed that GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified by CGH-array somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had 19q duplication only. These duplications were further confirmed by DNA FISH. Among the 5 genes encompassed in the smallest region of overlap, only GIPR was over expressed. We demonstrated in vitro that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in HEK293 T and H295R cells. Conclusion Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Missense loss-of-function mutations in TAC3 and TACR3, the genes encoding neurokinin B and its receptor NK3R, respectively, were recently discovered in kindreds with nonsyndromic normosmic congenital hypogonadotropic hypogonadism (CHH), thus identifying a fundamental role of this pathway in the human gonadotrope axis.The objective of the study was to investigate the consequences on gonadotrope axis of TAC3 deletion and TACR3 truncation in adult patients with normosmic complete CHH.We identified three unrelated patients with the same homozygous substitution in the TAC3 intron 3 acceptor splicing site (c.209-1GC) and three siblings who bore a homozygous mutation in the TACR3 intron 2 acceptor splicing site (c.738-1GA). We demonstrated that these two mutations, respectively, deleted neurokinin B and truncated its receptor NK3R. We found in three patients with TAC3 mutation originating from Congo and Haiti a founding event in a more distant ancestor by means of haplotype analysis. We calculated that time to this common ancestor was approximately 21 generations. In several patients we observed a dissociation between the very low LH and normal or nearly normal FSH levels, this gonadotropin responding excessively to the GnRH challenge test. This particular hormonal profile, suggests the possibility of a specific neuroendocrine impairment in patients with alteration of neurokinin B signaling. Finally, in these patients, pulsatile GnRH administration normalized circulating sex steroids, LH release, and restored fertility in one subject.Our data demonstrate the hypothalamic origin of the gonadotropin deficiency in these genetic forms of normosmic CHH. Neurokinin B and NK3R therefore both play a crucial role in hypothalamic GnRH release in humans.

We investigated whether mutations in the gene encoding gonadotropin-releasing hormone 1 (GNRH1) might be responsible for idiopathic hypogonadotropic hypogonadism (IHH) in humans. We identified a homozygous GNRH1 frameshift mutation, an insertion of an adenine at nucleotide position 18 (c.18-19insA), in the sequence encoding the N-terminal region of the signal peptide-containing protein precursor of gonadotropin-releasing hormone (prepro-GnRH) in a teenage brother and sister, who had normosmic IHH. Their unaffected parents and a sibling who was tested were heterozygous. This mutation results in an aberrant peptide lacking the conserved GnRH decapeptide sequence, as shown by the absence of immunoreactive GnRH when expressed in vitro. This isolated autosomal recessive GnRH deficiency, reversed by pulsatile GnRH administration, shows the pivotal role of GnRH in human reproduction.

Progressive familial intrahepatic cholestasis (PFIC) and to a lesser extent, Alagille syndrome, often lead to end-stage liver disease during childhood. We report our experience of DNA-based prenatal diagnosis of PFIC1-3 and Alagille syndrome.Four molecular antenatal diagnoses were performed in 3 PFIC families and 17 in 11 Alagille syndrome families. DNA was isolated from chorionic villus or cultured amniocyte samples from women, without pregnancy complications.All four foetuses with a family history of PFIC1, 2, or 3 were heterozygous for an ATP8B1, ABCB11, or ABCB4 mutation and pregnancies were continued. Three of the infants were healthy after birth, and 1 premature infant, who had an ABCB4 mutation, experienced transient neonatal cholestasis. Among the families with a history of de novo JAG1 mutation, none of the foetuses was mutated, versus 40% of those with a history of familial mutation. Of 4 pregnant women with a JAG1-mutated foetus, 3 cut short their pregnancy and 1 gave birth to a child with overt Alagille syndrome.Molecular antenatal diagnosis of PFIC1-3 and Alagille syndrome is reliable because clinical outcome after birth corresponded to molecular foetal data.

