Edición 77 - Enero 2020 / Subject Review

Subject Review – Ed. 77

Marco A. Rivarola and Alicia Belgorosky. Hospital de Pediatria Garrahan, Buenos Aires, Argentina.

For this issue Subject Review of Endocrinología Pediátrica On-Line, we have selected the following publication:
Hum Mol Genet. 2019 Apr 15;28(8):1357-1368. doi: 10.1093/hmg/ddy451.
EAP-1 (Enhanced at Puberty-1) regulation of GnRH promoter activity is important for human pubertal timing. 
Mancini A1, Howard SR1, Cabrera CP2, Barnes MR2, David A3, Wehkalampi K4, Heger S5, Lomniczi A6, Guasti L1, Ojeda SR6, Dunkel L1
1Centre for Endocrinology, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.2 Centre for Translational Bioinformatics, William Harvey Research Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, UK.3Centre for Integrative Systems Biology and Bioinformatics, Department of Life Sciences, Imperial College London, London, UK.4Children’s Hospital, Helsinki University Hospital and University of Helsinki, Helsinki, Finland.5Department of Pediatric Endocrinology, Children’s Hospital Auf der Bult, Hannover, Germany.6 Oregon National Primate Research Center/Oregon Health and Science University, Portland, OR, USA.

Note. The EAP-1 gene is also known as PTTG-1, HPTTG, and TUTRI. Chromosome location: 5q33.3, exon count: 6, the regulator of sister chromatid separation factor, named securin [Homo sapiens (human)]. The encoded protein is a homolog of yeast securin proteins, which prevent separins from promoting sister chromatid separation. It is an anaphase-promoting complex (APC) substrate that associates with a separin until activation of the APC. The gene product has transforming activity in vitro and tumorigenic activity in vivo, and the gene is highly expressed in various tumors.

The initiation of puberty is orchestrated by an augmentation of gonadotropin-releasing hormone (GnRH) secretion from a few thousand hypothalamic neurons. Recent findings have indicated that the neuroendocrine control of puberty may be regulated by a hierarchically organized network of transcriptional factors acting upstream of GnRH. These include enhanced at puberty-1 (EAP-1), which contributes to the initiation of female puberty through the transactivation of the GnRH promoter. However, no EAP-1 mutations have been found in humans with disorders of pubertal timing. The authors performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited delayed puberty (DP). Variants were analyzed for rare, potentially pathogenic variants enriched in case versus controls and relevant to the biological control of puberty. They identified one in-frame deletion (Ala221del) and one rare missense variant (Asn770His) in EAP-1 in two unrelated families; these variants were highly conserved and potentially pathogenic. Expression studies revealed Eap-1 mRNA abundance in the peri-pubertal mouse hypothalamus. EAP-1 binding to the GnRH1 promoter increased in monkey hypothalamus at the onset of puberty as determined by chromatin immunoprecipitation. Using a luciferase reporter assay, EAP-1 mutants showed a reduced ability to trans-activate the GnRH promoter compared to wild-type EAP-1, due to reduced protein levels caused by the Ala221del mutation and subcellular mislocation caused by the Asn770His mutation, as revealed by western blot and immunofluorescence, respectively. In conclusion, authors have identified the first EAP-1 mutations leading to reduced GnRH transcriptional activity resulting in a phenotype of self-limited delayed puberty.

