Mutation screening of SEMA3A and SEMA7A in patients with congenital hypogonadotropic hypogonadism.

Background:Congenital hypogonadotropic hypogonadism (HH), a rare disorder characterized by absent, partial, or delayed puberty, can be caused by the lack or deficient number of hypothalamic gonadotropin-releasing hormone (GnRH) neurons. SEMA3A was recently implicated in the etiology of the disorder, and Sema7A-deficient mice have a reduced number of GnRH neurons in their brains.Methods:SEMA3A and SEMA7A were screened by Sanger sequencing in altogether 50 Finnish HH patients (34 with Kallmann syndrome (KS; HH with hyposmia/anosmia) and 16 with normosmic HH (nHH)). In 20 patients, mutation(s) had already been found in genes known to be implicated in congenital HH.Results:Three heterozygous variants (c.458A>G (p.Asn153Ser), c.1253A>G (p.Asn418Ser), and c.1303G>A (p.Val435Ile)) were found in SEMA3A in three KS patients, two of which also had a mutation in FGFR1. Two rare heterozygous variants (c.442C>T (p.Arg148Trp) and c.1421G>A (p.Arg474Gln)) in SEMA7A were found in one male nHH patient with a previously identified KISS1R nonsense variant and one male KS patient with a previously identified mutation in KAL1, respectively.Conclusion:Our results suggest that heterozygous missense variants in SEMA3A and SEMA7A may modify the phenotype of KS but most likely are not alone sufficient to cause the disorder.

Responsiveness to a physiological regimen of GnRH therapy and relation to genotype in women with isolated hypogonadotropic hypogonadism.

CONTEXT: Isolated hypogonadotropic hypogonadism (IHH) is caused by defective GnRH secretion or action resulting in absent or incomplete pubertal development and infertility. Most women with IHH ovulate with physiological GnRH replacement, implicating GnRH deficiency as the etiology. However, a subset does not respond normally, suggesting the presence of defects at the pituitary or ovary. OBJECTIVES: The objective of the study was to unmask pituitary or ovarian defects in IHH women using a physiological regimen of GnRH replacement, relating these responses to genes known to cause IHH. DESIGN, SETTING, AND SUBJECTS: This study is a retrospective analysis of 37 IHH women treated with iv pulsatile GnRH (75 ng/kg per bolus). MAIN OUTCOME MEASURES: Serum gonadotropin and sex steroid levels were measured, and 14 genes implicated in IHH were sequenced. RESULTS: During their first cycle of GnRH replacement, normal cycles were recreated in 60% (22 of 37) of IHH women. Thirty percent of women (12 of 37) demonstrated an attenuated gonadotropin response, indicating pituitary resistance, and 10% (3 of 37) exhibited an exaggerated FSH response, consistent with ovarian resistance. Mutations in CHD7, FGFR1, KAL1, TAC3, and TACR3 were documented in IHH women with normal cycles, whereas mutations were identified in GNRHR, PROKR2, and FGFR1 in those with pituitary resistance. Women with ovarian resistance were mutation negative. CONCLUSIONS: Although physiological replacement with GnRH recreates normal menstrual cycle dynamics in most IHH women, hypogonadotropic responses in the first week of treatment identify a subset of women with pituitary dysfunction, only some of whom have mutations in GNRHR. IHH women with hypergonadotropic responses to GnRH replacement, consistent with an additional ovarian defect, did not have mutations in genes known to cause IHH, similar to our findings in a subset of IHH men with evidence of an additional testicular defect.

Insulin and NPY pathways and the control of GnRH function and puberty onset.

Energy balance exerts a critical influence on reproductive function. Leptin and insulin are among the metabolic factors signaling the nutritional status of an individual to the hypothalamus, and their role in the overall modulation of the activity of GnRH neurons is increasingly recognized. As such, they participate to a more generalized phenomenon: the signaling of peripheral metabolic changes to the central nervous system. The physiological importance that the interactions occurring between peripheral metabolic factors and the central nervous system bear for the control of food intake is increasingly recognized. The central mechanisms implicated are the focus of attention of very many research groups worldwide. We review here the experimental data that suggest that similar mechanisms are at play for the metabolic control of the neuroendocrine reproductive function. It is appearing that metabolic signals are integrated at the levels of first-order neurons equipped with the proper receptors, ant that these neurons send their signals towards hypothalamic GnRH neurons which constitute the integrative element of this network.

