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유전체 통합 분석을 통한 새로운 성조숙증 연관 유전자의 동정

Identification of novel genes involved in precocious puberty by integrated genomic analyses

초록/요약

Precocious puberty is a multifactorial disease that occurs in one out of 5,000-10,000 children with the appearance of secondary sexual characteristics before 8 years in girls and 9 years in boys. Early activation of the pituitary gland and gonadal (HPG) axis, regulated by hypothalamic Kiss1 and GnRH neurons, has been reported to result in precocious puberty. Both environmental and genetic factors are involved in precocious puberty. Currently, only few genes such as KISS1, KISS1R and MKRN3 have been demonstrated to be closely relate to the pathogenesis of precocious puberty; therefore, the genetic risk factors for precocious puberty remain to be identified. In this study, I aimed to identify the novel genetic biomarkers that are closely associated with an increased risk of precocious puberty and thereby, useful for the early diagnosis of the disease. Screening of the differentially expressed genes in the hypothalamus of female mice during the pubertal period was performed by whole-genome RNA sequencing using next-generation sequencing (NGS). I identified six genes that were significantly increased or decreased during the period of puberty; the expression of four genes, Gabrd, Opalin, Plg, and Trf, increased and that of two genes, Glra3 and Npw, decreased significantly in the mouse hypothalamus during the pubertal period (day 22 to day 30). These expression patterns were confirmed by quantitative RT-PCR using gene-specific primers. To verify whether the six identified genes are susceptible to precocious puberty, I conducted a candidate gene-based case-control association study using the SNP genotype and clinical data of total 669 Korean girls with precocious puberty and 300 Korean age-matched normal controls. Although no significant associations satisfying the Bonferroni-corrected significance level (p <0.0003), were observed, 43 of 1,183 SNPs located in the six genes were associated with precocious puberty (p <0.05): 8 of 245 in GABRD, 6 of 178 SNPs in GLRA3, 3 of 161 SNPs in NPW, 7 of 219 SNPs in OPALIN, 9 of 178 SNPs in PLG, and 10 of 202 SNPs in TRF. rs679749 (odds ratio=1.4160 and p=0.0034) and rs1197312 (odds ratio =0.7069 and p=0.0034) in the TRF gene were the most significant SNPs. Finally, I performed in silico pathway analysis of the six genes to investigate how these genes are directly and indirectly related to precocious puberty using the IPA program. All the six genes were closely related to precocious puberty via two or three nodes. This study provides insights into the genetic basis of precocious puberty and the identification of clinically useful genetic biomarkers for the disease.

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목차

I. INTRODUCTION 1
II. MATERIALS AND METHODS 6
A. Tissues extraction in female mice 6
B. Whole-genome RNA sequencing 6
C. RNA isolation, cDNA synthesis, and quantitative RT-PCR 8
D. Study subjects, genotyping, and SNP selection 9
E. Ingenuity Pathway Analysis 10
F. Statistical analysis 10
III. RESULTS 14
A. Expression changes of the known genes involved in precocious puberty in the hypothalamus of female mice during the pubertal period 14
B. Identification of six genes showing significant expression changes during pubertal periods in mouse hypothalamus 17
C. Validation of changes of the mRNA levels of Gnrh, Kiss1 and Mkrn3 in the mouse tissues during the pubertal period by gene-specific quantitative RT-PCR 22
D. Validation of the changes in the mRNA levels of the six genes identified during the pubertal period in mouse tissues by gene-specific quantitative RT-PCR 29
E. Candidate gene-based association analysis between SNPs in identified genes and precocious puberty in Korean girls 44
F. In silico network analysis of the six identified genes related to precocious puberty 48
IV. DISCUSSION 54
V. CONCLUSION 58
REFERENCES 59
국문요약 64

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