Excerpts extracted from this article:
EAP-1 mRNA has been shown to increase in the hypothalamus of rats and non-human primates at the time of puberty, and EAP-1 deficiency led to delayed puberty and disrupted estrous cyclicity in both rodents and nonhuman primates. Eap1 codes for a nuclear transcription factor, characterized by a dual transcriptional activity; it both transactivates the GnRH promoter, which facilitates GnRH secretion, and inhibits the preproenkephalin promoter, which represses GnRH secretion. Therefore, Eap-1 transcriptional activity facilitates the initiation of female puberty, in a manner that is independent of hypothalamic Kiss1 expression.  Notably, a study performed in rats showed that hypothalamic expression of Eap-1
is not directly regulated by ovarian steroids, as its expression in the peri-pubertal female hypothalamus changes even in the absence of ovaries (Matagne, V. et al, Endocrinology, 150:1870–1878). Despite this seemingly important role, no EAP-1 mutations have yet been identified in humans with pubertal disorders.
Delayed puberty usually resolves by the age of 18 years, and in this case is referred to as self-limited, or constitutional, delayed puberty. Self-limited delayed puberty is commonly familial and segregates with an autosomal dominant inheritance pattern in >70% of families, indicating a strong genetic basis of the trait. Such an inheritance pattern suggests that delayed puberty has a monogenic or oligogenic background, although very few underlying genes have been discovered.
Exome sequencing of families with self-limited delayed puberty identified variants in EAP-1. It has been highlighted as an important potential candidate for the hierarchical regulation of pubertal timing through system biology approaches and animal models. EAP-1 mRNA levels and protein expression are seen to increase
in the hypothalamus of primates and rodents at the time of pubertal onset.
Two identified variants in EAP-1 are rare, highly conserved, and potentially damaging to protein function. The two variants p.Asn770His and p.Ala221del, have both minor allele frequency, <0.5% in population databases. Both variants affect amino acids that are highly conserved among homologs, and multiple sequence alignment. The p.Asn770His missense variant is predicted to be deleterious to protein function by prediction tools, as the affected amino acid residue resides in a long loop region, which is C-terminal to the C3HC4 ring domain (residues 715-762). This region shows evolutionary conservation among different species.
Family members with classical self-limited delayed puberty from two pedigrees carry heterozygous EAP-1 variants inherited in an autosomal dominant pattern. The affected individuals from these two families have classical clinical and biochemical features of self-limited delayed puberty, with delayed onset of Tanner stage II and delayed peak height velocity. The proband from family A presented at the age of 15.7 years with delayed pubertal development. His father had had a similar delay in puberty onset. At initial evaluation, the proband had testes volumes of 4 ml and circulating testosterone concentration at early pubertal level (5.7 nmol/l). The proband from family B presented at the age of 15.3 years with prepubertal testes volumes of 3 ml bilaterally. Both probands had markedly delayed bone age at presentation and during follow-up, they had spontaneous pubertal development without testosterone therapy excluding idiopathic hypogonadotropic hypogonadism.

Eap-1 is expressed within key regions within the mouse adult hypothalamus.
They performed in situ hybridization on the peri-pubertal hypothalamus of male and female mice. An abundance of Eap-1 mRNA was detected in the ventromedial, paraventricular and arcuate nuclei of male and female mice. Eap-1 expression was also investigated concurrently with the detection of GnRH neurons using immunohistochemistry. GnRH neuronal axons were mainly detected at the level of the median eminence, whereas GnRH neuron bodies were interspersed in the median preoptic area in a positive Eap1 milieu.
Eap-1 expression specificity was tested with a sense probe, which resulted in no detectable staining. Hence, Eap-1 displayed strong expression in specific hypothalamic subregions of both female and male peri-pubertal mice. These results in mice are in keeping with Eap-1 expression in rat and non-human primate hypothalami.
Other indirect evidence includes previous evidence showing the expression of Eap-1 by GnRH neurons, and the increase of the binding of EAP-1 to GnRH promoter at puberty.
In summary, they identified two pathogenic mutations in the central transcriptional regulator EAP-1 as the likely cause for self-limited delayed puberty in two families. Mutations in several genes known to influence GnRH secretion have been identified in patients with pubertal delay, including TAC3, TACR3, KISS1 and, now, EAP-1. These results strengthen the concept that EAP-1 is a bona fide regulator of pubertal onset and add to the understanding of the regulatory neural network that controls the onset of human puberty.

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