Complex genetics in idiopathic hypogonadotropic hypogonadism.

Idiopathic hypogonadotropic hypogonadism (IHH) is an important human disease model. Investigations of the genetics of IHH have facilitated insights into critical pathways regulating sexual maturation and fertility. IHH has been traditionally considered a monogenic disorder. This model holds that a single gene defect is responsible for the disease in each patient. In the case of IHH, 30% of cases are explained by mutations in one of eleven genes. In recent years, several lines of evidence have challenged the monogenic paradigm in IHH. First, disease-associated mutations display striking incomplete penetrance and variable expressivity within and across IHH families. Second, each locus is responsible for only a small percentage of cases. Third, more than one disease-associated mutation seems to be segregating in some families with IHH, and their combined or separate presence in individuals accounts for the variability in disease severity. Finally, IHH is not strictly a congenital and life-long disorder; occasionally it manifests itself during adulthood (adult-onset IHH); in other cases, the disease is not permanent, as evidenced by normal activity of the hypothalamic-pituitary-gonadal axis after discontinuation of treatment in adulthood (IHH reversal). Together, these observations suggest that IHH is not strictly a monogenic mendelian disease, as previously thought. Rather, it is emerging as a digenic, and potentially oligogenic disease, in which hormonal and/or environmental factors may critically influence genetic predisposition and clinical course. Future investigations of IHH should characterize the extent of the involvement of multiple genes in disease pathogenesis, and elucidate the contributions of epigenetic factors.

Endometriosis: alternative methods of medical treatment.

Endometriosis is an inflammatory estrogen-dependent disease defined by the presence of endometrial glands and stroma at extrauterine sites. The main purpose of endometriosis management is alleviating pain associated to the disease. This can be achieved surgically or medically, although in most women a combination of both treatments is required. Long-term medical treatment is usually needed in most women. Unfortunately, in most cases, pain symptoms recur between 6 months and 12 months once treatment is stopped. The authors conducted a literature search for English original articles, related to new medical treatments of endometriosis in humans, including articles published in PubMed, Medline, and the Cochrane Library. Keywords included "endometriosis" matched with "medical treatment", "new treatment", "GnRH antagonists", "Aromatase inhibitors", "selective progesterone receptor modulators", "anti-TNF α", and "anti-angiogenic factors". Hormonal treatments currently available are effective in the relief of pain associated to endometriosis. Among new hormonal drugs, association to aromatase inhibitors could be effective in the treatment of women who do not respond to conventional therapies. GnRH antagonists are expected to be as effective as GnRH agonists, but with easier administration (oral). There is a need to find effective treatments that do not block the ovarian function. For this purpose, antiangiogenic factors could be important components of endometriosis therapy in the future. Upcoming researches and controlled clinical trials should focus on these drugs.

Differential Gene Expression between Fall- and Spring-Run Chinook Salmon Assessed by Long Serial Analysis of Gene Expression

Of all Pacific salmonids, Chinook salmon Oncorhynchus tshawytscha display the greatest variability in return times to freshwater. The molecular mechanisms of these differential return times have not been well described. Current methods, such as long serial analysis of gene expression (LongSAGE) and microarrays, allow gene expression to be analyzed for thousands of genes simultaneously. To investigate whether differential gene expression is observed between fall- and spring-run Chinook salmon from California's Central Valley, LongSAGE libraries were constructed. Three libraries containing between 25,512 and 29,372 sequenced tags (21 base pairs/tag) were generated using messenger RNA from the brains of adult Chinook salmon returning in fall and spring and from one ocean-caught Chinook salmon. Tags were annotated to genes using complementary DNA libraries from Atlantic salmon Salmo salar and rainbow trout O. mykiss. Differentially expressed genes, as estimated by differences in the number of sequence tags, were found in all pairwise comparisons of libraries (freshwater versus saltwater = 40 genes; fall versus spring = 11 genes: and spawning versus nonspawning = 51 genes). The gene for ependymin, an extracellular glycoprotein involved in behavioral plasticity in fish, exhibited the most differential expression among the three groupings. Reverse transcription polymerase chain reaction analysis verified the differential expression of ependymin between the fall- and spring-run samples. These LongSAGE libraries, the first reported for Chinook salmon, provide a window of the transcriptional changes during Chinook salmon return migration to freshwater and spawning and increase the amount of expressed sequence data.

