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Association between single nucleotide polymorphisms in the 3'untranslated region of the SRD5A2 gene and prostate cancer risk

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Association between single nucleotide polymorphisms in the 3'untranslated region of the SRD5A2 gene and prostate cancer risk

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Content ASSOCIATION BETWEEN SINGLE NUCLEOTIDE POLYMORPHISMS IN THE 3’UNTRANSLATED REGION OF THE SRD5A2 GENE AND PROSTATE CANCER RISK by Prutha Badiani A Thesis Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree MASTER OF SCIENCE (BIOCHEMISTRY AND MOLECULAR BIOLOGY) August 2003 Copyright 2003 Prutha Badiani Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 1417911 INFORMATION TO USERS The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleed-through, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. ® UMI UMI Microform 1417911 Copyright 2004 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES, CALIFORNIA 90089-1695 This thesis, w ritten by peutHA b ftrsihM_____ under the direction o f thesis committee, and approved by a ll its members, has been presented to and accepted by the D ire cto r o f Graduate and Professional Program s, in p a rtia l fu lfillm e n t o f the requirements fo r the degree o f D irector Date A u gu st 1 2 , 2003 Thesis Committee C hair Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DEDICATION -to my mother, who is now in heaven, has and is always blessing me -to my father, who has always inspired me -to my husband, who has always supported and encouraged me -to my sisters, who have always taken care of me.... Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I thank Dr. Juergen Reichardt, whose lab I worked in for 2 years. I appreciate his kindness for accepting me to his lab and guiding me all through these 2 years. I thank my committee member and my master’s thesis advisor Dr. Zoltan Tokes for guiding me during my M.S and providing me help whenever required. I thank Dr. Austin Mircheff for agreeing to be my committee member and taking time for my defense. I thank Dr. Nick Makridakis, who was my personal mentor, for teaching me all the new techniques and being patient at all stages of learning. I thank my collegues Lucio Ferraz, Troy Phipps, Claudia Weihe, Nancy C, Patricia Sharpe, Nancy H, Frank Luh, Dolly Foti, for being helpful always in my labwork as well as during writing my thesis. I thank Eugene Kim for helping me with the analysis data. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Dedication ii Acknowledgements iii List of Tables vii List of Figures v iii Abstract x 1 .Introduction 1 1.1 Prostate cancer 1 1.1.1 Prostate gland 1 1.1.2 Prostate cancer epidemiology 2 1.1.3 Risk factors 3 1.1.3.1 Age 4 1.1.3.2 Race 4 1.1.3.3 Family history/genetics 5 1.1.3.4 Hormones 6 1.1.3.5 Smoking 7 1.1.3.6 Body size and physical activity 7 1.1.3.7 Occupation 8 1.1.3.8 Diet 9 1.1.3.9 Vasectomy 9 1.1.4 Pathology 9 1.1.4.1 TNM classification 10 1.1.4.2 Gleason grading 11 1.1.5 Screening 13 1.1.6 Treatment 13 1.2 Androgens, the SRD5A2 gene and prostate cancer 14 1.2.1 Androgens 14 1.2.1.1 Testosterone 15 1.2.1.2 Dihydrotestosterone 16 1.2.1.3 Androgens and prostate cancer 20 1.2.1.4 Androgens and race 21 1.2.2 The SRD5A2 gene 22 1.2.2.1 Isozymes of steroid 5a-reductase 22 1.2.2.2 Gene structure of SRD5A2 23 1.2.2.3 The SRD5A2 gene, polymorphic markers, and prostate cancer 24 1.2.2.3.1 Pseudohermaphroditism 24 1.2.2.3.2 Mutations in the SRD5A2 gene and prostate cancer risk 25 1.2.2.4 3’UTR of the SRD5A2 gene 28 iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2.4.1 3’UTR and mRNA stability 28 1.2.2.4.2 3’UTR and (TA)n repeat 32 1.2.2.4.3 Single nucleotide polymorphisms and the SRD5A2 gene 33 1.2.2.5 The SRD5A2 gene, ethnicity and prostate cancer 34 1.2.2.6 Hypothesis 35 2 Materials and Methods 37 2.1 Study subjects and DMA samples 37 2.2Overview of the experimental methods 37 2.2.1 PCR 38 2.2.1.1 Primer design 38 2.2.1.2Thermal cycling 40 2.2.2 Gel electrophoresis and PCR purification 41 2.2.2 SNaPshot multiplex reaction 42 2.2.3 Sample preparation for ABI 3100 genetic analyzer 46 2.2.4 Data analysis 47 2.3 Experimental Difficulties 47 2.3.1 Study subjects 47 2.3.2 PCR 47 2.3.3 PCR purification 48 2.3.4 Genotyping 48 2.3.5 Primers for SNaPshot 49 3 Results 51 3.1 PCR results 3.2Genotyping results 3.3 Data analysis results 3.3.1 Association of the A3146T, A3174G, A3681C, and G3877A SNPs with prostate cancer risk 3.3.2 Hardy Weinberg equilibrium 51 51 53 53 56 4 Discussion and conclusion 4.1 Biological rationale 4.1.1 Importance of prostate cancer 4.1.2 Androgens and the SRD5A2 gene 4.1.3 3’UTR of the SRD5A2 gene 4.1.4 Ethnicity 59 59 59 60 61 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4.1.5 Clinical importance 64 4.2Conclusion 67 4.3 Future directions 69 References 74 vi Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES 1.1 TNM classification 10 1.2 Gleason grading system 11 1.3 Gleason score 12 2.1 PCR reagents, volume and concentration 2.2 Dye assignments for individual ddNTPs 2.3 Reagents for SNaPshot multiplex reaction 40 43 44 2.4 Thermal cycling for the SNaPshot reaction 45 3.1 Distribution of the A3146T, A3174G, A3681C, and G3877A genotypes and OR for the association between the genotypes and prostate cancer 54 3.2 OR for the association between the A3174G genotype and prostate cancer risk by racial/ethnic groups 55 3.3 OR for the association between the G3877A genotype and prostate cancer risk by racial/ethnic groups 56 3.4 Hardy Weinberg equilibrium 57 3.5 Frequencies of SNPs amongst different ethnic groups 57 Vll Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF FIGURES 1.1 Anatomy of the pelvis and prostate gland 1 1.2 Prostate cancer mortality rates 3 1.3 Incidence rates of prostate cancer by race, US, 1990-94 5 1.4 Simplified pathological picture of the Gleason grades for prostate cancer 12 1.5 Chemical structure of testosterone 15 1.6 Role of the SRD5A2 gene 16 1.7 Chemical structure of dihydrotestosterone 17 1.8 Androgen metabolic pathway 18 1.9 DHT-AR complex formation and gene transcription 19 1.10 The SRD5A2 gene 23 1.11 Effect of SNPs in the 3’UTR of a gene and exonucleolytic pathway 31 1.12 Effect of SNPs in the 3’UTR of a gene and endonucleolytic pathway 32 2.1 PCR primers annealing sites on 3’UTR sequence of SRD5A2 39 2.2 Diagram of the four SNaPshot multiplex primers and primer extension products 44 2.3 Orientation of primers before optimization 50 2.3 Electropherogram showing genotyping result with primers of different orientation 50 3.1 PCR products 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3.2 Homozygous wild type 52 3.3 Heterozygous mutant for A3146T 52 3.4 Heterozygous mutant for A3174G and G3877A 52 3.5 Heterozygous mutant for A3681C 53 3.6 Homozygous mutants for A3174G and G3877A 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT Prostate cancer is known to be androgen-dependent. Enzyme steroid 5a-reductase type II (encoded by the SRD5A2 gene) catalyzes the irreversible reduction of testosterone to dihydrotestosterone (DHT) in the prostate. 3’untranslated region (3’UTR) might possibly affect mRNA stability and hence might have a role in prostate carcinogenesis. Racial differences in their susceptibilities towards prostate cancer have been observed since long. Four SNPs (A3146T, A3174G, A3681C, and G3877A) in the 3’UTR of the SRD5A2 gene have been hypothesized to be probably associated with prostate cancer risk and a there is a racial variation in the association between these SNPs and prostate cancer. Genotyping of 848 genomic DNA samples (472 incident cases and 376 cohort controls) was done using SNaPshot multiplex technique. A non-significant association between A3681C and prostate cancer was found (OR=3.05, 95% Cl = 0.36-38.88) and a relatively higher association was found between A3174G and G3877A and prostate cancer among Whites (OR= 1.7 and 1.67, 95% Cl = 0.81-3.58 and 0.87-3.44 respectively). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1 INTRODUCTION 1.1 Prostate cancer 1.1.1 The prostate gland The prostate is a sex gland in men. It is an ovoid structure, about the size of a walnut, lies inferior to the urinary bladder and surrounds the urethra (www.umm.edu/prostate/panat.htm) (figure 1.1). It is partly muscular and partly glandular, with ducts opening into the prostatic portion of the urethra (www.umm.edu/prostate/panat.htm). It is made up of three lobes: one central and two lateral ones, and secretes a slightly alkaline fluid that forms part of the seminal fluid that carries sperm (www.umm.edu/prostate/panat.htm). S 5?' • ' It; flipSM P8t»l*§ „t~ 'J.' rectum it & l-V Wimzmmm Figure 1.1 Anatomy of the pelvis and prostate Gland (Reproduced with permission from Greystone.net, Atlanta, GA (http://www. umm.edu/prostate/panat)) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.2 Prostate cancer epidemiology Prostate cancer is the ninth most common cancer globally, with the highest rates occurring in North America, Europe, and Australia and lowest rates reported in Japan, India, Hong Kong, and China (World Cancer Research 1997). It is the most common noncutaneous cancer and the second leading cause of cancer-related deaths (first being lung cancer) among men in the United States (www.cancer.org 1998). In the US itself, nearly 220,900 new cases (-33% ) of prostate cancer and approximately 28,900 (-10% ) cancer-related deaths are estimated by the end of the year 2003 (www.cancer.org 2003). The incidence and mortality rates for prostate cancer for 2003 are estimated to be 168.9 per 100,000 and 33.9 per 100,000 respectively (www.cancer.org 2003). The incidence rates increased highly during the period 1988 to 1992, probably due to the prostate-specific antigen (PSA) blood test screenings, declined steeply between 1992 and 1995 and came to stagnant level between 1995 and 1998 (Howe, Wingo et al. 2001; Ries 2001). The mortality rates in the United States have been steadier with a little increase during the 1973 and 1991 period and a decrease between 1991 and 1995 term (Stanford 1999), as shown in figure 1.2. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. m Lung & Bronchus I 1 Stomach I $ I n a m m « s e Figure 1.2 Prostate cancer mortality rates (Modified from Cancer Facts and Figures, American Cancer Society (www.cancer.org 2003). The death rates are age-adjusted and depict the period from 1930- 1999, in the US) 1.1.3 Risk Factors There are multiple risk factors of prostate cancer. These are age (Stanford 1999), race (seer.cancer.gov 1973-1988; www.cancer.org 1998), family history (Cannon 1982), sex hormones (Wilding 1995), smoking (Hayes, Pottern et al. 1994), body size (Nilsen and Vatten 1999), physical 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. activity (Cerhan, Tomer et al. 1997), occupation (Sharma-Wagner, Chokkalingam et al. 2000), diet (Giovannucci, Rimm et al. 1993), and vasectomy (John, Whittemore et al. 1995). 1.1.3.1 Age Age has been one of the most important and substantial risk factor for prostate cancer (Ries 2001). The incidence as well as the mortality rates of prostate cancer increase exponentially with age (Stanford 1999). Prostate cancer is rare below the age of 30, but the risk increases gradually thereafter such that at the age of 90, the chances of suffering from prostate cancer becomes 100% (Peehl 1999). 1.1.3.2 Race At any age, African-American men are at higher risk (nearly two times) than white men for developing prostate cancer (seer.cancer.gov 1973-1988; www.cancer.org 1998). The incidence rate has remained higher for African-American than Whites as shown in figure 1.3 (www.cancer.org 1998). The risk is low in Japanese-Americans and Chinese-Americans but they are at higher risk than native Japanese and Chinese (reviewed by (Carroll 2002)). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 ® ~ 4 4 m-m $@-54 ss -st m-m ts-m ?