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BRCA1 mutations and polymorphisms in African American women with a family history of breast cancer identified through high throughput sequencing

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BRCA1 mutations and polymorphisms in African American women with a family history of breast cancer identified through high throughput sequencing

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Content INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. 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 bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send U M I a complete manuscript and there are missing pages, these w ill be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand comer and continuing from left to right in equal sections with small overlaps. ProQuest Information and Learning 300 North Zeeb Road, Ann Arbor, M l 48106-1346 USA 800-521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BRCA1 MUTATIONS AND POLYMORPHISMS IN AFRICAN AMERICAN WOMEN WITH A FAMILY HISTORY OF BREAST CANCER IDENTIFIED THROUGH HIGH THROUGHPUT SEQUENCING by Lucy Yining Xia 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 (APPLIED BIOSTATISTICS AND EPIDEMIOLOGY) May 2002 Copyright 2002 Lucy Yining Xia Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UM I Number: 1411815 __ ___ __< g ) UMI UMI Microform 1411815 Copyright 2003 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 O F S O U T H E R N CALIFORNIA TH E GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES. CA LIFO RN IA S 0 0 0 7 This thesis, written by Lucy X t’ a, under the direction of h is Thesis Committee, and approved by all its members, has been pre sented to and accepted by the Dean of The Graduate School, in partial fulfillment of the requirements for the degree of M . S . ,r> Dt mm Date **7 10» 2002 THESIStCOMMI Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGEMENTS I am very grateful to Dr. Roberta McKean-Cowdin for her patient guidance, sharing of knowledge, and expert advice in the preparation of this manuscript. I would like to express my deepest gratitude to my thesis committee, Drs. Brian Henderson, Giske Ursin, and Gerry Coetzee, for their comments and suggestions regarding the manuscript. Without their help, this would not have been possible. I would also like to thank Shiella Mangune for her excellent technical assistance. Last but not least, I wish to thank the many families who participated in this research study. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Acknowledgements ii List of Tables iv Abstract v Introduction 1 Materials and Methods 3 Results 1 1 Discussion 21 References 27 iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. LIST OF TABLES Table 1: Age Distribution at Diagnosis of Breast Cancer for Affected 11 Women and at Time of Blood Draw for Unaffected Women Table 2: Identified BRCA1 Sequence Variants in African American Women 13 Table 3: Estimated Allele Frequency and Relative Risk of Breast Cancer 14 (Rare Missense Variants/Mutations) Table 4: Characteristic of Rare Missense Variants/Mutations Carriers 15 Table 5: Estimated Allele Frequency and Relative Risk of Breast Cancer 18 (Polymorphisms) Table 6: Genotype Distribution of the Polymorphisms in 19 Affected and Unaffected Women Table 7: Estimated Allele Frequency of Frequent Variants 20 Among Different Study Populations iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ABSTRACT We developed an efficient high throughput system to sequence the entire BRCA1 coding region for 157 African American women from 87 families with a history of breast or breast and ovarian cancer. Eighteen sequence variants were identified, including 14 missense and four silent mutations. Seven of the 18 variants were relatively common (allele frequency 15% or greater), and the remaining were rare (allele frequency less than 5%). Of the 18 variants, 14 showed no statistically significant difference in allele frequencies between cases and non-cases. Four variants occurred in only cases, three occurred once and one (G2196A, Asp693Asn) occurred in seven cases. A total of 97% cases and 91% non-cases had one or more variants. All variants identified in this study were previously described in the BIC database among white women. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. INTRODUCTION Breast cancer is a leading cancer and the second most common cause of cancer-related deaths among American women, with approximately one in eight being affected in their lifetime (1,2). A family history of breast cancer substantially increases a woman’s risk of the disease, especially when two or more close relatives are affected (3,4). It is estimated that 5%-10% of all breast cancer cases may be due to inherited autosomal dominant susceptibility genes (5,6). Germline mutations in BRCA1 gene are believed to be a predisposing genetic factor in 15%-45% of hereditary breast cancers (3,6,7). Recent genetic epidemiologic studies indicate that BRCA1 mutation carriers have a lifetime risk of breast cancer that is greater than 80% (6, 8, 9). The identification of the first breast cancer susceptibility gene BRCA1 in 1994 provided the initial opportunity to study directly the relationship between a gene and breast cancer risk. The BRCA1 gene was mapped to chromosome 17q21 by genetic linkage of early-onset breast cancer families in 1990 (10) and Miki et al. isolated the gene in 1994 by positional cloning (11). BRCA1 is composed of 24 exons containing 5592 bp of coding sequence that encodes a protein of 1863 amino acids. The translated region begins in exon 2 (120 bp) while exon 4 is believed to be an artifact of the isolation method used. Thus, the actual coding sequence contains only 22 exons. The entire gene covers approximately 100 kilobases (kb) of genomic sequence (11). 