NOVEL MOLECULAR MECHANISMS UNDERLYING GNRH NEURON BIOLOGY AND ASSOCIATED REPRODUCTIVE DISORDERS

Gonadotropin-releasing hormone (GnRH) neurons regulate the neuroendocrine hypothalamuspituitary-
gonad axis which is the main regulator of reproductive function in many vertebrates. During
development, GnRH neurons originate in the nasal placode and migrate along olfactory nerves
towards the hypothalamus, where they finally set and integrate in a complex neural network that
regulates GnRH secretion. Isolated GnRH Deficiency (IGD) is a rare disorder affecting reproduction
characterized by absent puberty onset and infertility. IGD may be caused from either defective GnRH
neuron development or function, leading to a wide spectrum of phenotypes which includes
normosmic Hypogonadotropic Hypogonadism (nHH), Kallmann Sydrome (KS) and syndromic IGD.
Although 35 causative genes have been already identified, IGD genetics remains largely unknown,
with up to 50% of overall cases still idiopathic. The identification of novel genes implicated in IGD
is fundamental to ameliorate diagnosis and treatment. Here, we have combined screening of IGD
patients, together with in silico, in vitro and in vivo experimental models to identify novel IGD
causative genes. First, we found a novel homozygous variant in Semaphorin 3G (SEMA3G) gene in
two brothers affected by an unrecognized form of syndromic IGD, displaying nHH, facial
dysmorphisms and mental retardation. We demonstrated both in GN11 cells and in Sema3g-null mice
that SEMA3G underlies nHH, whereas additional mutations in Rhotekin (RTKN) and Natural Killer
cell Triggering Receptor (NKTR) genes may potentially explain the complex phenotype observed in
the two brothers. Then, we showed that Plexin a1 (Plxna1) and Plexin a3 (Plxna3) cooperatively act
as signal transducing receptors for Sema3a in the context of GnRH neuron and olfactory system
development. Indeed, compound Plxna1;Plxna3-null mice, but not single Plxna1 and Plxna3 knock
out mice, displayed an abnormal phenotype suggesting that the loss of a single Plxna isoform is not
sufficient to phenocopy Sema3a deletion. Hence, we proposed that not only PLXNA1 but also
PLXNA3 should be considered as a candidate gene to be screened in IGD patients and in particular in
whose affected by KS. Finally, we investigated the genetic causes that lead to an IGD-related disorder
known as familial self-limited delayed puberty (DP). By analysing exome sequencing data from a
large number of patients who belong to a large and well-characterised cohort of patients, we linked a
deleterious heterozygous variant in Heparan Sulphate 6-O Sulpho-Transferase 1 gene (HS6ST1) to
the onset of familial self-limited DP. These results were corroborated by in vivo experiments on
Hs6st1+/- and wild type showing that haploinsufficiency of Hs6st1 is sufficient to delay puberty onset
in mice. Overall, the results presented in this thesis have provided novel insights into the molecular
mechanisms that control GnRH neuron biology and into the genetics underlying associated
reproductive disorders. These results also provided evidences that the combined access to mutational
screenings of patients with the application of in silico, in vitro and in vivo experimental models are
effective in the discovery of novel genes implicated in rare and complex inherited disorders such as
IGD.