BACKGROUND: Pleiotropy is a biological phenomenon of a single gene exhibiting influence over several different seemingly disparate phenotypes. This phenomenon poses significant challenges to fully understanding the etiologies of many different Mendelian diseases. Two such Mendelian diseases are Idiopathic Hypogonadotropic Hypogonadism (IHH) and Craniosynostosis (CS). IHH results from the failure of differentiation, migration, secretion, or action of the GnRH neurons resulting in absent puberty and infertility. CS is characterized by premature fusion of one or more of the cranial sutures resulting in dysmorphic shape of the skull that can lead to life-threatening raised intercranial pressure requiring surgical intervention. Thus far, 77 genes have been implicated in IHH and 128 genes have been implicated CS, both representing ~50% of the cases in their respective diseases. Recent research has suggested a shared molecular landscape in CS and IHH but the full ensemble of this overlap is not known. OBJECTIVE: This study will attempt to utilize human genetics, bioinformatics, statistics, phenotype data of IHH patients, and the prior literature in order to ascertain the full extent of the shared biology of IHH and CS. METHODS: The gene sets of both IHH and CS were used in gene overlap statistical analysis to investigate shared genetics. Whole exome sequencing data from 1,395 patients from the IHH cohort of the Massachusetts General Hospital were used for gene-variant burden analysis to determine genetic overlap with CS. Detailed physician notes from this cohort were used to determine phenotypic presence of CS in IHH. Conversely, evidence of reproductive phenotypes in genetically characterized CS patients was gathered from the reported CS gene literature. The CS and IHH gene sets were also bioinformatically analyzed using both the Metascape and DAVID bioinformatic platforms for pathway annotation, protein-protein interaction (PPI), and functional interactions to provide evidence for the mechanism of shared biology. RESULTS: Of the 128 CS genes and 77 IHH genes, 4 were determined to be causal for both diseases with a further 3 considered as potentially causal candidates for both diseases. The 4 overlapping causal genes were tested using three different methods and this overlap was determined to be of statistical significance (p<0.05). Furthermore, the phenotypic review revealed that while there was not a significant enrichment for CS phenotypes in the IHH cohort, the literature review yielded 49 of 128 CS genes that were reported with phenotypic evidence of failure of the hypothalamic-pituitary portion of the HPG axis. Gene-variant burden analysis yielded nominal (p<0.05) enrichment in the IHH cohort for 17 CS genes, of which 3 were significant after Bonferroni multiple testing correction (p<0.00039). The CS/IHH gene sets were both enriched in 44 shared pathways according to Metascape and 17 shared pathways according to DAVID. PPI analysis yielded 3 shared communities between the two disorders with enrichment in fibroblast signaling, ossification, and cardiac chamber development. CONCLUSIONS: The shared biology between IHH and CS was significantly greater than what was previously appreciated. Shared pathways of the two gene sets point toward the neural crest origin of subpopulations of the GnRH neuron and cranial suture osteoblast as a possible foundation for this shared biology, as well as the migratory nature of these two cells and the role that many genes in both gene sets play in cellular motility. Several CS genes emerge as candidates for IHH and must be individually evaluated. Functional studies should be used to confirm and further unravel the underlying mechanisms for the biological overlap between these two diseases. This study may provide a model for preemptive in silico work prior to more expensive in vitro or in vivo studies of pleiotropy.

The genes that regulate pubertal timing in the general population are not well understood. Recently, genome-wide association studies have demonstrated that genetic variants near LIN28B associate with variation in pubertal timing in humans. To investigate where within the hypothalamic-pituitary-ovarian (HPO) axis Lin28b, and its homologue Lin28a, regulate pubertal timing, expression of these genes was assessed across the pubertal transition. The finding that Lin28a/b expression decreases only in the ovary suggests that the Lin28 pathway may exert its regulatory effects with respect to puberty in the ovary. Another aim of this thesis was to examine the effect of estrogen on Lin28b expression in immortalized GnRH neuronal cells, but the data remains equivocal and detailed future studies are needed to make definitive conclusions. The ovarian expression data lay the foundation for further studies using conditional knockout mice to verify the importance of the tissue and age specific developmental pattern that was identified.

The genes that regulate pubertal timing in the general population are not well understood. Recently, genome-wide association studies have demonstrated that genetic variants near LIN28B associate with variation in pubertal timing in humans. To investigate where within the hypothalamic-pituitary-ovarian (HPO) axis Lin28b, and its homologue Lin28a, regulate pubertal timing, expression of these genes was assessed across the pubertal transition. The finding that Lin28a/b expression decreases only in the ovary suggests that the Lin28 pathway may exert its regulatory effects with respect to puberty in the ovary. Another aim of this thesis was to examine the effect of estrogen on Lin28b expression in immortalized GnRH neuronal cells, but the data remains equivocal and detailed future studies are needed to make definitive conclusions. The ovarian expression data lay the foundation for further studies using conditional knockout mice to verify the importance of the tissue and age specific developmental pattern that was identified.