ESPE Abstracts

Objectives: Several different pathogenic mechanisms may converge on a final common pathway to produce the phenotype of delayed pubertal timing. In our cohort of patients with familial self-limited delayed puberty (DP) we have demonstrated mutations in IGSF10 leading to mis-regulation of the embryonic migration of GnRH neurons (Howard et al, EMBO MM 2016). We aimed to discover novel genetic mutations in pathways regulating GnRH neuronal migration and development in our DP cohort. In these patients with significantly delayed pubertal onset, their extreme phenotype may be inherited via one or a few genetic variants discoverable through next generation sequencing (NGS).

Methods: We performed whole exome sequencing (WES) in 160 members of 67 families from our self-limited DP patient cohort, and analysed the data looking for genes with rare, predicted deleterious variants that segregated with trait, and that were significantly enriched for pathogenic variants in our cohort by whole gene rare variant burden testing (RVBT). These data were filtered by biological relevance to GnRH neuronal development via pathway analysis: by comparison with microarray expression data from GnRH neurons and by integration of data from multiple annotation tools (e.g. Uniprot, KEGG, OMIM, Genego MetaCore and Ingenuity Variant Analysis), expression and variant databases.

Results: Rare, potentially pathogenic variants were found in 19 genes related to GnRH neuronal migration or development in our cohort of self-limited DP patients. Variants in 7 genes were excluded by Sanger sequencing due to lack of segregation within families. 3 of these 19 genes were highly enriched by RVBT: HS6ST1, ZC3H3 and SHANK1. 2 genes were already known to be associated with GnRH deficiency: FEZF1 and HS6ST1. Biochemical analysis showed that the pathogenic variant identified in HS6ST1 led to reduced biological activity of the mutant protein in vitro. Hs6st1 mRNA was expressed in peri-pubertal wild type mouse hypothalamus. Vaginal opening,a proxy of pubertal onset, was delayed in Hs6st1^+/− mice despite normal postnatal growth. Additionally, we identified as novel candidates for DP regulation the G-protein coupled receptor LGR4, SIX Homeobox 6, and several modulators of cell migration and adhesion including Neurocan, Fibulin 2 and a member of the semaphorin family.

Conclusions: These data lend further weight to the evidence that abnormalities of GnRH neuronal development, migration and function can present as a phenotype of delayed puberty without defects in fertility. Extracellular matrix proteins and chemotaxtic factors may be particularly relevant to aberrant development of the GnRH neuroendocrine network.