Neuron-Derived Neurotrophic Factor Is Mutated in Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disorder characterized by infertility and the absence of puberty. Defects in GnRH neuron migration or altered GnRH secretion and/or action lead to a severe gonadotropin-releasing hormone (GnRH) deficiency. Given the close developmental association of GnRH neurons with the olfactory primary axons, CHH is often associated with anosmia or hyposmia, in which case it is defined as Kallmann syndrome (KS). The genetics of CHH are heterogeneous, and >40 genes are involved either alone or in combination. Several CHH-related genes controlling GnRH ontogeny encode proteins containing fibronectin-3 (FN3) domains, which are important for brain and neural development. Therefore, we hypothesized that defects in other FN3-superfamily genes would underlie CHH. Next-generation sequencing was performed for 240 CHH unrelated probands and filtered for rare, protein-truncating variants (PTVs) in FN3-superfamily genes. Compared to gnomAD controls the CHH cohort was statistically enriched for PTVs in neuron-derived neurotrophic factor (NDNF) (p = 1.40 x 10(-6)). Three heterozygous PTVs (p.Lys62*, p.Tyr128Thrfs*55, and p.Trp469*, all absent from the gnomAD database) and an additional heterozygous missense mutation (p.Thr201Ser) were found in four KS probands. Notably, NDNF is expressed along the GnRH neuron migratory route in both mouse embryos and human fetuses and enhances GnRH neuron migration. Further, knock down of the zebrafish ortholog of NDNF resulted in altered GnRH migration. Finally, mice lacking Ndnf showed delayed GnRH neuron migration and altered olfactory axonal projections to the olfactory bulb; both results are consistent with a role of NDNF in GnRH neuron development. Altogether, our results highlight NDNF as a gene involved in the GnRH neuron migration implicated in KS.Peer reviewe

Mutations in FGF17, IL17RD, DUSP6, SPRY4, and FLRT3 Are Identified in Individuals with Congenital Hypogonadotropic Hypogonadism

Congenital hypogonadotropic hypogonadism (CHH) and its anosmia-associated form (Kallmann syndrome [KS]) are genetically heterogeneous. Among the >15 genes implicated in these conditions, mutations in FGF8 and FGFR1 account for ∼12% of cases; notably, KAL1 and HS6ST1 are also involved in FGFR1 signaling and can be mutated in CHH. We therefore hypothesized that mutations in genes encoding a broader range of modulators of the FGFR1 pathway might contribute to the genetics of CHH as causal or modifier mutations. Thus, we aimed to (1) investigate whether CHH individuals harbor mutations in members of the so-called "FGF8 synexpression" group and (2) validate the ability of a bioinformatics algorithm on the basis of protein-protein interactome data (interactome-based affiliation scoring [IBAS]) to identify high-quality candidate genes. On the basis of sequence homology, expression, and structural and functional data, seven genes were selected and sequenced in 386 unrelated CHH individuals and 155 controls. Except for FGF18 and SPRY2, all other genes were found to be mutated in CHH individuals: FGF17 (n = 3 individuals), IL17RD (n = 8), DUSP6 (n = 5), SPRY4 (n = 14), and FLRT3 (n = 3). Independently, IBAS predicted FGF17 and IL17RD as the two top candidates in the entire proteome on the basis of a statistical test of their protein-protein interaction patterns to proteins known to be altered in CHH. Most of the FGF17 and IL17RD mutations altered protein function in vitro. IL17RD mutations were found only in KS individuals and were strongly linked to hearing loss (6/8 individuals). Mutations in genes encoding components of the FGF pathway are associated with complex modes of CHH inheritance and act primarily as contributors to an oligogenic genetic architecture underlying CHH

FGFR1 and PROKR2 rare variants found in patients with combined pituitary hormone deficiencies

