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行政院國家科學委員會專題研究計畫 期中進度報告

(子計畫六)膀胱癌易罹癌基因之多發家族遺傳連鎖分析

（2/3）

計畫類別： 整合型計畫

計畫編號： NSC92-3112-B-038-002-

執行期間： 92 年 05 月 01 日至 93 年 04 月 30 日 執行單位： 臺北醫學大學公共衛生學系

計畫主持人： 邱弘毅

計畫參與人員： 陳建仁,薛玉梅,蒲永孝,沈正煌,王淵宏

報告類型： 完整報告

報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢

中 華 民 國 93 年 3 月 12 日

基因體醫學國家型科技計畫

National Research Program for Genomic Medicine National Science Council, the Executive Yuan, ROC.

砷與移行細胞癌之毒理基因體研究─（子計畫六）

膀胱癌易罹癌基因之多發家族遺傳連鎖分析（2/3）

Linkage Analysis of Susceptibility Genes for Bladder Cancer in Multiplex Families (2/3)

報告類別：

□ 新進研究計畫 □ 修正後計畫書 ; 年度成果報告 (New Proposal) (Revised Proposal) (Progress Report) 計畫類別：□ 個別型計畫 ; 整合型計畫

(Individual Project) (Program Project) 計畫編號： 91GMP012-6

計畫主持人 (Principle Investigator)：邱弘毅 ( Hung-Yi Chiou ) 執行單位 (Institution)： 台北醫學大學公共衛生學系

( School of Public Health, Taipei Medical University )

中華民國 92 年 12 月 24 日

National Research Program for Genomic Medicine National Science Council, the Executive Yuan, ROC.

Progress Report—Research Project

基因體醫學國家型科技計畫

國科會延續性計畫進度報告

Program Classification:

; Genomic Medicine … Bioinformatics

… Proteomics & Structural Genomics … ELSI Project Number

91GMP012-6

NSC Funding Number

NSC93-3112-B038-002

(in Chinese) 中文

膀胱癌易罹癌基因之多發家族遺傳連鎖分析

Title of Project 計畫名稱

(in English) 英文

Linkage Analysis of Susceptibility Genes for Bladder Cancer in Multiplex Families

(in Chinese) 中文

台北醫學大學 公共衛生學系

Institution

研究（執行）單位

(in English) 英文

School of Public Health, Taipei Medical University

(in Chinese) 中文

邱弘毅

Principle Investigator 計畫主持人

(in English) 英文

Hung-Yi Chiou

FY 2002 2003 2004 Total Budget

(in NT dollars: 1USD = 34 NTD) Signature of the PI：_______________Date：__12/24/2003____

Part B. Progress Report of

Component Projects

TABLE OF CONTENTS

Project Number

91GMP012-6

Part B. Progress Report

B1. Response to previous reviewer’s critiques B2. Progress Report

Abstract (in Chinese and English) Background and Significance B2a. Specific Aims

B2b. Studies & Results B2c. Personnel

B2d. Projected Timeline B2e. Publications (Optional) B2f. Patents (Optional)

B3. Request for Modifications of the Project (Optional)

You do not have to finish this section except your budgets or items of requests are modified.

(請求經費或項目變動者才須加填此一部份，否則直接附上去年全程計畫書當附件 即可)

B3a. Background & Statement (including literature cited) B3b. Summary Budget Requested

B3c. Postdoctoral Fellows Requested B3d. Detailed Budget for Personnel

B3e. Detailed Budget Requested for Equipments B3f. Detailed Budget Requested for Travel to Overseas B3g. Detailed Budget Requested for Attending Conferences B3h. Detailed Budget Requested for Other Categories B3i. Use of Core Facilities Requested

B3j. Biographical Sketches of New Personnel

Progress Report

B1. Response to previous reviewers’ critiques

Please describe the previous reviewers’ critiques and how based on the critiques, you made modifications to specific aims, experimental design, or resource

allocation etc.

