Title Page
ACKNOWLEDGDMENT
CONTENTS
ABBREVIATIONS 9
ABSTRACT 10
Ⅰ. INTRODUCTION 12
Ⅱ. MATERIALS AND METHODS 16
Ⅱ. 1. Sample Collection 16
Ⅱ. 2. DNA Extraction and Laboratory Analysis 16
Ⅱ. 3. TSER VNTR and G/C SNP Genotyping 17
Ⅱ. 4. 3 ′UTR 6bp Insertion Genotyping 19
Ⅱ. 5. Statistical Analyses 21
Ⅲ. RESULTS 22
Ⅲ. 1. TS Polymorphisms in Stomach Cancer Patients 22
Ⅲ. 1. 1. Genotyping Results of 3 Polymorphism- TSER VNTR, G/C SNP and 6 bp Insertion 22
Ⅲ. 1. 2. Analysis of overall combined genotype frequencies 27
Ⅲ. 1. 3. Combined Genotype Frequency Comparison between stomach patients and controls 29
Ⅲ. 1. 4. Analysis of Histologic Type of Stomach Cancer 33
Ⅲ. 1. 5. Haplotype Analysis 37
Ⅲ. 2. Thymidylate synthase Gene Polymorphisms in Colonl Cancer Patients 39
Ⅲ. 2. 1. Genotypes and Combined Genotype in a Whol Colon Cancer Patients 39
Ⅲ. 2. 2. Polymorphic Genotype Analysis by Proximal and Distal Localization of Colorectal Cancer 42
Ⅲ. 2. 3. Gender Difference of Genotype Distributions 48
Ⅲ. 3. Thymidylate Synthase Gene Polymorphisms and Homocysteine Level in Stroke Patients 53
Ⅲ. 4. Ethnic Characteristics of Thymidylate Synthase Gene Polymorphisms Distributions 57
Ⅲ. 5. Novel TSER VNTR Variants 59
Ⅳ. DISCUSSION 61
REFERENCE 66
SUMMARY IN KOREAN 71
Table 1. Genotype distributions of thymidylate synthase gene polymorphisms in 126 stomach cancer patients (a), female group (b) and male group (c) of stomach cancer patients 24
Table 2. Comparison of Observed and Expected Numbers of Combined Genotypes 28
Table 3. Combined genotypes of TSER VNTR-6 bp insertion polymorphism or TSER VNTR-G/C SNP-6 bp insertion polymorphism in stomach cancer patients (a), stomach cancer female group (b) and male group (c) 30
Table 4. Genotype frequencies in 30 diffuse type patients and 54 intestinal type patients. TSER VNTR and 6bp insertion polymorphism (a) and TSER VNTR-G/C SNP (b) 35
Table 5. Differences between Observed and Expected Numbers of Haplotypes 38
Table 6. Distribution comparison of thymidylate synthase polymorphism genotype (A) and combined genotype (B) between 111 colon cancer patients and 201 control groups 40
Table 7. An individual genotype and combined genotype distribution in proximal colon cancer patients (a, c) and distal colon cancer patients (b, d) 44
Table 8. An individual genotype and combined genotype distributions in 56 female groups (a, c) and 55 male group (b, d) of colorectal cancer patients 49
Table 9. Comparisons of homocysteine level by an individual genotype between stroke patients and controls (a) and gender difference of homocysteine level (b) 55
Table 10. Ethnic distribution of TSER VNTR and 6bp insertion polymorphism 58
Figure 1. Schematic diagram of thymidylate synthase gene polymorphisms- TSER VNTR (double or triple), G/C SNP and 6 bp insertion polymorphism. This diagram shows 3R (triple repeat) of thymidylate synthase mRNA. In case of 2R, 1st repeat is absent. G/C SNP exists at 12th base of the 2nd repeat in 3R (triple repeat) 15
Figure 2. TSER VNTR and G/C SNP genotyping. VNTR of 2R/2R, 2R/3R and 3R/3R were distinguished by amplification fragment size (`Plane). G/C SNP genotyping was performed by Hae III digestion (` H' lane). Each genotype with 3C allele showed digested fragments of 94 bp and 44 bp, while 3G and 2R allele showed digested fragments of 66 bp and 44 bp 18
Figure 3. Genotyping of 6 bp insertion polymorphism. Left 3 columns of the marker lane show a 6 bp insertion polymorphism PCR product and right side 3 columns show a genotyping by Dra I digestion. Heterozygote 0bp/6bp had an undigested 0bp allele and digested 6bp allele of 70 bp and 88 bp. Homozygote 0bp/0bp or 6bp/6bp had only undigested 152 bp or digested 70 bp and 88 bp, respectively 20
Figure 4. 5′ UTR tandem repeat variants 60