The genetic aetiology of congenital hypopituitarism (CH) is not entirely elucidated. FGFR1 and PROKR2 loss-of-function mutations are classically involved in hypogonadotrophic hypogonadism (HH), however, due to the clinical and genetic overlap of HH and CH; these genes may also be involved in the pathogenesis of CH. Using a candidate gene approach, we screened 156 Brazilian patients with combined pituitary hormone deficiencies (CPHD) for loss-of-function mutations in FGFR1 and PROKR2. We identified three FGFR1 variants (p.Arg448Trp, p.Ser107Leu and p.Pro772Ser) in four unrelated patients (two males) and two PROKR2 variants (p.Arg85Cys and p.Arg248Glu) in two unrelated female patients. Five of the six patients harbouring the variants had a first-degree relative that was an unaffected carrier of it. Results of functional studies indicated that the new FGFR1 variant p.Arg448Trp is a loss-of-function variant, while p.Ser107Leu and p.Pro772Ser present signalling activity similar to the wild-type form. Regarding PROKR2 variants, results from previous functional studies indicated that p.Arg85Cys moderately compromises receptor signalling through both MAPK and Ca2 + pathways while p.Arg248Glu decreases calcium mobilization but has normal MAPK activity. The presence of loss-of-function variants of FGFR1 and PROKR2 in our patients with CPHD is indicative of an adjuvant and/or modifier effect of these rare variants on the phenotype. The presence of the same variants in unaffected relatives implies that they cannot solely cause the phenotype. Other associated genetic and/or environmental modifiers may play a role in the aetiology of this condition

Differential response to exogenous and endogenous activin in a human ovarian teratocarcinoma-derived cell line (PA-1): regulation by cell surface follistatin.

The activin/follistatin system is implicated in growth and differentiation of various cell types. Follistatin (FS), through binding and neutralizing activin, plays a major role in the regulation of activin bioavailability. We previously reported that ovarian PA1 cells constitutively secrete FS and show a decreased proliferation rate in response to exogenous activin only if cell surface associated FS is first removed by heparin treatment. These observations suggest that cell-associated FS prevents exogenous activin from accessing its receptor. We hypothesized that cell surface FS would differentially regulate the bioavailability of endogenous and exogenous activin in these cells. To examine the effect of endogenous activin, PA1 cells were stably transfected with an activin betaA-subunit complementary DNA (cDNA). The proliferation rate of five activin-secreting clones was measured by [3H]thymidine incorporation and compared with the proliferation rate of untransfected cells. In clones secreting levels of activin ranging from 22.6 +/- 7.1 to 42.4 +/- 9.9 ng/ml, proliferation was decreased by 31-72% at 96 h of culture, whereas one cell line secreting lower levels of activin (0.4 +/- 0.1 ng/ml) proliferated similarly to the untransfected cells, in which activin was not detectable. To further assess activin signaling, wild-type PA1 cells and activin-secreting clones were transiently transfected with an activin response element-luciferase reporter construct. Basal luciferase activity was 6-fold higher in activin-secreting clones than in wild-type PA1 cells. Exogenous activin (100 ng/ml) increased the transcriptional response of wild-type PA1 cells by 3-fold but did not increase reporter activity in activin secreting clones. Interestingly, the transcriptional response in activin secreting clones was always greater than the basal or activin-stimulated response in wild-type cells. Furthermore, we found that FS was removed from the cell surface by lipofectamine used for these transfections. Therefore, these results show that activation of the luciferase reporter gene occurs under conditions in which proliferation is affected, suggesting that the antiproliferative effect of activin could be due to a direct stimulation of activin signaling pathways. In summary, as opposed to exogenous activin, endogenous activin decreased proliferation of PA1 cells even in the presence of cell surface associated FS. These results are consistent with a model in which FS acts as a barrier for exogenous (endocrine-paracrine) but not for endogenous (autocrine) activin. In addition, the higher PA1 cell responsiveness to endogenous compared with exogenous activin, suggests that activin overexpression in PA1 cells may up-regulate an activin signaling component, or down-regulate an activin signaling inhibitor.Journal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.info:eu-repo/semantics/publishe