Study question What is the exact prevalence of Kisspeptin Receptor (KISS1R) mutations in the population of patients with normosmic congenital hypogonadotrophic hypogonadism (nCHH) by comparison with other genes, involved in gonadotrophin-releasing hormone (GnRH) release or action? Summary answer KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases and were less prevalent than GnRH Receptor (GNRHR) mutations. What is known already The respective prevalence of each of the genetic causes of nCHH is unclear. Large series of patients are very rare and suffer from heterogeneity of the population of CHH studied. Study design, size, duration Patients with nCHH were consecutively enrolled in a single French referral centre and were gradually tested for KISS1R between January 2006 and April 2015. Participants/materials, setting, methods A total of 603 patients with nCHH (399 men and 204 women) were diagnosed at the Bicetre Hospital and underwent KISS1R analysis. The GNRHR, tachykinin receptor 3 (TACR3), gonadotrophin-releasing hormone 1 (GNRH1), tachykinin 3 (TAC3) and KISS1 genes were also sequenced. Functional characterization of KISS1R mutations included a study of signal transduction using a reporter gene (serum response element-luciferase (SRE-Luc) involved in the mitogen-activated protein (MAP) kinase pathway. Main results and the role of chance We detected 15 KISS1R variants (10 novel), in 12 of the 603 patients (2.0%, 95% CI [0.9-3.1]. KISS1R mutations were less prevalent than GNRHR (4.7%) and TACR3 (2.6%) mutations but more prevalent than GNRH1 (1.5%), TAC3 (1.0%) and KISS1 (0%) mutations. KISS1R mutants were present in the biallelic state in 8 of the 12 patients concerned. Among 5 men with biallelic KISS1R mutations, 4 had either micropenis or cryptorchidism. In vitro analysis of the 5 new variants present in the biallelic state (C95W, Y103*, C115W, P176R and A287E) showed a loss of function. Limitations, reasons for caution The prevalence of TACR3, GNRH1, TAC3 and KISS1 mutations was calculated from a smaller number of nCHH patients than KISS1R and GNRHR. This should prompt caution concerning the reported prevalence of mutations in these four genes. Wider implications of the findings We show that KISS1R mutants are responsible for the nCHH phenotype in only a small minority of cases. Together, the genes analysed here were mutated in fewer than 15% of patients, suggesting a role of other genes in nCHH. The presence of cryptorchidism and/or micropenis in the majority of men with biallelic KISS1R mutations strongly suggests that this gene is essential for prenatal GnRH secretion. Study funding, competing interests This work was supported in part by grants from Paris-Sud University (Bonus Qualite Recherche, and Attractivite grants) to J.B., French Ministry of Health, Hospital Clinical Research Program on Rare Diseases. Assistance Publique Hopitaux de Paris, Programme Hospitalier de Recherche Clinique (PHRC # P081212 HYPOPROTEO) to J.Y. C.P. was supported by student fellowships 'Annee Recherche' from Agence Regionale de Sante Provence Alpes Cotes d'Azur. The authors have nothing to disclose.

A 28 year old male with a background of abdominal tuberculosis living in Tekhand slum district of Delhi presented with two and half year history of recurrent loose stools following tingling sensation in the upper and lower limbs, as well as weakness in the lower limbs. He also reports erectile dysfunction and postural hypotension. He had lost weight : kilos. Upon examination the patient has lost all sensory perception in the lower limbs up to the upper thigh with severe wasting and dull lower limb reflexes. There was pes cavus and a high stepping gait. Investigations revealed persistent circumferential thickening in the abdomen on CT. Sural nerve biopsy showed inflammation with demyelination and amyloidosis. There was a loss of parasympathic/sympathetic reactivity. Bone marrow biopsy revealed amyloid deposits compatible with secondary AA with increase in anti-LKM antibodies. Endoscopy revealed increased inflammation in the lamina propia. Familial amyloid polyneuropathy was suspected, and TTR Sanger sequencing revealed detection of a missense mutation of Val30Ala at a heterozygous state. Transthyretin familial amyloid polyneuropathy (TTR-FAP) is a rare systemic disease due to endoneurial amyloid deposit, and is one of the most severe disabling hereditary peripheral neuropathies in adults. It has been described clinicopathologically in 1952 by Corino Andrade in north of Portugal and genetically in 1984 by Maria Joao Saraiva. TTR-FAP has long been considered an endemic disease in Portugal, Sweden and Japan, although sporadic cases have also been reported in Europe initially in France and the UK. There is genetic heterogeneity in TTR-FAP, with Val30Met being the most frequent genotype in the world. Val30Ala is a rare mutation, which have previously been described in the Chinese population. In this manuscript, we document to our knowledge the first genetically proven case of FAP in India, a populated country of more than 1.2 billion inhabitants, and a detailed description of the patient case, as well as family investigations are provided. The patient is awaiting for anti-amyloid treatment. Written informed consent for publication of their clinical details and/or clinical images was obtained from the patient /parent/guardian/ relative of the patient.

