The Mutation Analysis of GnRH1 and GnRH Receptor Gene in Korean Girls woth Central Precocious Puberty

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윤소영의 의학 석사학위 논문을 인준함.

심사위원장

황

진

순

인

심 사 위 원

정

조

원

인

심 사 위 원

윤

계

순

인

아 주 대 학 교

대 학 원

년

월

일

2008

12

22

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-Fig. 1. Schematic representation of the GnRH1(A) and GnRH-R(B) ··· 16 Fig. 2. Correlation of age of breast budding with polymorphism ··· 20 Fig. 3. The hypothalamic-pituitary-gonadal axis ··· 27 Fig. 4. Ligand-induced GnRH receptor activation and signal transduction,

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vi

-Table 1. GnRH1 primer ··· 9 Table 2. GnRH-R primer ··· 10 Table 3. Clinical characteristics of the total patients··· 14 Table 4. Results of the Gonadotropin Releasing Hormone (GnRH)

Stimulation test ··· 15 Table 5. The genotype and allelic frequencies of polymorphism

in the GnRH1 gene ··· 17 Table 6. Correlation of clinical characteristics with GnRH1 genotype

in CPP patients··· 18 Table 7. Correlation of GnRH stimulation test with GnRH1 genotype

in CPP patients ··· 19 Table 8. Correlation of age of breast budding with polymorphism ··· 20 Table 9. SNPs in GnRH1 and GnRH-R ··· 29

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vii -Central precocious puberty : CPP

Gonadotropin Releasing Hormone : GnRH

Gonadotropin Releasing Hormone Receptor: GnRH-R Luteinizing Hormone : LH

Follicle-stimulating hormone: FSH Dichlorodiphenyl dichoroethane: DDE Polychlorinated biphenyls: PCB

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9

-GnRH1 For PCR forward For PCR reverse

Exon 1a 5'-GCAGGAAAGATTTCAATGTCC-3' 5'-GATTTAGCCCTTGGGCTGTC-3'

Exon 1b 5'-CCATCTTCTGCAGGGTTAGTG-3' 5'-GCCTTATCTCACCTGGAGCA-3'

Exon 2 5'-CCCCACTCTCCACAATTTTT-3' 5'-CAGGAATGTAAGCCCCACAG-3'

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10

-GnRH-R For PCR forward For PCR reverse

Exon1 5'-CAGGGACAAAATTTGACATACG-3' 5'-CTGACTTCCAGAACCCAAGC-3' Exon2 5'-GGCTAGCAGAGTACCAAAGAG-3' 5'-TGCCACTCTGTTTTTGACATTG-3' Exon3 5'-TCCTTTTTGTCCACTTTGGTTT-3' 5'-TCCCAGATGGAGAGATCCA-3'

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14

-Table 3. Clinical characteristics of the total patients

Patient (n=101)

Normal control (n=51) Chronological age (year) 7.93±0.98

Bone age (year) 10.24±1.38

Bone age SDS 2.90±1.20

Bone age

-chronological age (year) 2.85±0.40

Weight (kg) 31.57±7.46 51.49±4.95

Weight SDS 1.0±0.80

Height (cm) 131.78±8.25 161±4.43

Height SDS 1.20±1.00

Body mass index (kg/m2₎ _17.90±2.40

BMI SDS 0.60±0.80 Faternal height (cm) 171.43±5.61 170±4.12 Maternal height (cm) 158.29±5.66 158.29±4.43 Target height (cm) 158.30±5.70 Target height SDS -0.50±1.00 Birth weight (kg) 3137.90±479.20 Birth weight SDS -0.40±1.00

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15

-Table 4. Results of the Gonadotropin Releasing Hormone (GnRH) stimulation test

LH

FSH

0 min ( ) 2.23±0.92 2.76±1.51 30 min 17.39±14.55 10.41±4.61 45 min 15.66±12.50 11.51±4.59 60 min 13.36±10.04 11.97±4.64 90 min 10.26±6.87 11.56±4.70 peak ( ) 17.50±14.50 11.50±5.70

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16 -(A) Human GnRH1 gene (Chromosome 8p11.2-p21)

(B) Human GnRH receptor gene (Chromosome 4p21.2)

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17

-Table 5. The genotype and allelic frequencies of polymorphism in the GnRH1 gene

Each P value was calculated with co-dominant(co), dominant(do), and recessive(re) model. Logistic regression analysis was applied to control for age and sex as covariable. OR: odds ratio CI: confidence interval, q.: minor allele frequency.

CPP (N=101) NC (N=51) CPP vs. NC P value OR (95% CI) 16 G>C GG 27 (26.70%) 8 (15.70%) co: 0.178 GC 61 (60.40%) 32 (62.70%) do: 0.165 1.862 (0.768~4.513) CC 13 (12.90%) 11 (21.60%) re: 0.127 1.961 (0.818~4.699) q. 0.437 0.529

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-Table. 6 Correlation of clinical characteristics with GnRH1 genotype in CPP patients

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19

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20

-Fig. 2. Correlation of age of breast budding with polymorphism

Table. 8 Correlation of age of breast budding with polymorphism

§ _{:breast budding}

GnRH1

exon 1b N Mean±S.D.

95% Confidence Interval for Mean

Min. Maxi. ANOVA Lower Bound Upper Bound BB§ GG 35 8.3±2.0 7.6 9.0 4.3 13.0 0.196 GC 93 8.9±2.3 8.5 9.4 1.8 15.0 CC 24 9.4±2.7 8.2 10.5 4.5 15.0 Total 152 8.9±2.3 8.5 9.2 1.8 15.0

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23

-α β γ

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27

-Fig. 3. The hypothalamic-pituitary-gonadal axis. Secretion of GnRH from the

hypothalamus occurs in a pulsatile fashion and is partially responsible for controlling

the number of GnRH-Rs in the pituitary gland and controls synthesis and secretion of

FSH and LH. These gonadotropins in turn regulate function of the testes and ovaries.

E, estrogen; P, progesterone; T, testosterone.

Harrison GS et al., Endocrine-Related

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-Fig. 4. Ligand-induced GnRH receptor activation and signal transduction, leading to LH and FSH gene expression and secretion. Seven transmembrane helices (TMH1±7) of the GnRH receptor are schematically illustrated. Karges, B. et al., Human Reproduction

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-Table 9. SNPs in GnRH1 and GnRH-R

Gene Region SNP ID Chromosome

GnRH1 exon1 rs6185(16G>C) chr8 exon1 rs35542850(46G>C) chr8 exon2 rs6186(61C>T) chr8 GnRH-R exon1 rs35400155(48G>C) chr4 exon1 rs4986942(151C>T) chr4 exon2 rs13130501(183A>C) chr4 exon2 rs13149772(184G>C) chr4 exon3 rs28933074(284A>G) chr4

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