An association study between the genetic polymorphisms within GnRHI, LHβ and FSHβ genes and central precocious puberty in Chinese girls

Neuroscience Letters 486 (2010) 188–192

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An association study between the genetic polymorphisms within GnRHI, LHˇ and FSHˇ genes and central precocious puberty in Chinese girls Ying Zhao a,b , Ting Chen a , Yuxun Zhou a , Kai Li a , Junhua Xiao a,∗ a b

Institute of Biology Science and Technology, DongHua University, 2999 North Ren Min Road, Shanghai 201620, China Institute of Bioengineering, College of Biochemistry and Molecular Biology, East China University of Science and Technology, 130 Mei Long Road, Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 15 September 2009 Received in revised form 21 June 2010 Accepted 17 September 2010 Keywords: SNP Linkage disequilibrium CPP LH␤ GnRHI FSH␤

a b s t r a c t Objectives: Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are three hypothalamic–pituitary–gonadal axis expressing hormones. They play critical roles in the onset of puberty. Here we report the relationship between the three hormones and Central Precocious Puberty (CPP) in Chinese Han girls. Methods: We analyzed the single nucleotide polymorphisms (SNPs) of 5 -flanking regions of these genes by DNA sequencing in 27 CPP samples. Then the SNPs sites were genotyped by ligase detection reaction in a total of 283 Chinese Han CPP cases and 284 matched controls. Distributions of the polymorphisms and haplotypes were calculated for statistical evaluation. Results: Nine SNPs (One in GnRHI gene: −2003 C/T; Five in LHˇ gene: −1456 C/G, −1424 C/G, −238 G/A, −164 G/A and −34 T/A; Three in FSHˇ gene: −1825 T/C, −261 G/T and −132 T/A.) were found. A quantitative genetic association study was made. −1825 T/C in FSHˇ gene was related with CPP with a weak effect (P = 0.025). A haplotype in the 5 -flanking region of LHˇ gene was significantly associated with CPP in Chinese Han girls (P = 8.25 × 10−09 ). However, analysis software showed that none of SNP was found in the regulating control element of these genes. Conclusions: Our finding implies that the polymorphisms in the 5 -flanking regions of FSHˇ gene and LHˇ gene probably were related to the puberty onset time of these girls. Further studies on the polymorphisms are needed for the exact mechanism. © 2010 Elsevier Ireland Ltd. All rights reserved.

Precocious puberty is a common development abnormal phenomenon characterized by early onset of puberty. Precocious puberty in China is currently defined as the secondary sexual characteristics appear before the age of 8 years old in girls [6,24]. In 2005, an epidemiological survey on precocious puberty showed that the prevalence of precocious puberty among girls from Shanghai was about 1% [1]. Central precocious puberty (CPP), triggered by the abnormal function of hypothalamic–pituitary–gonadal axis, can accelerate the onset of puberty in children. Series of evidence demonstrated that the interaction between the hypothalamus and the anterior pituitary was crucial to the onset of puberty in human [2,4,15]. GnRHI (gonadotropin-releasing hormone) was one of the most important hormones for the activation of the pituitary–gonadal axis at puberty. It stimulated the pituitary to synthesize and secrete luteinizing hormone (LH) and follicle-stimulating hormone (FSH) [8,22]. When the quantity of pulsatile secretion reached the critical threshold, GnRH promoted the onset the puberty [3,15]. The pituitary hormone LH and FSH were hetero-dimers. The unique beta-subunits helped LH and FSH to aim at the special target and induce a series of downstream reactions [25].

