Genómica funcional y disección molecular de FOXD1 para la identificación de nuevos biomarcadores genéticos asociados a patologías de la reproducción de origen endometrial y placentario

Abstract

Recurrent pregnancy loss (RPL) is defined as the loss of two or more consecutive and spontaneous miscarriages before the 20th week of gestation. This pathology affects ~1% to 5% of the couples. The RPL aetiology is classified as non-genetics and genetics. However, the 50% of the cases remains idiopathic. Similarly, the aetiology of the recurrent implantation failure (RIF), defined as the implantation failure in at least 2 or more consecutive cycles of in vitro fertilization (IVF), is poorly understood. To note, the molecular RPL and RIF aetiology is potentially associated with hundreds of genes which participate in the physiological molecular pathways related to the implantation and gestation. The candidate gene approach using Sanger sequencing has been useful to identify some genes associated with RPL. However, the next generation sequencing (NGS) has been an effective tool to overcome this limitation because it allows the simultaneous study of multiple genes related to complex diseases. In first part of this thesis, we used the NGS-exome approach to identify new genes and mutation potentially implicated in the development of RPL. Some of the mutations found by this approach were tested by functional in vitro assays to determine their possible deleterious effect within the pathology. The identification of the variant THBD p.Trp153Gly in Colombian women with RPL and its validation through functional in vitro assays suggested, for the first time, a direct association with the RPL pathophysiology. Therefore, it may be considered as a molecular biomarker for the RPL diagnosis in Colombian patients. In the second part of this thesis, we identified and studied the potential implication of new FOXD1 mutations, a relevant gene in the endometrium and placenta physiology, in patients with RIF, RPL, preeclampsia (PE) and intrauterine growth restriction (IUGR). The functional in vitro assays demonstrated that FOXD1 p.His267Tyr and FOXD1 p.Arg57del mutations modify the C3 promoter transactivation, thus contributing to the phenotype. Therefore, FOXD1 could be considered a molecular biomarker for the diagnosis of RPL, RIF, PE and IUGR patients. The chromatin immunoprecipitation assays (ChIP) allowed to determine new direct FOXD1 target genes (CTSC, CD86, CMA1 y TRPC6) within the placenta context. The information generated during this thesis contributes to the general knowledge about the origin and development of RPL, RIF, PE/IUGR. These results might be useful for the clinical specialist in a context of translational medicine.