1 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. While the normal function of the BRCA1 gene remains unknown, data accumulated since the isolation of BRCAl gene suggest that it has several important functional activities. It is believed to be a tumor suppressor gene (12,13), playing a role in transcription, cell cycle control, DNA damage repair pathways and regulating apoptotic cell death (14-19). Since the isolation of BRCAl, more than S00 sequence variations have been identified within the coding region of the gene and more than 100 distinct highly penetrant mutations have been described in a Breast Cancer Information Core (BIC) database (20). The majority are frameshift mutations, identified using mutation detection techniques (SSCP, PTT) other than direct sequencing. Most of these mutations are predicted to result in a truncated BRCAl protein. Some missense mutations are also known to alter protein function (21-23). Population genetic studies have shown that the proportion of high-risk families with breast or ovarian cancer attributable to BRCAl mutations varies widely across populations (24). However, studies of hereditary breast cancer have largely focused on Caucasian populations of European decent as summarized in the BIC database. The spectrum of mutations in African American women has been characterized in only a few studies (25-27). Most of these studies involved 54 or fewer breast cancer cases, with the largest study (28) including 88 cases. Few variations have been described in African American women because the populations under study were either small or not Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. selected for high-risk individuals with a family history of breast or breast and ovarian cancer. In the present study, we sequenced the BRCAl coding sequences among a large collection of African American families with a history of multiple cases of breast or breast and ovarian cancer. To complete this aim, we developed an efficient, high throughput method to directly sequence the BRCAl coding region. Using the method, we were able to identify and describe the frequency of sequence variants in our unique African American population. Further, we described the characteristics of individuals with select variants and calculated odds ratios and their 95% confidence interval to estimate relative risks of breast cancer for those carrying the variants. MATERIALS AND METHODS Subjects The family study of BRCAl and BRCA2 is a sub-study of an ongoing large population- based multiethnic cohort (MEC) study conducted in Los Angeles and Hawaii (29). The MEC study was designed to emphasize diet and other lifestyle characteristics in the etiology o f cancer. The Los Angeles sub-cohort includes large numbers o f African American (35,107) and Latino (47,438) participants, aged 45 to 75 years at enrollment into the cohort. The MEC family study includes those MEC participants that reported a family history of breast and/or ovarian cancer on their first questionnaire. The eligibility 3 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. criteria were that a family includes at least two breast cancer cases or one of breast and one of ovarian cancer in first-degree relatives. No other selection criteria were used to determine eligibility. The focus of this thesis is BRCAl alterations in African American women enrolled in the MEC family study. At the time of enrollment into the cohort, a total of 848 African American MEC members reported a family history of breast or ovarian cancer These potential members were contacted to determine eligibility for inclusion in the family study. Of the 848 contacted members, 87 African American families met eligibility requirements at that time and agreed to participate in the study. A total of 157 African American female MEC members and eligible sisters were recruited into the study. Each participant completed a family history questionnaire and provided blood sample for genetic analysis. The protocol was approved by the local Institutional Review Board (IRB). An IRB approved informed consent was obtained from each participant. The recruited women in this study include 97 individuals affected with breast cancer, three individuals with ovarian cancer and 57 unaffected sisters. The baseline age is the age at time of blood draw. The diagnosis age is the age at time of diagnosis of breast or ovarian cancer for individuals affected with the disease. The baseline age of this study population was between 45 and 86 years and the average is 66.3. The diagnosis age was from 26 to 78 years and the average is 57. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Laboratory Methods We developed a high throughput method for sample preparation and DNA sequencing to sequence the entire BRCAl gene as part of a larger study of BRCAl and BRCA2 alterations in 450 African American and Hispanic participants. The samples in this study represented a subset of this larger sample. The sample preparation and DNA sequencing procedure consisted of the following 6 steps: 1) DNA extraction, 2) polymerase chain reaction (PCR) amplification, 3) PCR product purification, 4) fluorescent dye labeling and extension, 5) extension product purification, and 6) sequencing. 1) DNA Extraction Genomic DNA was extracted from 300ul buffy coat using Gentra systems’ Puregene DNA isolation kit (Puregene blood kit, catalog number D-5000) following the manufacturer’s protocol. Briefly, 300ul of buffy coat was mixed with 900ul of RBC (red blood cell) Lysis Solution and centrifuged at 14,000 rpm for 10 min. The white cell pellet was incubated at 37°C with 300ul of Cell Lysis Solution for 30 min. Then 1.5ul RNaseA Solution was added to the cell lysate, and incubated at 37°C for 15 min. Subsequently 200ul o f Protein Precipitation Solution was added to the cell lysate, and centrifuged at 14,000 rpm for 10 min. The supernatant was transferred to a clean tube, mixed with 600ul of isopropanol and centrifuged at 14,000 rpm for 3 min to precipitate the DNA. The DNA pellet then was washed with 500ul of 70% ethanol. After 5 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. discarding the ethanol, the pellet was air dried and dissolved in 200ul of DNA Hydration Solution. After incubation at room temperature overnight, or at 65°C for lhr, the DNA was ready for PCR amplification. 