® ~« t s - t * » - « » + Figure 1,3 Incidence rates of prostate cancer by race, US, 1990-94 (Modified from Cancer Facts and Figures, American Cancer Society (www.cancer.org 1998)) 1.1.3.3 Family History/Genetics The risk of prostate cancer, specifically early-onset disease, is strongly affected by family history (reviewed by (Bratt 2002)). The risk is two to three times higher for men with a first-degree relative (father, son or brother) with prostate cancer and the risk increases with increasing number of relatives with a positive history (Carter, Bova et al. 1993; Whittemore, Wu et al. 1995). This familial clustering reflects genetic mechanisms. Twin studies, complex segregation analysis as well as linkage studies have been done to understand the underlying genetic mechanism (Gronberg, Damber et al. 1997; Lange, Chen et al. 1999; Lichtenstein, Holm et al. 2000). Seven 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. commonly susceptibility loci have been found while doing genome wide linkage analysis such as HPC1 (Smith, Freije et al. 1996), PCaP (Berthon, Valeri et al. 1998), HPCX (Xu, Meyers et al. 1998; Lange, Chen et al. 1999), CAPB (Gibbs, Stanford et al. 1999), HPC20 (Berry, Schroeder et al. 2000), HPC2 (Tavtigian, Simard et al. 2001), and HSD3B (Chang, Zheng et al. 2002). These multiple distinct loci that have been found, and the high likelihood that additional loci will be identified, underlines the vast and potentially complex genetic heterogeneity that characterizes hereditary prostate cancer (Isaacs 2001). 1.1.3.4 Hormones The prostate is an androgen-regulated gland; the growth and maintenance of normal prostate epithelium depends on androgens (reviewed by (Wilding 1995)). Studies have shown that sex hormones have initiating as well as promoting effects on prostate cancer development (reviewed by (Wilding 1995)). It can regress when androgen stimulation is removed (reviewed by (Sternberg 2003)). Prostate cancer is rare among men who have been castrated at an early age (reviewed by (Carroll 2002)) and the development of prostate cancer increases after administration of high levels of testosterone in rats (Noble 1977). Majority of the androgens are formed in the testes and the adrenal gland, while some amount is also formed from peripheral tissues such as prostate and skin (reviewed by 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Hsing, Reichardt et al. 2002)). In males the two most important androgens are testosterone (T) and dihydrotestosterone (DHT) (reviewed by (Hsing, Reichardt et al. 2002)). Apart from androgens, Insulin-Like Growth Factor-1 (IGF-1) levels have also been proved to be a risk factor for prostate cancer (Mantzoros, Tzonou et al. 1997; Chan, Stampfer et al. 1998; Wolk, Mantzoros et al. 1998; Stattin, Bylund et al. 2000). 1.1.3.5 Smoking Few studies have shown a positive correlation between smoking and prostate cancer (Hayes, Pottern et al. 1994; Vander Gulden, Verbeek et al. 1994), while no association have been shown by others (Lumey, Pittman et al. 1997; Furuya, Akimoto et al. 1998; Hsieh, Thanos et al. 1999). While some researchers have shown relation between smoking and prostate cancer mortality (Coughlin, Neaton et al. 1996; Yu, Ostroff et al. 1997; Giovannucci, Rimm et al. 1999), some others have found a relation between smoking and tumor grade (Hussain, Aziz et al. 1992; Daniell 1995). 1.1.3.6 Body size and physical activity There have been studies done to find relation between body size and prostate cancer. According to some of the researchers, taller men have 7 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. moderately higher risk of prostate cancer (Giovannucci, Rimm et al. 1997; Nilsen and Vatten 1999). There is an association between obesity and higher estrogen levels and lower T levels, a hormonal situation that might be protective for prostate cancer (Pasquali, Casimirri et al. 1991; Wilding 1995; Gann, Hennekens et al. 1996). Body Mass Index (BMI) has been positively correlated with prostate cancer in some studies (Gronberg, Damber et al. 1996; Cerhan, Tomer et al. 1997), while no relation has been found in few others (Giovannucci, Rimm et al. 1997; Hsing, Deng et al. 2000). Physical activity also has conflicting results, but majority of studies have shown a beneficial effect (Cerhan, Torner et al. 1997; Clarke and Whittemore 2000; Lund Nilsen, Johnsen et al. 2000). 1.1.3.7 Occupation Positive correlations between several occupational exposures and risk of prostate cancer have been reported. Some of these occupational carcinogens are diesel exhaust (Seidler, Heiskel et al. 1998), metallic dust (Aronson, Siemiatycki et al. 1996; Weston, Aronson et al. 2000), calcium carbonate (Weston, Aronson et al. 2000), pesticides (Sharma-Wagner, Chokkalingam et al. 2000), leather (Sharma-Wagner, Chokkalingam et al. 2000), polycyclic aromatic hydrocarbons (Krstev, Baris et al. 1998), fire fighting (Krstev, Baris et al. 1998), electrical industry (Krstev, Baris et al. 1998) and others. 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.3.8 Diet Epidemiological studies have shown a relationship between diet and prostate cancer. Excess intake of animal fat, meat (specially red), saturated fats, dairy products, calcium, a-linolenic acid elevate the risk of prostate cancer (Giovannucci, Rimm et al. 1993; Whittemore, Kolonel etal. 1995; Chan, Stampfer et al. 2001), whereas vegetables (specially tomatoes), lycopene, vitamin E, selenium have beneficial effect on the development of prostate cancer (Giovannucci, Ascherio et al. 1995; Hayes, Ziegler et al. 1999; Tzonou, Signorello et al. 1999; Nomura, Lee et al. 2000). 1.1.3.9 Vasectomy Vasectomized men have higher levels of circulating testosterone and DHT and a higher ratio of DHT: T than non-vasectomized men (John, Whittemore et al. 1995). This could be a possible reason for a positive association between vasectomy and moderate risk of prostate cancer (John, Whittemore et al. 1995). However some studies have reported contradictory results (Lesko, Louik et al. 1999). 1.1.4 Pathology The gross (TNM classification) as well as microscopic (Gleason grading) pictures of a cancer are necessary to decide the treatment and judge the prognosis (Carroll 2002). 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.4.1 TNM classification One of the common gross pathological staging systems is the TNM (tumor-nodes-metastasis) classification system (Carroll 2002). Table 1.1 shows the TNM staging system for prostate cancer. able 1.1 TNM classification Tx Primary tumor cannot be assessed TO No evidence of Primary tumor T1 Tumor is an incidental histologic finding (i.e. not palpated or imaged) T1 a 5% or less of specimen involved by tumor T 1 b More than 5% of specimen involved by tumor T 1 c Tumor found in needle biopsy (i.e. after elevated serum PSA) T2 Tumor present clinically or grossly but limited to gland T2a Tumor involves one lobe only T2b Tumor in more than one lobe T3 Tumor invades beyond the prostate capsule but is not fixed T3a Extracapsular extension (unilateral or bilateral) T3b Tumor invades seminal vesicles T4 Tumor fixed to or invades adjacent structures other than seminal vesicles (i.e. bladder neck, external sphincter, rectum, levator muscles, and/or pelvic wall). Invasion into the prostatic apex or into (but not beyond) the prostatic capsule is not classified as T3 but as T2. Nx Regional lymph nodes cannot be assessed NO No regional lymph node metastases N1 Metastasis in a single or multiple lymph node(s) Mx Distant metastasis cannot be assessed MO No distant metastasis M1 Distant metastasis M1a Nonregional lymph node(s) M1b Bone metastasis M1 c Other site(s). If more than one site of metastasis is present, the most advanced category is used. M1c is most advanced. (Modified from ‘ Prostate Cancer’ by Carroll and Grossfeld (Carroll 2002)) 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.4.2 Gleason grading The Gleason system (table 1.2) has been the most approved and widely used microscopic grading system for prostate cancer (Gleason, Mellinger et al. 2002). The system recognizes five growth patterns of invasive adenocarcinoma representing a progressive scale of decreasing glandular differentiation finally reaching undifferentiated growth (Carroll 2002). A simplified pathological figure of the grading system is also shown (figure 1.4). Gleason scores are used to predict the prognosis and they are a sum of primary and secondary pattern (table 1.3). Table 1.2 Gleason grading system________________________ Pattern 1 A circumscribed nodule of uniform, single, separate, closely packed glands Pattern 2 Fairly circumscribed nodule with minimal extension of tumor glands into benign prostate tissues. Glands are single and separated by stroma and more loosely arranged than in pattern 1 Pattern 3 Tumor glands infiltrate in and among the benign prostate glands, with the glands having marked variation in size and shape. Many glands are smaller than those of patterns 1 and 2. Smoothly circumscribed cribriform nodules are consistent with pattern 3 Pattern 4 Tumor glands are fused with ragged infiltrating edges (i.e. glands are no longer separate as in patterns 1, 2, and 3) Pattern 5 Tumor shows no glandular differentiation with either solid masses of cells or individually infiltrating cells or tumor nests with central comedolike necrosis (Modified from ‘ Prostate Cancer’ by Carroll and Grossfeld (Carroll 2002)) 1 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 1.4 Simplified pathological picture of the Gleason grades for prostate cancer. (Numbers indicate the grades. Modified from (http://www.phoenix5. org/lnfolink/Gleason Grading, html)) ' 'able 1.3 Gleason score Gleason Score (Modified from ‘ Prosta 2-4(well differentiated) 5-7(moderately differentiated) 8-10(poorly differentiated) e cancer’ by Carroll and Grossfeld (Carroll 2002)) 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.1.5 Screening Prostate cancer being a widespread and very common cancer, it is important for early prostate cancer screening. The incidence of prostate cancer is rising since past few years due to increase in the screening process, whereas mortality has been reducing (Coldman 2003). Serum prostate specific antigen (PSA) is the key biomarker used to detect early prostate cancer (Carroll, Coley et al. 2001). PSA related tests are PSA density (PSAD), PSA velocity, age-specific PSA and free-to-total PSA (reviewed by (Carroll 2002)). However, there are various schools of thought regarding the use of screening (Ransohoff, McNaughton Collins et al. 2002). There are a lot of occult prostate cancer patients, and early detection will lead to early treatment which itself leads to post-operative complications; while various other surgeons weight screening much more so that chances of patient remaining undetected are reduced (Ransohoff, McNaughton Collins et al. 2002). Other screening methods are digital rectal examination (DRE) (American Urological Association 2000) and transrectal ultrasound (TRUS) (May, Treumann et al. 2001). 1.1.6 Treatment Various treatment modalities are present and are given based on the pathological and clinical staging. Early stage cancer is curable but not advanced (metastatic) cancers (reviewed by (Carroll 2002)). Traditional 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treatment methods include radical prostatectomy (retropubic, perineal, laproscopic), radiation therapy (external beam radiation therapy, intensity- modulated radiation therapy, particle beam radiotherapy), pharmacological treatment (e.g. finasteride), hormonal treatment (androgen ablation/depletion, orchiectomy, estrogen treatment, nonsteroidal antiandrogen monotherapy, steroidal antiandrogen), second-line hormone therapy, brachytherapy and others (reviewed by (Carroll 2002)). Novel methods of treatment are cryotherapy, thermal ablation, alcohol injection, PSA-dependent oncolytic adenovirus therapy, chemotherapy (mitoxantrone, estramustine), and some other newer modalities like use of matrix metalloproteinase inhibitors, growth factor inhibitors, antiangiogenesis, gene therapy, immunotherapy, dendritic cell vaccination therapy, vitamin D, biphosphonates, and others (reviewed by (Sternberg 2003)). 1.2 Androgens, the SRD5A2 gene, and prostate cancer 1.2.1 Androgens The prostate is an androgen-regulated gland; the growth and maintenance of prostate depends on the androgens (Coffey 1979). Androgens are steroid hormones responsible for the differentiation and maturation of the male reproductive organs and the development of male secondary sexual characters (reviewed by (Hsing, Reichardt et al. 2002)). Though mainly formed in the testes and adrenal gland, some percentage of 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. androgen is also formed from peripheral tissues such as prostate and skin (reviewed by (Hsing, Reichardt et al. 2002)). The two most important androgens in adult males are testosterone (the principal androgen in circulation) and dihydrotestosterone (DHT, the most potent and the primary nuclear androgen) (reviewed by (Hsing, Reichardt et al. 2002)). 