International audience; BACKGROUND: Pituitary adenomas are rare in children and adolescents. The response of macroprolactinomas to dopamine agonists (DA) in this age group has been less extensively studied than in adults. OBJECTIVE: We retrospectively analyzed data on a large cohort of young patients with macroprolactinomas. PATIENTS AND METHODS: Patients aged younger than 20 years at macroprolactinoma diagnosis and seen in three tertiary referral centers between 1983 and 2013 were studied by analyzing their clinical and genetic (AIP and MEN1) characteristics. Hormonal and tumoral responses to DA were analyzed, and the patients' status at their last visit, after a mean (+/-SD) follow-up of 8.2 +/- 5.8 years, was assessed. RESULTS: The cohort comprised 77 patients (26 males, 51 females). Mean age at diagnosis was 16.1 +/- 2.5 years (range, 4.5-20 y). In both sexes, the most frequent revealing symptom was a pubertal disorder (49%), followed by visual problems (24%) and growth retardation (24%). Basal prolactin (PRL) levels and maximal tumor diameter were significantly higher in boys than in girls (7168 ng/mL, 202-40 168 vs 1433 ng/mL, 115-20 000, P = .002; and 33 +/- 14 mm, 15-64 vs 19 +/- 9 mm; 10-50, P \textless .001, respectively). PRL levels normalized in 74% of the patients treated with DA. A mutation of AIP or MEN1 was found in 14% of the patients. Factors associated with resistance to DA were young age, higher PRL levels, larger volume, and the presence of a MEN1 (but not an AIP) mutation. CONCLUSION: Macroprolactinomas are rare below the age of 20 years, mainly occurring in girls and during adolescence. Like adults, young patients are very sensitive to DA, which should therefore be considered the first-line treatment. DA resistance is associated with a higher PRL level and larger tumor size, both parameters being closely linked together. About 14% of these young patients have an AIP or MEN1 mutation, this latter being an independent predictor of DA resistance.

We report on four patients with severe polyneuropathy associated with intestinal pseudoobstruction (MNGIE). Three patients presented characteristic supranuclear ophthalmoplegia, and hyperdense signals on T2 weighted cerebral MRI and dystrophic mitochondria in Schwann cells and in endothelial cells in nerve biopsy specimens. Two of these patients had a Charcot-Marie-Tooth (CMT) presentation. All three were heterozygous for a recessively transmitted double substitution in the TP gene: Glu286Lys/Glu289Ala, Asp156Gly/Leu177Pro and Glu289Ala/Gly387Asp. The fourth patient, who was the only patient of this series with an affected sib, had no oculomotor manifestations, nor T2 hyperdense signals on brain MRI, and no TP gene mutation and or morphological abnormalities of mitochondria on electron microscopic examination. He was the only patient of this series with an affected sib. The three patients with the full MNGIE syndrome died before the age of 30 years. Detailed results of nerve pathology show that severe axonal degeneration is associated with segmental abnormalities of the myelin sheath in this syndrome which appears genetically heterogeneous. Our findings suggest that only ophthalmoplegia and hyperdense signals on cerebral MRI are directly related to the mitochondriopathy.

The new members of the tumour necrosis factor (TNF) receptor-ligand family, receptor activator of nuclear factor-kappaB ligand (RANKL) and its receptor RANK, play a crucial role in osteoclast differentiation and activation. An increased expression of RANKL and/or RANK may be involved in the excessive bone resorption observed in multiple myeloma (MM). We used immunohistochemistry to study RANK and RANKL expression in bone marrow (BM) biopsies obtained at diagnosis in 15 MM patients, six patients with monoclonal gammopathy of undetermined significance (MGUS) and 10 normal BM biopsies. Plasma cells were not labelled with anti-RANKL or anti-RANK antibodies. In all biopsies, RANKL was expressed in endosteal bone surface, around vessels and in cells characterized by cytoplasmic expansions. These last cells did not express CD45 and were vimentin positive, corresponding to bone marrow stromal cells. Numerous stromal cells expressed RANKL in MM and MGUS specimens, with a greater expression in MM than in MGUS. Very few cells were stained with anti-RANKL in normal BM specimens. With the anti-RANK antibody, small mononuclear cells in the bone microenvironment were positive and were identified as erythroblast cells. In conclusion, we showed that RANKL was expressed in reticular stromal cells, with a greater intensity in myeloma specimens. These results suggest that RANKL overexpressed by bone marrow stromal cells may contribute to the high rate of bone resorption observed in MM.