2) Polymerase Chain Reaction (PCR) Amplification We amplified the entire coding sequence and all intron-exon boundaries of the BRCAl gene from each of the DNA samples. Polymerase chain reaction (PCR) with 22 pairs of primers was used to amplify exons 2, 3 and 5 through 24. Exon 1 was not amplified because the translation start site is in exon 2. Exon 4 was excluded because it is known to be a variant exon not seen in normal BRCAl messenger RNA. Primer sequences used were described in the BIC database (20). PCR reactions for all coding exons except exon 11 were carried out in a total volume of 25ul. Each PCR reaction mix contained 30ng of genomic DNA as template, 40 pmoles of each exon-specific forward and reverse primer, lOOuM dNTPs, 2 units o f Taq polymerase (Amersham Pharmecia Biotech) and lx reaction buffer. The PCR amplification consisted of 30 cycles with denaturation at 94°C for 1 min, annealing from 52°C to 58°C for 1 min (depending on the melting temperature of specific primer sequence pairs) and extension at 72°C for 1 min. An initial denaturation step o f 3 min at 94°C and a final extension at 72°C for 10 min were employed. We used the GeneAmp XL PCR kit (PE Applied Biosystems) to amplify exonl 1. The PCR reaction was carried out in a total volume o f lOOul to account for the large size of this exon. All PCR 6 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. products were amplified using 96-well plates, on a MJ research, Inc. PTC-100 thermal cycler. 3) PCR Product Purification We purified the PCR products by ultrafiltration using MultiScreen FB filter plates (Millipore Corporation). The FB filter plate was placed on the top of a standard 96 well plate, then the plate containing PCR products was flipped over and placed on the top of the FB filter plate, centrifuged at 1000 x g for 5 min to transfer the PCR products and bind the DNA to the FB filter plates. The filter-bound DNA was washed twice in 200ul of 80% ethanol per well, by centrifuging at 1000 x g for 5 min. The purified DNA was eluted by adding 50ul o f TE buffer to each well of the FB filter plate, placing it on top of a clean 96-well plate, and centrifuging at 1000 x g for 5 min. This procedure removes salts, proteins, detergents, unincorporated dNTPs and excess primers. 5ul of each purified PCR product was tested on a 1% agarose gel containing ethidium bromide to ensure that the product was present for each subject. 4) Fluorescent Dye Labeling and Extension The fluorescent dye terminator labeling and extension reactions were performed using the ABI PRISM® BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems). The labeling and extension reactions were carried out in a total volume of 20ul, containing 5 tolOng of purified PCR fragments as template, 6 pmoles of exon specific forward or reverse primer as used in the PCR reaction, 1.34ul of Ready Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reaction Premix, and lx reaction buffer. Labeling and extension reactions were run for 25 cycles with denaturation at 96°C for 10 seconds, annealing at 50°C for 5 seconds and extension at 60°C for 4 min. 5) Extension Product Purification Excess dye terminators obscure data at the beginning part of the sequence and can interfere with base calling. To generate high quality DNA sequence data, unincorporated dye terminators must be removed from the extension product prior to analysis by electrophoresis. We used MultiScreen-HV 96-well Filtration Plates (Millipore Corporation) for large-scale throughput. The filtration plates were prepared following the manufacture’s directions. G-50 superfine sephadex powder was used to make a mini-column gel in each well of the filtration plate. The extension products were then added to the center of each well, the plates were placed on top of a clean 96-well plate and centrifuged at 910 x g for 5 min. This gel filtration procedure can desalt and remove unincorporated dye terminators and other contaminants present in the extension products. The resulting filtrates contained the purified extension products. These samples were denatured at 90°C for 2 min, and immediately placed on ice for I min. This completed the preparation process and the samples were ready for sequencing. 6) Sequencing Sequencing was performed on an ABI PRISM® 3700 DNA Analyzer (PE Applied Biosystems), using capillary electrophoresis technology. The ABI PRISM® 3700 DNA 8 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Analyzer is a fully automated, multi-capillary instrument designed for large-scale DNA sequencing. The plates containing the purified DNA samples from step 5) were placed on the instrument worksurface, and the sample plate layout structure was loaded into the attached ABI computer. The autoloader transferred aliquots of 2.5ul from the sample plates into the 4ul injection wells of the load bar. The 96 samples were transferred simultaneously into the parallel capillary array by electrokinetic injection. This was followed by electrophoretic separation of the dye-labeled nucleic acid fragments. Data were collected through the ABI detection system and read by the sequencing analysis software in the attached ABI computer. Data Cleaning and Analyses The ABI sample files containing the sequencing results were transferred from the ABI computer. Because some long stretches of introns are amplified with exons, the exported data were edited to remove them. We used the ABI Prism DNA Sequencing Analysis software on a Windows NT computer to edit out the intron sequences before importing the data to the PolyPhred software program (developed by Phil Green at the University of Washington, described in detail in (30)). PolyPhred was used to identify mutations and polymorphisms. This software was designed to process large volumes of data with high sensitivity for mutations and polymorphisms. The high sensitivity designed into the program results in a high false- positive rate. To increase the specificity of our findings, we manually reviewed Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. electropherograms of all variants identified by PolyPhred. All variants we identified through this procedure was re-amplified and re-sequenced in both the forward and reverse directions to eliminate false positives. After the data were reviewed and cleaned, they were processed by PHRAN software, developed by Drs. Dan Stram, Roberta McKean-Cowdin and others at the University of Southern California, into a SAS data set that creates variables for all sequence variants and allele frequencies. PHRAN names the variants according to the exon base number where the variant is located and indicates whether the variant results in an amino acid change, and whether the change is silent, missense or nonsense. A silent mutation alters a codon but it codes for a same amino acid due to the redundancy of the genetic code. A missense mutation alters a codon so that it codes for a different amino acid. A nonsense mutation alters a codon into a stop codon, resulting in premature protein termination. Statistical Methods Associations between specific alleles and breast cancer risk were assessed by odds ratios, using unaffected sisters as controls. Prevalence estimates, allele frequencies, odds ratios and their 95% confidence intervals (Cl’s) were calculated using SAS software. The allele frequency of the variant for the study population was estimated from unaffected sisters. Sib-pair relationships were not maintained in the analysis due to the low number of probands with qualifying siblings. 