Endocrine Abstracts

The ovarian reserve naturally declines with age, however, 1–2% of women before 40 years experiences a premature exhaustion of the ovarian function and suffers from a fertility defect named Primary Ovarian Insufficiency (POI). The genetic origin of POI is well established and strongly supported by multiple reports of familial cases. To date, thanks to the candidate gene-discovery approach, few X-linked and autosomal genes have been associated to POI onset, but most of 46,XX cases still remain idiopathic suggesting the involvement of new genetic mechanisms. Whole-Exome Sequencing (WES) has been performed in ten POI patients with the extreme phenotype of absent pubertal development (primary amenorrhea), of which 6 familial and 4 sporadic, to reveal rare variants affecting genes implicated in ovarian function. Their relatives have been similarly analyzed as control population, for a total of 24 exomes. Genomic DNA was first extracted by patients and their relatives, then sheared into random fragments of roughly 300 base pairs and those fragments falling in exome regions were enriched by capturing, and finally sequenced. Data analysis consisted of image recognition, base calling, demultiplexing and trimming of adapter sequences, quality control of generated reads and estimation of coverage, alignment of the clean reads to the reference genome. In order to prioritize variants with potentially pathogenetic role in POI, first we focused on rarity and assessed the frequency of each variant in the general population by using the information provided by ongoing large genome projects (such as ExAC, Exome Variant Server, dbSNP browsers). Variants’ frequencies >1% have been considered not rare and filtered out. A total of 18 570 rare variants (including: missense, 86%; nonsense, 3.5%; small indels, 5.6%) have been identified in our selected cohort of patients. The pathogenic level of each identified variant has been predicted in silico. A pathway-based analysis was performed through the Reactome software on 1916 genetic identifiers occurring mutated at least once. The overrepresentation analysis revealed enrichment among altered genes in: the Chromatin organization pathway and, consequently, the cell cycle and meiosis pathways; the Extracellular matrix organization and cell-cell communication pathways. Further, one hundred fifty-two coding variants in 117 genes participating in key biological processes within the ovary were identified by WES and therefore result potentially related to the POI onset in our patients. Finally, we observed the presence of at least two variants in distinct genes in all the patients in agreement with the oligogenic nature of POI.