中文摘要

關鍵詞:移形上皮細胞癌、砷、家族聚集研究

無機砷是確認之人體肺癌與皮膚癌致癌物，包括台灣、美國、日本、阿根廷、智利及 芬蘭等國家，均有研究指出砷暴露與泌尿道癌症的產生具有統計顯著相關存在。我們最近 於台灣東北角的蘭陽盆地亦發現砷暴露與泌尿道癌，特別是移形上皮細胞癌（TCC）具 有明顯的劑量效應關係存在。不過，引起膀胱癌之主要易感受基因及其功能目前並不清 楚。因此本計畫的研究目的包括（1）透過癌症登記系統及各合作醫院確認的膀胱癌病例，

以家族疾病史問卷作電話或家戶訪視，以尋找膀胱癌多發家庭。（2）以問卷訪視蒐集指標

個案及其家庭成員之研究資訊並收集其血液、尿液等生物檢體。（3）分離、純化及分裝研

究對象 DNA 並儲存在超低溫冷凍庫以供各子計畫使用。（4）針對過去 CGH 及 LOH 研 究所獲得在染色體上與膀胱癌發生相關之 DNA 增加與缺失的部位，進行易感受基因多型

性分析。（5）針對砷引起與非砷引起之膀胱癌，利用多發家庭連鎖分析，對相關易感受性

基因進行定位。（6）分析可能的易感受基因與環境因子對膀胱癌發生危險性之交互作用。

（7）建立遺傳流行病學與生物統計支援中心，協助各子計畫進行資料分析工作。本研究 共分為兩個部分，第一部分預備以三年時間在台南、嘉義、宜蘭三縣各合作醫院收集 150 位多發性家族之膀胱癌指標個案，及每一指標個案的十位一等親成員，合計 1500 位多發 家庭成員。第二部分針對 150 個多發家庭（每一家庭除指標個案外，至少一名成員罹患膀 胱癌）以問卷蒐集環境暴露等危險因子及生活史資料，以判定為砷暴露及非砷暴露組。每 一位研究對象亦將蒐集 35c.c.血液及 50c.c.尿液，以獲得研究所需的 DNA，DNA 將被分 離、純化、儲存在超低溫冷凍庫中，以供各子計畫使用。本計畫亦將針對過去 CGH 及 LOH 研究所獲得在染色體上與膀胱癌發生相關之 DNA 增加與缺失的部位，進行易感受 基因定位之連鎖分析，比較砷引起及非砷引起膀胱癌的易感受基因的部位之異同，供日後 做預防與治療的基礎資料。

Abstract

Keywords: Transitional Cell Carcinoma; Inorganic arsenic; multiplex family study

Inorganic arsenic has been well documented as a human carcinogen of skin and lung. A significant association between arsenic exposure and risk of urinary cancer has also been reported in many epidemiological studies carried out in many countries of the world. Our recent study had also found a significant dose-response relationship between risk of cancers of urinary organs, especially for transitional cell carcinoma (TCC), and arsenic exposure through drinking well water in Lanyang Basin. However, the major susceptible gene(s) of bladder cancer from arseniasis-endemic and non-endemic areas and their functional changes that make a person to be a victim of arsenic-induced bladder cancer is still unclear. The objective of this subproject is to identify and differentiate the major susceptibility gene(s) for arsenic-induced and non-arsenic-induced bladder cancers through the linkage analysis of genetic markers in members of multiplex families. It will include following specific aims: 1) the ascertainment of multiplex families through the telephone or home-visit interview, based on a family history questionnaire, of bladder cancer cases reported to the national cancer registry and cases diagnosed and treated in collaborative medical centers; 2) the recruitment of probands and families members through home-visit personal questionnaire interview and biospecimen collection; 3) the purification, depository and inventory of DNA samples in central biospecimen bank; 4) the typing of genetic markers on chromosomes in which loss or gain have been observed through previous comparative genomic hybridization and loss of heterozygosity studies; 5) the mapping of susceptibility gene(s) through linkage analysis of multiplex family data for bladder cancer cases in arseniasis -endemic and non-endemic areas; 6) the examination of the effects of possible candidate genetic marker(s) and their synergistic interactions with environmental factors on bladder cancer; and 7) the establishment of Genetic Epidemiology and Biostatistics Supporting Core to provide methodological support to other subprojects. A total of 150 multiplex families of bladder cancer including 150 probands and 1500 first-degree relatives from arseniasis -endemic and non-endemic area will be recruited by the end of three year grant period. An informed consent will be obtained from each participant for the collection of risk factor information through questionnaire interview. A 35 mL blood and buccal cell specimen will be obtained from each consenting participant. DNA samples will be extracted from peripheral lymphocytes and buccal cells, aliquoted and frozen at -70℃. Polymorphisms of genetics markers closely linked to the major susceptible gene(s) of bladder cancer will be typed.

