ESPE Abstracts

Background: The initiation of puberty is orchestrated by the augmentation in the secretion of gonadotropin-releasing hormone (GnRH) from a few thousand neurons located in the hypothalamus. Recent findings identified that the neuroendocrine control of puberty is regulated by a network of transcriptional factors hierarchically organized, but this still remains not fully elucidated. Enhanced At Puberty 1 (EAP1) is one of the main regulators of pubertal onset and it is expressed in the hypothalamus of pubertal rats and non-human primates. Its role has been described as being involved in the initiation of female puberty by transactivating GnRH promoter. Its inhibition in the hypothalamus, causes disrupted estrous cyclicity and delayed puberty in rats. Self-limited delayed puberty (DP) (i.e. constitutional delay of puberty) runs in families with either autosomal dominant or complex inheritance patterns in >70% of families, indicating a strong genetic basis of the trait. However, only a few genes have been identified underlying DP, and to date, no EAP1 mutations have been found in humans.

Objective, Hypotheses and Methods: Whole exome sequencing was performed on DNA from 160 individuals of 67 multi-generational families affected with DP. Variants returned were analysed to identify rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. We identified two EAP1 variants whose pathogenicity was validated in vitro and tissue expression was examined in mouse hypothalamus.

Results: After filtering we identified one in-frame deletion and one rare missense variant in EAP1 in two unrelated families (five affected individuals) and all segregated with DP trait with the expected autosomal dominant pattern of inheritance. These variants are highly conserved and were predicted to be deleterious by the main prediction software tools (i.e. SIFT and POLYPHEN 2). Expression studies on Eap1 revealed its broad expression in the hypothalamus of peri-pubertal mice and specifically in the nuclei expressing GnRH neurons, such as the arcuate and periventricular nuclei. The pathogenicity of each variant was investigated using a promoter-reporter assay in a HEK293T cell line. For verifying the EAP1 transactivating ability on GnRH promoter, we employed a luciferase reporter gene whose expression is driven by the GnRH promoter. EAP1 mutant proteins showed a significantly reduced transcriptional activity compared to wild-type, thus impairing its function on GnRH promoter.

Conclusion: We have identified, for the first time, two human EAP1 mutations leading to a reduced GnRH transcriptional activity resulting in the phenotype of self-limited DP.