∗ Corresponding author. Tel.: +86 21 67792652; fax: +86 21 67792647. E-mail address: [email protected] (J. Xiao). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.09.049

It is clear that the expression levels of these three hormones are regulated by their upstream factors. Some mutations in the regulating elements of these genes would induce the CPP by change the transcription level of these genes. The GnRHI gene expression was a promoter-dependent phenomenon, mediated by the interaction between Oct-1 and the regulatory elements in the NSE (neuron specific enhancer) region. The mutation at NSE (−1787/−1783) region would block the pulsatile GnRHI promoter activity in GT1–7 neuronal cells [26]. In 1999, Jiang found eight point mutations in 661 bp 5 -flanking region of V-LHˇ gene. Compared with the wild-type, the promoter activity of the mutants increased 40% and 36% by luciferase report gene assay in HEK 293 and L␤T2 cells. At the same time, more qualitative differences were found in the promoter of V-LHˇ in response to various hormonal regulations. It was also indicated that these mutants had relationship with the early onset of puberty [9]. It was proved that there were more SNPs in the 5 -flanking regions than the coding regions [23]. So the SNPs in this region could be used as informative genetic markers. We carried out the analysis about the relationship between these three candidate genes (GnRHI, LHˇ and FSHˇ) and CPP in Chinese Han girls. Our association study was based on single nucleotide polymorphisms (SNPs) and haplotypes within 5 -flanking regions of these three genes in a total of 283 Chinese Han CPP cases and 284 matched controls. The 5 -flanking regions were resequenced for the discovery of SNPs in

Y. Zhao et al. / Neuroscience Letters 486 (2010) 188–192

27 CPP samples. By this way, we expect to find potential active SNPs on gene regulation elements and/or informative genetic markers for associated study based on linkage disequilibrium principle. Subjects, Chinese Han female, are composite of 567 individuals with 283 CPP and 284 controls. The individuals with CPP were from Ruijin Hospital, Harbin Children’s Hospital and Children’s Hospital Affiliated to Shanghai Jiaotong University. They were mainly from Shanghai and Heilongjiang Province, 64.7% and 20.5%, respectively, and the remains were from the other provinces of China. The CPP patients at the age of 5.9–8.5 were all diagnosed with clinical symptoms. All of them were found Tanner breast stage scores R2 based on standardized Tanner breast stage assessment. And bone ages of the 79.04% of subjects were found to be at least 1 year ahead of their true ages. A GnRH stimulation test was performed in all subjects to make sure that the hypothalamic–pituitary–gonadal axis had been activated. After 40 min of the injection, significantly high luteinizing hormone (LH) peak values (23.2 ± 12.7 mIU/ml) were observed compared with the cut-off values of 15 mIU/ml [31], and the LH/follicle-stimulating hormone (FSH) ratios were all above 0.66 [17]. Both traumatic brain injury and brain tumors were eliminated. Normal controls were recruited from several universities in Shanghai. Their ages of menarche were questioned by a freewill questionnaire. The mean age of the control subjects was 18.24 ± 1.38 and their menarche mean age was above 12 years. 53.2% samples of controls were local Shanghai inhabits and the remains came from the other regions of China. The study protocols were approved by the Ethics Committees for Human Research of the hospitals and the colleges, and

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informed consents were obtained from all the subjects or their parents. Peripheral blood samples were collected from all subjects, and genome DNA samples were isolated with phenol–chloroform method. 27 samples were randomly chosen from the case group for resequencing. Six pairs of overlapping primers covering the 2000 bp long 5 -flanking region (from 2000 bp upstream of the translation start site) of GnRHI, LHˇ and FSHˇ genes were designed and synthesized (Sangon, Shanghai, China). To avoid the error of sequencing due to the LHˇ/CGˇ cluster, the 3 -flanking sequences of the primers were designed to locate in the special regions which are different between LHˇ and CGˇ. The PCR was performed in a 20 ␮l system by 35 cycles of amplification, and then the purified products were sequenced on an ABI 3100 sequencer (Applied Biosystems, Foster City, CA, USA). In all patients and normal girls, single nucleotide polymorphisms found in the resequencing were genotyped with a method developed upon ligase detection reaction (LDR) [14,28]. All primers and probes were synthesized by Sangon. We tested all SNPs in cases and controls for Hardy–Weinberg equilibrium. If the P values from the test are smaller than 0.05 in the samples of control, the corresponding SNPs will be discarded from further analysis. Fisher’s exact test was used to compare allele and genotype distributions in patients and controls. OR values, as estimates of the relative risk of disease, were calculated with 95% CIs. Based on genotype data for the polymorphisms, LD analysis was carried out using the SHEsis program [21]. A 2 -test and odds ratio test were used to compare the discrepancies of allele and genotype frequencies on single locus and multi-loci haplotypes between