10 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. RESULTS A total of 157 African American women originated from 87 familial breast cancer families were in this study. O f these, 97 individuals were affected with breast cancer and three individuals with ovarian cancer, 57 were unaffected sisters. Table 1 gives the distribution of age at diagnosis of breast cancer for affected individuals and age at blood draw for unaffected individuals. The distributions of baseline age of affected and unaffected individuals were similar (data not shown). The mean diagnosis age for this study population was 57 (95% Cl: 54.8-59.3) and the mean baseline age for unaffected individuals was 65.1 (95% Cl: 62.2-67.9). About 63% of the affected individuals were diagnosed at age 55 or older, more than 26% were diagnosed at 65 or older. Only 12 (12.4%) cases were diagnosed before the age of 45. Of the unaffected women, none were under age of 45, 79% were 55 years or older and 60% were 65 or older at time of blood draw. Table 1: Age Distribution at Diagnosis of Breast Cancer for Affected Women And at Time of Blood Draw for Unaffected Women Age (Years) Number of Individuals at Diagnosis of Breast Cancer" (%) Number of Unaffected Women at Blood Drawb (%) <45 12 (12.4) 0 (0) 45-54 24 (24.7) 1 2(21.1) 55-64 35 (36.1) 11 (19.3) >65 26 (26.8) 34 (59.6) Total 97c(100) 57(100) d The mean age at diagnosis was 57 years, 95% Cl: (54.8, 59.3). bThe mean age at blood draw was 65.1 years, 95% Cl: (62.2,67.9). cThere are three additional cases of ovarian cancer. 11 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Sequencing the entire coding region of the BRCAl gene for the 157 study participants resulted in the identification of 18 different BRCAl sequence variants (Table 2). All alterations were single-base substitutions and all were transitions (substitution of one purine for another purine or one pyrimidine for another pyrimidine). These single-base substitutions resulted in missense mutations or silent mutations. The majority of sequence variants were detected in exon 11 (total of 12); three were in exon 16 and one each in exons 9,13, and 22. A total of 97 (97%) of the cases and 52 (91%) of the non cases carried at least one of the variants. Among the 18 variants, fourteen were missense and four were silent. Seven of these sequence variants were relatively common (allele frequency 15% or greater). O f these common variants, four were missense and three were silent. All these common variants and another low frequency silent mutation were recognized as polymorphisms in BIC. A polymorphism is referring to coexistence of two or more genetic forms presented in readily noticeable frequencies in the same population. In this report, we defined rare missense variants/mutations as missense variants with an allele frequency < 5%, and polymorphisms as frequent sequence variants with an allele frequency > 5%. All silent mutations were also classified as polymorphisms. Subsequent discussions are organized according to these two categories: rare missense variants/mutations (UV in table 2) and polymorphisms (P in table 2). 12 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 2: Identified BRCAl Sequence Variants in African American Women Exon Nucleotide Position3 Base Change Codon Amino Acid Change Mutation Type Mutation Effectb 9 676 C-»A 186 Ser->Tyr Missense UV 11 1186 A-»G 356 Gln->Arg Missense u v 11 1546 A-»G 476 His->Arg Missense UV 11 1831 T-»C 571 Ile-»Thr Missense u v 11 2196 G-»A 693 Asp->Asn Missense u v 11 2201 C-»T 694 Ser->Ser Silent p 11 2430 T-»C 771 Leu->Leu Silent p 11 2577 A-»G 820 Lys->Glu Missense u v 11 2731 C-»T 871 ProLeu Missense p 11 3232 A-»G 1038 Glu-»Gly Missense p 11 3238 G-»A 1040 Ser->Asn Missense u v 11 3537 A-»G 1140 Ser->Gly Missense u v 11 3667 A-»G 1183 Lys->Arg Missense p 13 4427 T-»C 1436 Ser->Ser Silent p 16 4810 T-»C 1564 LeuPro Missense u v 16 4931 A-»G 1604 Gln->Gln Silent p 16 4956 A-»G 1613 Ser->Gly Missense p 22 5467 T-»C 1783 Met“>Thr Missense u v “Nucleotide numbering starts at the first transcribed base according to GenBank entry U14680. b UV=Rare missense variants/mutations (Unclassified variant), P=Polymorphism. Rare Missense Variants/Mutations Ten BRCAl rare missense variants/mutations were identified (Table 3). Table 3 presents the allele frequency for each variant by disease status, the estimated variant allele frequency in the study population and the estimated relative risk of breast cancer for carrying the variant. Table 4 describes the characteristic of these rare missense variants/mutations carriers. These rare missense variants/mutations were detected in 37 (24%) of our study participants, including 23 (24%) women with breast cancer, 13 (23%) unaffected women and one woman with ovarian cancer (Table 4). 13 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 3: Estimated Allele Frequency and Relative Risk of Breast Cancer* (Rare Missense Variants/Mutations) Variant0 Affected with Breast Cancer (%) Unaffected (% ) Estimated Rare Allele Frequency (%) RR1 of Breast Cancer (95% Cl) C676A' Serl86Tyr 1/194 (0.5) 2/114(1.75) 1.75 0.29 (0.03, 2.86) A1186G' Gln356Arg 3/188(1.6) 2/110(1.8) 1.8 0.88 (0.14, 5.34) A1546Gd His476Arg 1/188 (0.5) 1/110(0.9) 0.9 0.59 (0.04, 9.19) T1831Cd Ile571Thr 3/188(1.6) 2/110(1.8) 1.8 0.88 (0.14, 5.34) G2196Ad Asp693Asn 7/194 (3.6) 0/110(0) 0 - A2577G' Lys820Glu 4/194(2.1) 5/110(4.5) 4.5 0.45 (0.12, 1.68) G3238A' Serl040Asn 1/194 (0.5) 0/112(0) 0 - A3537G' Seri 140Gly 3/194(1.5) 2/110(0) 1.8 0.85 (0.14, 5.17) T4810C' Leul564Pro 1/194 (0.5) 0/114(0) 0 - T5467C1 Metl783Thr 1/194 (0.5) 0/114(0) 0 - The differences in the total number of alleles observed are due to variability in the PCR product. bSequence variants are listed in order of location from the S’ to 3' end o f BRCAl. 