Identificación y validación funcional de nuevos genes y mutaciones asociadas a la etiología de la insuficiencia ovárica primaria (IOP) : implicación de los genes BMP15, BMPR2, MSH4, ATG7, ATG9A y NOTCH2

Abstract

Primary ovarian insufficiency (POI) is a frequent pathology affecting 1% - 3% of women from the general population under 40 years old. There are not specific data related Colombian population, however, this could be an interesting point to be determinate taking account the variation of POI prevalence according ethnical origin. POI is characterized by the absence (primary amenorrhea) or the cessation of ovulation (secondary amenorrhea) for at least four months, as well as high plasmatic levels of FSH (Follicle Stimulating Hormone). Autoimmune, metabolic, infectious and iatrogenic causes have been related to the disease pathogenesis. However, in the majority of cases, the aetiology remains undiscovered. Despite numerous attempts to identify these genetic causes, only a few genes have been functionally related to POI’s etiology. This Ph.D. thesis focuses on the identification and functional validation of new variants and/or new genes related to POI’s etiology. This manuscript is divided in two chapters. The first one describes the identification of new BMP15 variants and the evaluation of how these variants may contribute to ovarian dysfunction. The effect of ten BMP15 mutations in mature peptide production, activation of SMAD signaling and GDF9 synergy were assessed. The BMP15 screening on 35 Colombian patients led to the identification of a new variant (c.986C>G-p.Arg329His) and a previously reported one (c.581T>C, p.Phe194Ser) associated with POI. Assessing the expression/activity of these and other 8 BMP15 mutants allowed to describe that: (1) multiple variants, including p.Leu148Phe, p.Phe194Ser and p.Tyr235Cys reduced mature protein production; (2) other variants (p.Arg138His, p.Ala180Thr and p.Arg329His) displayed lower the activity than wild-type BMP15; and (3) some variants (p.Arg68Trp, p.Phe194Ser and p.Asn196Lys) reduced GDF9 synergy. These results showed that BMP15 mutations can disrupt ovarian function through different mechanisms. The second chapter focuses on the identification of rare variants with a moderate/severe effect potentially related to POI’s etiology. For this purpose, we used NGS (Next Generation Sequencing). We used account three approaches: the first one was performed on a gene subset (n=70). The second one was based on whole exome sequencing (WES) assays in a family affected by POI. The third one was performed using WES in non-related patients (non-familial cases). These approaches led to the identification of approximately 60 variants possibly related to POI’s etiology. Furthermore, the identification of patients with at least two mutations in different genes argued in favor of a polygenic nature of POI. Finally, we performed the functional validation of some mutations. These in vitro assays confirmed that mutations in BMPR2 (c.2960C>T-p.Ser987Phe), MSH4 (c.2355+1G>A-p.Ile743_Arg785del), ATG7 (c.1209T>A-p.Phe403Leu), ATG9A (c.2272C>T-p.Arg758Cys) and NOTCH2 (c.5411C>T-p.Ser1804L, c.6947>T-p.Ala2316Val and c.7075C>G-p.Pro2359A) genes could modify the WT protein function and suggesting contribute to POI etiology.