1.2.1.1 Testosterone Testosterone is one of the most important androgens in an adult male (reviewed by (Hsing, Reichardt et al. 2002)). The chemical structure of testosterone is as shown in figure 1.5. It is a 19-carbon steroid with a 4,5 double bond. OH Figure 1.5 Chemical structure of testosterone (Reproduced from (www.people.vcu.edu/~urdesai/adg.htm)) In blood, only 1 -2% of testosterone is found in a free (unbound) state; nearly 44% of testosterone is bound with high affinity to sex hormone- binding globulin (SHBG) and about 54% is bound with low affinity to albumin (reviewed by (Hsing, Reichardt et al. 2002)). The free, unbound 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. testosterone in circulation enters the prostate cells by passive diffusion, whereas the albumin-bound testosterone, because of its low affinity, can dissociate from albumin and can also enter the prostatic cells (reviewed by (Hsing, Reichardt et al. 2002)). Some data also suggest that SHBG bound testosterone also may enter the prostatic cells (Hryb, Khan et al. 1990). In the prostate, the T converts irreversibly to DHT by the enzyme steroid 5a- reductase with NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) as the cofactor (Cheng 1993) (figure 1.6). Figure 1.6 Role of the SRD5A2 gene (Modified from (www.people.vcu.edu/~urdesai/adg.htm)) 1.2.1.2 Dihydrotestosterone DHT is the most potent and primary nuclear androgen in adult males (reviewed by (Hsing, Reichardt et al. 2002)). The chemical structure of DHT is shown in figure 1.7. NADPH TMcwterarw Dtiptrcieilostsrone 16 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. OH. Figure 1.7 Chemical structure of dihydrotestosteronefMocf/f/ed from (www.people, vcu.edu/~urdesai/adg. him)) Approximately 65-75% of DHT is formed from the irreversible reduction of testosterone, in the prostate, by the enzyme steroid 5a- reductase with NADPH (nicotinamide adenine dinucleotide phosphate, reduced form) as the cofactor (Cheng 1993); or formed from circulating inactive androgens, such as androstenedione, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS). The testes secrete the rest 25% of the DHT in the circulation (reviewed by (Hsing, Reichardt et al. 2002)). Figure 1.8 shows the androgen metabolic pathway within the prostate. DHT can also be formed from androstenedione by a two-step reduction reaction, catalyzed by the steroid 5a-reductase and 17p- hydroxysteroid dehydrogenase (encoded by the HSD17B gene) (reviewed by (Hsing, Reichardt et al. 2002)). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. M tK n n * m a a S S D 5 A 2 T esttstw ii 5 lt H |lt lia * w^tl) n i m mMA M i r JM aireim M ^ jjiu c t t r o iid c f S a - iM r i G , i Jp-MwtawIM to K krm ^ 3 p *M » S h l» iM 3 s - A s d r J W t s a w l W i a d f t t e g lttw w M c H S D i/B S * lfl la . ) . ' t . . ~ y ? p ^ d r ® ^ £ g ? is mm I W f 4 |i» ijS s r « M | fe ty fta g M w ionkKy A riro5iif»8iii*«r«sliit SM/,1 k - r d a r t s s * i y - p i 2 ■ * M m iirfiiM A rt« ite « m lte * m m Ufrhydicsystere'i d e U y d r ^ j a j . ^ ! m m Figure 1.8 Androgen metabolic pathway (Reproduced with permission from Wiley-Liss, Inc. (Hsing, Reichardt et al. 2002)) DHT further undergoes a reversible reduction reaction either to form 5a-androstane-3a, 17p-diol (3a-dioI) via the enzyme 3oc-hydroxysteroid dehydrogenase, or 5oc-androstane-3(3, 17(3-diol (3p-diol) via the enzyme 3p-hydroxysteroid dehydrogenase (reviewed by (Hsing, Reichardt et al. 2002)). 3a- and 3P- diol irreversibly conjugate to produce 3a-androstanediol glucuronide (3a-diol G) and 3P-androstanedio! glucuronide (3P-diol G) respectively in the presence of glucuronyl transferase; and 3a- androstanediol sulfate and 3p-androstanediol sulfate respectively via the enzyme sulfuryl transferase (reviewed by (Hsing, Reichardt et al. 2002)). 1 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The-product 3a- and 3(3- diol G are two of the final metabolites of DHT. This inactivation of DHT through the reduction reactions, determines the intracellular DHT concentration (reviewed by (Hsing, Reichardt et al. 2002)). Within the prostate, the DHT binds to a protein receptor called androgen receptor (AR) and forms an intracellular DHT-AR complex; which further translocates to the cell nucleus and binds to the androgen response element (ARE), leading to transactivation of many genes resulting in cellular proliferation in the prostate as in figure 1.9 (reviewed by (Hsing, Reichardt et al. 2002)). Hence DHT is a potential modulator of androgenic activity in the prostate. ■* O T o s t o s t - e i'o r i© | * < 3 ------ ► •O' °h t At < !rr v.pn-n»spcrfcsuv8 e s a lt “r K *n ° | ; arJ> Qirnerteatidn and ^ - ‘ . H-SP ' AR \ AR Figure 1.9 DHT-AR complex formation and gene transcription (Reprinted by permission from Nature Reviews Cancer (Feldman and Feldman 2001) copyright (2001) Macmillan Magazines Ltd.) 1 9 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.1.3 Androgens and prostate cancer Steroid hormones play a critical role in controlling cell proliferation in their primary target organs (reviewed by (Ross, Coetzee et al. 1999)); this increased cell proliferation is important in the pathogenesis of most human cancers (Preston-Martin, Pike et al. 1993). Prostate cancer is androgen- dependent and androgen ablation has been the mainstay of early prostate cancer therapy (reviewed by (Ross 1996)). It has also been shown that men with highly underdeveloped prostates, such as eunuchs or men with constitutional steroid 5a-reductase deficiency, never develop prostate cancer (reviewed by (Ross 1996)). A study has shown a strong trend of increasing prostate cancer risk with increasing levels of circulating free testosterone (Gann, Hennekens et al. 1996). The amount of DHT also influences the risk of prostate cancer (Ross, Bernstein et al. 1992). The steady state levels of DHT in the prostate are determined by its biosynthesis from testosterone (catalyzed by the enzyme steroid 5a- reductase), and by its degradation to its final metabolites (catalyzed by enzymes 3a- and 3p- hydroxysteroid dehydrogenases) (reviewed by (Reichardt 1999)). Therefore, variation in the DHT levels may play a significant role in the risk of prostate cancer. Also, the ratio of DHT:T is nearly 1:10 in the serum whereas DHT concentration is much higher than Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. testosterone in the prostate (reviewed by (Hsing, Reichardt et al. 2002)). This underlines the importance of DHT in the development of prostate cancer. 1.2.1.4 Androgens and race The androgen profile for various race/ethnicity varies, which explains the difference in the prostate cancer epidemiology amongst various ethnic groups of people (reviewed by (Ross, Coetzee et al. 1999)). It has been reported that testosterone levels are higher in African-American than White men (Ross, Bernstein et al. 1986). Native Japanese have not been shown to have significant difference of testosterone levels from African-American or Whites; but they have been shown to have 25-35% lower levels of 3a- androstanediol glucuronide (an index of in vivo steroid 5a-reductase) than African-American and White males (Ross, Bernstein et al. 1992). The native Chinese were also shown to have similar androgen profile as native Japanese (Lookingbill, Demers et al. 1991). Another study shows the androgen dihydroepiandrosterone sulfate (DHEAS) levels to be almost double amongst African-Americans than Caucasians (Girgis, Abrams et al. 2000). These differences in hormonal profile could explain the difference in prostate cancer incidence among various ethnic groups. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2 The SRD5A2 gene The reduction reaction of T to DHT is carried out by the enzyme steroid 5a-reductase in the presence of NADPH as the cofactor as shown in figure 1.6 (Cheng 1993). Hence, the enzyme steroid 5a-reductase plays an important role in regulating the amount of DHT produced and hence the DHT-AR complex and ultimately the transcriptional activity of the prostate DNA (reviewed by (Hsing, Chen et al. 2001)). This significant role of steroid 5a-reductase makes it necessary to learn in detail during the study of prostate cancer. 1.2.2.1 Isozymes of steroid 5a-reductase There are two different isozymes of steroid 5a-reductase enzyme: type I and type II, respectively encoded by SRD5A1 and SRD5A2 genes (Thigpen, Silver et al. 1993). There is 50% homology and 45% identity between the amino acid sequences (Andersson, Berman et al. 1991; Ross, Coetzee et al. 1999). Type I enzyme, with an alkaline pH, is expressed primarily in the newborn scalp, skin and liver, while type II enzyme, with an acidic pH, is expressed primarily in genital skin, liver and the prostate (Thigpen, Silver et al. 1993). This suggests that type I enzyme is primarily responsible for virilization and male pattern baldness, and type II enzyme is mainly involved in prostate development and growth (Reichardt, Makridakis 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. et al. 1995). It is the type II isozyme that catalyses the conversion of T to DHT in the prostate (Cheng 1993). 1.2.2.2 Gene structure of SRD5A2 The SRD5A2 gene encoding the steroid 5a-reductase type II enzyme is located on the short arm of human chromosome 2 (band 2p23) spanning over 56 kb of genomic DMA (http://snpper.chip.org/bio/find-gene 2001). It consists of five exons and four introns, is highly expressed in the prostate, and encodes a hydrophobic protein of 254 amino acids (Andersson, Berman et al. 1991; Thigpen, Davis et al. 1992). Figure 1.10 shows the schematic diagram of the gene structure of the SRD5A2 gene. 350 1 E 3 1 0 1 150 1576 46.B -if— 3.0 II III IV V Figure 1.10 The SRD5A2 gene (The schematic diagram shows the five exons, numbered I to V, shown as boxes and the introns which are straight lines between the exons. Sizes are reproduced from http://snpper. chip, org (http://snpper.chip.org/bio/find-gene 2001). Exon size is in bp and Intron size is in kb) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2.3 The SRD5A2 gene, polymorphic markers, and prostate cancer The identification of genetic variants in genes that control androgen biosynthesis or metabolism (Ross, Pike et al. 1998), which can also affect the risk of prostate cancer, has important implications for understanding of the biology of prostate cancer, for identification of at-risk men, before symptoms arise and for development of chemopreventive stratergies (Makridakis, Ross et al. 1999). 1.2.2.3.1 Pseudohermaphroditism Inherited defects in the SRD5A2 gene cause a rare autosomal recessive disorder, male pseudohermaphroditism, due to steroid 5a-reductase type 2 deficiency (Andersson, Berman et al. 1991). The males suffering from this disorder are 46,X Y, have feminine phenotypical characteristics at birth, but develop masculine secondary sexual characters at puberty (Thigpen, Silver et al. 1993). However, the prostate remains underdeveloped and DHT levels are low inspite of a rise in testosterone at puberty (Thigpen, Silver et al. 1993). The disease is now known as steroid 5a-reductase type 2 deficiency (reviewed by (Wilson, Griffin et al. 1993)). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2.3.2 Mutations in the SRD5A2 gene and prostate cancer risk Genetic polymorphism that may be associated with prostate cancer risk are much more common in the population than hereditary prostate cancer susceptibility genes (reviewed by (Coughlin and Hall 2002)). It has been suggested that the activity of the steroid 5a-reductase type 2 (encoded by the SRD5A2 gene) may be associated with prostate cancer risk (Hsing, Chen et al. 2001). More than 22 mutations have been reported for the SRD5A2 gene (Wigley, Prihoda et al. 1994). Mutations in the SRD5A2 gene may either reduce the enzyme activity or increase it (Makridakis, di Salle et al. 2000). Increased enzymatic (steroid 5a- reductase type 2) activity may increase DHT levels, while decreased activity decreases the DHT levels (reviewed by (Coughlin and Hall 2002)). Suppression of DHT may reduce the carcinogenic transformation of prostate cells; and elevated levels of testosterone and intraprostatic DHT may account for the increased risk of prostate cancer (reviewed by (Coughlin and Hall 2002)). Four of the 22 known mutations- A49T (alanine replaced by threonine at codon 49), V89L (valine replaced by leucine at codon 89), R227Q (arginine replaced by glutamine at codon 227), and a (TA)n dinucleotide repeat, have been studied for their association with prostate cancer in several epidemiologic studies most of which have produced negative results (reviewed by (Hsing, Reichardt et al. 2002)). 