'Previously detected in African American. d Detected in Caucasian or no information on ethnicity. rRR: Relative Risk. 14 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 4: Characteristic of Rare Missense Variants/Mutation Carriers Variant Subject* Ager i Breast Cancer Ovarian Cancer Types of Cancers in (yrs) In the Family (n) In the Family (n) Other Family Member C676A R0392 (Co) 56 2 1 Cervix, Lung R0410 (Co) 71 2 1 Cervix, Lung R0451 (BC) 61 2 I Cervix, Lung A1186G R0148 (Co) 73 2 0 Stomach Colon R0249 (BC) 44 2 0 Stomach R0250 (Co) 45 2 0 Stomach R0317 (BC) 48 3 0 - R0366 (BC) 52 3 0 - A1546G R0352 (BC) 51 2 0 Uterus R0440 (Co) 65 2 0 Two Unknown Cancers T1831G R0215 (BC) 57 1 1 - R0217 (BC) 47 2 0 Colon, Hemtopoetic R0220 (Co) 51 1 1 - R0248 (Co) 54 2 0 Pancreas R0287 (BC) 74 2 0 Lung G2196A R0307 (BC) 64 2 0 - R0309 (BC) 56 2 0 Stomach R0332 (BC) 72 3 0 Stomach colon prostate R0377 (BC) 78 5 0 3 Prostate R0396 (BC) Unk 5 0 3 Prostate R0406 (BC) 48 2 0 - R0423 (BC) 46 2 0 Brain A2577G R0008 (BC) 63 2 0 Colon, Lung, Lymph R0035 (Co) 73 2 0 Three Lung R0062 (BC) 63 3 0 - R0095 (Co) 67 2 0 Three Lung R0121 (Co) 69 3 0 Two Stomach R0152 (BC) 64 3 0 Two Stomach R0352 (BC) 51 2 0 Uterus R0378 (Co) 46 2 0 - R0440 (Co) 65 2 0 Two Unknown Cancers G3238A R0155 (BC) 70 2 0 Cervix, Liver A3537G R0076 (BC) 49 2 0 Two Stomach R0106 (Co) 65 3 0 Two Stomach R0216 (OV) 56 3 1 - R0280 (BC) 55 2 0 Colon, Hematopoietic R0282 (Co) 78 2 0 Lung R0313 (BC) 54 2 0 - T4810C R0357 (BC) 52 2 0 - T5467C R0045 (BC) 48 2 1 - BC: Breast Cancer, OV: Ovarian Cancer, Co: Unaffected. b Age is age at diagnoses for affected women and age at blood draw for unaffected womon. 15 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Of the 10 rare missense variants/mutations, six (C676A, A1186G, A1546G, T1831G, A2577G and A3537G) were found in both women with and without breast or ovarian cancer, while four rare missense variants/mutations G2196A, G3238A, T4810C and T5467C were found only in breast cancer affected individuals. For the six rare missense variants present among both affected and unaffected individuals, the frequencies of the variants did not differ significantly between affected and unaffected individuals. None of the breast cancer odds ratios were statistically significant. The estimated allele frequency of the variants in the population were less than 2% for all except one (A2577G) at 4.5% (Table 3). The G2196A rare missense variant/mutation was detected in seven breast cancer affected individuals, but not seen in any o f the unaffected women (Table 4). The seven affected carriers came from six families. Two carriers were sisters from a family with five cases of breast cancer and three cases of prostate cancer diagnosed in first-degree relatives. The other carriers came from different families, only one of which had more than one family member enrolled in the study who was an unaffected sister. The rare variants/mutations G3238A, T4810C and T5467C were each detected once in breast cancer affected individuals but not in unaffected women (Table 4). Polymorphisms Eight BRCAl polymorphisms were identified (Table 5). Four were missense variants (C2731T, A3232G, A3667G and A4956G) showing an estimated variant allele Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. frequency 15% or greater and four were silent mutations (C2201T, T2430C, T4427C and A4931G). All these polymorphisms were present among both affected and unaffected individuals. A total of 89 (89%) affected individuals and 52 (91%) unaffected individuals carried at least one of these polymorphisms. Table5 presents the allele frequency for these variants by disease status, the estimated variant allele frequency in the study population and the estimated relative risk of breast cancer for carrying the variant allele. Table 6 presents the genotype distribution of these polymorphisms. With the exception of the silent mutation A4931G, the other seven polymorphisms showed an estimated frequency of the variant allele equal to or greater than 15%. The frequencies of these variants did not differ significantly between affected and unaffected individuals. The estimated relative risks of breast cancer for carrying any of the variant alleles varied from 0.76 to 1.24, but none were statistically significant (Table 5). However, we observed a higher risk of breast cancer for homozygous carriers of the Leu allele (C2731T, Pro871Leu). The estimated relative risk of breast cancer for homozygous carriers of the Leu alleles compared to Pro (wildtype) alleles was 2.3 (95% Cl 0.88-6.0) (Table 6). There appeared to be linkage disequilibrium between these frequent variants. Variants C2201T, A3667G and A4956G were jointly present in 60 out of 68 individuals carrying at least one of these three variants. The C2201T and A3667G located in exon 11, and 17 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 5: Estimated Allele Frequency and Relative Risk of Breast Cancer” (Polymorphisms) Variant Affected with Breast Cancer (%) Unaffected (%) Estimated Rare Allele Frequency (%) RRC of Breast Cancer (95% Cl) C2201T1 Ser694Ser 39/194 (20) 27/110(24.5) 25 0.82 (0.47, 1.41) T2430Cd Leu771Leu 33/194(17) 19/110(17.3) 17 0.99 (0.53, 1.82) C2731T Por871Leu 96/194(50) 44/110 (40) 40 1.24 (0.8, 1.9) A3232G Glul038Gly 30/194 (15.5) 17/112(15.2) 15 1.02 (0.53, 1.93) A3667G Lysl 183Arg 42/194(21.6) 29/110 (26.4) 26 0.82 (0.48, 1.39) T4427Cd Serl436Ser 36/194(18.6) 20/114(17.5) 18 1.06 (0.58, 1.92) A4931Gd Glnl604Gln 2/194(1) 1/114(0.9) 1 1.18 (0.11, 13.12) A4956G Serl613Gly 43/194 (22.2) 30/114 (26.3) 26 0.84 (0.5, 1.42) Frequency of the less common allele on the number of chromosomes observed. The differences in the total number of alleles observed are due to variability in the PCR product. Sequence variants are listed in order of location from the S’ to 3’ end of BRCA1. C RR: Relative Risk. dSilent mutation A4956G located in exon 16. Despite the fact that these three polymorphisms are parted between distances o f ~1.5 kb to ~30 kb of the genomic DNA, they appeared to be in linkage disequilibrium. All three had estimated frequencies of 25%-26% (Table 5). 