Analysis of Lod score, and transmission disequilibrium test will be carried out to map susceptible gene(s) of arsenic-induced and non-arsenic-induced bladder cancer based on multiplex family study.

Background and Significance

Our previous studies have shown that capability of arsenic methylation is associated with the risk of arsenic-induced skin cancer (12). Null genotypes of glutathione S-transferase (GST) M and T1 and variant genotype of GST P1 have been found to increase the risk of arsenic-induced skin cancer (13). Whether the genotypic and phenotypic polymorphisms of GSTs and other enzymes related to arsenic methylation may modify the risk of arsenic-induced TCC remain to be elucidated. Furthermore, genetic polymorphisms of some DNA repair enzymes have been observed to be associated with the risk of arsenic-induced skin cancer (14). It is also important to assess whether the DNA repair enzymes, either their genotypes or gene expression profiles, are related to the risk of arsenic-induced bladder cancer. The existence of genetic susceptibility to arsenic-induced bladder cancer may be explored through the microarray analysis of differential expression profiles and single nucleotide polymorphisms of related genes. It may also be investigated by the genetic epidemiological study on familial TCC through linkage analysis.

Tobacco smoke is another important etiological agent of urinary bladder cancer in Taiwan. It plays an important role in the induction of urinary bladder cancer in non-arseniasis-endemic areas. The tobacco-related bladder cancer risk is modified by the genetic polymorphisms of N-acetyltransferase 1 and 2 (15). The comparison of toxicological genomic characteristics of TCC induced by tobacco and arsenic may help the elucidation of the arsenic-induced carcinogenicity. This program project is organized to study the difference in toxicological genomic characteristics between TCC induced by arsenic and tobacco smoke.

Due to the lack of animal models for arsenic carcinogenicity, arsenic is a unique carcinogen in humans. Therefore, the high prevalence of urinary TCC in arseniasis-endemic area in Taiwan is a unique chance to investigate the carcinogenicity of arsenic. Since cancer development is mainly attributable to environmental factors and genes, it is of fundamental importance to understand the interaction between environment and genes. In this PPG, epidemiological components will establish the association of environmental factors and incidences of urinary TCC. Molecular, cellular and genetic components will identify the genetic factors involved in formation of urinary TCC. The joint efforts of epidemiological and molecular genetic studies will successfully dissect the interactions between environmental factors and genes. Novel findings of this PPG will allow us designing new strategy for prevention, diagnosis, and treatment of urinary TCC.

Though arsenic is a human carcinogen, there is no good animal model for the carcinogenicity of inorganic arsenic. Arsenic is inactive or extremely weak to induce gene mutations at specific loci (1,16). The modes of action for arsenic-induced carcinogenicity might include the induction of chromosome abnormality, inhibition of DNA repair, induction of oxidative stress, and increase of cell proliferation (17). Inorganic arsenic has several genotoxic effects including the induction of changes in chromosome structure and number, increases in sister chromatid exchanges and micronuclei, gene amplification, cell

transformation, aneuploidy, and chromosome-type chromosome aberration (1,17-22). The role of inorganic arsenic in the carcinogenesis has also been hypothesized as a co-carcinogen such as promoter or progressor rather than an initiator (23,24). However, the evidence is far from adequate to draw a definite conclusion on the exact mechanism of inorganic arsenic to induce various cancers in humans.