An ancient founder mutation in PROKR2 impairs human reproduction.

Congenital gonadotropin-releasing hormone (GnRH) deficiency manifests as absent or incomplete sexual maturation and infertility. Although the disease exhibits marked locus and allelic heterogeneity, with the causal mutations being both rare and private, one causal mutation in the prokineticin receptor, PROKR2 L173R, appears unusually prevalent among GnRH-deficient patients of diverse geographic and ethnic origins. To track the genetic ancestry of PROKR2 L173R, haplotype mapping was performed in 22 unrelated patients with GnRH deficiency carrying L173R and their 30 first-degree relatives. The mutation's age was estimated using a haplotype-decay model. Thirteen subjects were informative and in all of them the mutation was present on the same ~123 kb haplotype whose population frequency is ≤10%. Thus, PROKR2 L173R represents a founder mutation whose age is estimated at approximately 9000 years. Inheritance of PROKR2 L173R-associated GnRH deficiency was complex with highly variable penetrance among carriers, influenced by additional mutations in the other PROKR2 allele (recessive inheritance) or another gene (digenicity). The paradoxical identification of an ancient founder mutation that impairs reproduction has intriguing implications for the inheritance mechanisms of PROKR2 L173R-associated GnRH deficiency and for the relevant processes of evolutionary selection, including potential selective advantages of mutation carriers in genes affecting reproduction.

When genetic load does not correlate with phenotypic spectrum: lessons from the GnRH receptor (GNRHR).

CONTEXT: A broad spectrum of GnRH-deficient phenotypes has been identified in individuals with both mono- and biallelic GNRHR mutations. OBJECTIVE: The objective of the study was to determine the correlation between the severity of the reproductive phenotype(s) and the number and functional severity of rare sequence variants in GNRHR. SUBJECTS: Eight hundred sixty-three probands with different forms of GnRH deficiency, 46 family members and 422 controls were screened for GNRHR mutations. The 70 subjects (32 patients and 38 family members) harboring mutations were divided into four groups (G1-G4) based on the functional severity of the mutations (complete or partial loss of function) and the number of affected alleles (monoallelic or biallelic) with mutations, and these classes were mapped on their clinical phenotypes. RESULTS: The prevalence of heterozygous rare sequence variants in GNRHR was significantly higher in probands vs. controls (P < 0.01). Among the G1-G3 groups (homozygous subjects with successively decreasing severity and number of mutations), the hypogonadotropic phenotype related to their genetic load. In contrast, subjects in G4, with only monoallelic mutations, demonstrated a greater diversity of clinical phenotypes. CONCLUSIONS: In patients with GnRH deficiency and biallelic mutations in GNRHR, genetic burden defined by severity and dose is associated with clinical phenotype. In contrast, for patients with monoallelic GNRHR mutations this correlation does not hold. Taken together, these data indicate that as-yet-unidentified genetic and/or environmental factors may combine with singly mutated GNRHR alleles to produce reproductive phenotypes.

Expression of the GABAA receptor associated protein Gec1 is circadian and dependent upon the cellular clock machinery in GnRH secreting GnV-3 cells.

The timely regulation of gonadotropin-releasing hormone (GnRH) secretion requires a GABAergic signal. We hypothesized that GEC1, a protein promoting the transport of GABA(A) receptors, could represent a circadian effector in GnRH neurons. First, we demonstrated that gec1 is co-expressed with the GABA(A) receptor in hypothalamic rat GnRH neurons. We also confirmed that the clock genes per1, cry1 and bmal1 are expressed and oscillate in GnRH secreting GnV-3 cells. Then we could show that gec1 is expressed in GnV-3 cells, and oscillates in a manner temporally related to the oscillations of the clock transcription factors. Furthermore, we could demonstrate that these oscillations depend upon Per1 expression. Finally, we observed that GABA(A) receptor levels at the GnV-3 cell membrane are timely modulated following serum shock. Together, these data demonstrate that gec1 expression is dependent upon the circadian clock machinery in GnRH-expressing neurons, and suggest for the first time that the level of GABA(A) receptor at the cell membrane may be under timely regulation. Overall, they provide a potential mechanism for the circadian regulation of GnRH secretion by GABA, and may also be relevant to the general understanding of circadian rhythms.

