학위논문(박사)--서울대학교 대학원 :의과대학 의학과,2020. 2. 박영주.Thyroid cancer is the most common endocrine cancer and thyroid nodule is most common endocrine problem in Korea. Both phenotypes show a high degree of heritability. Several genome-wide association studies (GWAS) for thyroid cancer were conducted in European descendants and identified susceptibility loci of differentiated thyroid cancer (DTC). However, there is no GWAS for thyroid cancer in Asian population, and inherited genetic risk factors for thyroid nodules and their associations with thyroid cancer remain unknown.
Here, GWAS and replication study was performed using a total of 1,085 DTC cases and 8,884 controls of Koreans and these results were validated with an expression quantitative trait loci (eQTL) analysis and clinical phenotypes. The most robust associations were observed in the NRG1 gene (rs6996585, P=1.08×10-10), and this SNP was also associated with NRG1 expression in thyroid tissues. In addition, three previously reported loci (FOXE1, NKX2-1, and DIRC3) were confirmed and seven susceptibility loci (VAV3, PCNXL2,INSR, MRSB3, FHIT, SEPT11, and SLC24A6) associated with DTC were newly identified. Furthermore, I identified specific variants of DTC that have different effects according to the cancer type or ethnicity.
Furthermore, a three-stage GWAS for thyroid nodules was performed. The discovery stage involved a genome-wide scan of 811 subjects with thyroid nodules and 691 subjects with a normal thyroid from a population-based cohort. Replication studies were conducted in an additional 1981 cases and 3100 controls from the participants of a health check-up. Expression quantitative trait loci (eQTL) analysis was also performed using public data. The most robust association was observed in TRPM3 (rs4745021) in the joint analysis (OR=1.26,
P = 6.12 × 10-8) and meta-analysis (OR = 1.28, P = 2.11×10-8). Signals at MBIP/NKX2-1 were replicated but did not reach genome-wide significance in the joint analysis (rs2415317; P = 4.62 × 10-5, rs944289; P = 8.68 × 10-5). The eQTL analysis showed that TRPM3 expression was associated with the rs4745021 genotype in thyroid tissues. The results of GWAS for DTC provide deeper insight into the genetic contribution to thyroid cancer in different populations. And GWAS for thyroid nodule suggest that thyroid nodules have a genetic predisposition distinct from that of thyroid cancer.갑상선암은 한국에서 가장 흔한 내분비암이며 갑상선 결절은 가장 흔한 내분비 질환이다. 두가지 질환 모두 높은 유전성을 보인다. 몇몇의 갑상선암에 대한 전장 유전체 연관 연구가 서양인들에게서 이루어졌고, 분화갑상선암에 대한 감수성 유전자좌를 발굴하였다. 그러나 아시아인에 대한 전장 유전체 연관 연구는 수행된 바 없으며, 갑상선 결절에 대한 유전적 연구는 없었으며 이와 관련된 유전자와 갑상선암과의 관련성도 여전히 알 수 없는 상태이다.
따라서, 본 연구에서는 1,085 명의 분화 갑상선암과 8,884 명의 대조군으로 전장 유전체 연관 분석 및 재현 연구를 수행하였고, 그 결과를 발현 양적 형질 유전자좌 연구 및 임상 발현형질을 통해서 검증하였다. 가장 뚜렷한 관련성은 보이는 유전자좌는 NRG1 유전자였으며 (rs6996585, P=1.08×10-10), 이 SNP 은 NRG1 의 발현과도 관련성이 있었다. 부가적으로 이전에 보고되었던 유전자좌 (FOXE1, NKX2-1, DIRC3)를 확인하였으며 7 개의 유전자좌 (VAV3, PCNXL2, INSR, MRSB3, FHIT, SEPT11, SLC24A6)를 새롭게 발견하였다. 또한,
분화갑상선암과 관련된 유전변이가 암의 종류 및 인종에 따라서 다른 영향을 가지는 것을 확인하였다.
또한 갑상선결절에 대한 3 단계의 전장 유전체 연관 분석을 시행하였다. 발견 단계의 전장 유전체 스캔을 인구 기반 코호트의 811 명의 갑상선 결절군과 691 명의 정상 갑상선군에서 수행되었다. 재현 연구는 건강검진 대상자에서 총 1981 명의 결절군과 3100 명의 정상군에서 수행되었으며 발현 양적 형질 유전자좌 분석도 공공데이터를 통해서 수행되었다. 가장 유의한 관련성은 결합분석 (OR=1.26, P = 6.12 × 10-8) 및 메타분석 (OR = 1.28, P = 2.11×10-8) 결과 TRPM3 (rs4745021) 유전자에서 관찰되었다. MBIP/NKX2-1 변이는 재현이 되었으나 전장 유전체 유의성을 보이지 못했다. 발현 양적 형질 유전자좌 분석에서 TRPM3 의 발현은 갑상선조직에서 rs4745021 유전자형과 관련성이 있었다.
분화갑상선암에 대한 전장 유전체 연관 분석 결과는 갑상선암의 발생에서 유전적 기여에 대한 이해할 수 있게 해주었으며, 갑상선
결절에 대한 전장 유전체 연구를 통해 갑상선 결절은 갑상선암과 차별되는 유전적 특징을 가지고 있음을 확인하였다.Introduction 1
1. Epidemiology of thyroid cancer 1
2. Risk factors of differentiated thyroid cancer 1
3. Heritability of differentiated thyroid cancer 3
4. Familial syndromes associated with thyroid cancer and germline mutation of differentiated thyroid cancer 4
5. Epidemiology of thyroid nodule 4
6. Clinical significance and heritability of thyroid nodule 4
7. Genome-wide association study for differentiated thyroid cancer 5
8. Genetic studies for thyroid nodule 6
9. Hypothesis 9
10. Aims of study 9
Chapter I. Genome-wide association and expression quantitative trait loci studies for thyroid cancer 10
Materials and methods 11
Study participants for the Stage 1 genome scan 11
Study participants for the Stage 2 follow-up 11
Discovery SNP genotyping and imputation 15
Replication SNP selection and genotyping 16
RNA sequencing and eQTL analysis 18
Statistical analysis 18
Ethics statement 20
Results 21
Stage 1 genome scan 21
Stage 2 follow-up and joint Stages 1 and 2 analyses 24
Validation of the candidate SNPs with cis-eQTL and GSEA analyses 29
Association between candidate SNPs and clinical phenotypes 35
The most significantly associated variant in the NRG1 locus 38
Other known associated variants in the NKX2-1, DIRC3, or FOXE1 loci 44
Novel candidate variants in the VAV3, PCNXL2, INSR, MRSB3, FHIT or SEPT11 loci 48
A comparison with the European GWAS results 51
Chapter II. Genome-wide association and expression quantitative trait loci studies for thyroid nodule 55
Materials and methods 56
Discovery series and thyroid ultrasonography 56
First replication series and ultrasonography 59
Second replication 59
Discovery GWAS and Imputation 60
Candidate SNP and genotyping of first replication 61
Genotyping of second replication 64
Comparison of allele frequencies between DTC, thyroid
nodules, and normal thyroid 64
Expression quantitative trait loci analysis 64
Statistical analysis 65
Ethics statement 66
Results 67
Discovery GWAS 67
Replication studies, joint analysis and meta-analysis 71
Comparison of allele frequencies between DTC, thyroid nodules, and normal thyroid 76
Expression quantitative trait loci analysis 80
Discussion 82
GWAS for DTC 82
GWAS for Thyroid nodule 92
Summary and conclusions 100
References 101
Abstract in Korean 116Docto
