Eur J Endocrinol
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

DOI: 10.1530/EJE-07-0061
European Journal of Endocrinology, Vol 157, Issue 1, 113-118
Copyright © 2007 by European Society of Endocrinology
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Luan, X.
Right arrow Articles by Xiao, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Luan, X.
Right arrow Articles by Xiao, J.
CLINICAL STUDY

Association study of the polymorphisms in the KISS1 gene with central precocious puberty in Chinese girls

Xiaohui Luan1,2, Yuxun Zhou2, Wei Wang3, Hong Yu4, Pin Li5, Xiaohong Gan2, Dongzhi Wei1 and Junhua Xiao1,2

1 State Key Laboratory of Bioreactor Engineering, Institute of Biochemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People’s Republic of China, 2 Institute of Bioscience and Technology, Donghua University, 1882 West Yan’an Road, Shanghai 200052, People’s Republic of China, 3 Department of Pediatrics, Ruijin Hospital, Shanghai Jiaotong University, 197 No. 2 Ruijin Road, Shanghai 200025, People’s Republic of China, 4 Department of Adolescent Clinic, Harbin Children’s Hospital, 57 Youyi Road, Harbin 150010, People’s Republic of China and 5 Department of Endocrinology, Shanghai Jiaotong University Affiliated Children’s Hospital, 1400 West Beijing Road, Shanghai 200040, People’s Republic of China

(Correspondence should be addressed to J Xiao; Email: xiaojunhua{at}dhu.edu.cn)


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
Objective: The kisspeptin/GPR54 pathway has been proven to be crucial in the process of puberty onset, yet the polymorphisms in the KISS1 gene and their relationships with central precocious puberty (CPP) have not been investigated. This study was performed to reveal the relationship between the gene and the disease.

Design and Methods: 272 Chinese Han girls diagnosed to be CPP patients were recruited as Case Group I, 43 unrelated African women as Case Group II, and 288 unrelated normal Chinese Han girls as Control Group. Polymorphism scans of the KISS1 gene were performed for the first time by bidirectional resequencing of the whole gene in a subset of the patients, and then by ligase detection reaction some of the polymorphisms identified were typed in the two groups and the respective haplotypes were constructed. The relationships of the typed polymorphisms and the haplotypes with CPP were evaluated by an association study between genotypes and phenotypes.

Results: By resequencing, eight polymorphisms were identified, five of which were typed forming 18 haplotypes. Although one novel nonsynonymous single nucleotide polymorphism substituting one amino acid in kisspeptin (P110T) was found to be statistically related to the disease (P = 0.025), no further supporting evidence has yet been found. The other polymorphisms and all the haplotypes were not found to be related.

Conclusion: The polymorphism scanning and typing of KISS1 uncovered several potentially meaningful polymorphisms, but the conclusion was not solid and further studies are necessary for function validation of these polymorphisms.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
When kisspeptin was first identified in 1996 (1), it was mainly considered as a tumor metastasis suppressor. Since it was found to be the ligand of orphan G-protein coupled receptor GPR54 in 2001 (2), researchers came to notice the possible role of kisspeptin/GPR54 signal pathway in the regulation of endocrine function, which was proved by recent studies to demonstrate that kisspeptin would directly activate the onset of puberty in mammals via GPR54 by stimulating the secretion of gonadotropin-releasing hormone (GnRH; (3–5)).

The onset timing of puberty varies depending on numerous variables. Generally, it would be defined as precocious puberty if any signs of secondary sexual maturation appears at an age more than 2 standard deviation units below the population mean (<8 for girls and <9 for boys in China), and would be further defined as central precocious puberty (CPP) if the process is driven by activation of hypothalamic GnRH secretion.

There have been reports indicating that CPP patients would be shorter in stature when fully grown and more susceptible to breast cancer (6). The incidence of CPP in Chinese children was about 1% according to epidemiologic data in 2003 (7). Some studies have identified several associated genes or polymorphisms involved in development, body composition, gonadal steroid metabolism, and many other biological processes (8–10), yet the exact mechanism remains a mystery. The important function of kisspeptin in puberty onset makes it necessary to investigate the polymorphisms in the gene and their relationships with CPP.

The KISS1 gene consists of three exons, of which only part of the second and third exons are finally translated into a 145-amino acid protein with a putative signal sequence, from which three forms of kisspeptins containing 54, 14, and 13 amino acids are derived (Fig. 1Go). The three peptides exhibit the same affinity for the receptor since they share a common C-terminal decapeptide designated kisspeptin-10, which is the minimum sequence necessary for receptor activation.


Figure 1
View larger version (26K):
[in this window]
[in a new window]

  		Figure 1 Amino acid sequence of the KISS1 product and derived peptides. The predicted signal peptide of KISS1 is in italicized and bold letters. Dibasic motifs representing potential cleavage sites by furins and prohormone convertases are underlined and in bold letters. The two amino acids changed by 54650055 G/T and 54650141 G/C are marked within rectangles.