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Out of the many studies done, two studies reported a positive correlation between A49T polymorphic marker and risk of prostate cancer (Makridakis, Ross et al. 1999; Jaffe, Malkowicz et al. 2000). The frequency of the A49T T allele among healthy subjects in most populations is low (Hsing, Chen et al. 2001). However, the significance of the T allele in prostate cancer is high because of various reasons: (a) a previous study found that almost 10% of prostate cancer cases had the T allele (Ross, Pike et al. 1998); (b) the T allele was linked to progression and severity of prostate tumors (Jaffe, Malkowicz et al. 2000); and (c) A49T substitution increased the in vitro steroid 5a-reductase activity the most (~5 times) than all the other known mutations in the SRD5A2 gene (Makridakis, di Salle et al. 2000). Because other studies have shown no association between A49T substitution and prostate cancer (Mononen, Ikonen et al. 2001), more work needs to be done to prove its significance in prostate cancer. Most studies reported no association between the V89L mutation and prostate cancer risk (reviewed by (Hsing, Reichardt et al. 2002)). V89L is the most common mutation on the SRD5A2 gene (reviewed by (Hsing, Chen et al. 2001)). Compared to the W genotype (homozygous wild type), the LL V89L genotype (homozygous polymorphism) causes a 42% reduction in steroid 5ot-reductase activity in vitro (Makridakis, di Salle et al. 2000). Serum testosterone levels are higher among men with the LL genotype (compared with men with the VV genotype) (Hsing, Chen et al. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2001). This suggests the lower steroid 5a-reductase activity, because reduced steroid 5a-reductase activity in the prostate, results in less conversion of T to DHT, and hence the T gets accumulated in the prostate, increasing the leaking of T back into circulation increasing the serum T levels (Hsing, Chen et al. 2001). The R227Q mutation is related to male pseudohermaphroditism, has been found only in Asians (reviewed by (Hsing, Reichardt et al. 2002)). The R227Q Q allele reduces the steroid 5a-reductase activity significantly (Makridakis, di Salle et al. 2000). But a significant association between R227Q and prostate cancer risk has not yet been found (Hsing, Chen et al. 2001). (TA)n dinucleotide repeats have been found in the 3’UTR of the SRD5A2 gene (Davis and Russell 1993). Three major (TA)n alleles have been reported, viz., (TA)0, (TA)9 and (TA)iS , with (TA)0 being the most common in most populations (Hsing, Chen et al. 2001). Men carrying copies of the longer (TA)n allele had a nonsignificatly lower risk of prostate cancer (Kantoff, Febbo et al. 1997). Serum levels of DHT are found to be higher among men with the (TA)o/(TA)g genotype, though the biological significance is not yet clear (Hsing, Chen et al. 2001). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 1.2.2.4 3’ UTR of the SRD5A2 gene 1.2.2.4.1 3’UTR and mRNA stability Changes in the transcription, mRNA splicing, mRNA stability, translation, and post-translation, all can have effect in the expression of a gene (reviewed by (Day and Tuite 1998)). Both the level of the encoded mRNA as well as its ability to efficiently translate its intended protein product play a role in the regulation of normal gene expression (Bilenoglu, Basak et al. 2002). Hence post-transcriptional events occurring in the nucleus as well as in the cytoplasm are important (Bilenoglu, Basak et al. 2002). The primary sequence of a nascent mRNA transcript contains all of the information necessary for its appropriate nuclear processing and efficient nuclear-to-cytoplasmic transport (Bilenoglu, Basak et al. 2002). This signifies the importance of the mRNA sequence. Hence mRNA stability is very important from the point of view of a normal protein production. The regulation of mRNA stability is one component of posttranscriptional control mechanism (Zaidi and Malter 1994). Various factors/elements involved in regulation mRNA stability are: the 7 mGpppN cap structure, 5’UTR sequences, premature termination codons, open reading frame sequences, 3’UTR sequences, AU-rich elements, and the poly(A) tail (Staton, Thomson et al. 2000). On the whole, the determinants of mRNA stability are its rate of synthesis as well as decay rate (reviewed by (Day and Tuite 1998)). Increased mRNA decay prevents sufficient cytoplasmic mRNA 28 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. accumulation preventing significant protein synthesis (Zaidi and Malter 1994). Whereas stabilized mRNAs accumulate to high levels increasing the protein synthesis (Zaidi and Malter 1994). mRNA degradation can be prevented by binding of RNA-binding proteins and cap-binding proteins, protecting the mRNA from exo and endo nucleases (Staton, Thomson et al. 2000). Since past several years, importance of 3’UTR has increased tremendously. This is because 3’UTR may have a significant role in the stability of mRNA and hence regulation of gene expression and ultimately the amount of protein (steroid 5a-reductase enzyme) produced (reviewed by (Day and Tuite 1998)). The poly(A) tail protects the RNA chain from degradation by 3’ to 5’ exonucleases, resulting in enhancement of translation, and a number of motif sequences or cis-acting elements that control the translation, degradation and localization of transcripts or regulate mRNA stability and/or translational efficiency (reviewed by (Day and Tuite 1998; Wilson and Brewer 1999). Protein factors acting in trans (cleavage/polyadenylation specificity factor, poly(A) polymerase, etc.) to modulate mRNA stability are being characterized (Zaidi and Malter 1994; Staton, Thomson et al. 2000). Binding of these regulatory proteins to 3’UTR mRNA sequences either play a role in stabilizing the transcripts (Zaidi and Malter 1994) or hastening their degradation by triggering deadenylation (Shyu, Belasco et al. 1991) or serving as an anchor for 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. endonuclease cleavage (Binder, Horowitz et al. 1994). However, mutations in the 3’UTR of a gene, can interfere with the interaction of specific trans acting factors with the cis elements either directly or indirectly and hence can affect the mRNA stability and hence the expression of the gene (Bilenoglu, Basak et al. 2002). It has also been suggested that the 3’UTR may have signals specifying the nuclear-cytoplasmic transport apart from just maintaining cytoplasmic stability (Bilenoglu, Basak et al. 2002). This is explained in figure 1.11 where the SNPs in the AU rich elements (ARE) in the 3’UTR can either lead to binding of a stabilizing factor (e.g. HuR in this case) leading to stability of the mRNA (possibility 2) and hence increased protein production; or a destabilizing factor (e.g. AUF1) (possibility 1) leading to detachment of the poly (A) binding protein (PABP), hence exposing the poly (A) tail, and hence chewing up of the poly (A) tail by the exonucleases. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. MMi(MAUAMMMW S w io a -e l MmMMW W M W M W A *A M A A A A A M A A A M M * possfeity 1 pas sMt y 2 Figure 1.11 Effect of SNPs in the 3’UTR of a gene and exonucleolytic pathway (Modified from (Wilusz, Wormington et al. 2001)) In the other case, as shown in figure 1.12, the pathway shows how endonucleolytic decay can take place or the mRNA is protected from the endonuclease attack. As shown, there is a endonucleolytic cleavage recognition site and there is a stabilizing factor binding site. If there is a SNR in the endonucleolytic cleavage recognition site, then it can either lead to binding of the stabilizing protein (possibility 1) and hence protecting the cleavage site from the endonuclease, leading to increased stability of the mRNA and hence increase in the protein production; or it can lead to a 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. situation where the protein cannot bind to the mRNA and hence exposing the endonucleolytic recognition site (possibility 2), leading to internal cleavage by the endonuclease and generating fragments that are easily accessible to exonucleases, leading to rapid degradation. Praf&n-&fK8ng s s t& SNP ' a * « dy ___ RscogrfSen tv tefaifear E i'w o rw c fs a s s ' «CQ§fiiKrs site »< < S ta b le m R N A AMAW ^AAAAAAAA Rboossnltksti Ov #f¥jo n tcteassi AAAAAAAWW^MA posa**yt p ossto«y 2 II— a a A A A M A M M M A fc'w jnu cfe oM ie < te csy Figure 1.12 Effect of SNPs in the 3’UTR of a gene and endonucleolytic pathway (Modified from (Wilusz, Wormington et al. 2001)) 1.2.2.4.2 3’UTR and (TA)„ repeat (TA)n dinucleotide repeats, (TA)o, (TA)g & (TA)i8 have been found in the 3’UTR of the SRD5A2 gene (Davis and Russell 1993). The (TA)0 allele family is the most common and the (TA)is allele family is found exclusively in African-American men (Reichardt, Makridakis et al. 1995). Although no 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. significance has been found between (TA)n dinucleotide repeats and prostate cancer, it has been proved that certain steroid 5a-reductase enzyme variants encoded by the SRD5A2 genes marked by particular (TA)n repeat alleles may result in increase in enzyme activity, and hence elevating prostatic level of DHT (Reichardt, Makridakis et al. 1995). A nonsignificant reduction in risk of prostate cancer has been observed in men carrying copies of the longer (TA)n allele (Kantoff, Febbo et al. 1997). However, (TA)n in the 3’ untranslated region of the SRD5A2 gene has been associated with other cancer (Bharaj, Scorilas et al. 2000). Because the (TA)n marker is situated in the 3’UTR of the SRD5A2 gene, its functional consequences are thought to be due to the instability of mRNA transcripts, which in turn may affect the steroid 5a-reductase activity levels (Hsing, Chen et al. 2001). Again, an association was found between the (TA)n marker and serum androgen levels, supporting a role of (TA)n marker and prostate cancer risk (Hsing, Chen et al. 2001). 1.2.2.4.3 Single nucleotide polymorphisms (SNPs) and the SRD5A2 gene Single-nucleotide polymorphism (SNR) is the most common form of polymorphism (Halushka, Fan et al. 1999). The human genome has nearly 1.8 million SNPs (http://snp.cshl.org/ 2002). Such SNPs on coding region may lead to amino acid substitution and may tend to alter the 33 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. structure of the encoded protein (Majewski 2003). Some of the substitutions have conservative effect, i.e. have minimal impact on the protein structure, while some others have severe changes in its properties (Majewski 2003). SNPs are very important in the genetic association studies and in linkage analysis (Goto, Yue et al. 2001). These single-nucleotide variants are also directly associated with familial/hereditary diseases (Goto, Yue et al. 2001). There are nearly 57 SNPs in the SRD5A2 gene (http://snpper.chip.org/bio/find-gene 2001). Five SNPs, T2584C (T replaced by nucleotide C), A3146T (nucleotide A replaced by T), A3174G (nucleotide A replaced by G), A3681C (nucleotide A replaced by C), and G3877A (nucleotide G replaced by A), have been identified in the 3’UTR of the SRD5A2 gene (identified by Nick Makridakis, personal communication, not in the database). 1.2.2.5 The SRD5A2 gene, ethnicity, and prostate cancer The SRD5A2 gene plays a role in the racial/ethnic variation in prostate cancer risk (Reichardt, Makridakis et al. 1995). Varying levels of testosterone and intraprostatic DHT could determine this variation in the racial/ethnic incidence of prostate cancer (Ross, Bernstein et al. 1992; Gann, Hennekens et al. 1996). The levels of testosterone and DHT are determined partly by the enzyme steroid 5a-reductase encoded by the SRD5A2 gene in the prostate. Any mutation in the SRD5A2 gene affecting 34 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the activity or amount of enzyme produced may affect the risk of developing prostate cancer. The A49T mutation has a significant association with risk of advanced prostate cancer in African-American and Hispanic males (Makridakis, Ross et al. 1999). An association between A49T and more aggressive disease among Caucasian patients has also been reported (Jaffe, Malkowicz et al. 2000). The valine allele of the V89L substitution is most common in high-risk African-American men while the leucine allele of the V89L substitution is most common in low-risk Asians (Makridakis, Ross et al. 1997). This reflects the high and low steroid 5a-reductase activity amongst the African-Americans and Asians respectively (Makridakis, Ross et al. 1997). Longer TA alleles, (TA)1 8 , are exclusively found in African- American men whereas the (TA)o allele is the predominant allele among African-American, Asian-American as well as non-Hispanic Whites (Reichardt, Makridakis et al. 1995). These studies provide plausible molecular rationale for the difference in prostate cancer risk among various racial/ethnic groups. 