IMPE Abstracts

The genetic defects underlying hypogonadotrophic hypogonadism (HH) are known in approximately 50% of cases. In normosmic patients, abnormal GnRH production or action may be due to defects in regulatory factors or in the genes encoding GnRH or GnRHR. Although many gene variants have been described in GNRHR (4q13.2), the potential impact of structural alterations of the resulting mutant proteins have not been studied. In this study, we analysed the 3D-structure of the variant GnRHR Arg139His found in a boy with pubertal delay due to HH. A 15-year-old boy consulted for short stature and pubertal delay. Serum LH, FSH, T and AMH were low for age. He was diagnosed with isolated HH following a GnRH infusion test (LH and FSH peaks: 0,57 IU/L and 0,19 IU/L respectively). After ruling out organic or functional HH, a congenital aetiology was suspected. Genomic DNA study by next-generation sequencing (NGS), using the TruSight One capture kit (4811 genes) and filters related to variant frequency and quality of the reads, identified the homozygous variant in GNRHR: NC_000004.11(NM_000406.3):c.416G>A, NP_000397.1:p.(Arg139His), (alt/total alleles: 86/90, GnomAD genome frequency:1/3138 and exome frequency:1/6966). The variant was classified as pathogenic according to ACMG criteria. Both parents were heterozygous for the variant. The GnRHR is a special type of the seven transmembrane helices G-protein-coupled receptor (GPCR) superfamily, which uses primarily the Gq protein for downstream signalling. The conserved Asp-Arg-Tyr motif in most GPCRs corresponds to the Asp-Arg-Ser motif in GnRHR, containing Arg139. The positively charged Arg139, located between the third transmembrane helix and the second intracellular loop, is conserved among species. The 3D crystallographic structure of the GnRHR-GnRH antagonist (elagolix) at 2.80 Å of resolution (PDB 7BR3) was analysed using Pymol. The Arg139 of the wild-type GnRHR forms an intrahelical salt bridge with Asp138 and a polar interaction with Thr265. Instead, in the Arg139His variant, the histidine side chain is shorter as compared to the arginine one. Consequently, the His139-Thr265 pair is further apart, which is predicted to weaken the His139-Thr265 interhelix interaction. In addition, the absence of the positive charge of Arg139 predicted a loss of the intrahelical salt bridge with Asp138, resulting in further intrahelix destabilisation. In summary, through NGS and protein structure analysis, we identified a GnRHR Arg139His variant in a boy with HH and characterised the underlying 3D structural alteration destabilising the conformational structure of the receptor.