In 1926, Roussy and Levy described a large family whose members manifested an early onset dominantly inherited gait ataxia, pes cavus, and areflexia, which was eventually associated with distal muscle atrophy, postural tremor, and minor sensory loss. Slow nerve conduction and demyelination of nerve fibers with onion bulb formations in nerve biopsy specimens led to the Roussy-Levy syndrome (RLS) being considered a variant of demyelinating Charcot-Marie-Tooth disease (CMT-1). In the present article, we report on the long-term follow-up, on nerve biopsy findings, and on the underlying molecular genetic defect in members of the original family studied by Roussy and Levy. All patients were able to walk during their seventh decade of life. Morphologically, a chronic demyelinating neuropathy with the remarkable aspects of a focally hypertrophic myelin sheath and major loss of myelinated fibers was observed in nerve biopsy specimens of 3 members of this family. Molecular genetic testing identified a previously unknown heterozygous missense point mutation which yielded an Asn131Lys substitution in the extracellular domain of the myelin protein zero (P0). These findings show that the Roussy-Levy family belongs to the CMT-1B subtype and has original morphological and genetic features.

The existence of recessive transmission of Dejerine-Sottas disease, a severe demyelinating neuropathy of childhood, has been questioned, because only heterozygous mutations of the myelin proteins P0 or PMP22 genes have been identified in virtually all patients with this phenotype. We report on a family with 3 affected children with this phenotype, born to clinically and electrophysiologically unaffected parents. All 3 children carried a previously unknown homozygous missense point mutation (Arg157Trp) of the PMP22 gene. The parents were heterozygous for the same mutation. These findings demonstrate the occurrence of recessive transmission in this setting. Ann Neurol 1999;45:518–522

Background: Except after neck surgery, hypoparathyroidism is a rare disease caused by defects in genes involved in parathyroid gland development (TBX1/22q11.2 del, GCMB, GATA3, TBCE) or function [calcium sensing receptor (CASR), GNA11, PTH], or the autoimmune polyglandular syndrome type 1 (AIRE). Approximately 90% of sporadic cases and 30% of familial cases of isolated hypoparathyroidism remain unexplained. Recurrent missense mutations in AP2S1, a calcium-sensing receptor regulator, have been recently identified in familial hyperparathyroidism. Aim: The aim of the study was to investigate AP2S1 as a putative hypoparathyroidism-causing gene. Methods: Sequencing analysis and quantitative genomic PCR of the AP2S1 gene in a large cohort of 10 index cases (from nine families) and 50 sporadic cases affected with isolated hypoparathyroidism were investigated. Results and Conclusions: None of the 60 patients presented with nucleotidic changes or copy number variation in the AP2S1 gene, thereby excluding AP2S1 def...

Objective The objective of this study was to examine the association of plasma estradiol and testosterone with risk for dementia in elderly men. Methods Within the population based Three-City study, including 3650 men age 65 years and older, a case–cohort design was set up after 4-years of follow-up. Baseline plasma levels of total 17-β estradiol (Total-E2), total testosterone (total-T) and bioavailable testosterone (bio-T) were measured for all cases of incident dementia (n = 105) and for a random sample of the cohort (n = 413). Cox regression models were used to estimate multivariate steroid sex hormone-associated hazard ratios (HR) and 95% confidence intervals of dementia. Results There was a reverse J-shaped relationship between total-T and risk for dementia ( P = .007). Compared with the median tertile, the HRs associated with total-T in the lower and upper tertile were increased (HR, 2.33; P = .026; HR, 1.9, P = .126; respectively). Low bio-T was associated with a greater risk for dementia (HR for one standard deviation of decreasing log(bio-T), 1.29; 95% confidence interval, 1.03–1.62). An interaction was found between bio-T and age ( P P = .044). Risk for dementia associated with low bio-T was greater in older men (80 years or older) than in younger men (younger than 80 years; HR, 3.11; P = .011 vs. HR, 1.07, P = .715, respectively) and in men with high level of education compared with those with low level of education (HR, 2.32; P = .0002 vs. HR, 0.95; P = .790, respectively). No significant association was found between Total-E2 and dementia. Conclusions Low levels of testosterone are associated with a risk for dementia in elderly men. The association between low bio-T and dementia may be more relevant to men 80 years or older and men with a high level of education.