 
In this study, the three exons, exon–intron boundaries, and 1.4 kb of 5' flanking region of the KISS1 gene were amplified and sequenced in a cohort of 36 Chinese CPP girls, by which the mechanism of how how the kisspeptin/GPR54 system functions in the onset of puberty may be better understood.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Experimental subjects

Three groups of subjects were involved in this study. Case Group I consisted of 272 Chinese sporadic CPP girls recruited from three hospitals in Shanghai and Harbin, China; Case Group II consisted of 43 normal African adult female subjects; and Control Group consisted of 288 independent normal Chinese Han girls recruited from East China University of Science and Technology. The study protocols were reviewed and approved by the Ethics Committees for Human Research of the hospitals and the college, and informed consent was obtained from all the subjects or their guardians.

The CPP patients were all diagnosed according to the same criteria and at diagnosis they were between 5.9 and 8.5 years of age. The patients all showed dramatic growth acceleration, with Tanner breast stage scores ≥2 based on standardized Tanner breast stage assessment. Bone ages were measured and data of 215 subjects (79.04%) were found to be at least 1 year ahead of their true ages, and a few of the patients even had vaginal bleeding. A GnRH stimulation test was carried out in all subjects to confirm that the hypothalamic–pituitary–gonadal axis had been activated, by which significantly high luteinizing hormone (LH) peak values (23.2 ± 12.7 mIU/ml) were observed after 40 min of the injection, when compared with the cut-off value of 15 mIU/ml (11), and the LH/follicle-stimulating hormone (FSH) ratios were all above 0.66 (12). To exclude any possible brain abnormalities, magnetic resonance imaging was performed and no brain diseases were found. Most of the parents of the patients were questioned about their pubertal ages, yet none of them recalled precocious puberty in childhood.

The DNA samples of the African subjects were obtained from the Chinese National Human Genome Center at Shanghai. The 43 subjects were all normal African women with a mean age of 36.8 ± 12.5 years. The puberty onset timing of the subjects was not obtained.

The control subjects were selected from female volunteers who were first grade students in East China University of Science and Technology based on a freewill questionnaire regarding their age of menarche. The mean age of the control subjects was 18.24 ± 1.38 and the mean age of menarche was above 12 years.

In total, 36 CPP subjects were randomly selected from Case Group I for sequencing.

Peripheral blood samples were collected from all subjects and genome DNA samples were isolated with phenol–chloroform method.

Sequencing

Six pairs of overlapping primers covering all the sequenced regions were designed and synthesized (Sangon, Shanghai, China), with which PCR was performed in a 20 µl system by 35 cycles of amplification. The products were purified for bidirectional sequencing on an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA).

Genotyping

Typing of the selected single nucleotide polymorphisms (SNPs) was performed with a method developed by ligase detection reaction (LDR; (13)), the mechanism of which is shown in Fig. 2Go. Multiplex PCR was performed to amplify four fragments containing the five SNPs with HotStarTaq polymerase (Qiagen Inc.) in a volume of 20 µl by 40 cycles of amplification.


Figure 2
View larger version (24K):
[in this window]
[in a new window]

  		Figure 2 Flow chart of genotyping method based on ligase detection reaction. A sample which was wild-type homozygous at 54 650 055 and heterozygous at 54 650 141 was selected to explain the mechanism of the genotyping method. The trace map at the bottom was generated with the software GeneMapper ID version 3.2.

 
The PCR products were used as templates for LDR. For each polymorphism, one universal fluorescence-labeled probe and two allele-specific probes of different lengths were designed according to the flanking sequence. The LDR was carried out in a volume of 20 µl containing probes, PCR products and thermostable Taq DNA ligase (New England Biolabs, Ipswich, MA, USA) according to the instructions. Recognizing respective alleles, the probes would be joined by ligase into corresponding products distinguishable on ABI 3100 sequencer. The electrophoresis results were analyzed by GeneMapper ID software version 3.2 (Applied Biosystems). Primers and probes were also synthesized by Sangon, and oligo sequences and detailed reaction conditions can be supplied on request.

Deviations from the Hardy–Weinberg equilibrium were tested by comparison of observed and expected genotype frequencies with {chi}2 test.

Haplotype construction and statistical analysis

Haplotypes were estimated from genotype data for individual participants using the PHASE program version 2.0 (14).

The comparison of the allele frequencies among the three groups was performed by Fisher’s exact test. The SPSS 11.5 software package (SPSS Inc., Chicago, IL, USA) was used to perform statistical analysis with P < 0.05 as the minimal level of statistical significance.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Sequencing of the KISS1 gene revealed eight SNPs as listed in Table 1Go, among which four were identified for the first time (sequencing traces shown in Fig. 3Go). Two SNPs (54 650 055 G/T and 54 650 141 G/C) were found to be nonsynonymous, leading to substitution of P110T and P81R respectively.


