2022254NRT - Next Generation Sequencing and Drosophila modeling to unveil the molecular basis of Primary Ovarian Insufficiency (NGSDro-POI)

Ovarian deficiency represents one of the main causes of female infertility and includes diminished ovarian reserve and premature ovarian insufficiency. The prevalence of Primary Ovarian Insufficiency (POI) and premature menopause in women is considerable and accounts for up to 3.7%. Notwithstanding a wide spectrum etiology has been considered for POI, the genetic causes are thought to prevail in the pathogenesis of idiopathic POI women, as indicated by families with several affected women, with 4 to 31% incidence. Though recurrent genetic defects have been identified in several POI cases, the precise pathogenic mechanism is not yet fully understood. In addition many POI patients still lack a molecular diagnosis, indicating that further unrecognized molecular alterations on unknown genes contribute to POI aetiology. Currently, the underlying genetic architecture of POI appears heterogeneous resulting from defects in different complementary pathways. Thanks to omics approaches the POI causative gene lists are expanding, including genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and genes safeguarding cell maintenance. The objective of this proposal is to upgrade and enhance the knowledge of the molecular bases of POI to improve molecular diagnosis and patients management, in the transition to aging, and to identify new compensatory and therapeutic interventions. Main aim of the current project is to detect new disease genes and/or variants in known genes associated with primary ovarian failure by using both WES and WGS and then clarify the role of selected core genes in POI etiology by a functional approach. Starting from our previous WES data obtained on an Italian POI cohort, we will first pursue this objective by searching for pathogenic sequence variants applying a dual targeted and non-targeted approach. Since WES cannot offer sequence information in regulatory or intronic regions, the entire genome of an additional selected cohort of 40 patients with POI characterized by an extreme phenotype (non-syndromic early onset (<25 years) POI or primary amenorrhea) will be screened focusing on 914 selected genes and evaluating the presence of new variants in regulatory regions of the known and candidate POI genes. To corroborate our WES results and improve the knowledge on pathways involved in POI aetiopathogenesis we plan to use Drosophila models to investigate the role in ovary function of 17 identified human genes. Drosophila modelling can document the role of candidate genes in ovary development and function by assessing the variability in the ovariole number. Considering the presumptive highly polygenic/oligogenic basisof POI, double mutants will be used to investigate the combined effect of variants. The innovative use of WGS and functional approaches will increase the diagnostic rate for POI patients and will help to elucidate the POI molecular pathways.