ANALISI MOLECOLARE E FUNZIONALE DI NUOVE VARIANTI PATOGENETICHE E IDENTIFICAZIONE DI NUOVI GENI CANDIDATI, NELLA PI� VASTA CASISTICA ITALIANA DI IPOGONADISMO IPOGONADOTROPO E SINDROME DI KALLMANN.
Creators: name: LIBRI, DOMENICO VLADIMIRO
Publisher: name: Universit� degli Studi di Milano
Year: 2012

description: Idiopatic Central Hypogonadism (ICH) is a rare pathology with a strong genetic component, in which hypothalamic and pituitary dysfunctions involving development and/or functionality of GnRH neurons, cause a reduced or absent gonads functionality. This disease can occur in association with anosmia or hyposmia, (Kallmann Syndrome, KS) o with a normal sense of smell (normosmic idiopathic hypogonadotropic hypogonadism, nIHH) and it shows an extreme phenotypic variability. Despite the identification of 14 genes implicated in the pathogenesis of the disease, approximatively 70% of ICH cases remains idiopathic. Among the causative genes, a role of particular importance is covered by the the Prokineticin pathway, in particular the Prokineticin-2 (PROK2) and its receptor (PROKR2). In fact, in approximately 10% of cases of ICH is possible to identify a genetic variant in one of these two genes such as pathogenetic event of the disease. The receptor PROKR2 belongs to the family of G-protein coupled receptor (GPCR). Its activation, through the binding with PROK2, determines the activation of protein Gq, Gs and Gi, a consequent production of IP3, cAMP and subsequently the mobilization of intracellular calcium. To date in literature have been described 27 PROKR2 mutations and the functional studies performed on a minority of them have only evaluated the effects on the Gq-IP3 signal transduction pathway. Nevertheless a growing number of works in the field of GPCRs demonstrates the importance of the functional studies of all the possible pathways related to a single receptor in order to interpret the functional consequences of genetic variants identified. 
In the present work we have developed two main lines of research starting from the wider availability of Italian cohort of ICH patients.
In the first part of this work we have carried out studies of genetic screening of a cohort of 217 patients, considering the main causative known genes for that pathology, including PROKR2. Genetic variants identified in PROKR2 were then characterized by a functional point of view to test their potential pathogenic role. This screening allowed the identification of seven PROKR2 missense variants (V158I, L173R,T260M, R268C, V274D, V331M and V334M) of which 3 have not yet been described in the literature; in addition to 2 variants nonsense (15fsX45, 20fsX43). The variants identified have been inserted by site-directed mutagenesis into vectorsSPRT-PROKR2-pcDNA3, characterized by the presence of a Rhodopsin tag at the N-terminus of the receptor. This allows the display of the cellular localization of the mutants by binding with an anti-rhodopsin antibody. The constructs thus generated were transfected into HEK 293 cells and CHO for the following functional studies. The FACS analysis revealed that all variants have a reduced membrane expression (reduction of 11-55%), with the exception of the mutation V334M, which shows an expression slightly exceeding that of the wild-type receptor. Functional assays were then performed with the generation of concentration-effectcurves for both IP1 that for cAMP. The results obtained show how the mutation T260M, R268C, V274D, V331M and V334M cause a strong reduction of the signal mediated by the Gq protein , while the signal of cAMP mediated by the Gs protein is significantly reduced in the mutant L173R andV334M. The V334M and V274D mutations are characterized by a marked inactivation of both pathways. Finally analyzing the homology model of PROKR2 it appears evident that the variant V331M is localized at the level of a highly conserved domain (the motif NPXXY), involved in signal transduction. These are the first experiments that analyze both transduction pathways activated by PROKR2 receptor and showing how the different variants associated with ICH can affect signal transduction pathways in a very variable manner. In particular, some variants causing inability to stimulate the two pathways, suggesting that the integrity of both is necessary for normal development and function of GnRH-secreting neurons.
The second part of this thesis, it was instead intended to further clarify the genetic mechanisms (and eventually epigenetic) about the ethiopathogenesis underlying ICH. To conduct these studies we used the techniques of SNPs and CNVs genotyping , on a selected series of familial cases of ICH. For each patient were analyzed 660,000 SNPs and CNVs 100,000, then comparing them with a large database of apparently healthy controls in our possession, in a case / control analysis. The analyzes focused on the identification of SNPs and on the presence of CNVs significantly correlated with ICH and on an family type analysis to detect extended regions of homozygosity in patients (LOH = Loss of Heterozigosity regions). A first macroscopic analysis of the data obtained shows that the number and extent of the deletions in single copy is significantly higher in cases of ICH, compared to controls. The analysis of SNPs and CNVs showed, among the 30 identified loci four genes (CNTNAP2, GPC, RAB39B and PPFIA2) that for expression and molecular function seem to be good candidates for direct sequencing screening in ICH patients. Furthermore we have identified three clusters of microRNA (mir4275, mir507/508/509,mir320D2), suggesting for the first time a potential involvement of these molecules in ICH pathogenesis. Analyzing instead the genes that reside in LOH areas , it is possible to observe an enrichment for certain pathways or protein families, such as: the FGFR pathway; cadherins and cell adhesion molecules (CAM); receptors and ligands involved in the differentiation of the central nervous system (CNS), hypothalamus and pituitary; genes associated with midline defects or with Prader-Willi and Angelmann syndrome; plexins and RAB proteins.