18 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Table 6: Genotype Distributions of the Polymorphisms in Affected* and Unaffected Women C2201T (Ser694Ser) CC No. (%) Unaffected 30 (54) Affected 62 (64) CT No. (%) 23 (42) 31(32) TT No. (%) 2(4) 4(4) RRb of Breast Cancer Heterozyg (95% Cl) RRb of Breast Cancer Homozyg (95% Cl) T2430C (Leu771Leu) TT No. (%) Unaffected 36 (65.5) Affected 67 (69) TC No. (%) 19(34.5) 27 (28) CC No. (%) 0(0) 3(3) C2731T (Por871Leu) ProPro No. (%) Unaffected 19(34.5) Affected 31 (32) Pro Leu No. (%) 28(51) 36(37) LeuLcu No. (%) 8 (14.5) 30(31) 0.8 (0.37, 1.68) 2.3 (0.88, 6.0) A3232G (Glul038Gly) GluGlu No. (%) Unaffected 39 (69.6) Affected 70 (72) GluGly No. (%) 17(30.4) 24(25) GlyGly No. (%) 0(0) 3(3) 0.8 (0.38, 1.64) - A3667G (Lysll83Arg) LysLys No. (%) Unaffected 28(51) Affected 59(61) LysArg No. (%) 25(45) 34(35) ArgArg No. (%) 2(4) 4(4) 0.65 (0.32, 1.28) 0.95 (0.16, 5.6) T4427C (Serl436Ser) TT No. (%) Unaffected 37 (65) Affected 64 (66) TC No. (%) 20 (35) 30 (31) CC No. (%) 0(0) 3(3) A4931G (Glnl604Gln) AA No. (%) Unaffected 56 (98.2) Affected 95 (98) AG No. (%) 1 (1.8) 2(2) GG No. (%) 0(0) 0(0) A4956G (Serl613Gly) SerSer No. (%) Unaffected 30 (52.6) Affected 57 (59) SerGly No. (%) 24 (42) 37 (38) GlyGly No. (%) 3 (5.4) 3(3) 0.8 (0.41, 1.6) 0.4 (0.08, 1.82) Affected: affected with breast cancer. bRR: Relative Risk. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. We compared the allele frequencies of the frequent variants found in this African American population to those reported in Caucasian and Japanese populations in the literature. The estimated allele frequency for variants C2201T, T2430C, A3232G, A3667G, T4427C and A4956G were lower in the African Americans included in this study than what has been reported in Caucasians or Japanese (28,31,32). The variant C273 IT was, however, higher in this African American population than previously reported for Caucasians (31) (Table 7). Table 7: Estimated Allele Frequency of Frequent Variants Among Different Study Populations Variant Allele Frequencies C2201T (Ser694Ser) O ur Study (African American) 0.25 King’s Study (Caucasian") 0.33 Durocher’s Study Inoue’s Study (Caucasian1 ) (Japanese) 0.27 NE T2430C (Leu771Leu) 0.17 0.33 0.30 NE C2731T (Por871Leu) 0.40 NE 0.31 NE A3232G (Glul038Gly) 0.15 0.33 0.24 0.38 A3667G (Lysl 183Arg) 0.26 0.33 0.32 0.38 T4427C (Serl436Ser) 0.18 NE 0.33 NE A4956G (Serl613Gly) 0.26 0.33 0.30 0.33 ■ ‘ Northern European origin 20 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. DISCUSSION In this study, we found 18 sequence variants in the coding region of BRCA1 from African American women through direct sequencing of the gene. More sequence variants were identified in our population than have been reported in the literature from previous, smaller studies of African Americans. Shen et al. (25) screened 54 African American women breast cancer patients; they reported three missense sequence variants and one frameshift mutation. Panguluri et al. (26) screened 45 African American women at high-risk for hereditary breast cancer and found five missense sequence variants and two frameshift mutations in the BRCA1 coding region. Gao et al. (27) screened 28 African American women breast cancer patients, finding two missense sequence variants and one frameshift mutation. King et al. (28) screened 88 African American women with sporadic breast cancer and reported seven missense sequence variants and no frameshift mutation. To our knowledge, the present study is the largest study of BRCA1 in African American women, and is the first study of African Americans using direct sequencing method to scan for mutations in BRCA1 coding regions. The previous studies of African American did not identify as many sequence variants possibly due to several factors: I) they screened a smaller sample; 2) they did not select subjects based on family history of breast cancer; and 3) (some studies) only screened the so-called “hot-region” for mutations, specifically they only screened selected exons. In addition, the screening techniques used in these earlier studies were either protein truncation tests (27, 28) or single-stranded conformation polymorphism 21 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. (SSCP) analysis (25, 26) followed by DNA sequencing of variant bands. The low sensitivity and technical limitations of these screening methods might have resulted in fewer variants/mutations being detected. The direct sequencing method that we used is more sensitive and accurate for detecting variants and mutations in genes than these other screening techniques. The wide spectrum of variations reported here was expected, given the genetic diversity in people of African American ancestry, however, no novel sequence variants were found. It is interesting to note that, even though this study was relatively large, no frameshift mutations or high risk disease related mutations were found. The absence of frameshift mutations in this study population may result from the fact that this population is a group of older postmenopausal women who were not selected for early-onset disease. We would expect that women with early-onset breast cancer are more likely to harbor severe mutations, resulting in protein truncation (10, 33). The absence of severe mutations also may be related to the overall fewer number of mutations described in African American women. There is some evidence that BRCA1 mutations may be less frequent among African American than Caucasian women. Two population-based studies of African American women with breast cancer also identified multiple variants, none of which appeared to be strong predictors of disease (28, 30). Our study results are consistent with this. However, more studies are necessary before a conclusion on frequency and spectrum o f BRCA1 mutations in African American women can be made. 22 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Preliminary analysis of our data suggests that the sequence variants