Alagille syndrome is a rare congenital multisystem disorder that may involve heart disease and pulmonary or peripheral artery stenosis. We report the clinical and radiological presentation of five adult patients with Alagille syndrome, hypertension and renal artery stenosis. All had systolic hypertension and a narrowing of the abdominal aorta, corresponding to a secondary midaortic syndrome. Renovascular disease progressed during follow-up, with increases in blood pressure, decreases in glomerular filtration rate and/or kidney atrophy. A literature review identified several anecdotal reports of Alagille syndrome associated with hypertension, renal artery stenosis and/or midaortic syndrome. We discuss this condition, focusing on diagnosis, differential diagnosis, associated conditions and management. Cardiologists, nephrologists and radiologists should be aware of this rare cause of renovascular hypertension and of the need for clinical, biological and echographic follow-up.

International audience; Context:Pituitary stalk interruption represents a frequent feature of congenital hypopituitarism, but only rare cases have been assigned to a known genetic cause.Objective:Using a candidate gene approach, we tested several genes as potential causes of hypopituitarism with pituitary stalk interruption. We hypothesized that ectopic posterior pituitary may be a consequence of defective neuronal axon projections along the pituitary stalk or defective angiogenesis of hypophyseal portal circulation. Considering the role of the prokineticin 2 pathway in angiogenesis and neuronal migration, we screened PROK2 and PROKR2 genes.Design:PROK2 and PROKR2 and all genes previously known to be involved in hypopituitarism with pituitary stalk interruption (LHX4, HESX1, OTX2, and SOX3) were screened in 72 index cases with pituitary stalk interruption syndrome from the GENHYPOPIT database. In vitro studies were performed to assess the functional consequences of allelic variants.Results:We identified two heterozygous PROKR2 mutations (p.Leu173Arg and p.Arg85His) previously reported in isolated hypogonadotroph hypogonadism and a novel PROKR2 variant (p.Ala51Thr) that, in contrast with both other mutations, did not impair receptor signaling activity. Three allelic variants of HESX1 were identified: the heterozygous p.Phe156Ser and the homozygous p.Arg109X mutations were functionally deleterious, whereas p.Ser67Thr was found as a rare allelic variant in association with p.Arg85His PROKR2 mutation in the same patient.Conclusions:We report PROKR2 variants in congenital hypopituitarism with pituitary stalk interruption, suggesting a potential role of the prokineticin pathway in pituitary development.

Menopause represents the final stage of the continuous process of reproductive aging in a woman’s life, marking the end of her fertility. According to the World Health Organization (WHO), the natural menopause is defined as the permanent cessation of menstruation resulting from the loss of ovarian follicular activity (WHO Report, 1996). Preceded by endocrine and menstrual cycle changes described as “menopausal transition”, natural menopause occurs at an average age of approximately 51 years, although a high inter-individual variability is supported by results from epidemiological studies. However, occurrence of menopause outside the estimated normal age interval (45-55 years) is associated with increased morbidity, either when a late or on the contrary, a premature cessation of menstruation appears. A late menopause implies a longer exposure to estrogens and a possible increased risk for breast (Colditz, 1993; Kelsey & Bernstein, 1996) and endometrial cancer (Dossus et al., 2010; McPherson et al., 1996) or for venous thromboembolism (Simon et al., 2006). On the other hand, women entering menopause earlier are facing a hypo-estrogenic state for a longer period compared to women undergoing normal menopause. That is the case for about 1% of women, which are confronted with the diagnosis of primary ovarian insufficiency (POI). POI is defined by the presence of amenorrhea associated with elevated follicle-stimulating hormone (FSH) levels in the menopausal range in women younger than 40 years (Bachelot et al., 2009). Women facing a premature cessation of the ovarian function were shown to be at increased risk for premature death, cardiovascular disease, neurologic disease, mood disorders, osteoporosis or psychosexual dysfunction (Shuster et al., 2010). As the main rationale for these disorders was linked to hormonal changes, maintaining a certain level of ovarian steroids for a given period of time arose as an essential condition for conserving life quality in women (Wilson, [1966]). Accentuated by the increasing life span, researches related to menopause and its treatment have provided scientific community with an increased body of data during the last decades. However, different aspects regarding the benefit/risk balance or the ideal doses and routes of administration of hormone replacement therapy (HRT) in menopausal women remain uncertain (Grodstein et al., 1997; Rossouw et al., 2002).