Most of the genetic variants which have been studied using the design of case-control study were only associated with a modest increased genetic risk for developing cancer, although the magnitude of their attributable risk is large because there are quite frequent in the population. Many rare cancer syndromes due to a mutated gene which confer very high risk of cancer have been well-documented, including the rare Li Fraumeni symdrom in which persons inhriting a gerlime mutation in the p53 gene are at almost 100 % risk of breast caner and other cancers by the age of 60 years (25,26), the familial adenomatous polyposis symdrome associated with mutation in the APC gene (27), and the breast-ovary cancer syndrome associated with an inherited mutation in BRCA1 (28).

Human genome sequence have been cloned successfully by “Human Genome Project”. A total of 3 x 10⁵ of an estimated 3-10 x 10⁶ single nucleotide polymorphisms which distinguish individuals and their disease traits and risk will be identified by 2002 (29).

Classical family, twin and adoptee studies have shown substantial heritabilities for many disease traits, but except in rare instances the pattern is polygenic rather than monogenic.

Knowledge and technology have become sufficient to enable molecular geneticists to study megaphenic disorders in single families, with the exceptation of isolating ‘the genes’

understanding the pathology and deriving clinically applicable test of status (30,31).

For polygenic traits, knowledge is sufficient to initiate tests of hypotheses, but the technology is yet insufficient to measure the contribution of genetic diversity to disease liabilities. Linkage analysis of multiplex family has an important role in genetic epidemiology because it identifies a biologic mechanism for transmission of a trait or disease (31). A large family displaying a clear-cut segregation pattern for a disease is examined at polymorphic sites representing each part of each chromosome (30). Recently, most sites of cancer has been recognized as multigene disorders. It means that many regions of each chromosome have been examined intensively to identify susceptible genes and then to differentia their functions on risk of various cancers. With a availability of a fairly complete human genome sequence, this approach is reducing to searching the sequence for the possible culprit gene on criteria such as its tissue expression pattern, apparent function predicted from sequence, etc. For example, haemochromatosis was shown by positional cloning to be attributable to an HLA-related genes and it has immediately been possible to examine the relationship between genetic and iron status diagnostics, population prevalence of mutations, and prevalence of HFE mutations in haemochromatosis-associated disease groups such as diabetics (32). In addition, given the widespread roles of iron, for various phenotypes and investigation of HFE interactions in other disorders such as porphyries, haemoglobinopathies and coronary disease and

environmental variables such as diet, lead poisoning and infections, is now proceeding to explore gene-environment and gene-gene interactions through epidemiological design (33-35).

The transformation of a normal cell into a malignant cell is a multistep mechanism, which involves various alterations on the molecular and genetic level. These molecular alterations occur spontaneously or are induced by carcinogens. As in most other malignancies the development of bladder cancer is caused by the accumulation of various molecular changes. The expression of oncogenes (ras, erbB-2 and EGF receptor), tumor-suppressor genes (Rb, p53),

cell-cycle genes (p15, p16) and DNA-repair genes is altered mostly by mutation or chromosomal aberration. Loss of heterozygosity of chromosome 9p and 9q has been shown to be a crucial event in the transition of normal urothelium to papillary transitional cell carcinoma while p53 is primarily involved in the development of carcinoma in situ (36). A study based on 44788 pairs of twin from Sweden, Denmark, and Finland showed that heritability was estimated to account 31 percent of the variation in susceptibility to the risk of bladder cancer. It was implied that genetic component might not be ignored in the carcinogeneicity of bladder cancer (37).

In this study, by the aid of linkage analysis, we plan to map the susceptibility gene(s) through multiplex family data of bladder cancer cases from arseniasis-endemic and non-endemic areas, to differentiate the difference between gene profiles of residents from arseniasis-endemic and non-endemic areas and to clarify the genetic and functional changes that make a person affected with arsenic-induced bladder cancer.

PROGRAM PROJECT: Component Project ___6__

B2a. Specific Aims

Please state the overall goals of the project, and specific aims, as reviewed and approved by the Study Section and actually awarded. If these specific aims as actually funded did not differ in scope from those actually pursued during the grant period, and if the aims have not been modified, state this. If they have been modified, give the revised aims and the reasons for the modifications.