Fig. 1. Sequencing results of single nucleotide polymorphisms. Typical sequencing traces of the 9 SNPs are shown: (a) GnRHI gene; (b)–(f) LH␤ gene; and (g)–(i) FSH␤ gene. The red arrows indicate the positions of polymorphisms.

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Y. Zhao et al. / Neuroscience Letters 486 (2010) 188–192

Table 1 Genotype and allelic distributions of the GnRHI, LHˇ and FSHˇ SNP in patients with CPP and controls.

a

No.

Gene

1 2 3 4 5 6 7 8 9

GnRHI LH␤ LH␤ LH␤ LH␤ LH␤ FSH␤ FSH␤ FSH␤

Positiona −2003 −1456 −1424 −238 −164 −34 −1825 −261 −132

Allele

dbSNP ID

T/C C/G C/G G/A G/A T/A T/C G/T T/A

rs2321049 rs2287756 – rs2387590 rs1960072 rs3752210 rs639403 – –

The translation start site is defined as 1.

cases and controls. P values <0.05 were considered statistically significant. After the resequencing of 2 kb 5 -flanking regions of GnRHI, LHˇ and FSHˇ genes in 27 Chinese CPP girls randomly selected from 283 CPP patients, nine SNPs were found as shown in Fig. 1. In these SNPs, three (−1424 C/G allele in LH␤ gene, −261 G/T allele and −132 T/A allele in FSH␤) were the first time reported (Table 1). In GnRHI gene, only one SNP, −2003 C/T, was found in the upstream region. However, there was no significant difference in genotype and allele distributions between controls and patients (Table 2). In the LH␤ gene, −1424 C/G did not fit the expectation of HWE in the patients (P = 0.007). And no significant differences were found for rs2287756 C/G allele (P = 0.51), rs2387590 G/A allele (P = 0.98), rs1960072 G/A allele (P = 0.823) and rs3752210 T/A allele (P = 0.643) between case and controls (Table 3). Based on the five SNPs, haplotype analysis was carried out by SHEsis software. The distribution frequencies of haplotypes were globally significant (2 = 43.48, df = 4, P = 8.25 × 10−09 ) (Table 5). We found one risk haplotype, GCAAA (OR = 1.69, 95% CI = [1.24–2.29]) positively linked with abnormal puberty, and two haplotypes, GCGGT (OR = 4.35,

95% CI = [2.17–8.33]) and CCAAA (OR = 3.45, 95% CI = [1.79–6.67]), negatively correlated with abnormal puberty (Table 5). Three SNPs in FSHˇ gene were genotyped. The genotypic distributions in both cases and controls were all in Hardy–Weinberg equilibrium. The rs639403 T/C allele showed statistical association with CPP (P = 0.025). Notably, rs639403 T/C allele deviated from HWE (P < 0.05) in the case group. No significant differences were found in −261 G/T (P = 0.302) and −132 T/A (P = 0.488) between case and control (Table 4). More than four haplotypes were constructed based on the typing results, yet none of them showed significant correlation. Alibaba of version 2.1, a regulator binding site predicting software, was selected to analyze the 500 bp sequences around the nine polymorphisms to predict their probable effect. However, none of SNP was on the regulating control element. We examined the 5 -flanking regions of GnRHI, LHˇ and FSHˇ genes for genetic variants that might lead to early pubertal development. The GnRHI is an important factor in the process of puberty onset [3,15]. It was reported that genetic variation in the coding region of GnRHI was not related to the onset time of puberty in the general population [20]. In our experiment, only one SNP (−2003 T/C) was found in the 5 -flanking region of GnRHI gene and the distribution of this SNP was not significantly different in case and control. Our result complemented the conclusion of the previous study and suggested that genetic variation in GnRHI would not be a substantial modulator of pubertal timing in Chinese girls. FSH␤ is a pituitary gonadotropin that plays a key role in the regulation of gonadal function [12]. The nucleotide changes in the coding region FSHˇ would loose its bioactivity [11–13,16,18]. In two Caucasian populations, FSHˇ gene was highly conserved, especially in its promoter region [10]. In our research, we found rs639403 T/C allele in FSHˇ gene was weakly correlated with CPP (P = 0.025) in Chinese population. Alibaba of version 2.1 was selected to analyze the 500 bp sequence around the polymorphism to predict its probable effect. We found rs639403 T/C allele was not

Table 2 Genotype and allelic distributions of the GnRHI SNP in patients with CPP and controls. Inter-SNP distance (bp)

Group

−2003 (T/C)

Case Control

Genotype frequency (GF)

Allele frequency (AF)

C/C

C/T

T/T

C

T

37 (0.14) 41 (0.15)

140 (0.51) 136 (0.50)

98 (0.36) 95 (0.35)

214 (0.39) 218 (0.40)

336 (0.61) 326 (0.60)

Odds ratio (95% CI) 0.75–1.21 0.95

2 -test HWE (df = 1)

GF (df = 2)

AF (df = 1)

2 = 1.38, P = 0.240 2 = 0.46, P = 0.499

P = 0.864 2 = 0.29

P = 0.694 2 = 0.16

HWE, Hardy–Weinberg equilibrium; CI, confidence interval.

Table 3 Genotype and allelic distributions of the LHˇ SNP in patients with CPP and controls. Odds ratio (95% CI)

2 -test HWE (df = 1)

GF (df = 2)

AF (df = 1)

338 (0.648) 345 (0.663)

0.83–1.39 1.07

2 = 0.43, P = 0.510 2 = 0.19, P = 0.666

P = 0.641 2 = 0.89

P = 0.588 2 = 0.29

G 11 (0.021) 7 (0.014)

C 519 (0.979) 489 (0.986)

0.57–3.85 1.48

2 = 7.17, P = 0.007 2 = 0.05, P = 0.822

P = 0.582 2 = 1.08

P = 0.418 2 = 0.66

A/A 27 (0.099) 17 (0.062)

G 374 (0.685) 400 (0.727)

A 172 (0.315) 150 (0.273)

0.63–1.06 0.82

2 = 0.00, P = 0.980 2 = 1.10, P = 0.294

P = 0.222 2 = 3.01

P = 0.124 2 = 2.36

A/G 129 (0.471) 121 (0.442)

G/G 109 (0.398) 123 (0.449)

A 201 (0.367) 181 (0.330)

G 347 (0.633) 367 (0.670)

0.92–1.51 1.17

2 = 0.05, P = 0.823 2 = 0.00, P = 0.976

P = 0.439 2 = 1.65

P = 0.205 2 = 1.61

T/A 130 (0.474) 123 (0.447)

A/A 34 (0.124) 30 (0.109)

T 350 (0.639) 367 (0.667)

A 198 (0.361) 183 (0.333)

0.69–1.13 0.88

2 = 0.21, P = 0.643 2 = 0.01, P = 0.904

P = 0.588 2 = 1.06

P = 0.320 2 = 0.99

Inter-SNP distance (bp)

Group

Genotype frequency (GF)

Allele frequency (AF)

G/G

G/C

C/C

G

C

−1456 (C/G)

Case Control

30 (0.115) 31 (0.119)

124 (0.475) 113 (0.435)

107 (0.410) 116 (0.446)

184 (0.352) 175 (0.337)

−1424 (C/G)

Case Control

G/G 1 (0.004) 0 (0.000)

G/C 9 (0.034) 7 (0.028)

C/C 255 (0.962) 241 (0.972)

−238 (G/A)

Case Control

G/G 128 (0.469) 142 (0.516)

G/A 118 (0.432) 116 (0.422)

−164 (G/A)

Case Control

A/A 36 (0.131) 30 (0.109)

−34 (T/A)

Case Control

T/T 110 (0.401) 122 (0.444)

HWE, Hardy–Weinberg equilibrium; CI, confidence interval; P values of <0.05 are underlined.

Y. Zhao et al. / Neuroscience Letters 486 (2010) 188–192

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Table 4 Genotype and allelic distributions of the FSHˇ SNP in patients with CPP and controls. Inter-SNP distance (bp)

Group

Genotype frequency (GF) C/C

C/T

Allele frequency (AF) T/T

C

Odds ratio (95% CI)

2 -test

T

HWE (df = 1)

GF (df = 2)

AF (df = 1)

−1825 (T/C)

Case Control

20 (0.08) 30 (0.12)

131 (0.50) 94 (0.39)

111 (0.42) 117 (0.49)

171 (0.33) 154 (0.32)

353 (0.67) 328 (0.68)

0.79–1.34 1.03

 = 4.93, P = 0.026 2 = 2.56, P = 0.110

P = 0.025 2 = 7.38

P = 0.817 2 = 0.05

−261 (G/T)

Case Control

G/G 110 (0.40) 124 (0.46)

G/T 134 (0.49) 109 (0.40)

T/T 31 (0.11) 37 (0.14)

G 354 (0.64) 357 (0.66)

T 196 (0.36) 183 (0.34)

0.72–1.19 0.93

2 = 1.06, P = 0.302 2 = 2.65, P = 0.104

P = 0.143 2 = 3.89

P = 0.545 2 = 0.37

−132 (T/A)

Case Control

A/A 30 (0.11) 37 (0.14)

A/T 129 (0.47) 109 (0.40)

T/T 115 (0.42) 124 (0.46)

A 189 (0.34) 183 (0.34)

T 359 (0.66) 357 (0.66)

0.80–1.32 1.03

2 = 0.48, P = 0.488 2 = 2.65, P = 0.104

P = 0.256 2 = 2.72

P = 0.835 2 = 0.04

2

HWE, Hardy–Weinberg equilibrium; CI, confidence interval; P values of <0.05 are underlined.

Table 5 Haplotype analysis of the association between LHˇ gene and the CPP (LFT ≥ 0.05). Haplotype

Distribution frequency Case

GCGGT 11.32 (0.023) CCGGT 286.69 (0.585) GCGAA 19.44 (0.040) GCAAA 139.15 (0.284) CCAAA 11.79 (0.024) Others 21.56 (0.044) Global Chi-square test of HF (df = 4)

Odds ratio

95% CI

Control 42.38 (0.094) 252.33 (0.558) 22.99 (0.051) 88.85 (0.197) 36.44 (0.081) 8.58 (0.019)

0.23 1.19 0.79 1.69 0.29 – 2 = 43.48

0.12–0.46 0.92–1.55 0.43–1.47 1.24–2.29 0.15–0.56 – P = 8.25 × 10−09

2 -test of HF (df = 1) 2

P

21.00 1.70 0.55 11.31 14.80 –

4.67 × 10−06 0.192 0.457 7.77 × 10−04 1.21 × 10−04 –

LFT, lowest frequency threshold; CI, confidence interval; P values of <0.05 are underlined.

in the region, which could be bound and recognized by transcription factors. And this site was far from the translation start position, which suggested it was probably not in the normal regulation elements. It was supposed that there would be other active SNPs, which related to the CPP, at the downstream region of rs639403 T/C allele. Analyzed by HWE, the departure of data was appeared in the case group. It also implied that rs639403 T/C allele probably was a genetic marker of CPP by linking to other active SNPs. More samples in Chinese girls should be collected and genotyped for validation in the future. It was reported that the mutants in the LHˇ gene coding region caused its bioactive lost [5,7,19,27]. But the LHˇ promoter with mutations showed higher basal activity than the wild-type LHˇ promoter [9]. In our research, none of positive results appeared in the 5 -flanking region of LHˇ gene by Fisher’s exact test for each allele. However, haplotype analysis indicated that this region was associated with CPP. In this research, those Chinese girls with haplotype GCAAA in LHˇ gene had much higher possibility to get CPP than those with the other haplotypes. Interestingly, there were more girls with haplotype GCGGT and CCAAA in control group than in case group, which probably indicated that the hormone with these two haplotypes were related to the normal onset time of puberty. It is not rare that some markers, such as SNPs or haplotypes, have positive association with the control group in some important bioprocesses, which are critical for individual development or survival [29,30]. Many human diseases, such as hypertension, hyperglycemia and precocious puberty, are related to quantitative trait. All of these diseases are affected by multiplex loci, which contain several alleles. In these loci, some alleles contribute to these traits by increasing protein expression, while others decreasing. The collective influence of these alleles makes the phenotypes distribute randomly, and some extremely high or low value may be obtained from the distribution. At last, not only does the extreme value related alleles appear, but also the non-extreme value related alleles will be obtained. Provided that these loci take part in the special bioprocesses of diseases in human being, the results are significantly important

in finding out the mechanisms of the diseases. In this research, extremely low P-value and high odds ratio were obtained in analysis of the relationship between haplotypes of LHˇ gene and normal onset of puberty. It indicated that the haplotypes GCGGT and CCAAA were protective in the puberty onset of the girls. They might link to comparatively late onset of puberty and avoid CPP. In summary, the positive analytic result in LHˇ and FSHˇ genes gives out some clues of the mechanism of the puberty onset in Chinese girls, and more researches with larger sample sizes are necessary to further explore such principles. Acknowledgements This work was supported by grants from the National Nature Science Foundation of China (Grant No. 30771191) and Science and Technology Commission of Shanghai Municipality (Grant No. 071409004). References [1] D.P. Cai, Progress of precocious puberty in children, J. Appl. Clin. Pediatr. 6 (2005) 1. [2] L.S. Cheryl, L.F. Douglas, The neural basis of puberty and adolescence, Nat. Neurosci. 7 (2004) 1040–1047. [3] W.H.A. Delemarre-van, Regulation of puberty, Best Pract. Res. Clin. Endocrinol. Metab. 16 (2002) 1–12. [4] F.J. Ebling, The neuroendocrine timing of puberty, Reproduction 129 (2005) 675–683. [5] K. Furui, N. Suganuma, S. Tsukahara, Y. Asada, F. Kikkawa, M. Tanaka, T. Ozawa, Tomoda S Y., Identification of two point mutations in the gene coding luteinizing hormone (LH) beta-subunit, associated with immunologically anomalous LH variants, J. Clin. Endocrinol. Metab. 78 (1994) 107–113. [6] M.M Grumbach, D.M. Styne, Puberty: ontogeny, neuroendocrinology, physiology, and disorders, in: J.D. Wilson, D.W. Foster, H.M. Kronenberg, P.R. Larsen (Eds.), Williams Textbook of Endocrinology, 9th ed., WB Saunders Co., Philadelphia, 1998, pp. 1509–1625. [7] A.M. Haavisto, K. Pettersson, M. Bergendahl, A. Virkamaki, I. Huhtaniemi, Occurrence and biological properties of a common genetic variant of luteinizing hormone, J. Clin. Endocrinol. Metab. 80 (1995) 1257– 1263. [8] D.J. Haisenleder, A.C. Dalkin, G.A. Ortolano, J.C. Marshall, M.A. Shupnik, A pulsatile gonadotropin-releasing hormone stimulus is required to increase transcription of the gonadotropin subunit genes: evidence for differential reg-

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