1.2.2.6 Hypothesis Sequence variation in the 3’UTR of a gene can interfere with the interaction of specific trans-acting factors with the cis-elements on the 3’UTR, either directly or indirectly and hence can affect the mRNA stability and hence the expression of the gene (Bilenoglu, Basak et al. 2002). 35 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Based on the knowledge of how sequence variation can affect the mRNA stability, it was hypothesized that the SNPs in the 3’UTR of the SRD5A2 gene may lead to increased mRNA stability by binding a stability factor protecting it from either exo- or endonucleases and hence preventing degradation (figures 1.11 possibility 2 and 1.12, possibility 1), increasing the amount of the protein produced (enzyme steroid 5ot-reductase) and leading to increase in the DHT production, which ultimately leads to increased transcription of genes involved in cellular growth and leading to cancer formation (reviewed by (Day and Tuite 1998; Hsing, Reichardt et al. 2002)), hence I examined the hypothesis that SNPs in the 3’UTR of the SRD5A2 gene are associated with increased risk of prostate cancer. I also hypothesize that the SNPs in the 3’UTR of the SRD5A2 gene partly explain the variation in the susceptibility to prostate cancer among various ethnic groups. Hence my study included men from four different ethnic groups: African-American, Caucasian-American, Latino-American, and Japanese- American. Out of the five SNPs in the 3’UTR of the SRD5A2 gene, because the frequency of T2584C (T nucleotide replaced by C) was low (as known from Nick Makridakis), I screened four SNPs: A3146T (nucleotide A replaced by T), A3174G (nucleotide A replaced by G), A3681C (nucleotide A replaced by C), and G3877A (nucleotide G replaced by A) in the study population, to find out their frequency as well as to check for association between the SNPs and prostate cancer risk. 36 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2. MATERIALS AND METHODS 2.1 Study subjects and DNA samples A large multiethnic cohort (MEC) study was started in 1993, comprising of 215,251 men and women from Hawaii and Los Angeles (Kolonel, Henderson et al. 2000). A subcohort of this study was taken in my study, consisting of a total of 884 people to conduct a case-cohort study. However due to failure in amplification or insufficient DNA samples, few samples were not analyzed. Analysis was done on 848 samples. These were blood samples from 472 number of cases (incident prostate cancer patients) and 376 number of cohort controls. It had an ethnic distribution as follows: 203 African-American, 247 Latino, 181 Whites, and 217 Japanese- American. DNA was extracted from white blood cells of the patients’ blood. To avoid observational bias during screening genotypes, I was blinded by the disease status of samples during the screening of genotypes. 2.2 Overview of the experimental methods I genotyped four SNPs in the 3’untranslated region of the SRD5A2 gene. They are A3146T (nucleotide A replaced by T), A3174G (nucleotide A replaced by G), A3681C (nucleotide A replaced by C), and G3877A (nucleotide G replaced by A). The basic experimental pattern carried out was as shown in the following flowchart. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Amplify genomic DNA I Remove dNTPs and primers Obtain purified template SNaPshot reaction Perform Thermal Cycling 1 CIP reaction (Remove u To r a t e d d d N T P S ) Electrophorese samples on ^31 3100 Analyze the data with GeneScan 3.1 software Template Preparation Reaction Preparation Thermal Cycling & Post-extension treatment > GeneScan Analysis 2.2.1 PCR Polymerase chain reaction (PCR) was done to amplify the genomic DNA. 2.2.1.1 Primer design Primers were designed myself and custom ordered from Invitrogen (Carlsbad, CA) to amplify a part of the 3’UTR of the SRD5A2 gene that included the four SNPs, viz., A3146T, A3174G, A3681C and G3877A. They 38 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. amplified a region of 937 bp. The sequence of the forward as well as the reverse primers was as follows: 3’UTR/Forward (5’ to 3’): GGT GGC TTA TGA GAG AGT AGA G 3’UTR/Reverse (5’ to 3’): CAC CCC AAT ACC T IG TGA AAA TC The lengths of forward and reverse primers were 22 and 23 bp respectively, while the melting temperature for both was 66° C. These primers annealed to the region of the 3’UTR of the SRD5A2 gene as shown in figure 2.1, where the sequence of the template is shown too. c g g a a a a c tg c a a a a a g g c a c c c tg g a tg g a a a c t c t c a t g ta a a a c a a a g a t g t c c a c a t c c t g g a g c t c c ta g g g g c a a a a a tt g c a g c t c a g a c a t a t c a g g a g a t t t g g a g g a t c t a c g ta g c ta g g t a t g g c t a t t g a a c c t g gg c ta c t a a g a g g t t t t t g g a a g a g a a t t a a t a c a a t a t g a a t c t t c c t t g t g c c tg a a tg c a c a g a g g g g c t a g t t t t t t t c c t t c a t a a a g a a c a ta c a g t a a g c a t t t g t c t t g a a g g c c a g g t t t t a a ta a c c a c a a a t g g c t t a t g a g a g a g ta g a g a a c a a c a tg a t a a t t g g a a t t g c a t t g t c a c ta a g c a c a g g a g g a tg a tg t g t t t g a a t t g g g tg t g c t t t a g t g t c g t t a c a tg g a a a t a t a t t t a g c a a a a c t c a c t a t a g a t t a c t g j t t c a t t a a a a a a a t a t c c t c c c t g t c c c g t c c t t t c t t c a c tG g tg g c a a t a r c o a a a t a a tg a g t'a g t g a t g a g g t t a c a t g c t g c t t g c c tc c a c c a g ta c a g c a g a a g c c c c a a g c a a c t t t c c t t a g t t c t c a g g a c c t g t t c a a g a a g g t g t c t c t c c c t c a a a g g a c c tg c a g g c a g a g a c tg a c g tc tg g g c a g a a a a c c tg t t t t g t t t g g t t t t t t t a c a a a g t t t c a a a a a c t t a a a a a a c t c t a g c a t t c t a g t t t c a t t t a a a a a g t a g c c c a c a t t tc c a c a c c a g a a c tg g a a c t a g t t t g c a a a c t c t t g t g a a g g g g tc a c c c . ’ 1 . A gg aag gg g c c g a a c g c t tg a t t t g t c t t c t c a g g c c t a t g t t t g c g g a a a a c t g t t t t g a c a a a tg a g c a c c a g tg g t a t a t a a g t c a c a ta g g a a a c t t g a a a g g t c t a a a a a tA . > ■ ; - i ’ . - - ■ t a c c t t t a a a t c a c g g a t g t t a a g c a g t t x g g g a c t t c t g g a g a t t t t c a c a a g g t a t t g c c t g t c c c t c a t t c c c a t t c a c c c t g t c t g t c t t c t c t c t Figure 2.1 PCR primers annealing sites on 3’UTR sequence of SRD5A2 (Sequence reproduced from (http://www.nchi.nlm.nih.gov/). The primer annealing sequences for PCR are shown as bold and underlined. The SNP positions are shown in bold, underlined, capitals. The sequences in grey boxes are primers for SNaPshot for each SNP position.) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.1.2 Thermal cycling The PCR reagents were optimized to obtain required PCR product as in table 2.1 and table 2.2. The 10X PCR buffer, Taq DNA polymerase (recombinant), MgCI2 and dNTPs all were from Invitrogen. The PCR was conducted in a Robocycler® PCR machine (Stratagene, La Jolla, CA). Table 2.1 PCR reagents, volume and concentration Reagents Volume Final Concentration 1 0 X PCR Buffer 5 pi 1X 50 mM MgCI2 1.5 pi 1.5 mM 10 mM dNTP mixture 0.5 pi 0.2 mM each Forward primer 0.05 pi 0.1 pM Reverse primer 0.05 pi 0.1 pM Taq DNA polymerase (recombinant) 0.3 pi 1.5 U Genomic DNA 0 .5 -1 .5 pi - Total (add deionized water) 50 pi (Nick Makridakis, personal communication) Thermal cycling was carried as follows: After 3:00 minutes of initial denaturing at 95°C, 29-32 cycles were done for denaturing, annealing and extension for 1:00 min, 1:00 min, and 2:30 min, at 95°C, 56°C and 72°C respectively, with a final extension for 5:00 min at 72°C. 40 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.2 Gel electrophoresis and PCR purification After the DNA templates were amplified, the amplified products were electrophoresed on a 1% agarose gel (GibcoBRL, Grand Island, NY) in a gel electrophoresis device (Owl Separation Systems, Portsmouth, NH). TAE buffer was used as running buffer. 10 pi of the PCR products each (without oil) were loaded on the agarose gel along with 2 pi of blue/orange 6X loading dye (Promega, Madison, Wl) for tracking migration during electrophoresis. Along with the PCR products, a «j)X174 DNA-Haelll marker (New England Biolabs, Beverly, MA) was loaded to check for the product size. The electrophoresed samples in gel were exposed to 254 nm UV light (Dual Light™ Transilluminator, Ultra-Lum Inc., Claremont, CA). After confirming amplification, the remaining PCR products (40 pi) were purified, to reduce the amount of unused primers, dNTPs and primer byproducts that would interfere with subsequent primer extension (Makridakis and Reichardt 2001), using QIAquick® PCR purification kit (Qiagen, Valencia, CA) with minor modification as follows: ❖ 5 volumes (200 pi) of buffer PB (binding buffer: Guanidine hydrochloride + isopropranol) was added to 1 volume (40pl) of PCR samples and mixed (without removing the oil). ❖ The samples were applied to QIAquick column, which 41 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. themselves were placed over a 2ml collection tubes provided, and centrifuged at the rate of 13,000 rpm in a conventional tabletop microcentrifuge for 1 minute. ❖ The flow-through were discarded and the QIAquick columns were placed back on the 2ml collection tubes. ❖ 750 pi of buffer PE (wash buffer: containing 100% ethanol) was added to the QIAquick columns and centrifuged again for 1 minute at 13,000 rpm, to wash the DNA. ❖ The flow-through were discarded again and tubes recentrifuged for one more minute at 13,000 rpm. ❖ Now, the QIAquick columns were placed on clean 1.5 eppendorf tubes and 35-50 pi of buffer EB (elution buffer: 10 mM Tris-CI) was added and centrifuged again for 1 minute at 13,000 rpm, to elute the DNA. ❖ The purified DNA were stored at -2 0 °C till further use. 2.2.3 SNaPshot multiplex reaction Multiplex automated primer extension analysis of the SNPs was done using the commercially available protocol called SNaPshot™ (Applied Biosystems, Foster City, CA)(Makridakis and Reichardt 2001). The kit comes with a ready reaction mix called SNaPshot™ multiplex ready 42 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. reaction mix. The mix contains Amplitaq® DNA polymerase and flourescently labeled ddNTPs. Different fluorescent dyes are assigned to individual ddNTPs as shown in table 2.2. able 2.2 Dye assignments for individual ddNTPs ddNTP Color of analyzed data A Green C Black G Blue T(U) Red (Applied Biosystems) To conduct the SNaPshot reaction, the purified DNA template and four primers each for the respective SNPs were used with the SNaPshot ready reaction mix. The chemistry is based on the dideoxy single-base extension of an unlabeled oligonucleotide primer/primers (Makridakis and Reichardt 2001). Each primer binds to the 937 bp complementary template in the presence of fluorescent labeled ddNTPs and Amplitaq® DNA polymerase. The polymerase extends the primer by one nucleotide, adding a single ddNTP to its 3’end (ABI Prism® SNaPshot™ Multiplex Kit protocol). The four primers used for my experiments were as follows: 3’UTR-F/3146(5’-»3’): GAG AAC AGT TTT ACA ATA GAC 3’UTR-R/3174(5'-*3’): CTA CTC ATT ATT TGG ATA TTG CCA C 3’UTR-F/3681(5’-»3’): GCA TGA GTG CTG AGA TAT GGA CTC TCT A 3’UTR-R/3877(5’-»3’): CAA CAA AAA CAC TTA TTT ATA TGA TTG CAA TTT G 43 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The primers were designed myself and custom ordered from Invitrogen. The sizes for the primers 3’UTR-F/3146, 3’UTR-R/3174, 3’UTR- F/3681 and 3’UTR~R/3877 were 21 bp, 25 bp, 28 bp and 34 bp, respectively. The primers bind to the template as shown in figures 2.1 and 2.2. A3146T A3174G A3681C G3877A ■■■► A /C A/G* — -------— ►A/C G/A* .......... 939 bp template Figure 2.2 Diagram of the four SNaPshot multiplex primers and primer extension products (The four arrows indicate the position of the primers for the respective SNP, and their directions indicate the orientation o f the primers, forward or reverse) The SNaPshot protocol had been modified for optimization as in table 2.3. Table 2.3 Reagents for SNaPshot multiplex reaction REAGENTS QUANTITY SNaPshot multiplex ready reaction mix 5 pi Pooled Primers (all four primers together) 1 pi (0.25 pi each) (with concentration of 0.1 pmol/pl each) DNA template 1 pi Deionized water 3 pi Total 10 pi reaction (Applied Biosystems) 44 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The thermal cycling was done in Peltier Thermal Cycler (PTC-100™, MJ Research, Inc., Waltham, MA) and was optimized as shown in table 2.4. Table 2.4 Thermal cycling for the SNaPshot reaction Temperature Time Initial Denaturing: 96°C 2 minutes Denaturing: 96°C Annealing: 50°C Extension: 60°C 10 seconds 5 seconds 30 seconds ^ 25 cycles The samples were kept at 4°C till post-extension treatment. (Applied Biosystems) The unincorporated fluorescently labeled ddNTPs can co-migrate with the fragment(s) of interest, if left untreated. Hence, the removal of 5’phosphoryl groups by phosphatase treatment alters the migration of the unincorporated ddNTPs and thus prohibits interference (Applied Biosystems). I used Calf Intestinal Phosphatase (CIP)(New England Biolabs, Beverly, MA) to remove the unincorporated ddNTPs. The post extension treatment was done according to the protocol by ABI as follows: ❖ Master mix of CIP was made by mixing CIP (10U/jil), Buffer 3 (100 mM NaCI, 50 mM Tris-HCL, 10 Mm MgCI2, 1 mM dithithreitol pH 7.9 @ 25°C) and deionized water in the ratio of 1:1:8 respectively. 45 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ❖ 1 pi of the master CIP mix (equivalent to 1.0 unit of CIP) was added to the reaction mixture, mixed thoroughly and incubated at 37°C for 1 hour. ❖ The enzyme was deactivated at 75°C for 15 minutes. ❖ Samples were then stored at 4°C for use within 24 hours or kept at -20°C for storage longer than 24 hours. 2.2.4 Sample preparation for ABI 3100 genetic analyzer The CIP treated samples, Hi-Di formamide (highly deionized formamide) and a GeneScan-120 LIZ size standard (Applied Biosystems, Foster City, CA) were thawed. 9 pi of Hi-Di formamide, 0.05 pi of GeneScan-120 LIZ size standard and 0.5 pi of the sample were added together and the final reaction made to 10 pi. They were placed in a 96-well plate provided by Applied Biosystems. The reaction was then heat denatured at 95°C for 5 minutes and then kept on ice till they were loaded in the ABI 3100 Genetic Analyzer. The whole plate was loaded in the automatic genetic analyzer to be processed. The analyzer underwent a pre run to ensure that the parameters were ideal for gathering data using the GeneScan analysis software version 3.1 (Applied Biosystems). Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 2.2.5 Data Analysis Though the study was a case-cohort study, analysis for such a study is difficult. Hence, statistical analysis method used to analyze the data was a MEC standard case-control study design, carried out by Peggy Wan (Dr. Malcom Pike’s lab, USC) with the help of Eugene Kim (Dr. Juergen Reichardt’s lab, USC). 2.3 Experimental difficulties 2.3.1 Study subjects This study included a large sample size (n=884) in the beginning. However, either due to PCR amplification failure or insufficient DNA sample, analysis was done finally on 848 subjects, which itself is a large sample size to conduct a case-cohort study. There were 472 incident prostate cancer cases and 376 cohort controls. The racial/ethnic distribution was: 203 African-American, 247 Latino, 181 Whites, and 217 Japanese- American, to investigate the relationship between the four SNPs and prostate cancer risk amongst the different ethnic groups. 2.3.2 PCR PCR conditions were optimized to amplify the genomic DNA samples. Since I had difficulty amplifying few samples, a number of variables were changed to get optimal amplification, including changes in 47 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. concentration of the PCR reagents, number of cycles and melting temperature. The dilution of samples (1:10, 1:50, and 1:100) was also carried out in a step-wise manner. For few difficult samples, usage of AmpliTaq Gold (Perkin Elmer, Branchburg, NJ) was considered. All these changes resulted in successful amplification of many samples. 2.3.3 PCR purification To get rid of the primer-dimers, primer by products, and dNTPs, the PCR products were purified using PCR purification kit (Qiagen, Valencia, Ca), which yielded good result with very little background (Makridakis and Reichardt 2001). This technique was preferred over gel extraction as the former is less time consuming at the same time, producing good results. To avoid any interference of primer-dimers and hence to avoid using gel extraction, the amount of primers used was kept as low as possible (Makridakis and Reichardt 2001). For this a gradient of primer amount was used to find the minimum amount of primer required for optimal amplification. 2.3.4 Genotyping The multiplex automated primer extension analysis (MAPA) based on SNaPshot technique was used to genotype the SNPs. It is a relatively newer technique that is much less laborious and works faster than 48 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. sequencing for already known SNPs. With this technique, as many as ten SNPs at a time can be genotyped (Makridakis and Reichardt 2001). It is reliable, accurate and fast and hence was preferred over sequencing. 2.3.5 Primers for SNaPshot The samples were electrophoresed in ABI 3100 genetic analyzer and were analyzed using the GeneScan 3.1 software. While analyzing the SNPs, I encountered a mobility shift problem (explained below). To overcome this problem, various combinations of primers had to be used. The difference in the size of the primers has to be 4-10 bases for proper separation of the peaks. The orientation of the primers is also an important aspect of getting optimal genotyping results. Keeping these factors in mind, parameters were changed to achieve optimization. Figure 2.3 shows an example of one of the sets of primers used before optimization was achieved. The primers used were reverse for A3681C and forward for G3877A in contrast to the orientation of the primers giving optimal results (figure 2.2). Figure 2.4 shows the genotyping result where overlapping of the alleles is seen. As seen, the C allele for the A3681C genotype runs with the A allele of the previous (A3174G) SNP position, giving it a false appearance of A3174C heterozygous mutant and making the genotyping difficult. This kind of mobility shift problem was tackled using various different primers in different 49 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. orientation, till satisfactory results were yielded. Nearly 15% of the samples were regenotyped as they did not produce satisfactory results. A3146T A3174G A3681C G3877A ► A/C AIG4 < ■ A/C G/A----------- ► 939 bp template Figure 2.3 Orientation of primers before optimization A3W 1 Figure 2.4 Electropherogram showing genotyping result with primers of different orientation (The two peaks A and C where the arrow points represent the heterozygous mutant alleles (A and C) for the SNP A3681C, but the C peak runs with the previous peak falsifying the genotyping as A3174G). 50 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 3 RESULTS 3.1 PCR results The PCR conditions were optimized to obtain strong amplification and without any nonspecific products. Several samples needed dilution (in the range of 1:10,1:50 and 1:100), while different Taq polymerases (including Amplitaq Gold (Perkin Elmer, Branchburg, NJ)) were used to obtain amplification. The PCR product was 937 bp in length as shown as a band on the agarose gel as in figure 3.1. M S37bp Band of Interest Figure 3.1 PCR products (M =<f>X174 DNA-Haelll Marker. The bands seen are PCR products of various DNA samples. There are two samples that did not amplify) 3.2 Genotyping results The genotyping results for the four SNPs, A3146T, A3174G, A3681C, and G3877A were shown together in the electropherogram as peaks of different colors. Figures 3.2, 3.3, 3.4, 3.5, and 3.6 shows examples of various genotypes for the four SNPs. Approximately 15% of the samples were regenotyped since they did not produce accurate data. 51 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. AA AA AA GG Figure 3.2 Homozygous wild type (All the four SNP positions are homozygous wild type) Figure 3.3 Heterozygous mutant for A3146T (Heterozygous mutant for the position 3146) Figure 3.4 Heterozygous mutant for A3174G and G3877A (Both A3174G and G3877A are heterozygous mutants) 52 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Figure 3.5 Heterozygous mutant for A3681C (Heterozygous mutant) m Figure 3.6 Homozygous mutants for A3174G and G3877A (Homozygous mutants at position 3174 and 3877) 3.3 Data analysis 3.3.1 Association of the A3146T, A3174G, A3681C, and G3877A SNPs with prostate cancer risk Out of the total 884 samples, the final analysis result consists of 848 samples. This is either because of insufficient amount of DNA available or PCR amplification failure. Table 3.1 shows the genotyping results and the 53 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. odds ratio for cases as well as controls. None of the samples analyzed were homozygous mutant for A3146T and A3681C (TT for A3146T and CC for A3681C respectively). However the GG genotype for A3174G and AA genotype for G3877A were found and have been included in the analysis. Since the frequency was low, the homozygous and heterozygous mutants were combined for data analysis. Table 3.1 Distribution of the A3146T, A3174G, A3681C, and G3877A genotypes and OR for the association between the genotypes and prostate cancer_______________________________________________ Marker Case (472) Control (376) Total (848) OR** 95% Cl p-vaiue A3146T*** A3174G AA 350 286 636 1.00 AG + 122 90 212 1.01 0.72-1.41 0.96 GG* A3681C AA 468 375 843 1.00 AC + CC* 4 1 5 3.05 0.36-38.88 0.33 G3877A GG 339 283 622 1.00 GA +AA* 133 93 226 1.07 0.77-1.48 0.71 (*Since frequencies of homozygous mutations are low and we assume there is no dose-response effect, homozygous mutations were combined with heterozygous mutations in the analyses ** Adjusted for age at diagnosis for cases/blood collection for controls and ethnic groups. *** Not analyzed since the frequency of the variant is too rare. OR and Cl were calculated using logistic regression analysis, p-value was calculated using Wald’s test) A non-significant association is found between A3681C and prostate cancer (OR=3.05, p=0.33).But the number of samples in which this polymorphism was found was less (n=5). Analysis was not done for the 54 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. A3146T since it was found only in 2 samples. I did not find any significant association between the other two genotypes and prostate cancer (A3174G and G3877A), as the OR remains ~ 1.0. Also, no association could be found between the genotypes and the prostate cancer amongst the different racial/ethnic groups (Caucasians, African-Americans, Latinos and Japanese-Americans) (table 3.2 and 3.3). The results were not changed when cases were included with advanced prostate cancer (on regional/remote stage) (as known from data analysis result from Eugene Kim). Table 3.2 OR for the association between the A3174G genotype and prostate cancer risk by racial/ethnic groups Genotype AA AG + GG Ethnicity (Ca/Co) Case Control Case Control OR* 95% Cl White (95/86) 67 70 28 16 1.71 0.81-3.58 Black (134/69) 87 42 47 27 0.82 0.44-1.50 Latino (130/117) 109 101 21 16 1.29 0.62-2.69 Japanese (113/104) 87 73 26 31 0.72 0.37-1.40 (* Adjusted by age at the diagnosis for cases/blood collection for controls) Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3.3 OR for the association between the G3877A genotype and Genotype GG GA +AA Ethnicity (Ca/Co) Case Control Case Control OR* 95% Cl White (95/86) 66 69 29 17 1.67 0.87-3.44 Black (134/69) 81 40 53 29 0.88 0.48-1.60 Latino (130/117) 105 101 25 16 1.53 0.75-3.11 Japanese (113/104) 87 73 26 31 0.72 0.37-1.40 (*Adjusted by age at the diagnosis for cases/blood collection for controls) 3.3.2 Hardy Weinberg equilibrium In a population containing the genotypes BB, Bb and bb, the frequency of BB will be p2, bb will be q2, and that of Bb will be 2pq at equilibrium, where p is the frequency of B and q is the frequency of b (http://cancerweb.ncl.ac.uk/omd/). This is based on the Hardy-Weinberg equilibrium law, which states that a randomly-mating population will eventually reach these frequencies and be at this equilibrium as long as there are no selection pressures on the population (http://cancerweb.ncl.ac.uk/omd/). Calculations were done for my results (for the SNPs A3174G and G3877A) to see if the study subjects (controls only) were in Hardy Weinberg equilibrium. The frequency for the other two SNPs was found to be very low and so Hardy Weinberg equation was not 56 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. calculated for them. The distribution of the alleles from the Hardy Weinberg equation can be summarized as in table 3.4. Table 3.4 Hardy Weinberg equilibrium A3174G ALLELE OBSERVED EXPECTED Up value AA 286 288.73 >0.1 AG 87 81.51 GG 3 5.75 G3877A GG 283 285.24 >0.1 GA 89 84.49 AA 4 6.25 (*p-values are calculated from the Chi-Square test) This shows that the population is in Hardy Weinberg Equilibrium. Frequencies of the homozygous and heterozygous mutants were calculated for the A3174G and G3877A genotypes in cases as well as controls together, amongst the four ethnic groups, as shown in table 3.5. Table 3.5 Frequencies of SNPs amongst different ethnic groups GENOTYPE ETHNIC GROUP AG+GG IN A3174G GA+AA IN G3877A Whites 24.3% 25.4% Blacks 36.5% 40.4% Latino 14.9% 16.6% Japanese 26.3% 26.3% 57 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. These frequencies were calculated to see if any SNP appears to be more frequent in any ethnicity. These results show that the frequencies of both the A3174G and G3877A are highest in African-Americans whereas they are low amongst the Latinos. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 4 DISCUSSION AND CONCLUSION 4.1 Biological rationale The aim of the study was to find association between four single nucleotide polymorphisms (SNPs) A3146T, A3174G, A3681C, and G3877A, found on the 3’UTR of the SRD5A2 gene, and prostate cancer risk. It is of deep concern to investigate the risk factors for prostate cancer, because of a very strong biological rationale. 3’UTR may affect the mRNA stability (as illustrated in figures 1.11 and 1.12) which can change the amount of enzyme steroid 5a-reductase produced affecting the amount of DHT and hence through increased DHT-AR complex formation, there will be change in the transcription of genes involved in cellular growth (reviewed by (Hsing, Reichardt et al. 2002)). Hence this cascade of events probably would ultimately result in developing prostate cancer. 4.1.1 Importance of prostate cancer Prostate cancer has gained significant importance since it is the second most common cancer and the second leading cause of death among men in the United States (www.cancer.org 1998). Men are highly concerned since prostate cancer developing chances increase with age, which is a physiological and irreversible risk factor (reviewed by (Ross, Coetzee et al. 1999)). Considering these factors, early diagnosis and 59 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. treatment would be a great boon to the society. Finding more risk factors and adding more information on prostate cancer facts would be considered a great contribution to it. The carcinogenic process of prostate cancer is extremely slow (reviewed by (Labrie 2000)). Prostate cancer starts asymptomatically and remains latent for years to develop into a metastatic and invasive stage (Soderstrom, Wadelius et al. 2002). This gives ample opportunity to intervene and treat early. Also, though the incidence rates for various populations have found to be different, the prevalence rate of latent prostate cancer has been quite similar amongst different population (Breslow, Chan et al. 1977; Yatani 1982). This gives a great hint that the prostate cancer initiates due to some endogenous factor and progresses differently according to various exogenous factors among different population. 4.1.2 Androgens and the SRD5A2 gene One of the exogenous factors playing a major role in the development of prostate cancer is the steady level of androgens (reviewed by (Ross, Coetzee et al. 1999)). Prostate is an androgen-regulated gland (reviewed by (Wilding 1995)). Ample evidence already exists explaining the significance of androgens in the process of prostate cancer (Gann, Hennekens et al. 1996). Testosterone and DHT are the two most potent androgens and any variation in their amount influences prostate cancer risk 60 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (Ross, Bernstein et al. 1992; Gann, Hennekens et al. 1996; Hsing, Reichardt et al. 2002). The enzyme steroid 5a-reductase catalyses the irreversible conversion of testosterone to DHT with NADPH as the cofactor (Cheng 1993) and hence contributes in maintaining a balanced level of testosterone and DHT. In the prostate, the steroid 5a-reductase type II enzyme is encoded by the SRD5A2 gene (Thigpen, Silver et al. 1993). Hence, SRD5A2 gene may play an important role in the susceptibility towards prostate cancer. It is a candidate gene of several studies investigating any association between the genetic variation in the SRD5A2 gene and prostate cancer risk (Reichardt, Makridakis et al. 1995). 4.1.3 3’UTR of the SRD5A2 gene Though the 3’untranslated region of a gene does not code for amino acids and may not directly affect the function of the protein, it may play a significant role in the mRNA stability, hence affecting gene expression and finally the amount of protein produced (reviewed by (Day and Tuite 1998))(figure 1.11). Protein factors acting in trans, that modulate mRNA stability, have been characterized (Zaidi and Malter 1994). Binding of these factors to the 3’UTR can also play a role in either stabilizing the transcript or hastening their degradation (Shyu, Belasco et al. 1991; Zaidi and Malter 1994). As seen in figure 1.11 possibility 2, and figure 1.12, possibility 1, binding of a stabilizing protein factor to the 3’UTR of the gene, leads to 61 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. protection from either the exonucleoiytic decay or endonucleolytic degradation. It means, that these SNPs may lead to increased stability of the mRNA and hence increase in the production of the protein/enzyme produced. These facts about the importance of 3’UTR in the regulation of gene expression has led several researchers in the study of 3’UTR of SRD5A2 and its relation to the risk of prostate cancer ((TA)n repeat) (Reichardt, Makridakis et al. 1995). (TA)n dinucleotide repeats: (TA)0, (TA)g and (TA)i8have been found in the 3’UTR of the SRD5A2 gene (Davis and Russell 1993). Variation in the 3’UTR of SRD5A2 may lead to changes in the amount of the steroid 5a-reductase produced (reviewed by (Day and Tuite 1998; Hsing, Reichardt et al. 2002)). This change in the amount of enzyme steroid 5a-reductase produced can have serious consequences clinically. If more enzyme is produced, then the conversion of testosterone to DHT will be increased, which will lead to more DHT-AR complex formation, and ultimately it will result in elevated transcription of genes involved in cellular growth and in increased survival of the cell and formation of cancerous cell (reviewed by (Hsing, Reichardt et al. 2002)) (figures 1.8 and 1.9). Researchers have been trying to find out the association between (TA)n repeat, androgen levels and prostate cancer risk. Though the (TA)n repeat on the 3’UTR of the SRD5A2 gene repeat does not significantly correlate with the risk of prostate cancer, but it has been shown to be associated with the risk of other hormone-dependent 62 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancer (breast cancer) in some of the studies (Bharaj, Scorilas et al. 2000; Yang, Hamajima et al. 2002). This shows that the 3’UTR might play a significant role in a carcinogenic process and hence might be important in prostate cancer too. It has been shown that serum levels of DHT are found to be higher among men with the (TA)0/(TA)9 genotype (Hsing, Chen et al. 2001) in the 3’UTR of the SRD5A2 gene. Apart from TA repeat, to the best of my knowledge, studies on none other polymorphisms in the 3’UTR of SRD5A2 have been done. SNPs in general represent the largest diversity in human genome (Marras 2003) and though very few SNPs are directly involved with human diseases, they are important since they help in mapping the human genome, acting as genetic markers (Sachidanandam, Weissman et al. 2001). Considering all the factors discussed above, my study was based on the hypothesis that four SNPs, A3146T, A3174G, A3681C, and G3877A, found on the 3’UTR of the SRD5A2 gene, affect increase the mRNA stability and hence increase the amount of steroid 5a- reductase type II enzyme, and thus the DHT produced and so are associated with prostate cancer risk. 4.1.4 Ethnicity Four different ethnic groups: African-Americans, Latino-Americans, Japanese-Americans and Whites, all of which have different susceptibilities to develop prostate cancer, with African-Americans at the highest risk level 63 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. and Japanese at the lowest, the other two at the intermediate level (Reichardt 1999) were taken in this study. Whether this racial diversity has any significance with the SNPs I genotyped was considered as a part of my hypothesis. Since ethnicity has been one of the biggest risk factors of prostate cancer (reviewed by (Ross, Coetzee et al. 1999)), since androgen levels have been different in different races (Makridakis, Ross et al. 1997; Makridakis, Ross et al. 1999), and since certain mutations in the SRD5A2 gene have been proved to be associated with difference in prostate cancer risk among different races (Makridakis, Ross et al. 1999), it was considered relevant and reasonable to include subjects of various ethnicities in my study. 4.1.5 Clinical importance The study has great clinical importance too. The carcinoma of the prostate often progresses asymptomatically, until it reaches the metastatic, non-curable stage (Soderstrom, Wadelius et al. 2002). This emphasizes the importance of early detection of the disease. If the SNPs that were genotyped were found to be contributing to carcinogenesis or accelerating tumorigenesis and if they had been found with higher frequency among the prostate cancer patients, then those SNPs would be considered as markers for screening prostate cancer. Hence, the knowledge of variation in 64 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. the SRD5A2 gene may have potential applications in the prostate cancer screening. Apart from screening, the genetic variants of this gene might play a very important role in the prostate cancer prognosis. It has been shown by a study group that the A49T mutation on the SRD5A2 gene is associated with a greater frequency of extracapsular prostate cancer disease (Jaffe, Malkowicz et al. 2000). Also, the same study has shown that the A49T mutation is overrepresented in poor prognostic group of patients. The rationale that variation in the 3’UTR of the SRD5A2 gene might have influence on the pathological presentation of the tumor is the change in the androgen metabolism (Jaffe, Malkowicz et al. 2000).It is the fact that since 3’UTR of the SRD5A2 gene might be involved in mRNA instability, it would affect the production of the steroid 5a-reductase type II enzyme and hence the production of DHT from testosterone would change (reviewed by (Day and Tuite 1998)). If there were increase in the amount of DHT, which is a potent androgen in the prostate, then it would promote cell proliferation (reviewed by (Coughlin and Hall 2002)). Hence, the SNPs studied could have impact on the clinical manifestation of the disease. Thus it is biologically plausible that the SNPs increase the severity of the prostate cancer. Clinically speaking, it suggests that sequence variation in the SRD5A2 gene can affect the pathological characteristics of the prostate 65 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. cancer and in turn affect the prognosis of the patient suffering from prostate cancer. It is necessary to find more preventive measures for prostate cancer since the incidence rate is increasing and more men are diagnosed with prostate cancer. Finasteride, which is a steroid 5a-reductase enzyme inhibitor (Stoner 1996), is a potent chemopreventive drug (Irani, Ravery et al. 2002). Pharmacogenetic variation in the enzyme steroid 5a-reductase has been known (Makridakis, di Salle et al. 2000) when this drug was used. Variation in the 3’UTR of the SRD5A2 gene does not affect the activity of the enzyme, however, it might affect the amount of enzyme produced. If the SNPs lead to significant increase in the amount of the enzyme produced, then the concentration necessary for finasteride to act might have to be increased, and vice versa. However this depends on the magnitude of effect of the polymorphisms. This fact should be kept in mind while prescribing drugs (steroid 5a-reductase inhibitor) to patients. This signifies that SRD5A2 variability has therapeutic aspect to consider. Conclusively, the clinical importance of the SRD5A2 gene and its variability is underlined. The mutations in the SRD5A2 gene are just not important in the etiology of prostate cancer, but are important in various other clinical aspects like screening as well as indicators in the prognosis of the disease. 