Human puberty is initiated by an increase in the amplitude and frequency of pulses of gonadotropin-releasing hormone (GnRH) after a quiescent period during childhood. The resurgence of pulsatile GnRH release from specialized hypothalamic neurons induces the secretion of gonadotropin by the pituitary gland, which leads to gonadal maturation and the attainment of reproductive capacity (1). Pubertal onset occurs across a wide range of ages in normal, healthy adolescents and is influenced by complex interactions among genetic, nutritional, environmental, and socioeconomic factors (2). The genetic factors influence pubertal timing in 50%–80% of the general population (3).

Precocious puberty is defined as the appearance of secondary sexual characteristics in girls younger than 8 years old and in boys younger than 9 years old. Central precocious puberty (CPP) is caused by the premature activation of the hypothalamic-pituitary-gonadal axis. CPP occurs more frequently in girls than in boys, and most of these cases are considered idiopathic (456). Numerous studies have suggested that genetic factors play important roles in CPP. Familial CPP can occur in up to 27.5% of cases and segregation analysis has suggested autosomal dominant transmission with incomplete sex-dependent penetrance (7). Recently, mutations in MKRN3, the gene encoding makorin RING-finger protein 3, have been shown to cause familial CPP (8). However, the genetic determinants of CPP are largely unknown.

Over the last decade, the kisspeptin/kisspeptin receptor (KISS1R) signaling complex has emerged as a fundamental regulator of puberty and reproductive function. KISS1R, one of the G protein-coupled receptors, is a 398 amino acid protein going through the membrane seven times and is activated by its ligand, kisspeptin. The receptor is expressed widely in the brain, pituitary gland, and placenta (9). In 2003, two research groups independently reported inactivating mutations of the KISS1R gene in patients with idiopathic hypogonadotropic hypogonadism (IHH) (1011). These studies confirmed the critical role played by the kisspeptin/KISS1R system in hypothalamic-pituitary-gonadal axis activation. Navarro et al. (12) showed that binding to the G protein-coupled receptor in hypothalamic GnRH neurons enables kisspeptin to act as a powerful elicitor of GnRH-dependent luteinizing hormone (LH) secretion. In our previous study (13), we demonstrated that serum kisspeptin levels were significantly higher in girls with CPP than in controls. The KISS1 gene encoding kisspeptin and the KISS1R gene have recently been implicated in the pathogenesis of sexual precocity in humans. Activating mutations in KISS1 and KISS1R were detected as genetic causes of CPP (1415). Accordingly, it is assumed that genetic variations in the KISS1R gene can lead to higher or lower risk of CPP. However, few mutations in the KISS1R gene that cause CPP have been identified in previous studies, with the exception of the study mentioned above.

The aims of our study were to evaluate the occurrence of genetic variations, including mutations and single-nucleotide polymorphisms (SNPs), of the KISS1R gene in 194 girls with CPP and to investigate the correlation between KISS1R gene variations and CPP in Korean girls.

In this study, we evaluated a population of 194 girls with CPP for KISS1R gene mutations and polymorphisms, and we identified seven SNPs. Six polymorphisms except the intron variant c.505+42T>G were detected in both the CPP and control groups. Two polymorphisms, c.1091T>A and c.738+64G>T, were found to be statistically correlated to CPP. The missense variant (c.1091T>A) was a nonsynonymous polymorphism that induces amino acid substitution of p.Leu364His. The haplotype CAGTGTC was also significantly associated with CPP patients.

