Novel homozygous splice acceptor site GnRH Receptor (GnRHR) mutation: human GnRHR “knockout”

Mutations in the GnRH receptor (GnRHR) have been shown to be responsible for a significant number of autosomic recessive and, less commonly, sporadic cases of idiopathic hypogonadotropic hypogonadism. We describe a woman with complete GnRH resistance secondary to a novel homozygous GnRHR gene mutation, transmitted as an autosomal recessive trait. The propositus presented with primary amenorrhea and absent thelarche and pubarche. Dynamic tests demonstrated absent spontaneous gonadotropin pulsatility, and no response to either exogenous pulsatile (10 μg/pulse at 90-min intervals over 6 h) or acute (100 μg) GnRH administration. However, she responded to exogenous gonadotropin administration, with a resulting normal pregnancy. Genomic DNA extracted from peripheral blood was PCR amplified using amplimers spanning intron-exon boundaries for the three exons of GnRHR and revealed a homozygous splice junction mutation (G to A transversion) at the intron 1-exon 2 boundary. Her unaffected sister, with a totally normal phenotype, was heterozygous for this mutation. After lymphocyte Epstein-Barr virus transformation, RNA was extracted and subjected to RT-PCR, using primers located in the first and third exons. Results showed a transcript lacking all of exon 2 (exon 2 skipping), with splicing of exon 1 to exon 3. This created a frame shift, generating a coding sequence for three new amino acids, followed by a stop codon. Although it is not clear whether the mutant receptor is actually expressed, the resultant mRNA sequence was presumed to produce a truncated receptor with no binding or signaling capacity

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Heparan sulphate proteoglycans (HSPGs) exist in pancreatic beta cells, and HS seems to modulate important interactions in the islet microenvironment. However, the intra-islet structures of HS in health or altered glucose homeostasis are currently unknown. Here we show that distinct spatial distribution of HS motifs is present in islets in the adult, that intra-islet HS motifs are mostly conserved between rodents and humans, and that HS is abundant in glucagon producing islet alpha cells. In beta cells HS is characterised by 2-O, 6-O and N-sulphated moieties, whereas HS in alpha cells is N-acetylated, N-, and 2-O sulphated and low in 6-O groups. Differential expression of three HS modifying genes in alpha and beta cells was observed and may account for the different HS patterns. Furthermore, we found that FGF1 and FGF2 were present in alpha cells, whereas functional FGFRs exist in beta cells, but not in the alpha cell line aTC1-6, or in primary alpha cells in islets. FGF1 induced signalling was dependent on 2-O, and 6-O HS sulphation in beta cells, and HS desulphation reduced beta cell proliferation and potentiated oxidant induced apoptosis. In leptin resistant animals and in islets from streptozotocin treated rats there was a reduction in alpha cell HS expression. These data demonstrate the distinct HS expression patterns in alpha and beta islet cells and propose a novel role for alpha cells as a source of paracrine FGF ligands to neighbouring beta cells with specific cell-associated HS domains mediating the activation and diffusion of paracrine ligands

DCC/NTN1 complex mutations in patients with congenital hypogonadotropic hypogonadism impair GnRH neuron development.

Congenital hypogonadotropic hypogonadism (CHH) is a rare genetic disease characterized by absent puberty and infertility due to GnRH deficiency, and is often associated with anosmia [Kallmann syndrome (KS)]. The genetic etiology of CHH is heterogeneous, and more than 30 genes have been implicated in approximately 50% of patients with CHH. We hypothesized that genes encoding axon-guidance proteins containing fibronectin type-III (FN3) domains (similar to ANOS1, the first gene associated with KS), are mutated in CHH. We performed whole-exome sequencing in a cohort of 133 CHH probands to test this hypothesis, and identified rare sequence variants (RSVs) in genes encoding for the FN3-domain encoding protein deleted in colorectal cancer (DCC) and its ligand Netrin-1 (NTN1). In vitro studies of these RSVs revealed altered intracellular signaling associated with defects in cell morphology, and confirmed five heterozygous DCC mutations in 6 probands-5 of which presented as KS. Two KS probands carry heterozygous mutations in both DCC and NTN1 consistent with oligogenic inheritance. Further, we show that Netrin-1 promotes migration in immortalized GnRH neurons (GN11 cells). This study implicates DCC and NTN1 mutations in the pathophysiology of CHH consistent with the role of these two genes in the ontogeny of GnRH neurons in mice