The overall goals of this subproject are to identify and differentiate the major susceptibility gene(s) for arsenic-induced and non-arsenic-induced bladder cancers through the linkage analysis of genetic markers in members of multiplex families. This subproject will also set up a Genetic Epidemiology and Biostatistics Supporting Core for this program project. It will include following specific aims: 1) the ascertainment of multiplex families through the telephone or home-visit interview, based on a family history questionnaire, of bladder cancer cases reported to the national cancer registry and cases diagnosed and treated in collaborative medical centers; 2) the recruitment of probands and families members through home-visit personal questionnaire interview and biospecimen collection; 3) the purification, depository and inventory of DNA samples in central biospecimen bank; 4) the typing of genetic markers on chromosomes in which loss or gain have been observed through previous comparative genomic hybridization and loss of heterozygosity studies; 5) the mapping of susceptibility gene(s) through linkage analysis of multiplex family data for bladder cancer cases in arseniasis-endemic and non-endemic areas; 6) the examination of the effects of possible candidate genetic marker(s) and their synergistic interactions with environmental factors on bladder cancer; and 7) the establishment of Genetic Epidemiology and Biostatistics Supporting Core to provide methodological support to other subprojects. We do not modified any specific aims of this component project. Base on experience of the first year, we have confidence of accomplishing these specific aims. However, the total number of multiplex families will be less than the proposed number because the modified budgets raised in the initial time of the second grant year was not proved. We expect a total of 60 multiplex families will be recruited during three year grant period.

PROGRAM PROJECT: Component Project ___6__

Describe the studies directed toward the specific aims during the current grant period and the results obtained. Indicate the extent to which the work accomplished has successfully met the specific aims. Include negative results. If technical problems were encountered in carrying out this project, describe how your approach was modified.

Ⅰ. Multiplex families ascertainment

A total of 730 urothelial carcinoma (UC) consenting participants have been recruited.

Among them, 131 consenting participants from Taiwan University Hospital, 469 consenting participants from Chi-Mei Hospital and 130 consenting participants from Chia-Yi Christian Hospital. An informed consent will be obtained from each participant for the collection related study information. through personal interview in centers for outpatient and inpatient by well trained interviewer. Risk factor questionnaire (RFQ) was used to collect risk factors of UC including cigarette smoking, alcohol drinking, well water consumption, nutrition status, and usage of dye and drug. Family history questionnaire (FHQ) was used to obtain disease history of various cancers especially for UC and chronic diseases including diabetes mellitus (DM) and hypertension. Flow chart for multiplex families recruitment was shown in Figure 1.

Ⅱ. Recruitment of proband and families members

A total of 21 probands whose first degree relatives with UC have been ascertained through FHQ. Now we have mailed invited letters to family members of probands to invite them to participate this study. Among consenting family members, we decided to recruit ten members of each multiplex family including parents, spouse, sibling, and offspring. Questionnaire interview and biospecimen (blood, buccal cell, urine) collection will be executed either in outpatient center of collaborative medical center or household of consenting family members. The flow chart of multiplex families recruitment was shown in figure 2. Socio-demographic characteristics of probands and their first -degree relatives were shown in Table 1 and 2. Pedigrees of eight additive multiplex families newly recruited were illustrated in Figure 3-1 to Figure 3-8.

PROGRAM PROJECT: Component Project ___6__

Initially, among consenting family members, we decided to recruit ten members of each multiplex family including parents, spouse, sibling, and offspring. However, after trying to invite these relatives to participate in this study , about half of them can not be completely interviewed or even refused to be recruited. Reasons for not recruiting or not completing the questionnaire and biospecimen collection were: “no contact with relatives,” “too much effort,” “too emotional,” “too complicated to retrieve all data on relatives” ,“no permission from relatives to give information.”,

”expired address and telephone number” and “too far or no one can take them to the hospital” (44).

Especially, some probands already died for several years ago. In order to overcome these difficulties, we devoted to several strategies and did more intensive efforts to contact them for interview to achieve the goal as followed: [1] outreach to interview those unable to be the hospital

; [2] recheck their medical records and get the correct address and telephone number; [3] instead of face-to-face interview, we try to get the information by postal questionnaire. [4] give some kinds of presents or additional information and a reply card to encourage them to participate in this study.

