Mutations in a G protein-coupled receptor cause hypogonadotropic hypogonadism in humans and mice

The GPR54 gene as a regulator of puberty

Seminara et al. (2003)

The New England Journal of Medicine 349: 1614–1627

Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54

Roux et al. (2003)

PNAS 100: 10972–10976

Both genetic and environmental factors regulate the timing of puberty, a complex biological process that is initiated by secretion of gonadotropin-releasing hormone (GnRH) by the hypothalamus. The steps in the resulting reproductive cascade are well understood, but the genetic factors that regulate secretion and activity of GnRH, and thus timing of puberty, remain elusive. The discovery of genetic variants regulating onset of puberty is therefore important to advance our understanding of normal reproduction and provide insight into disorders of sexual maturation.

Four such variants have now been identified in the GPR54 gene by two independent research teams studying consanguineous families affected by idiopathic hypogonadotropic hypogonadism (IHH). IHH affects one in 50,000 people and is characterized by the absence of spontaneous sexual maturation despite concentrations of gonadotropins in the low-normal range. It belongs to the spectrum of disorders known as isolated hypogonadotropic hypogonadism (OMIM 146110) but lacks the anosmia (absence of smell) observed in Kallmann's syndrome (KMS) and the pituitary or hypothalamic anatomical lesions that cause other forms of hypogonadism. Patients who suffer from IHH are commonly treated with GnRH delivered subcutaneously in pulses injected by a small pump, mimicking the natural intermittent release of the protein in the brain.

Both groups performed genome-wide scans on large IHH families where the consanguinity resulted from matings between first cousins. Roux et al. reported a maximal two-point logarithm of the adds ratio (LOD) score of 3.5 at marker D19S886, and Seminara et al. referenced a recent publication in which they reported a maximal two-point LOD score of 2.94 at marker D19S209, which increased to 5.17 at two novel polymorphic markers on chromosome 19p13.3. The high degree of consanguinity facilitated homozygosity mapping strategies, resulting in candidate regions of 1.65 Mb and 1.06 Mb, respectively.

The GPR54 gene (also known as AXOR12) encoding a G protein-coupled receptor was immediately identified as a candidate gene potentially involved in reproduction, due to its expression in brain, pituitary gland, and placenta, and due to the elevation of its ligand, the KiSS1 protein, during pregnancy. Roux et al. were the first to publish, describing a homozygous deletion of 155 nucleotides, encompassing the splicing acceptor site of the intron 4–exon 5 junction and part of exon 5 (c.739-13_879del). Seminara et al. described a homozygous c.443T > C (p.L148S) mutation, but as this was not an obvious loss-of-function mutation, they were required to express the mutant GPR54 protein in transfected COS-7 (SV40 transformed African green monkey kidney) cells and show that the mutation decreased the ligand-stimulated production of inositol phosphate by 65%.

Each group then went on to sequence the gene in other patients with isolated hypogonadotropic hypogonadism (HH). A homozygous p.L102P mutation was detected in one of three unrelated familial cases of isolated HH, and compound heterozygous [p.R331X] + [p.X399R] mutations were identified in one of 63 unrelated cases of IHH. The function of the missense mutation was not tested, but the other mutations were shown to have reduced function and substantially reduced transcript levels, presumably due to nonsense and nonstop-mediated decay, respectively. No mutations in GPR54 were detected in 20 patients with KMS.

Seminara et al. were able to begin to address genotype–phenotype correlations by analyzing the results of previous dose–response studies that used exogenous, pulsatile GnRH. The patient carrying the compound heterozygous mutations had a left-shifted dose–response curve for GnRH as compared with six patients who had IHH without GPR54 mutations, suggesting that this patient was more sensitive to exogenous GnRH. His neuroendocrine profile included low-amplitude pulses of luteinizing hormone, further indicating that the phenotype might be due to an upstream defect, such as reduced secretion of endogenous GnRH (Fig. 1).

In an elegant example of human–mouse phenotypic concordance, the same group of researchers provided complementary data from a mouse model with a targeted mutation in GPR54. Mice deficient in GPR54 mirrored their human counterparts with isolated HH (small testes or delay in vaginal opening and absence of follicular maturation), demonstrating a conserved function of the gene among mammalian species. Like the affected humans, the mice remained responsive to stimulation by GnRH. Additionally, the generation of GPR54-deficient mice permitted the quantitation of their hypothalamic GnRH, which was normal despite their hypogonadotropism, similar to prepubertal rats and monkeys that display normal hypothalamic levels of GnRH. Although they have yet to provide definitive evidence, the authors hypothesize that GPR54 must be regulating the release of GnRH at the level of the hypothalamus.

The only other gene that has been implicated in normosmic IHH in humans is the GnRH receptor gene (GNRHR), which accounts for 10% of cases. A disruption of GnRH in mice leads to an isolated HH phenotype, but no mutations have yet been identified in humans, underlining the importance of the GPR54-deficient mouse as a model system for studying pathology, genetics, and pharmacologic intervention in HH. Other genes associated with human HH have been identified, but all are found combined with other phenotypic features, including anosmia, adrenal insufficiency, sex reversal, and obesity.

Although it appears that the frequency of GPR54 mutations as a cause of familial and sporadic IHH is not high, the identification of this gene as a regulator of GnRH physiology and puberty may well constitute an important milestone, illuminating other candidate genes involved in sexual maturation and puberty. These may include the genes that regulate availability of the GPR54 ligand or regulate the activity of the GPR54 receptor itself, or genes that belong to signaling pathways downstream of the receptor. Furthermore, drugs that increase GPR54 signaling (a readily attainable goal for proteins belonging to this family of proteins) may offer a less intrusive therapeutic alternative for patients with IHH, whereas GPR54 antagonists could potentially be used in new forms of medical castration, without the transient stimulation of sex steroids observed in current treatments with GnRH agonists.