66 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. It is very important that patient suffering should be the least after he has been diagnosed of a disease. Finding out more and more risk factors, prognostic factors, and treatment modes will help alleviating the sufferings of the people. Therefore, this study was designed keeping a great clinical contribution in mind. 4.2 Conclusion: The results of this study found a non-significant association between the A3681C polymorphism and prostate cancer risk (odds ratio=3.05)(95% Cl= 0.36-38.88) (table 3.1) i.e the AC heterozygous polymorphism at position 3681 is non-significantly associated with prostate cancer risk. The number of samples in which this polymorphism was found was low (n=5), and it needs future studies to be done with more prostate cancer samples to support the conclusion. Also, biochemical analysis should be done in order to test the changes occurring in the amount of enzyme steroid 5a- reductase and difference in mRNA stability due to this substitution. At this point of time, it seems that this polymorphism is very rare (only 5 out of 848 samples analyzed), but whenever present, suggests an inclination towards prostate cancer risk. However, the study could not support the hypothesis that the other SNPs A3174G, and G3877A are associated with the risk of prostate cancer (OR for A3174G and G3877A are 1.01 and 1.07 respectively)(table3.1). 67 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Statistical analysis was not done for the A3146T SNP, because this SNR was found only in two samples. The A3174G and G3877A SNPs are seen frequently, both amongst the cases as well as controls (122 AG+GG and 90 AG+GG for A3174G among cases and controls respectively; 133 GA+AA and 93 GA+AA for G3877A among cases and controls respectively)(table 3.1). While genotyping, I observed that these SNPs in majority of the samples were seen together. Hence, they were initially thought to be linked; however in some samples, they did not appear together. The odds ratio (OR) for A3174G and G3877A remained ~1.0 for both the wild type alleles as well as the polymorphic alleles, hence statistically non-significant and therefore not associated with prostate cancer risk. Since the A3174G and G3877A were seen frequently and the other two SNPs (A3146T and A3681C) were seen only in few samples, further analysis was done only for A3174G and G3877A. Since the study had hypothesized ethnic differences for the SNPs genotyped and prostate cancer risk, analysis was done to see if the two frequent SNPs A3174G and G3877A had any association with prostate cancer among the four racial/ethnic groups (African-Americans, Latinos, Caucasians and Japanese-Americans). The results indicated a non-significantly higher OR (OR=1.7 and 1.67, 95% Cl = 0.81-3.58 and 0.87-3.44, for A3174G and G3877A respectively) amongst the Whites (tables 3.2 and 3.3). Currently this does not signify anything since it does not concord with any older 68 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. findings (since Whites are at lower risk of developing prostate cancer) (Reichardt 1999), but future studies with more White subjects can produce better results in understanding the relation between the SNPs and prostate cancer risk in the White population. No significant correlation was found between either of the two SNPs and prostate cancer in any of the four ethnic groups. There was one more finding that could be taken into consideration. The results show that the polymorphisms AG+GG of A3174G and GA+AA of G3877A are most frequent among the African-American population (cases and controls taken together) than all the other three ethnic groups (Caucasians, Japanese-Americans and Latinos)(tabte 3.5). The frequencies are 36.5% and 40.4% for alleles AG+GG in A3174G and GA+AA in G3877A respectively. However since a positive association was not found between the SNPs A3174G and G3877A and prostate cancer among African-Americans, currently this result does not hold any significance. To summarize, this study produced several non-significant results on the basis of which several future studies can be done. It has opened paths for many novel studies for the other researchers. 4.3 Future directions Biochemical analysis of the four SNPs has not been done previously. The 3’UTR may play an important role in the mRNA stability and may affect 69 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. gene expression (reviewed by (Day and Tuite 1998)). More the mRNA stability, longer the time will it stay in the cell, and more will be the production of protein; and lesser the stability of the mRNA, faster will it be degraded and hence lesser the amount of protein produced. Therefore, polymorphisms in the 3’UTR of the SRD5A2 gene could affect the protein (enzyme steroid 5a-reductase type II) amount by affecting the mRNA stability and hence it is worth conducting biochemical assays for all the four SNPs (reviewed by (Day and Tuite 1998)). Especially, since the results have suggested a positive association between A3681C and prostate cancer, it is highly necessary to conduct biochemical analysis of the steroid 5a-reductase type II for this SNP to see if this SNP causes any change in the amount of the enzyme steroid 5oc-reductase type II. For this, a variety of assays can be done, like luciferase assay, alkaline phosphatase assay, and p-galactosidase assay. I describe here the luciferase assay, by which the difference in the amount of protein between a wild type gene and a mutant gene can be measured. Firstly, the 3’UTR of the SRD5A2 gene is ligated to the luciferase reporter vector using restriction digestion to produce a recombinant plasmid. To insert the desired variants, oligonucleotides should be designed containing the SNPs of interest (A3146T, A3174G, A3681C and G3877A) and should be reconstructed in the 3’UTR containing 70 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. recombinant plasmid using a standard site-directed mutagenesis kit in separate experiments. Cells are treated with DpN1 restriction enzyme in order to digest the parental (methylated) vector. Large quantities of recombinant plasmids are produced by doing maxiprep. Direct sequencing should be done to confirm the DNA sequences of the newly introduced fragments in the recombinant plasmids. After the sequence is confirmed, the mutant plasmids are transfected into mammalian cells with little or no endogenous SRD5A2 expression, using electroporation. 24-48 hours after the transfection of genetic constructs into the cell, the cells are sonicated to break-open, the extract is isolated and expression analysis done. Luciferase activity is measured by monitoring the production of light by a luminometer or scintillation counter. The amount of light produced is compared with that of the wild type (similar amount of protein has to be used for wild type and mutant). The more the light, the more is the luciferase activity and indicates more protein production, if there is difference in the amount of luciferase activity, it indicates that the particular polymorphism might cause a change in the mRNA stability and hence changes the amount of the enzyme 5a-reductase type II, and hence could have effect on development or progression of prostate. Sometimes, the assay results in no change in the amount of protein. This could be due to no change in mRNA. However, it is not always necessary. The amount of mRNA does not only depend on the rate of 71 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. synthesis, but also depends on the degradation rate. The mRNA amount might have been produced in larger quantities but would have degraded faster. So to make sure about the actual mRNA stability, mRNA is measured using Northern blot technique. The wild type and the mutant mRNA are isolated from the cells prepared in previous experiment (luciferase assay). The purity and integrity of the mRNA has to be maintained. The mRNA is run on a denaturating gel (to avoid forming secondary structures) or heat denatured before running on an agarose gel. Hence they are separated according to the size. Next the mRNA is to be transferred to a nitrocellulose/nylon membrane and should be exposed briefly to UV light. A specific complementary radiolabeled 3’UTR probe is taken to detect the specific mRNA. Hybridization is followed by autoradiography to detect and quantify the mRNA. If there is no change in the mRNA amount, then it could indicate a change in the translation rate. Whereas if the mRNA amount changes, it indicates a change in either the mRNA stability or in the transcription. To understand whether it was due to mRNA instability or due to change in transcription process, further complex techniques like nuclear run-on could be done if the previous experiments do not produce satisfactory results. However, they will rarely be necessary. Since a role of a trans-acting factor is suspected, electrophoretic RNA mobility gel shift assay could be done. This type of assay should be 72 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. done if any change in the protein amount produced or change in the mRNA stability is seen, to see if this is due to a change in the binding of a trans acting factor. This type of assay is based on the principle that the RNA (or the DNA) migrates faster under electric field than the RNA-protein (DNA- protein) complex due to difference in their charge-to-mass ratio (Ronai 2003). The most commonly used separation systems for mobility shift assay are agarose or non-denaturing polyacrylamide gel slab-gel electrophoresis (Ronai 2003). RNA can be labeled with radioactive nucleotides or florescent dyes (Ronai 2003). So, as a part of future studies for the SNPs in the 3’UTR of the SRD5A2 gene, for each SNP position, both wild type and mutant should be run on the gel and electrophoresed to check for the shift in the mobility. If there is a difference in the mobility between the wild type and the polymorphic gene, then further analysis should be done to see if the particular SNP changes the amount of protein or not according to the change in the mobility. Apart from measuring the kinetics, further sequencing of the 3’UTR could be done in a large multiethnic population to find other SNPs and contribute to the existing database. As the result has shown a non- significantly increased risk of prostate cancer (OR~1.7) among the Whites, future experiments should include more White population to understand the relationship better. Genotyping more prostate cancer samples to see if the A3681C SNP is associated with prostate cancer should be done. 73 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES: American Urological Association. (2000). "Prostate-Specific Antigen (PSA) Best Practice Policy." Oncology 14(2). Andersson, S., D. M. Berman, et al. (1991). 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Creator Badiani, Prutha (author)

Core Title Association between single nucleotide polymorphisms in the 3'untranslated region of the SRD5A2 gene and prostate cancer risk

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Degree Program Biochemistry and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, genetics,biology, molecular,health sciences, oncology,OAI-PMH Harvest

Language English

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Advisor Reichardt, Juergen (committee chair), Mircheff, Austin K. (committee member), Tokes, Zoltan A. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-307340

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