View this table:
[in this window]
[in a new window]

  		Table 1 Single nucleotide polymorphisms (SNPs) identified by sequencing.
 

Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		Figure 3 Sequencing results of novel polymorphisms. Typical sequencing traces of the four novel single nucleotide polymorphism are shown, wild-type homozygous subjects on the top and heterozygous genotype on the bottom. The arrows indicate the positions of polymorphisms.

 
Five SNPs of relatively high heterozygosity were selected and genotyped in the three groups, and the results fitted the expectation given by the Hardy–Weinberg equilibrium. Allele counts and frequencies are shown in Table 2aGo. With Fisher’s exact test, the relationships between the polymorphisms and the phenotypes were evaluated, among which the nonsynonymous polymorphism 54 650 055 G/T showed statistical significance with CPP (P = 0.025), while the other four SNPs showed none.


View this table:
[in this window]
[in a new window]

  		Table 2 Genotyping results and haplotype analysis.
 
In total 18 haplotypes were constructed in Case Group I and Control Group based on the typing data, and their relationships with CPP were also investigated (Table 2bGo), yet none of them showed significant correlation.


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
GPR54 remained an orphan receptor until three independent groups (2, 15, 16) identified its natural ligand kisspeptin. Numerous studies on various species including mice, rats, sheep, primates and humans had reported that the kisspeptin/GPR54 pathway would stimulate the secretion of GnRH and start the onset of puberty (3, 17–19). Several mutations were found in the GPR54 gene and their functions demonstrated, yet no such studies had been carried out on the KISS1 gene. This study was the first time that the polymorphisms in KISS1 and their relationships with CPP were investigated in Chinese girls.

Whole gene resequencing was carried out in part of the CPP patients to screen polymorphisms, after which the frequencies of the eight SNPs found in the sequencing were calculated in the sequenced cohort, and five were considered informative due to their relatively high degree of heterozygosity and were selected as ‘tagging’ SNPs for genotyping. Though the frequency of 54 656 436 G/A was a little higher than that of 54 650 055 G/T in the sequenced cohort, the latter was selected for typing since it was an amino acid substituting polymorphism.

According to the statistical results calculated with the typing data, the nonsynonymous SNP 54 650 055 G/T substituting the 110th amino acid of KISS1 protein from proline to threonine was found to be statistically related to CPP. Though the P110T site was contained by all the three natural in vivo forms of kisspeptin, kisspeptin-54, 14, and 13 (Fig. 1Go), the association could not be validated without further support from functional data, especially considering that the sample scale was relatively small and that a P-value of 0.025 was not significant enough.

A different ethnic group was involved for investigation to provide more solid evidence. From the aspect of human evolution and migration, delayed puberty onset would be observed as humans moved to colder regions, as described in a recent review (20) which showed that African–American children had earlier pubertal milestones than white children, with the data of Mexican–American children in between. The facts can be explained by the assumption that individuals tend to delay the onset of puberty when facing tougher environments – less food and lower temperature. Forty-three healthy African female adults were then selected as Case Group II despite the lack of their pubertal data. The five tagging SNPs were also typed with the same method in all the subjects, and the results (also shown in Table 2Go) showed that four SNPs were of similar frequencies as observed in Chinese subjects, except 54 650 055 G/T, which was not found in any of the African subjects. The P-value between Case Group II and Control Group was 0.006. Based on the data, it may be reasonable to hypothesize that the mutation could in some degree protect the carrier from earlier puberty onset.

The other nonsynonymous SNP did not exhibit significant relationship either between Case Group I and Control Group, or between Case Group II and Control Group. The polymorphism introduced a substitution at the 81st site from proline to arginine, which was included by kisspeptin-54 but not by the other three forms. From the result, it may be assumed that kisspeptin-14 and 13 are possibly more important than kisspeptin-54 in humans; thus, the amino acid change would not seriously affect the bioactivity, supported by a previous report that kisspeptin-54 was unstable and would easily degrade into the other two forms (2).

The association study of haplotypes constructed with the five SNPs typed showed no significant relationship between the 18 haplotypes, which could be partially attributed to the relatively small scale of population typed, causing the correlated alleles to disperse into different haplotypes. Owing to this, it was more difficult to confirm the relationship between the haplotypes and the disease.

In brief, this study tried to reveal the relationships between the polymorphisms and haplotypes in KISS1 gene and CPP in Chinese girls. The results would deepen our understanding in both the KISS1 gene and the mechanism of the disease. Yet, the limit of association study calls for more research in the future to confirm the actual function of the polymorphism.


