Study On The Polymorphisms Of Pvu Ⅱ And Xba Ⅰ Gene In The Estrogen Receptor-α And The Serum Levels Of Bone Formation Makers In Girls With Idiopathic Central Precocious Puberty

1. Background and ObjectionBackgroundOnset of development of secondary sexual characteristics(as the first step toward attaining reproductive capacity)before the age of 8 years and 9 years in girls and boys respectively had traditionally been used for defining precocious puberty.It can be divided into central precocious puberty (central precocious puberty,CPP), incomplete or partial precocious puberty, peripheral precocious puberty categories.As abnormal pubertal development,idiopathic central precocious puberty is the hypothalamic-pituitary-gonadal axis shaft early start, leading to early epiphyseal fusion,it ultimately affects the adult height of children,even producing some psychological and behavioral problems such as fear and anxiety.The physiopathological mechanism is still unknown. In recent surveys from around the world shows that the rising incidence of sexual precocity,only second to childhood obesity, and has risen to the first two in children who with endocrine disorders,and 90% were female children,there continues to advance the trend of the puberty age.The existence of early breast development trend of the age,but children age at menarche earlier trend after the mid-20th century has slowed or stalled,A recent surveys in USA shows that there are 6.7 percent of white women and and 27.3 percent of African female children appeared breast development and pubic hair before 7 years old.A survey in china shows that there were more than 10% girls shows thelarche before the age of 8,and 1.65% shows menarche before the age of 10.CPP is based on hypothalamic-pituitary-gonadalaxis activation with progressive pubertal development.Various environmental factors such as environmental endocrinedisruptors,estrogen contamination of food,water and other lifestyle changes, and obesity in intercountry adopted children can affect HPGA axis or even through their impact on gene caused the occurrence of precocious puberty.Estrogen receptor a (ERa) play a key role in maintaining respect and influence fecundity of female secondary sexual characteristics and individual reproductive cycle.There are several polymorphisms in the upstream gene, more than 2000 kinds of ERa gene polymorphism have been reported currently,and its PvuⅡ and Xbal gene polymorphisms had attracted widespread attentions.ERa located on chromosome 6,locus 6q24-q27,comprises eight exons and seven introns.There are two common site polymorphisms in the first intron,located at the recognition sites of the restriction enzymes known as PvuⅡ and Xbal, respectively,T/C allele of PvuⅡ labeled p/P,A/G alleles of XbaⅠ labeled as x/X,P and X are the genotype PvuⅡ and XbaⅠ restriction sites,different genotypes may decide ER expression and its function in different individuals, thus affecting the biological role of estrogen in the body.Previous studies have suggested that the PvuII and XbaⅠ gene polymorphisms in the estrogen receptor-α are related to many diseases,such as breast cancer, gynecological cancer, osteoporosis an so on.Meanwhile,There are few reports at home and abroad shows a assosiciation between PvuII and XbaⅠ gene polymorphisms and central precocious puberty ,The relationship between the PvuII and XbaⅠ gene polymorphism and central precocious puberty is still controversial.Thus,we conducted this study to evaluate the relationship between PvuII and XbaⅠ gene polymorphisms and central precocious puberty,so as to provide a theoretical basis for further research and treatment.Idiopathic central precocious puberty (ICPP) is manifested by the secondary sexual characteristics,skeletal maturation,and physical development in advance,which had a rapid metabolism,more and more people doing research to find some potential biomarkers for assessing the skeletal growth.Classic specific markers of bone formation, including calcium, phosphorus,alkaline phosphatase, but there are some drawbacks,such as poor sensitivity,while modern bone specific markers had advantage of specificity and high sensitivity,include former I-carboxy-terminal peptide of procollagen,bone alkaline phosphatase (bone alkaline phosphatase, BAP).osteocalcin (osteocalcin,OC),etc,these products can be understood as measured functional status of bone cells,has been widely used in the diagnosis,treatment monitoring and pathogenesis of adult bone disease.Amino-terminal pro-C-type natriuretic peptide(NT-proCNP) is a stable produce of C-type natriuretic peptide(CNP),CNP is a natriuretic peptide family in which a member of the natriuretic peptide family includes three structurally related proteins:atrial natriuretic peptide (ANP),brain natriuretic peptide (BNP) and CNP. ANP and BNP are mainly distributed in the cardiovascular system, while CNP found primarily in cardiac tissue, such as the central nervous system, musculoskeletal system.As a paracrine factor,CNP plays an important role in the proliferation,differentiation and extracellular matrix synthesis regulate growth plate chondrocytes cells,but as reported previously,plasma concentrations of the biologically active forms of CNP are low in both humans and sheep,close to the level of the assay detection limit, and shows relatively small changes in physiological and pathological status,which limited its application in clinical.By contrast,NT-proCNP is readily detected and circulates at levels 10 to 50 fold those of CNP increasing the likehood that the plasma NT-proCNP will better reflect CNP synthesis within tissues provided that renal function is normal.Many study had showed a strong association of blood levels with growth velocity and makers of bone,In summary,there are few study about the relationship in between precocious puberty and bone formation makers,especially NT-proCNP, the relationship between precocious puberty has gradually drawn biologist and pediatric endocrinologists’s concern,which including osteocalcin(OC), bone alkaline phosphatase(BAP) and NT-proCNP,this study detected the levels of bone formation makers,measuring the effect of ERa gene polymorphism in ICPP girls on bone formation makers,in order to provide useful indicators observed in molecular biology and biochemistry for earlier diagnosis and treatment,expected to improve the therapeutic benefits.1.2 ObjectiveTo investigate the association between the estrogen receptor-agene variations(PvuⅡ and Xbal)polymorphisms in girls with idiopathic central precocious puberty(ICPP), explore its relationship with central precocious puberty; Osteocalcin,bonealkaline pho-sphatase,NT-proCNP and IGF-1 levels were detected in ICPP girls,and to explore theimpact of ERagene polymorphism on markers of bone formation,in order to provide useful indicators observed in molecular biology and biochemistry for earlier diagnosis and treatment,expected to improve the therapeutic benefits.2.Materials and Methods2.1 Materials100 girls with ICPP who accepted and hospitalization in pediatric endocrine clinic of Shenzhen Maternal and Child Health care Hospital during January 2013 to August 2014 were chosen as the ICPP group;Aged 4 to 10.5,the average was (6.690±1.580), The detailed history, physical examination, measurement of thyroid function, kidney function, pituitary MRI (plain+enhanced), pelvis (uterus,ovaries), liver and adrenal glands and skeletal ultrasound and other tests had been taken for all the patients,every patient after gonadotropin-releasing hormone (GnRH) stimulation test, all the simple had met the diagnostic criteria with ICPP.Application image had been taken for excluding second precocious puberty by intracranial and pelvis,liver,adrenal glands and other parts of lesions,also the other endocrine disease,such as McCune-Albright syndrome.100 children without sexual characteristics and chronic wasting disease,aged 3.83 to 10.00 years old whose physical development were normal were chosen as normal control group.There was no statistically significant difference (P> 0.05) of age in the groups.2.2 Methods(1) In idiopathic central precocious puberty and normal healthy children,cubital vein 6ml,3ml of blood to be used for detecting of serum’s bone formation makers levels,The other 3m] venous blood was kept using EDTA anticoagulant in-80℃ refrigerator, to be used for detecting PvuII and XbaⅠ genetic polymorphisms.(2) PvuⅡ and XbaⅠ genes polymorphisms in ERa were detected by restriction fragment length polymorphism polymerase chain reaction(PCR-RFLP) in 100 girls with ICPP and 100 healthy groups, And the frequency distribution of genotypes and alleles of PvuⅡ and Xbal were statistically analyzed between the ICPP groups and control groups.(3)The serum of bone formation makers and insulin-like growth factor-1(IGF-1) were detected by the double-antibody sandwich enzyme-linked immunosorbent assay (ELISA).