Background: The initiation of puberty is dependent upon an augmentation of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus. Development of the GnRH neuroendocrine network in embryonic life depends on coordinated migration of neurons from the vomeronasal organ in the nose to the forebrain. We have previously demonstrated that dysregulation in GnRH neuronal migration leads to delayed pubertal onset. Late puberty affects up to 2% of the population and results in adverse health outcomes. Self-limited delayed puberty (DP) (i.e. constitutional delay of puberty) runs in families, most commonly with autosomal dominant inheritance patterns, indicating a strong genetic basis of the trait. However, the genes underlying DP remain mainly unknown.
Aims and methods: To discover novel genetic mutations in pathways regulating GnRH neuronal migration and development in our large, accurately phenotyped cohort of patients with DP. 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 with biological relevance to GnRH neuronal development pathways. The candidate gene LGR4, identified via this strategy, was investigated via in silico and in vitro techniques and via a mouse model.
Results: We identified three rare missense variants in LGR4 in four unrelated families (14 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 (SIFT and POLYPHEN 2). In vitro analysis of Lgr4 revealed specific expression in mice olfactory epithelium and vomeronasal organ at different embryonic stages. The LGR4 mutants showed an impaired Wnt β-catenin signalling, due to defective protein expression, a shorter protein half-life (p.I96V and p.G363C mutants) and defective trafficking to the plasma membrane (p.G363C and p.D844G mutants). Moreover, we investigated the role of Lgr4 in a knock-out mouse model: Lgr4+/- mice had a delayed onset of puberty and fewer GnRH neurons compared to Lgr4+/+ mice, both in early embryogenesis and at the hypothalamus, whereas Lgr4-/- mice failed to enter puberty and showed a significant reduction in GnRH neurons.
Conclusions: Defects in LGR4, acting via the Wnt signalling pathway, affect GnRH neuron development in foetal life, resulting in a phenotype of self-limited DP. Our findings contribute to the ongoing exploration of genetic factors controlling pubertal timing.
19 Sep 2019 - 21 Sep 2019