Bruno Francou

TAC3 and TACR3 Defects Cause Hypothalamic Congenital Hypogonadotropic Hypogonadism in Humans

CONTEXT 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. OBJECTIVE 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. RESULTS We identified three unrelated patients with the same homozygous substitution in the TAC3 intron 3 acceptor splicing site (c.209-1G>C) and three siblings who bore a homozygous mutation in the TACR3 intron 2 acceptor splicing site (c.738-1G>A). 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. CONCLUSION 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.

Kisspeptin Restores Pulsatile LH Secretion in Patients with Neurokinin B Signaling Deficiencies: Physiological, Pathophysiological and Therapeutic Implications

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.

Non-syndromic congenital hypogonadotropic hypogonadism: clinical presentation and genotype–phenotype relationships

Congenital hypogonadotropic hypogonadism (CHH) results from abnormal gonadotropin secretion, and it is characterized by impaired pubertal development. CHH is caused by defective GNRH release, or by a gonadotrope cell dysfunction in the pituitary. Identification of genetic abnormalities related to CHH has provided major insights into the pathways critical for the development, maturation, and function of the reproductive axis. Mutations in five genes have been found specifically in Kallmanns syndrome, a disorder in which CHH is related to abnormal GNRH neuron ontogenesis and is associated with anosmia or hyposmia. In combined pituitary hormone deficiency or in complex syndromic CHH in which gonadotropin deficiency is either incidental or only one aspect of a more complex endocrine disorder or a non-endocrine disorder, other mutations affecting GNRH and/or gonadotropin secretion have been reported. Often, the CHH phenotype is tightly linked to an isolated deficiency of gonadotropin secretion. These patients, who have no associated signs or hormone deficiencies independent of the deficiency in gonadotropin and sex steroids, have isolated CHH. In some familial cases, they are due to genetic alterations affecting GNRH secretion (mutations in GNRH1, GPR54/KISS1R and TAC3 and TACR3) or the GNRH sensitivity of the gonadotropic cells (GNRHR). A minority of patients with Kallmanns syndrome or a syndromic form of CHH may also appear to have isolated CHH, but close clinical, familial, and genetic studies can reorient the diagnosis, which is important for genetic counseling in the context of assisted reproductive medicine. This review focuses on published cases of isolated CHH, its clinical and endocrine features, genetic causes, and genotype-phenotype relationships.

Normosmic Congenital Hypogonadotropic Hypogonadism Due to TAC3/TACR3 Mutations: Characterization of Neuroendocrine Phenotypes and Novel Mutations

Context TAC3/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. Objective To 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. Results From 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 (p<0.0001) higher mean FSH/LH ratio in 11 nCHH patients with TAC3/TACR3 biallelic mutations than in 47 nCHH patients with either biallelic mutations in KISS1R, GNRHR, or with no identified mutations and than in 50 Kallmann patients with mutations in KAL1, FGFR1 or PROK2/PROKR2. Three patients with TAC3/TACR3 biallelic mutations had an apulsatile LH profile but low-frequency alpha-subunit pulses. Pulsatile GnRH administration increased alpha-subunit pulsatile frequency and reduced the FSH/LH ratio. Conclusion The 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.

Two Families with Normosmic Congenital Hypogonadotropic Hypogonadism and Biallelic Mutations in KISS1R (KISS1 Receptor): Clinical Evaluation and Molecular Characterization of a Novel Mutation

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.

NR5A1 (SF-1) Mutations Are Not a Major Cause of Primary Ovarian Insufficiency

CONTEXT Primary ovarian insufficiency (POI) is a disorder affecting approximately 1% of women under the age of 40 years. NR5A1 (SF-1) mutations have been recently reported in association with POI. OBJECTIVE Our objective was to evaluate the frequency and functional impact of NR5A1 variants in POI. PATIENTS AND METHODS One hundred eighty patients diagnosed with idiopathic POI were screened for NR5A1 mutations and functional analysis was performed for the identified variants. The DNA-binding capacity of the variants was evaluated by means of EMSA, while their transcriptional activity was assessed using luciferase reporter assays. RESULTS Sequencing the NR5A1 gene revealed 4 missense variants in 3 patients. These patients were aged 20, 25, and 33 years at diagnosis and presented with secondary amenorrhea. None of them presented a syndromic form, although 2 had a familial history of POI. The functional analysis carried out for these missense variants showed no significant difference in DNA binding capacity or in transcriptional activity compared to wild-type NR5A1. CONCLUSIONS Our study in a large cohort of patients with POI showed the prevalence of NR5A1 mutations to be low (1.6%, upper 95% confidence interval 3.5%). Moreover, no functional impact was observed. Overall, in contrast with the initial report, our results exclude NR5A1 mutations as a major genetic cause of POI.

Genetic and Epigenetic Defects at the GNAS Locus Lead to Distinct Patterns of Skeletal Growth but Similar Early-Onset Obesity: DISTINCT PATTERNS OF FETAL AND POSTNATAL GROWTH IN GNAS-RELATED DISORDERS

Pseudohypoparathyroidism type 1A (PHP1A), pseudoPHP (PPHP), and PHP type 1B (PHP1B) are caused by maternal and paternal GNAS mutations and abnormal methylation at maternal GNAS promoter(s), respectively. Adult PHP1A patients are reportedly obese and short, whereas most PPHP patients are born small. In addition to parathyroid hormone (PTH) resistance, PHP1A and PHP1B patients may display early‐onset obesity. Because early‐onset and severe obesity and short stature are daily burdens for PHP1A patients, we aimed at improving knowledge on the contribution of the GNAS transcripts to fetal and postnatal growth and fat storage. Through an international collaboration, we collected growth and weight data from birth until adulthood for 306 PHP1A/PPHP and 220 PHP1B patients. PHP1A/PPHP patients were smaller at birth than healthy controls, especially PPHP (length Z‐score: PHP1A –1.1 ± 1.8; PPHP –3.0 ± 1.5). Short stature is observed in 64% and 59% of adult PHP1A and PPHP patients. PHP1B patients displayed early postnatal overgrowth (height Z‐score at 1 year: 2.2 ± 1.3 and 1.3 ± 1.5 in autosomal dominant and sporadic PHP1B) followed by a gradual decrease in growth velocity resulting in normal adult height (Z‐score for both: –0.4 ± 1.1). Early‐onset obesity characterizes GNAS alterations and is associated with significant overweight and obesity in adults (bodey mass index [BMI] Z‐score: 1.4 ± 2.6, 2.1 ± 2.0, and 1.4 ± 1.9 in PPHP, PHP1A, and PHP1B, respectively), indicating that reduced Gsα expression is a contributing factor. The growth impairment in PHP1A/PPHP may be due to Gsα haploinsufficiency in the growth plates; the paternal XLαs transcript likely contributes to prenatal growth; for all disease variants, a reduced pubertal growth spurt may be due to accelerated growth plate closure. Consequently, early diagnosis and close follow‐up is needed in patients with GNAS defects to screen and intervene in case of early‐onset obesity and decreased growth velocity.

Pharmacology of Hormone Replacement Therapy in Menopause

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).