(4)Data was analyzed by the SPASS13.0 statistical software,Chi-square test was used to inspect distribution of genotype frequency of case group and control group,calculate the OR and 95% CI to show the association strength,and using hierarchical analysis method to analyze the interaction between genotype;Mean and standard deviationwere given for data (X±s),Normal distribution of data between two groups were compared using t test while non-normal distribution using a non-parametric t test;Measurement data between multiple groups were compared using analysis of covariance (ANCOVA) controlling for covariates,non-normal distribution,or (and) variance missing persons using Kruskal-Wills H test.Pairwise comparisons using LSD test homogeneity of variance.missing persons using non-parametric test of Dunnett T3 test.These two both were caught based on a was equal 0.05 for the significant level of inspection, only the data P<0.05 can be used for the statistics significance.3 Results3.1 PvuⅡ restriction site polymorphism analysis:There existed significant differences in the genotype freguencies and the alle of ERa at PvuⅡ enzyme cutting sites between two groups.thedistribution was detected in two study groups(x2 =9.023,P=0.011,x2=6.938,P=0.011);P genetype frequency in ICPP group is 40.5%,and that in control group is 28.0%,the OR is 1.750(95%CI 1.152-2.659,P<0.05), Pp genotype have higher proportion of 55% in ICPP group, pp genotype have higher proportion of 53% in control group.3.2 Xbal restriction site polymorphism analysis:There existed significant differences in the genotype freguencies and the alle of ERa at Xbal enzyme cutting sites between two groups.the distribution was detected in two study groups.(x2=11.740,P=0.003;x2=11.408,P=0.001);X genetype frequency in ICPP group is 42.0%,and that in control group is 26.5%,the OR is 2.061 (95%CI 1.152-2.659,P<0.05),Xx genotype have higher proportion of 48% in ICPP group, xx genotype have higher proportion of 58% in control group.3.3 PvuII and Xbal polymorphisms joint analysis:Haplotypes distributed by chi-square test on the difference between the two groups was statistically significant.(x2=31.388,P=0.001),PpXX genetype constituent ratio was higher in case group (13%) than that control group (3%),but ppxx genetype constituent ratio was lowerin case group (11%) than that control group (39%).3.4 The serumlevels of BAP,OC,NT-proCNP and IGF-1 were significantly higher in ICPP group than the healthy groups.In ICPP group,the serum levels of NT-proCNP and IGF-1 were highest in respectively in Tanner Ⅱ stage and Tanner IV stage.Serum OC and BAP were higher in Tanner Ⅲ stage,but the difference was not statistically significant.3.5 Bone formation makers’s ROC curve’s characteristics showed that all of them had certain value for the diagnosis of CPP,the serum IGF-1 had a higher sensitivity and specificity compared with NT-proCNP, OC and BAP.3.6 In PvuⅡ genotype distribution,the serum levelsof NT-proCNP were significantly higher in Pp genotype compared with pp genotype (P=0.011),and there is no statistically significant among BAP,OC,IGF-lin the PvuⅡ genotype;And NT-proCNP,BAP,OC,and IGF-1 in XbaⅠ genotype differences not statistically significant (P>0.05).4 Conclusions4.1 PvuⅡ and Xbal gene polymorphisms in ERa may be related with ICPP. The risk of ICPP in girls with allele P or X was 1.75 (95%CI 1.152～2.659,P<0.05) or 2.061(95%CI 1.351～3.145,P<0.05) fold of that with allele p or x.Allele P or X may be a risk factor for girls with ICPP, And the girls with genotype Pp or Xx were susceptible to ICPP.The Ppxx genetype may be the potential risk factors for central precocious puberty.4.2 The serum level of BAP,OC,NT-proCNP and IGF-1 were significantly higher in ICPP group than the healthy group.In ICPP group,the serum levels of NT-proCNP and IGF-1 were highest in respectively in Tanner Ⅱ stage and Tanner IV stage.Serum OC and BAP were higher in Tanner Ⅲ stage,but the difference was not statistically significant.4.3 Bone formation makers’s ROC curve’s characteristics showed that all of them had certain value for the diagnosis of precocious puberty,the serum IGF-1 had a higher sensitivity and specificity compared with NT-proCNP,OC and BAP.4.4 In PvuⅡ genotype distribution,the serum levelsof NT-proCNP were significantly higher in Pp genotype compared with pp genotype (P=0.011), and there is no statistically significant among BAP,OC,IGF-1 in the PvuⅡ genotype.And NT-proCNP,BAP,OCand IGF-1 in XbaI genotype differences not statistically significant (P>0.05).