The pulsatile GnRH secretion mediated through the kisspeptin/KISS1R system appears to be a key event at the onset and during progression of puberty (1121). The timing of puberty varies greatly among individuals and races, and much of this variation is due to genetic factors. The genetic modulation of puberty is assumed to arise from the additive effect of multiple genes (22). Many researchers have tried to ascertain a molecular mechanism of kisspeptin/KISS1R system associated with development of CPP. Silveira et al. (15) by investigating 83 children with CPP reported two novel KISS1 missense mutations in 3 unrelated children with CPP. Moreover, Ko et al. (23) identified one polymorphism (p.P110T) relevant to CPP in KISS1 gene in 101 Koreans girls with CPP and the polymorphism was suggested to exert a protective effect. In our previous study, we demonstrated that three KISS1 polymorphisms were statistically correlated with CPP, but could not find any clinical importance of these SNPs (24). Recently, it was found that the KISS1R gene is relevant to CPP pathogenesis (14). An autosomal dominant missense mutation, Arg386Pro, in KISS1R, leading to prolonged activation of intracellular pathways in response to kisspeptin, has been suggested to cause CPP in an adopted girl. Since then, no other CPP cases with activating KISS1R mutations have been reported. On the other hand, a few KISS1R polymorphisms have been identified in CPP patients. Some researchers have found A/G synonymous SNP (c.24A>G) in girls with CPP, but it did not seem to have a pathogenic role in patients (1819). The polymorphism c.*120C>G has been reported in Chinese girls with CPP and the SNP was not statistically correlated with CPP (17). In the same study, the missense mutation p.P196H in the KISS1R was also identified in one patient, although functional study was not performed (17). Moreover, a missense variant c.1091T>A has been identified in studies of Korean girls with CPP and other ethnic group (1820). However, the researchers were unable to determine any disease associations with the SNP.

In the present study, the seven polymorphisms were detected via the sequencing of the KISS1R gene. According to the calculated statistical results, a missense variant c.1091T>A exhibited a statistical correlation with the phenotype (P = 0.031). This polymorphism induced amino acid substitution of p.Leu364His. This SNP was previously reported in a study of Korean CPP patients in 2011 (20) and the allele frequency in the study was 30.3%. This result was slightly different to that in our study, in which the allele frequency was calculated as 27.8%. Since both study subjects had same ethnic background, the reason of this discrepancy in the allele frequency is unclear. One possible reason is a different sample size. In the previous study, the results showed a similar allele frequency in patients (30.3%) and controls (30.8%) (20). However, in our study, the allele frequencies were significantly higher in the patients (30.7%) than in the controls (22.2%), although the polymorphism c.1091T>A was identified in both the patients and the controls. Our finding suggests that the missense variant c.1091T>A might affect the pathogenesis of CPP. In order to evaluate the clinical significance of p.Leu364His, we compared the clinical characteristics and hormone values between the subgroups with or without this SNP in the patient group. However, no significant differences were observed between the two subgroups.

The intron variant c.738+64G>T also exhibited a statistical correlation with CPP patients (P = 0.023). It was located in the fourth intron with G to T substitution at nucleotide +64 and did not result in any amino acid change. This SNP was not introduced in previous studies concerning CPP. Although introns are generally known as non-protein-coding sections in genes, it can produce genetic and phenotypical variety by regulating or facilitating the transposition of exons (25). Therefore, it can be hypothesized that the polymorphism c.738+64G>T may affect the gene expression of KISS1R relevant to the pathogenesis of CPP in Korean girls. In the comparison of clinical parameters and hormonal results, no significant differences were observed between the subgroups with or without this SNP in the patient group. More evidence will be necessary to confirm the association between intron variant in the KISS1R and CPP.

In the current study, we have also identified a synonymous SNP (c.24A>G) residing in exon 1 of the KISS1R gene. This SNP was not correlated with CPP in our study. Since this variant induced a synonymous change, it did not appear to affect the activity of the KISS1R. This polymorphism was previously identified by others in a population of male patients with IHH, although no association with this disorder was found (2627). It was also detected in CPP patients in previous studies, but it did not seem to have a pathogenic effect on CPP (1819). These findings were in accord with our results. However, this polymorphism has not been recorded in a large population of Chinese girls (24 girls randomly selected from 272 girls with CPP) (17). The discordance in data between Korean and Chinese populations is partly due to different scale of typed samples. Also, the genetic background appeared to differ significantly, even though Koreans and Chinese are both Asian.

We identified one LD block that consisted of seven SNPs. The value of LD (D′) between each SNP was very high and five haplotype were constructed using the LD block. The haplotype CAGTGTC was detected more frequently in the CPP group (30.2%) than in the control group (22.2%) (P = 0.042). Our findings suggest that the haplotype CAGTGTC contributes to pathogenesis of CPP, although the P value of 0.042 was not sufficiently significant.

In conclusion, we have attempted to identify sequence variations of the KISS1R gene associated with CPP in Korean girls, and have noted some possibilities that the sequence variants within the KISS1R gene might be inducible factors in the pathogenesis of CPP. A possible limitation of the present study was that the sample scale was relatively small, and that P values were not very significant. Therefore, larger scale studies are necessary in order to clarify the association between the sequence variations of KISS1R and CPP.