we found are not strong predictors of breast cancer risk. However, one rare missense variant/mutation, G2196A may confer an increased risk of breast cancer. This variant was detected exclusively among breast cancer affected individuals (7 out of 97). Furthermore, two of the carriers were sisters who came from a high-risk cancer family with five cases of breast cancer and three cases of prostate cancer diagnosed in first-degree relatives. This variant results in a G to A transition that converts codon 693 from GAC, encoding the acidic residue Aspartate, into AAC encoding the polar residue Asparagine. Aspartate is considered a charged polar amino acid, while Asparagine is an uncharged, polar poly amino acid; both considered hydrophilic. The effect this difference in charge may have on protein function is unknown. This variant has previously not been reported in African Americans; however, Durocher et al. (31) previously described this variant in an unselected group of women from Quebec, Canada. They reported that this variant was found in three out of 78 controls, but they did not specify its frequency in affected individuals. The fact that the variant was observed in the control group does not necessarily mean that it has a benign effect. Penetrance of any variant conferring increased risk would be dependent on both genetic and other unknown factors. The significance of the high incidence of this missense mutation in our African American breast cancer cases is not yet clear. There were three other rare missense variants/muations in our data set, each identified in only one breast cancer affected individual. The G3238A variant results in a transition 23 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. from G to A that converts codon 1040 from AGC (encoding Serine) into AAC (encoding Asparagine). The T4810C variant coding for a T to C transtion at nucleotide 4810, changes codon 1564 from CTG (encoding Leucine) to CCG (encoding Proline). Serine and Asparagine both are uncharged polar amino acids and Leucine and Proline both are nonpolar amino acids. In each case the change is to an amino acid in the same subclass that is chemically very similar. This suggests that any functional differences between these two variants could be minor. The G3238A variant has been previously reported in one breast cancer patient (28) whose ethnicity was not stated. The T4810C variant has been reported once in a breast cancer affected African American woman (25). A third variant, T5467C is caused by T to C substitution at nucleotide 5467 that changes codon 1783 from ATG (encoding Methionine) to ACG (encoding Threonine). This variant has not been previously reported in any studies of African Americans, although there are three entries in BIC, all with unknown race. Methionine is a nonpolar amino acid and Threonine is an uncharged polar amino acid, belonging to a different subclass, Methionine being hydrophobic and Threonine being hydrophilic. Based on these differences, the change from Methionine to Threonine may possibly have some effect on the protein, and consequently alter its function. These two variants were rare in our sample, and their contribution in the risk of breast cancer has not yet been determined. Functional studies will be required to determine their significance and verify the consequence of these sequence variants/mutations on BRCA1 activity. 24 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. The Al 186G (Gln356Arg) variant is a rare missense variant/mutation, reported frequently in Caucasian populations. The allele frequency in Caucasian populations ranged from 13% to 18% (21, 34) while our estimate in this African American population was ~2%. Other studies of African Americans also estimated the allele frequency to range from 2% to 3% (26,28), consistent with our findings and suggesting the Al 186G variant may be more common in Caucasians than in African Americans. More studies are needed to determine the true frequency of this variant in different populations. Conclusion We developed a high-throughput sequencing method to efficiently and accurately identify sequence variants in the BRCA1 gene of an African American population with high-risks of breast or breast and ovarian cancer. The method involved PCR amplification and Fluorescent Dye Labeling in a 96-well plate format. A multi-capillary DNA device was used to sequence the entire coding region of the BRCA1 gene. Using this approach, we identified 18 sequence variants in 157 African American women from 87 families, more than reported in any previous studies of African Americans. Most variants were present in similar frequency among breast cancer affected individuals and their unaffected sisters. However, our data suggest that the presence of the rare missense variant G2196A (Asp693Asn) or homozygosity for the polymorphic Leu87l (C2731T) allele may be associated with an increased breast cancer risk. Further, our data as well as findings from previous studies of African Americans suggest that the 25 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. missense variant A l 186G (Gln356Arg) may occur less frequently in African Americans than Caucasians although the effect of this variant on breast cancer risk is unknown. Estimated allele frequencies for most of the common variants identified in our samples were lower than published frequencies for Caucasians or Japanese. 26 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. REFERENCES 1. Cancer facts & figures-1995. Atlanta: American Cancer Society, 10-11. 2. Alberg AJ, and Helzlsouer KJ. Epidemiology, prevention, and early detection of breast cancer. Curr. Opin. Oncol. 1997;9:505-511 3. Narod SA, Ford D, Devilee GP, Barkardottir RB, et al. An evaluation of genetic heterogeneity in 145 breast-ovarian cancer families. Am J Hum Genet 1995;56: 254-264. 4. Easton DF, Bishop DT, Ford D, Crockford GP. The Breast Cancer Linkage Consortium. Genetic linkage analysis in familial breast and ovarian cancer. Am J Hum Genet 1993;52:678-701. 5. Claus EB, Risch N, Thompson WD. Genetic analysis of breast cancer in the cancer and steroid hormone study. Am J Hum Genet 1991;48:232-242. 6. Easton DF, Ford D, Bishop DT, et al. Breast and ovarian cancer incidence in BRCA1-mutations. Am J Hum Genet 1995;56:265-271. 