Maione, Luigi (2015) Congenital Hypogonadotropic Hypogonadism: novel clinical, genetic and molecular insights. [Tesi di dottorato]

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GnRH decapeptide and its receptor are two key-player of the neuroendocrine control of reproduction. Mutations in genes encoding these proteins (GNRH1 and GNRHR) are associated with Congenital Hypogonadotropic Hypogonadism (CHH). The aim of our work was to screen 450 individuals with CHH for mutations in GNRH1 and GNRHR genes and to determine their deleterious character. Two novel p.W26X and p.R31C mutations in GNRH1 and two novel p.T269M and p.Y290F mutations in GNRHR have been identified in eight CHH individuals from five different families. Molecular and functional characterizations have been performed. The mutation p.W26X totally disrupts GnRH structure. The occurrence of this variant in two sisters affected by Kallmann Syndrome is not explained by genetics, and offers novel insights to define the pathogenesis in these patients. The mutation p.R31C introduces a cysteine in position 8 within GnRH. This substitution dramatically alters protein structure and GnRH-dependent signaling, as assessed by impairment of binding, intracellular pathways as MAPK signaling or calcium mobilization, gene expression and biological activity. Moreover, the mutant pre-pro-GnRH is less expressed in various cell lines compared to wild type. Despite the deleterious effects on protein function of p.R31C product, the mode of inheritance suggests a negative dominance with intriguing yet elusive mechanisms. The mutation p.T269M in GNRHR completely abolishes the binding, and produces a complete impairment of GnRH-dependent signals, as evaluated by calcium and MAPK. The homozygous status fully explains disease in two sisters with CHH. The p.Y290F mutated GnRH receptor is less able to activate the main intracellular GnRH-dependent cascades. The girl with CHH harbors a homozygous mutation from consanguineous parents, consistent with disease. In conclusion, novel GNRH1 and GNRHR mutations with deleterious properties are reported with their molecular characterization. In two cases, the disease cannot be fully explained by genetic abnormalities, thus opening interesting opportunities for further investigations in this field. Finally, a section on sensorineural characterization of CHH patients is provided. To this aim, a novel and innovative diagnostic test (Flavor Identification Test) has been developed as part of my Ph.D. thesis. This test has received a National Patent.

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