Deciphering genetic disease in the genomic era: the model of GnRH deficiency.

Isolated gonadotropin-releasing hormone (GnRH) deficiency is a treatable albeit rare form of reproductive failure that has revealed physiological mechanisms controlling human reproduction, but despite substantial progress in discovering pathogenic single-gene defects, most of the genetic basis of GnRH deficiency remains uncharted. Although unbiased genetic investigations of affected families have identified mutations in previously unsuspected genes as causes of this disease in some cases, their application has been severely limited because of the negative effect of GnRH deficiency on fertility; moreover, relatively few of the many candidate genes nominated because of biological plausibility from in vitro or animal model experiments were subsequently validated in patients. With the advent of exciting technological platforms for sequencing, homozygosity mapping, and detection of structural variation at the whole-genome level, human investigations are again assuming the leading role for gene discovery. Using human GnRH deficiency as a paradigm and presenting original data from the screening of numerous candidate genes, we discuss the emerging model of patient-focused clinical genetic research and its complementarities with basic approaches in the near future.

Comparative functional analysis of two fibroblast growth factor receptor 1 (FGFR1) mutations affecting the same residue (R254W and R254Q) in isolated hypogonadotropic hypogonadism (IHH).

FGFR1 mutations have been identified in both Kallmann syndrome and normosmic HH (nIHH). To date, few mutations in the FGFR1 gene have been structurally or functionally characterized in vitro to identify molecular mechanisms that contribute to the disease pathogenesis. We attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patient phenotypes. Two unrelated GnRH deficient probands were found to harbor mutations in FGFR1 (R254W and R254Q). Mutant signaling activity and expression levels were evaluated in vitro and compared to a wild type (WT) receptor. Signaling activity was determined by a FGF2/FGFR1 dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot and cell surface expression was measured by a radiolabeled antibody binding assay. The R254W maximal receptor signaling capacity was reduced by 45% (p<0.01) while R254Q activity was not different from WT. However, both mutants displayed diminished total protein expression levels (40 and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). The p.R254W and p.R254Q are both loss-of-function mutations as demonstrated by their reduced overall and cell surface expression levels suggesting a deleterious effect on receptor folding and stability. It appears that a tryptophan substitution at R254 is more disruptive to receptor structure than the more conserved glutamine substitution. No clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

A genetic basis for functional hypothalamic amenorrhea.

Background: Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. Methods: We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. Results: Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been previously shown for PROKR2 L173R and GNRHR R262Q. Conclusions: Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism are found in women with hypothalamic amenorrhea, suggesting that these mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations provide evidence for the role of rare variants in common multifactorial disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00494169.)

A comparative phenotypic study of kallmann syndrome patients carrying monoallelic and biallelic mutations in the prokineticin 2 or prokineticin receptor 2 genes.

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.

Semaphorin7A regulates neuroglial plasticity in the adult hypothalamic median eminence.

Reproductive competence in mammals depends on the projection of gonadotropin-releasing hormone (GnRH) neurons to the hypothalamic median eminence (ME) and the timely release of GnRH into the hypothalamic-pituitary-gonadal axis. In adult rodents, GnRH neurons and the specialized glial cells named tanycytes periodically undergo cytoskeletal plasticity. However, the mechanisms that regulate this plasticity are still largely unknown. We demonstrate that Semaphorin7A, expressed by tanycytes, plays a dual role, inducing the retraction of GnRH terminals and promoting their ensheathment by tanycytic end feet via the receptors PlexinC1 and Itgb1, respectively. Moreover, Semaphorin7A expression is regulated during the oestrous cycle by the fluctuating levels of gonadal steroids. Genetic invalidation of Semaphorin7A receptors in mice induces neuronal and glial rearrangements in the ME and abolishes normal oestrous cyclicity and fertility. These results show a role for Semaphorin7A signalling in mediating periodic neuroglial remodelling in the adult ME during the ovarian cycle.