Background Despite evidence that estrogens may be involved in atherothrombosis, the role of endogenous sex steroid hormones in ischemic arterial disease among postmenopausal women remains uncertain. Methods and Results In the Three‐City prospective cohort study of subjects (n=9294) >65 years of age, we investigated the association of total 17β‐estradiol, bioavailable 17β‐estradiol, and total testosterone with the 4‐year incidence of ischemic arterial disease among postmenopausal women who did not use any hormone therapy. We designed a case–cohort study including a random sample of 537 subjects and 106 incident cases of first cardiovascular events. Weighted Cox proportional‐hazards models with age as the time scale were used to estimate hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) for ischemic arterial disease by a 1–standard deviation increase in sex steroid hormones. In univariate analysis, HR of ischemic arterial disease was positively and significantly associated with both total and bioavailable estradiol levels. These associations remained significant after adjustment for traditional cardiovascular risk factors, including body mass index, diabetes, hypercholesterolemia, hypertension, and smoking status (HR: 1.42, 95% CI: 1.12–1.79, P P P =0.01; and adjusted HR: 1.50, 95% CI: 1.11–2.04, P P =0.08; and HR: 1.32, 95% CI: 0.94–1.84, P =0.11, respectively). By contrast, no significant association was found between total testosterone and ischemic arterial disease in both univariate and adjusted analyses. Conclusions High plasma level of endogenous estradiol emerges as a new predictor of ischemic arterial disease in older postmenopausal women. ( J Am Heart Assoc . 2012;1:e001388 doi : 10.1161/JAHA.112.001388 .)

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.

Pulsatile gonadotropin-releasing hormone (GnRH) is crucial to normal reproductive function and abnormalities in pulse frequency give rise to reproductive dysfunction. Kisspeptin and neurokinin B (NKB), neuropeptides secreted by the same neuronal population in the ventral hypothalamus, have emerged recently as critical central regulators of GnRH and thus gonadotropin secretion. Patients with mutations resulting in loss of signaling by either of these neuroendocrine peptides fail to advance through puberty but the mechanisms mediating this remain unresolved. We report here that continuous kisspeptin infusion restores gonadotropin pulsatility in patients with loss-of-function mutations in NKB (TAC3) or its receptor (TAC3R), indicating that kisspeptin on its own is sufficient to stimulate pulsatile GnRH secretion. Moreover, our findings suggest that NKB action is proximal to kisspeptin in the reproductive neuroendocrine cascade regulating GnRH secretion, and may act as an autocrine modulator of kisspeptin secretion. The ability of continuous kisspeptin infusion to induce pulsatile gonadotropin secretion further indicates that GnRH neurons are able to set up pulsatile secretion in the absence of pulsatile exogenous kisspeptin.

Backgrounds: Charcot–Marie–Tooth neuropathy has been reported to be associated with renal diseases, mostly focal segmental glomerulosclerosis (FSGS). However, the common mechanisms underlying the neuropathy and FSGS remain unknown. Mutations in INF2 were recently identified in patients with autosomal dominant FSGS. INF2 encodes a formin protein that interacts with the Rho-GTPase CDC42 and myelin and lymphocyte protein (MAL) that are implicated in essential steps of myelination and myelin maintenance. We therefore hypothesized that INF2 may be responsible for cases of Charcot–Marie–Tooth neuropathy associated with FSGS., Methods: We performed direct genotyping of INF2 in 16 index patients with Charcot–Marie–Tooth neuropathy and FSGS who did not have a mutation in PMP22 or MPZ, encoding peripheral myelin protein 22 and myelin protein zero, respectively. Histologic and functional studies were also conducted., Methods: We identified nine new heterozygous mutations in 12 of the 16 index patients (75%), all located in exons 2 and 3, encoding the diaphanous-inhibitory domain of INF2. Patients presented with an intermediate form of Charcot–Marie–Tooth neuropathy as well as a glomerulopathy with FSGS on kidney biopsy. Immunohistochemical analysis revealed strong INF2 expression in Schwann-cell cytoplasm and podocytes. Moreover, we demonstrated that INF2 colocalizes and interacts with MAL in Schwann cells. The INF2 mutants perturbed the INF2–MAL–CDC42 pathway, resulting in cytoskeleton disorganization, enhanced INF2 binding to CDC42 and mislocalization of INF2, MAL, and CDC42., Conclusions: INF2 mutations appear to cause many cases of FSGS-associated Charcot–Marie–Tooth neuropathy, showing that INF2 is involved in a disease affecting both the kidney glomerulus and the peripheral nervous system. These findings provide new insights into the pathophysiological mechanisms linking formin proteins to podocyte and Schwann-cell function., Funded by the Agence Nationale de la Recherche and others.Supported by grants from the Association pour l'Utilisation du Rein Artificiel (to Dr. Antignac), Association Française contre les Myopathies (ANR-08-GENOPAT-017-01, to Dr. Antignac), Agence Nationale de la Recherche (PodoNet project number ANR-07-E-RARE-011-01 in the ERA-Net Consortium [JTC2007], to Dr. Antignac, and ANR-06-MRAR-024-01, to Dr. Leguern), Fondation pour la Recherche Médicale (project number DMP 2010-11-20-386, to Dr. Antignac, and doctoral funding, to Dr. Boyer), Association des Malades du Syndrome Néphrotique (to Dr. Mollet), Fonds de la Recherche en Santé du Québec (Fellowship Training Award to Dr. Benoit), and Ministerio de Ciencia e Innovación (BFU2009-07886 and CONSOLIDER COAT CSD2009-00016, to Dr. Alonso).