[5] recruit more collaborated hospitals to increase the number of multiplex families and first-degree relatives; [6] subsequent telephone calls were made for additional information of uncompleted questionnaires; [7] refer the first-degree relatives living in the catchment area of our collaborated hospital to receive the face-to face interview; [8] link medical records and cancer registry database to identify new UC cases and update associated mortality information.

Table 1. The distribution of age and sex 21 UC probands

Family ID Sex Age (year)

NTU10222 M 61 NTU10263 F 67 NTU10310 M 61 NTU10264 M 66

CM1010 M 71

CM1012 M 85

CM1071 M 57

CM1059 F 62

CM1123 M 73

CM1356 F 96

CM1352 M 64

CM1355 F 65

CM1485 M 58

CM1271 M 73

CM1272 F 48

CM1402 M 60

CY1001 F 50

CY1032 F 85

CY1000 F 64

CY1056 F 45

CY1131 F 58

mean age:65.2 years old; sex ratio (M:F)=11:10

Table 2. The distribution of first degree relatives among 21 UC multiplex families

First-degree relatives

Family ID members Father mother brother sister son daughter

NTU10222 11 1(1) 1 4 4 1 0

NTU10263 10 1(1) 1 1 2 4 1

NTU10310 6 1(1) 1 0 0 3 1

NTU10264 12 1(1) 1 1 5 3 1

CM1010 9 1(1) 1 2 1 3 1

CM1012 14 1 1 2(1) 2 2 6

CM1071 14 1 1 6(1) 2 2 2

CM1059 10 1 1(1) 3 3 1 1

CM1123 9 1 1(1) 2 1 1 3

CM1356 14 1 1 1 3 4(1) 4

CM1352 10 1 1 3 2 2 1

CM1355 11 1 1 3(1) 2(1) 1 3

CM1485 15 1(1) 1 7 3 1 2

CM1271 10 1 1 0 1 4(1) 3

CM1272 6 1 1 3(1) 0 1 0

CM1402 11 1(1) 1 3 1 1 4

CY1000 9 1 1 1(1) 1 4 1

CY1001 8 1 1 1(1) 2 1 2

CY1032 17 1 1 3 6(1) 1 5

CY1056 7 1 1 2(1) 1 0 2

CY1131 10 1 1 2 2 3(2) 1

* Number of first-degree relatives affected with UC are shown in parenthesis

7

PROGRAM PROJECT: Component Project ___6__

Figure-3.1

Figure-3.2

Figure-3.3

CY1032 85

551 85

401 45

402 80

501 86

502 UN

503 UN

504 62

505 61

506 60

507 59

508 58

509 57

601 56

602 53

603 49

604 47

605 44

606 42 UN

UN

CY1001 50

551 401

70

402 62

501 52

502 46

503 42

601 24

602 27

603 21 551

64

CY1000 64

401 67

402 UN

501 62

502 54

601 36

602 33

603 34

604 31

605 29

8

Figure-3.4

Figure-3.5

Figure-3.6

CM1271 73

551 59 401

UN

402 UN

501 78

601 59

602 49

603 48

604 46

605 44

606 42

607 40 604

CY1131 58

551

401 402

602

501 502 503 504

601 603

CY1056 45

551 46 401

69

402 76

501 53

502 48

503 41

601 19

602 16

8

PROGRAM PROJECT: Component Project ___6__

Figure-3.7

Figure-3.8

503 55

504 53 CM1402

60

551 42 401

68

402 56

501 12

502 71

605 28 601

36

602 34

603 32

604 30 601

15 551

48 CM1272

48 401

UN

402 UN

501 52

502 51

503 34

9

PROGRAM PROJECT: Component Project ___6__

Ⅲ.Risk Ratios for Relatives

In human genetics and genetic epidemiology, risk ratios can assume a number of different forms, including risk ratios for relatives, for candidate genes, and for genetic markers. The goal of many genetic studies is to quantify the risk of disease occurrence associated with particular genetic factors. Evidence for familial aggregation, which is the tendency of disease to cluster in families, provides a rationale for subsequent genetic studies intended to assess particular genetic factors or to search for disease susceptibility genes.A related measure is known as the recurrence risk for a type R relative of an affected individual. It occurs in the numerator of the ratio (40), for familial

aggregation in family case–control designs. For controls that are representative of the general population, the denominator of this ratio approximates the population risk.