   Acknowledgements
 
This work was supported by grants from the National Nature Science Foundation of China (Grant no. 30 300 198) and the State Key Basic Research Program (Grant no. 2002CB512 900).


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

    1. Lee J-H, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE & Welch DR. KiSS-1, a novel human malignant melanoma metastasis- suppressor gene. Journal of the National Cancer Institute 1996 88 1731–1737.[Abstract/Free Full Text]

    2. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Poul EL, Brezillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G & Parmentier M. The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the Orphan G Protein-coupled receptor GPR54. Journal of Biological Chemistry 2001 276 34631–34636.[Abstract/Free Full Text]

    3. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, Thresher RR, Malinge I, Lomet D, Carlton MBL, Colledge WH, Caraty A & Aparicio S. Kisspeptin directly stimulates gonado-tropin-releasing hormone release via G protein-coupled receptor 54. PNAS 2005 102 1761–1766.[Abstract/Free Full Text]

    4. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ, Gottsch ML, Clifton DK & Steiner RA. Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology 2004 80 264–272.[CrossRef][ISI][Medline]

    5. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA & Bloom SR. Kisspeptin-54 stimulates the hypothalamic–pituitary gonadal axis in human males. Journal of Clinical Endocrinology and Metabolism 2005 90 6609–6615.[Abstract/Free Full Text]

    6. Petridou E, Syrigou E, Toupadaki N, Zavitsanos X, Willett W & Trichopoulos D. Determinants of age at menarche as early life predictors of breast cancer risk. International Journal of Cancer 1996 68 193–198.[CrossRef][ISI][Medline]

    7. Cai DP. Precocious and Delayed Puberty in Children. 1 Shanghai: Fudan University Press, 2003 p 35.

    8. Yu WH, Kimura M & Walczewaka A. Role of leptin in hypothalamic–pituitary function. PNAS 1997 94 1023–1028.[Abstract/Free Full Text]

    9. Kadlubar FF, Berkowitz SG, Delongchamp RR, Charles W, Green LB, George T, Jatinder L, Erin S & Mary SW. The CYP3A4*1B variant is related to the onset of puberty, a known risk factor for the development of breast cancer. Cancer Epidemiology, Biomarkers and Prevention 2005 12 327–331.

    10. Xin X, Luan X, Xiao J, Wei D, Wang J, Lu D & Yang S. Association study of four activity SNPs of CYP3A4 with the precocious puberty in Chinese girls. Neuroscience Letters 2005 381 284–288.[ISI][Medline]

    11. Zhang LJ. Diagnosis and treatment of precocious puberty. Journal of Practical Training of Medicine 2005 33 5–6.

    12. Oerter KE, Uriarte MM, Rose SR, Barnes KM & Cutler GB Jr. Gonadotropin secretory dynamics during puberty in normal girls and boys. Journal of Clinical Endocrinology and Metabolism 1990 71 1251–1258.[Abstract]

    13. Xiao Z, Xiao J, Jiang Y, Zhang S, Yu M, Zhao J, Wei D & Cao H. A novel method based on ligase detection reaction for low abundant YIDD mutants detection in Hepatitis B virus. Hepatology Research 2006 34 150–155.[ISI][Medline]

    14. Stephens M, Smith NJ & Donnelly P. A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics 2001 68 978–989.[CrossRef][ISI][Medline]

    15. Ohtaki T, Shintani Y & Honda S. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 2001 411 613–617.[CrossRef][Medline]

    16. Muir AI, Chamberlain L & Elshourbagy NA. AXOR12, a novel human G proteincoupled receptor, activated by the peptide KiSS-1. Journal of Biological Chemistry 2001 276 28969–28975.[Abstract/Free Full Text]

    17. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR & Plant TM. Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates. PNAS 2005 102 2129–2134.[Abstract/Free Full Text]

    18. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MBL, Crowley WF, Aparicio S & Colledge WH. The GPR54 gene as a regulator of puberty. New England Journal of Medicine 2003 349 1614–1627.[Abstract/Free Full Text]

    19. de Roux N, Genin E, Carel J-C, Matsuda F, Chaussain J-L & Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54. PNAS 2003 100 10972–10976.[Abstract/Free Full Text]

    20. Herman-Giddens ME. Recent data on pubertal milestones in United States children: the secular trend toward earlier development. International Journal of Andrology 2006 29 241–246.[CrossRef][ISI][Medline]

Received 4 February 2007
Accepted 16 April 2007




This article has been cited by other articles:


Home page
 Biol. Reprod.Home page
M. E. Wilson and B. Kinkead
Gene-Environment Interactions, Not Neonatal Growth Hormone Deficiency, Time Puberty in Female Rhesus Monkeys
Biol Reprod, April 1, 2008; 78(4): 736 - 743.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Luan, X.
Right arrow Articles by Xiao, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Luan, X.
Right arrow Articles by Xiao, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS