Mutations of the KISS1 gene in disorders of puberty

Abstract

Context: Kisspeptin, encoded by the KISS1 gene, is a key stimulatory factor of GnRH secretion and puberty onset. Inactivating mutations of its receptor (KISS1R) cause isolated hypogonadotropic hypogonadism (IHH). A unique KISS1R-activating mutation was described in central precocious puberty (CPP).

Objective: Our objective was to investigate KISS1 mutations in patients with idiopathic CPP and normosmic IHH.

Patients: Eighty-three children with CPP (77 girls) and 61 patients with IHH (40 men) were studied. The control group consisted of 200 individuals with normal pubertal development.

Methods: The promoter region and the three exons of KISS1 were amplified and sequenced. Cells expressing KISS1R were stimulated with synthetic human wild-type or mutant kisspeptin-54 (kp54), and inositol phosphate accumulation was measured. In a second set of experiments, kp54 was preincubated in human serum before stimulation of the cells.

Results: Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in three unrelated children with idiopathic CPP. Both mutations were absent in 400 control alleles. The p.P74S mutation was identified in the heterozygous state in a boy who developed CPP at 1 yr of age. The p.H90D mutation was identified in the homozygous state in two unrelated girls with CPP. In vitro studies revealed that the capacity of the P74S and H90D mutants to stimulate IP production was similar to the wild type. After preincubation of wild-type and mutant kp54 in human serum, the capacity to stimulate signal transduction was significantly greater for P74S compared with the wild type, suggesting that the p.P74S variant is more stable. Only polymorphisms were found in the IHH group.

Conclusion: Two KISS1 mutations were identified in unrelated patients with idiopathic CPP. The p.P74S variant was associated with higher kisspeptin resistance to degradation in comparison with the wild type, suggesting a role for this mutation in the precocious puberty phenotype.

Figure 1

A, The propositus (P74S) at age 1.6 yr and detail of the genitalia showing enlarged penis and testes for age and Tanner stage III pubic hair. B, Nucleotide sequence of KISS1 gene showing the wild type and the two variants identified in patients with CPP. Both variants are located in exon 3 of the KISS1 gene, schematically represented in C. D, Precursor peptide kisspeptin-1 (KP-1) encoded by exons 2 and 3 of KISS1 and processed to generate the mature 54-amino-acid peptide, kp54 (KP-54) or metastin. The two variants are located in the amino-terminal portion of kp54, outside of the receptor-binding domain. E shows detailed kisspeptin amino acid sequence alignment among species, showing that proline in position 74 is highly conserved among mammals, whereas histidine in position 90 is not conserved. The p.P74S variant is within a PEST sequence [sequence rich in the aminoacids proline (P), glutamic acid (E), serine (S) and threonine (T)] (dotted line). The following were sequence sources: Homo sapiens (GenBank NP_002247.2), Pan troglodytes (GenBank XP_514123.1), Macaca mulatta (GenBank XP_001098284.1), Bos taurus (GenBank XP_872566.1), Felis catus (ENSFCAP00000013771) Rattus norvegicus (GenBank NP_859043.1), and Mus musculus (GenBank NP_839991.1).

Figure 2

Stimulation of IP production by wild-type or mutant kisspeptin in CHO-KISS1R cells. A, Total IP accumulation was measured after stimulation for 2 h with 10⁻¹⁰ to 10⁻⁷ m wild-type human kp54 (WT) or mutant P74S kp54. B, The same concentrations of wild-type and mutant P74S kp54 were also preincubated in 50% human serum for 2 h before using them to stimulate CHO-KISS1R cells. C and D, The effects of wild-type and mutant H90D kp54 on total IP accumulation were similarly assessed without (C) or with (D) serum preincubation. Data points represent mean ± se for triplicate samples from representative experiments. Dose-response curves for both kisspeptins were done in the same experiments so that conditions were identical.