In Risch’s (41) development of multilocus models of inheritance for complex traits that are useful in linkage analysis, he defines a risk ratio, λR, which compares the recurrence risk in relatives of type R of an affected individual with the population prevalence K. For example, when the relative type is a sibling, λs is defined as(42)

Pr( D in Relative who is a sibling|affected case) K

Risch(43) also establishes how the power of affected relative pair studies to detect linkage critically depends on the value of λR.

Table 4. The estimated Recurrence Risk Ratio(RRR) of 730 urothelial cell carcinoma (UC) consenting participants

Type of relatives number (affected) siblings only( λs) all relatives (λR) Parents 1460 ( 9 )

Sibling 3650 ( 9 ) 8.2 Offspring 2920 ( 5 )

All relatives 8030 (23) 9.5

* The population prevalence was estimated as 30/100,000.

A widely used measure of familial aggregation is the sibling recurrence-risk ratio, also referred to as λS , which is defined as the ratio of the risk of disease manifestation in siblings of cases

compared with the disease risk in the general population [4,5]. These values underestimate the population risk and therefore overestimate familial aggregation. A mathematical foundation for the choice of the general population risk as the reference value for calculating the sibling

recurrence-risk ratio as a tool for mapping genes for complex diseases was clarified by the work of Risch in the early 1990s [5,14]. As λS is used to design studies and to estimate the power to detect genes for complex human disease, it must be determined [15–18].

PROGRAM PROJECT: Component Project ___6__

IV. DNA purification, depository and inventory in central biospecimen bank

10 ml blood was obtained from each consenting participant. Total genomic DNA was purified using Viogene Blood and tissue Genomic DNA Miniprep System from buffy coat. In core of biospecimens bank, each DNA sample was deposited at -70 ℃ refrigerators and also be inventoried in computer files. Table 3 showed number of RFQ, FHQ, blood sample and urine sample collected from consenting participants through Taiwan university Hospital, Chi-Mei Hospital and Chia-Yi Hospital.

Table 3. The Questionnaires and biospecimens of consenting participants

Taiwan University Hospital

Chi-Mei Hospital Chia-Yi Christian Hospital

Item N (%)^a N (%)^a N (%)^a

RFQ 131 (18) 469 (64) 129 (18) FHQ 131 (18) 393 (54) 129 (18) Blood 92 (13) 381 (52) 130 (18)

Urine 110 (15) 38 ( 5) 130 (18)

a: percentage of 730 consenting participants

Although, the collection of questionnaire and biospecimen was not comprehensive, we still try to do more efforts to increase the comprehensive rate. Some cases or their relatives are difficult to get appropriate volume of blood or urine, we use the buccal swab to collect buccal cells instead of blood samples. In another method, because patients will return to the hospital to have a routine examination, we mentioned interviewers and staffs of the Laboratory Science Department to collect un-recruited samples in this routine process.

At the same time, we extracted DNA from buffy coat or buccal cellls from biospecimen bank. If the patients died for several years ago, we try to check the tissue bank of the collaborated hospitals to get appropriate amounts of tissues for DNA extraction. Then we will performed PCR-RFLP to analyze the genotype of several UC associated susceptible genes such as NQO1, iNOS, EGFR.

PROGRAM PROJECT: Component Project ___6__

Summarize the personnel involved in the project during the grant period. List the personnel in accordance to the following categories: (1) senior investigators, including visitors; (2) postdoctoral fellows; (3) graduate students; (4) technicians; and (5) other research assistants.

Specify for each individual the period of involvement and the percentage commitment of effort.

Name

In Chinese In English Position Title Education

Degree % of effort Job Description or Responsibilities

邱弘毅

陳建仁

邱文祥

王淵宏

徐憶驊

蕭睿祺

謝宜蓁

Hung-Yi Chiou Chien-Jen Chen

Wen-Hsiang Chiou Iuang-Hung Wang Yi-Hwa Hsu

Jui-Chi Hsiao

Yi-Chen Hsieh

Professor

Professor

Associate Professor Doctor student Full-time research assistant Full-time research assistant Master student

Ph.D.

