The Role of Gonadotropin Pulsations in the Regulation of Puberty and Fertility At one extreme of pubertal development, isolated deficiency of GnRH results in a spectrum of rare clinical disorders of isolated GnRH deficiency (IGD), also known as idiopathic hypogonadotropic hypogonadism (IHH), which presents with delayed, incomplete, or absent sexual maturation. Defining the physiology of GnRH secretion is critical to understanding the clinical heterogeneity of IGD, particularly in light of emerging gene discoveries that aim to elucidate genotype-phenotype correlations. Non-reproductive phenotypic features have been identified in some individuals, including anosmia, auditory defects, and skeletal, neurological and renal anomalies. These additional features may be the key to determining the developmental function of genes implicated in this spectrum of disorders. Our clinical protocol (http://gp.nichd.nih.gov), which is a multicenter study in collaboration with the REU at MGH has identified a broad range of LH pulsatility patterns and other features that are being investigated in the context of genetic variants, where identified, in order to increase our understanding of the ontogeny of these disorders. Our phenotyping efforts have identified that uterine anomalies and Chiari type 1 malformations may represent novel non-reproductive features of IGD, which is now being investigated in our genetic study (below) to determine whether there is a common molecular cause for these phenotypes. We have also initiated a pilot study to determine the prevalence of psychiatric disorders and symptoms of negative emotional states in our cohort, compared with healthy controls, in order to determine whether there are previously unidentified psychological features in need of further investigation. Preliminary results from this pilot study suggest that there is an increase in both anxiety and depression symptoms in the IGD group, and that this can be ascertained by online surveys to improve recruitment. As a result of the phenotyping efforts pioneered by our collaborators at MGH, several rare phenotypic categories have been described in men with IGD, including adult-onset IHH (Nachtigall, LB, et al, N Engl J Med, 1997) and reversal of the disorder after a period of treatment (Raivio, T, et al, N Engl J Med, 2007). We recently reported that subjects with confirmed reversal, or recovery from HH with sustained endogenous LH pulsatility, are responsive to IV bolus infusions of kisspeptin, a neuropeptide known to be a key stimulus of GnRH release in the human. On the other hand, subjects who relapse from their recovery are not kisspeptin responsive, while they do respond to GnRH, suggesting that pituitary responsiveness is intact. Thus, the acquisition and maintenance of kisspeptin responsiveness may play a role in reversal, and by extension in pubertal development (Lippincott, MF, et al, JCEM, 2016). Neurocognitive Effects of Sex Hormone Deficiency at or Before Puberty There is little existing evidence for the neurocognitive effects of delayed puberty. We have performed neurocognitive testing and structural and functional MRI on subjects with IGD, compared with healthy controls matched for age, sex, and race. Accounting for gender, our preliminary findings suggested that in both sexes, pubertal sex steroid deficiency contributes to persistent structural and functional brain differences as well as to neurocognitive deficits primarily involving spatial ability and recognition memory, providing direct evidence in humans about the critical spatiotemporal role played by appropriately timed pubertal sex steroids during normal brain development. The final analysis of this data is being completed with a manuscript in preparation. The Molecular Basis of Inherited Reproductive Disorders Human and animal models have identified a number of genes responsible for IGD, but more than half of patients with clinical evidence of the disorder do not have a detectable mutation. In addition, there is significant clinical heterogeneity among affected individuals, including members of the same family harboring the same mutations, which is often explained by oligo-digenic inheritance patterns. Exome sequencing (ES) has been performed in the Molecular Genomics Core (NICHD) on 28 probands participating in our genetic research protocol (http://ird.nichd.nih.gov), including several extended families to identify novel genes responsible for IGD. Data analysis is underway, and our findings are likely to yield important insights into additional pathways involved in the regulation of GnRH secretion. In one of our larger families, we have identified a novel candidate gene for Kallmann syndrome (KS), which is characterized by IHH and anosmia. All affected individuals in this family carry a heterozygous nonsense variant in a gene whose function has been indirectly associated in animal models with the neuronal migration defect affecting GnRH neurons is responsible for the KS phenotype. Functional validation of the role of this gene in IGD is being planned. In addition, through our collaboration with the REU at MGH, we have also performed ES in several families with IGD and known uterine anomalies, and we have enrolled a cohort of individuals with delayed pubertal development and Chiari type 1 malformations whose genomic DNA will soon be sent for ES, based on discoveries made through our phenotyping protocol. Analysis of these data has the potential to identify new non-reproductive features of IGD, as well as novel molecular pathways involved in the regulation of GnRH secretion and uterine or brain/skull development. We are also investigating the role of genetic variants in 14 genes known to cause IGD in a subgroup of individuals with functional hypothalamic amenorrhea (HA), a hormonally similar condition that occurs in association with risk factors such as nutritional deprivation, exercise, or significant stress.
We aim to determine whether variants in the IGD genes are over-represented in individuals with HA, compared to the general population, using controls from the ClinSeq study (Biesecker, LG, et al, Genome Res, 2009). If the data support our hypothesis, this would provide further evidence that heterozygous variants in these genes may confer an increased susceptibility to developing HA in the setting of physiologic stressors, such as nutritional deficiency, extreme exercise, or psychological stress. At the other extreme of pubertal development are patients with premature reactivation of hypothalamic GnRH secretion, resulting in idiopathic central precocious puberty (CPP). There is evidence that familial cases account for anywhere from 20-45% of CPP, with most studies describing autosomal dominant inheritance patterns. Far less is known about the molecular basis of CPP, and candidate gene approaches have not been successful in identifying the molecular basis of this disorder. Thus, an unbiased approach to gene discovery seems more likely to achieve the goal of identifying novel candidate genes responsible for premature GnRH secretion in CPP, as was recently shown with the identification of causative mutations in MKRN3, using ES (Abreu, AP, et al, N Engl J Med, 2013). We have established collaborations with investigators both locally and internationally to increase recruitment of families with idiopathic CPP, and have performed ES analysis on this cohort and data analysis is underway. Examining the genetic characteristics of subjects with pubertal disorders will reveal insights into the mechanisms underlying the reawakening of the hypothalamic-pituitary-gonadal axis at puberty. This will provide opportunities for new diagnostic capabilities and therapeutic interventions for disorders of puberty and reproduction.
|Lippincott, Margaret F; Chan, Yee-Ming; Delaney, Angela et al. (2016) Kisspeptin Responsiveness Signals Emergence of Reproductive Endocrine Activity: Implications for Human Puberty. J Clin Endocrinol Metab 101:3061-9|