Congenital hypogonadotropic hypogonadism (CHH) causes pubertal failure and infertility in both women and men due to partial or total secretory failure of the two pituitary gonadotropins lutropin (LH) and follitropin (FSH) during periods of physiological activation of the gonadotropic axis. Men and women with CHH frequently seek treatment for infertility after hypogonadism therapy. Some etiologies, such as autosomal dominant or X-linked Kallmann syndrome, raise the question of hereditary transmission, leading to increasing demands for genetic counseling and monitoring of medically assisted pregnancies. Diagnosis and treatment of newborn boys is, therefore, becoming an increasingly important issue. In male individuals with complete forms of CHH, the antenatal and neonatal gonadotropin deficit leads to formation of a micropenis and cryptorchidism, which could undermine future sexual and reproductive functions. Standard treatments, usually started after the age of puberty, often only partially correct the genital abnormalities and spermatogenesis. The aim of this Review is to examine the possible additional benefits of neonatal gonadotropin therapy in male patients with CHH. Encouraging results of neonatal therapy, together with a few reports of prepubertal treatment, support the use of this novel therapeutic strategy aimed at improving sexual and reproductive functions in adulthood.

Objective To evaluate the degree of E 2 deficiency in male congenital hypogonadotropic hypogonadism (CHH), and its response to different hormonal treatments. Design Retrospective and prospective studies. Setting Academic institution. Patient(s) Untreated or treated CHH, healthy men, untreated men with Klinefelter syndrome (KS). Intervention(s) Serum sex hormone-binding globulin (SHBG) and total E 2 (TE2) as well as bioavailable (BE2) and free (FE2) levels were measured and determined. Main Outcome Measure(s) Total, bioavailable, and free testosterone, TE2, BE2, FE2 were compared in normal men, untreated and treated CHH and in untreated KS. Result(s) TE2, BE2, and FE2 levels were very significantly lower in untreated patients with CHH (n = 91) than in controls (n = 63) and in patients with KS (n = 45). The TE2 correlated positively with serum total T in patients with CHH. The TE2 also correlated very positively with serum LH in the combined population of patients with CHH and healthy men, suggesting that low E 2 levels in CHH are due to severe LH-driven T deficiency. All fractions of circulating E 2 were very significantly higher in patients with CHH receiving T enanthate (n = 101) or the FSH–hCG combination (n = 88) than in untreated patients with CHH. Contrary to dihydrotestosterone (DHT), both T enanthate and combined FSH-hCG therapy significantly and prospectively increased TE2 levels in patients with CHH. Conclusion(s) Contrary to KS, the male hypogonadism observed in CHH is associated with profound E 2 deficiency, which can be overcome by aromatizable androgen or combined gonadotropin therapy.

International audience; Oral, but not transdermal, estrogen therapy increases the risk of venous thromboembolism (VTE) in women who are past menopause. Data from the Estrogen and Thromboembolism Risk (ESTHER) study were used to investigate the effects of the genetic polymorphism of NFE2L2 rs6721961, which may impair Nrf2-dependent hepatic conjugation of estrogen metabolites. As compared with nonusers, the odds ratio (OR) for VTE in current users of oral estrogens was 2.5 (95% confidence interval (CI): 1.3-4.8) in patients with wild-type NFE2L2 and 17.9 (95% CI: 3.7-85.7) in those with the polymorphism (interaction, P = 0.01).

