Two Novel Missense mutations in the GnRHR Gene in Two Siblings with Isolated Hypogonadotropic Hypogonadism

Introduction: Gonadotropin releasing hormone receptor (GnRHR) is a seven transmembrane G protein-copuled receptor (GPCR) that binds GnRH, the principal regulator of the reproductive function. Mutations in the GnRHR gene are associated with the autosomic recessive form of isolated hypogonadotropic hypogonadism (HH) characterized by variable forms of absent or incomplete pubertal development and infertility. The human GnRHR gene is located on chromosome 4q13.2-13.3. Its coding sequence comprises 3 exons and encodes a 328 amino acid protein with some particular features that distinguish it from other GPCRs. Objective: We report GnRHR molecular analysis in two Italian siblings presenting clinical signs of HH. Patients and Methods: The female was referred at age 14 for pubertal delay (no sexual secondary signs). The male was followed since prepuberty. He started pubertal development at age 13 and the testis reached a volume of 8 ml with no subsequent progression. Hormonal evaluation showed very low levels of both gonadotropins (LHRH test) and sexual steroids in both patients. GnRHR gene was amplified in three different fragments with primers complementary to flanking intronic sequences and directly sequenced by CEQ2000 (Beckman-Coulter) on both strands. Results: Direct sequencing of exon 1 revealed in the two siblings the presence of two new single nucleotide substitutions resulting in the Thr104Ile and the Tyr108Cys substitution in the first extracellular loop (ECL1), confirmed on both strands in two different PCR reactions. The molecular analysis confirmed the carrier status of the parents. Conclusions: We identified two new missense mutations in the GnRHR gene in two siblings with HH. The nature of substitutions (polar Thr with hydrophobic Ile, and aromatic Tyr with a Cys) in the ECL1 involved in the ligand-receptor interaction, as well as the high conservation of the two residues in all mammalian GnRHR, are suggestive of some implications in the phenotype observed.