Sc.D.

MD, Ph.D.

MPH

MPH

BS

BS

30%

5%

10%

40%

100%

100%

40%

Director of the component project

Consultant of the component project

UC patients diagnosis and recruitment

Set-up of flow-chart of UC patient recruitment and data management

Questionnaire interview and biospecimens collection Questionnaire interview and biospecimens collection

DNA purification, depository and inventory

Component Project: 6

Data Analysis and Statistical Power:

Genetic linkage analysis will be carried out to identify major susceptible genes related to arsenic-induced bladder cancer based on the data of polymorphisms of microsatellite markers obtained from electrophoresis of ABI PRISM 377 sequencer. Nonparametric multipoint analysis using affected sib pairs, which is robust even when the mode of inheritance is not known, will be performed with program GENEHUNTER version 2.0 (38) to calculate normalized Z scores and associated P value. Traditional LOD score methods will be also performed by means of GENEHUNTER version 2.0 for two-point parametric analysis. Multipoint analysis has the advantage of increasing the power of detecting linkage by utilizing information from two flanking markers rather than a single marker (36). TDT will also be used to analyze linkage data when sufficient number of case-parental pairs is available. Gene markers will be identified as major susceptible gene(s) for arsenic-induced bladder cancer based on following criteria of P-value < 0.05 for Sibpal regression analysis and TDT, and LOD score >1.

Human subject

In this study, a total of 1650 study subjects will be recruited. Each study subject will sign an informed consent on a voluntary basis at recruitment. The consent form will follow the guidelines recommended by the Joint Working Group on Ethical, Legal, and Social Implications of Human Genome Research(39). Only the study subjects, themselves, will be permitted to give consent, not relatives or surrogates. The patients’ care will not be affected by whether they participate in the study or not.

The research procedures consist of the collection of 35 ml of venous blood, mucous cell, and 50 ml of urine sample for each study subject. All study information collected through various questionnaire including ARF, RFQ, and FHQ will be used for research purposes only. Disposable syringes with sterilized needles will be used for blood collection to prevent any blood-born transmission of infectious agents.

PROGRAM PROJECT: Component Project ___6__

Provide a reasonable timetable for the execution of the work outlined in the project. Highlight appropriate milestones that you might use to target the studies. Indicate technical hurdles that might slow down the execution of the work and discuss any contingencies that you have or might have built in the research plan in anticipation of these difficulties. Do not exceed one page.

Component projected timetable ( second year)

The second Year

Month Order

Item

1st Month 2nd Month 3rd Month 4th Month 5th Month 6th Month 7thMonth 8th Month 9th Month 10thMonth 11thMonth 12thMonth

Remark

Recruitment of more collaborative medical centers

Ascertainment of multiplex families (FHQ, RFQ) Biospecimens collection from consenting participants Set up biospecimens bank Recruitment first degree relatives of multiplex families

Data keying, checking, and analysis of FHQ and RFQ

During the second grant year of the component project, we worked very hard to recruit every UC patients we could found. Until now, we interview 730 consenting participants from Taiwan university Hospital ,Chi-Mei hospital. and Chia-Yi Christian Hospital. We also collected blood and urine from consenting participants if they agree us to do it. First degree relatives of twenty-one multiplex families have been recruitment (family history prevalence of UC =2.9%), which implied that we have good opportunities to examine the effects of possible candidate genetic marker(s) and their synergistic interactions with environmental factors on UC. However, based on our limited personnel, it would be very difficult to recruit 150 multiplex families during grant period. In order to speed recruitment of multiplex families, we need more research assistants to recruit at least 60 multiplex families each year in the following two grant year. The detail arrangement of personnel in this project and related modified budget will be further described in the section B3. I hope the modified budget could be funded. Then, we feel confident of accomplishing the component study.

Component Project: 6

Research Plan (continuation)- Literature Cited

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arsenic-induced skin cancer for genetic polymorphisms of glutathione

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College of Public Health, National Taiwan University. Taipei: National Taiwan

Component Project: 6

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Component Project: 6

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