The neuropeptide gonadotropin-releasing hormone (GnRH) plays a key regulatory role in mammalian reproduction. It is synthesized by hypothalamic neurons and released from nerve endings into the portal circulation. After binding to membrane GnRH type 1 receptors on gonadotropic cells of the anterior pituitary, it stimulates the synthesis and release of luteinizing hormone and follicle-stimulating hormone. These two peptides travel through the general circulation to the gonads, where they stimulate the synthesis and secretion of sex steroid hormones and trigger gametogenesis. The discovery in 1977 of a hypogonadal mouse lacking GnRH (hpg mice) and, in 1986, that the gnrh1 gene was deleted in this mouse, suggested that GNRH1 mutations might also cause human congenital idiopathic (or isolated) hypogonadotropic hypogonadism. This was finally demonstrated in 2009.

International audience; Context: Both biallelic and monoallelic mutations in PROK2 or PROKR2 have been found in Kallmann syndrome (KS). Objective: The objective of the study was to compare the phenotypes of KS patients harboring monoallelic and biallelic mutations in these genes. Design and Patients: We studied clinical and endocrine features that reflect the functioning of the pituitary-gonadal axis, and the nonreproductive phenotype, in 55 adult KS patients (42 men and 13 women), of whom 41 had monoallelic mutations and 14 biallelic mutations in PROK2 or PROKR2. Results: Biallelic mutations were associated with more frequent cryptorchidism (70% vs. 34%, P < 0.05) and microphallus (90% vs. 28%, P < 0.001) and lower mean testicular volume (1.2 +/- 0.4 vs. 4.5 +/- 6.0 ml; P < 0.01) in male patients. Likewise, the testosterone level as well as the basal FSH level and peak LH level under GnRH-stimulation were lower in males with biallelic mutations (0.2 +/- 0.1 vs. 0.7 +/- 0.8 ng/ml; P = 0.05, 0.3 +/- 0.1 vs. 1.8 +/- 3.0 IU/liter; P < 0.05, and 0.8 +/- 0.8 vs. 5.2 +/- 5.5 IU/liter; P < 0.05, respectively). Nonreproductive, nonolfactory anomalies were rare in both sexes and were never found in patients with biallelic mutations. The mean body mass index of the patients (23.9 +/- 4.2 kg/m(2) in males and 26.3 +/- 6.6 kg/m(2) in females) did not differ significantly from that of gender-, age-, and treatment-matched KS individuals who did not carry a mutation in PROK2 or PROKR2. Finally, circadian cortisol levels evaluated in five patients, including one with biallelic PROKR2 mutations, were normal in all cases. Conclusion: Male patients carrying biallelic mutations in PROK2 or PROKR2 have a less variable and on average a more severe reproductive phenotype than patients carrying monoallelic mutations in these genes. Nonreproductive, nonolfactory clinical anomalies associated with KS seem to be restricted to patients with monoallelic mutations.

International audience; Hormone therapy (HT) increases venous thromboembolism (VTE) risk among postmenopausal women. Data on the influence of steroids receptors polymorphisms on this association remain scarce. Since progesterone receptor (hPR) is expressed in human veins and specific progestogens increase VTE risk, we investigated the impact of the functional +331G/A hPR polymorphism on the association of VTE with HT. Using the data of the ESTHER study, we showed that ORs for VTE in current users of progesterone or progestins were not significantly different by hPR+331G/A genotype status. hPR polymorphism appears not to have a significant effect on VTE risk related to HT.

ObjectivePremature ovarian failure (POF) encompasses a heterogeneous spectrum of conditions, with phenotypic variability among patients. The etiology of POF remains unknown in most cases. We performed a global phenotyping of POF women with the aim of better orienting attempts at an etiological diagnosis.Design and methodsWe performed a mixed retrospective and prospective study of clinical, biological, histological, morphological, and genetic data relating to 357 consecutive POF patients between 1997 and 2008. The study was conducted at a reproductive endocrinology referral center.ResultsSeventy-six percent of the patients presented with normal puberty and secondary amenorrhea. Family history was present in 14% of the patients, clinical and/or biological autoimmunity in 14.3%. Fifty-six women had a fluctuating form of POF. The presence of follicles was suggested at ultrasonography in 50% of the patients, and observed in 29% at histology; the negative predictive value of the presence of follicles at ultrasonography was 77%. Bone mineral density alterations were found in 58% of the women. Eight patients had X chromosomal abnormalities other than Turner's syndrome, eight other patients evidenced FMR1 pre-mutation. Two other patients had autoimmune polyendocrine syndrome type 2 and 1.ConclusionA genetic cause of POF was identified in 25 patients, i.e. 7% of the whole cohort. POF etiology remains most often undiscovered. Novel strategies of POF phenotyping are in such content mandatory to improve the rate of POF patients for whom etiology is identified.