7. Couch FJ, DeShano ML, Blackwood MA, Calzone K, et al. BRCA1 mutations in women attending clinics that evaluate the risk of breast cancer. N Engl J Med 1997;336:1409-1415. 8. The New York Breast Cancer Study Collaborative Group (2001). Breast and ovarian cancer risks among women with BRCA1 and BRCA2 mutations in the New York Breast Cancer Study (NYBCS) (Abstract). Am J Hum Genet 2001;68:292. 9. Ford D, Easton DF, Bishop DT, Narod SA and Goldgar DE. Risks of cancer in BRCA1 mutation carriers. Lancet 1994;343:692-695. 10. Hall JM, Lee MK, Newman B, Morrow JE, Anderson LA, Huey B, and King MC. Linkage of early-onset familial breast cancer to chromosome 17q. Science 1990;250:1684-1689. 11. Miki Y, Swinsen J, Shattuck-Eidens D, Futreal PA, Harshman K, et al. A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 1994;266:66-71. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 12. Smith SA, Easton DF, Evans FG, Ponder BA. Allele losses in the region 17ql2- 21 in familial breast and ovarian cancer involve the wild-type chromosome. Nat genet 1992;2:128-131. 13. Chamberlain JS, Boehnke M, Frank TS, et al. BRCA1 map proximal to D17S579 on chromosome 17q2l by genetic analysis. Am J Hum Genet 1993; 52:792-798. 14. Scully R, Anderson SF, Chao DM, Wei W, Ye L, et al. BRCA1 is a component of the RNA polymerase II holoenzyme. Proc Natl Acad Sci USA 1997;94:5605- 5610. 15. Anderson SF, Schlegel BP, Nakajima T, Wolpin ES, Parvin JD. BRCA1 protein is linked to the RNA polymerase II holoenzyme complex via RNA hilicase A. Nat Genet 1998;19:254-256. 16. Hsu LC, White RL. BRCA1 is associated with the centrosome during mitosis. Proc Natl Acad Sci USA 1998;95:12983-12988. 17. Xu X, Weaver Z, Linke SP, Li C, Gotay J, et al. Centrosome amplification and a defective G2 -M cell cycle checkpoint induce genetic instability in BRCA1 exon 11 isoform-deficient cells. Mol Cell 1999;3:389-395. 18. Scully R, Chen J, Plug A, Xiao Y, Weaver D, Feunteun J, et al. Association of BRCA1 with Rad51 in mitotic and meiotic cells. Cell 1997;88:265-275. 19. Chen JJ, Silver D, Cantor S, Livingston DM, Scully R. BRCA1, BRCA2, and Rad51 operate in a common DNA damage response pathway. Cancer Research 1999;59(7 Suppl):1752s-1756s. 20. Breast Cancer Information Core. Available at: http://www.nhgri.nih.gov/Intramural_research/Lab_transferBIC. Accessed Nov.29, 2001. 21. Friedman LS, Ostermeyer EA, et al. Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nat Genet 1994;8:399-404. 22. Castilla LH, Couch FJ, et al. Mutations in the BRCA1 gene in families with early-onset breast and ovarian cancer. Nat Genet 1994;8:387-391. 23. Martin AM and Weber BL. Genetics and hormonal risk factors in breast cancer. J. Nat. Cancer Institute 2000; Vol.92 No.14 1126-1135. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. 24. Szabo Cl and King MC. Population genetics of BRCAl and BRCA2. Am J Hum Genet 1997;60:1013-1020. 25. Shen D, Wu Y, Subbarao M, et al. Mutation analysis o f BRCAl gene in African American patients with breast cancer. J. Nat. Med. Assoc. 2000;92:29-35. 26. Panguluri R, Brody LC, et al. BRCAl mutations in African Americans. Am J Hum Genet 1999;105:28-31. 27. Gao Q, Tomlinson G, Das S, et al. Prevalence of BRCAl and BRCA2 mutations among clinic-based African American families with breast cancer. Am J Hum Genet 2000;107:186-191. 28. Newman B, Mu H, King MC, et al. Frequency of breast cancer attributable to BRCAl in a population-based series of American women. JAMA. 1998; 279:915-921. 29. Kolonel LN, Henderson BE, et al. A multiethnic Cohort in Hawaii and Los Angeles: Baseline Characteristics. Am J Epidemiol 2000;151:346-357. 30. Nickerson D, et al. Phred and Phrap assembly program. Nucleic Acids Res. 1997 Jul 15; 25(14):2745-2751. 31. Durocher F, Sattuck-Eidens D, McClure M, et al. Comparison of BRCAl polymorphisms, rare sequence variants and/or missense mutations in unaffected and breast/ovarian cancer populations. Hum Mol Genet 1996;5:835-842. 32. Inoue R, Fukutimi T, et al. Germline mutation of BRCAl in Japanese breast cancer families. Cancer Research 1995;55:3521-3524. 33. Langston AA, Malone KE, Thompson JD, Daling JR, Ostrander EA. BRCAl mutations in a population-based sample o f young women with breast cancer. N Engl J Med 1997;334:137-142. 34. Dunning AM, Chiano M, et al. Common BRCAl variants and susceptibility to breast and ovarian cancer in the general population. Hum Mol Genet 1997; 6:285-289. 29 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. BIBLIOGRAPHY Alberg, A.J. and K J . Helzlsouer. “Epidemiology, prevention, and early detection of breast cancer.” Curr. Opin. Oncol. 1997;9:505-511 American Cancer Society. “Cancer facts & fignres-1995”. Atlanta: American Cancer Society. 1995;10-11. Anderson, S.F. et al. “BRCAl protein is linked to the RNA polymerase II holoenzyme complex via RNA hilicase A.” Nat Genet 1998;19:254-256. Breast Cancer Information Core. “The Breast Cancer Information Core Database.” [Online] 29 November 2001. <http://www.nhgri.nih.gov/Intramural_research/Lab_transferBIC>. Castilla, L.H. et al. “Mutations in the BRCAl gene in families with early-onset breast and ovarian cancer”. Nat Genet 1994;8:387-391. Chamberlain, J.S. et al. “Boehnke M, Frank TS, et al. BRCAl map proximal to D17S579 on chromosome 17q21 by genetic analysis.” Am J Hum Genet 1993; 52:792-798. Chen, J.J. et al. “BRCAl, BRCA2, and Rad51 operate in a common DNA damage response pathway.” Cancer Research 1999;59(7 Suppl):1752s-1756s. Claus, E.B. et al. “Genetic analysis of breast cancer in the cancer and steroid hormone study.” Am J Hum Genet 1991;48:232-242. Couch, F.J. et al. “BRCAl mutations in women attending clinics that evaluate the risk of breast cancer.” N Engl J Med 1997;336:1409-1415. Dunning, A.M. et al. “Common BRCAl variants and susceptibility to breast and ovarian cancer in the general population.” Hum Mol Genet 1997; 6:285-289. Durocher, F. et al. “Comparison of BRCAl polymorphisms, rare sequence variants and/or missense mutations in unaffected and breast/ovarian cancer populations.” Hum Mol Genet 1996;5:835-842. Easton, D.F. et al. “Breast and ovarian cancer incidence in BRCAl-mutations.” Am J Hum Genet 1995;56:265-271. 30 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Easton, D.F. et al. “The Breast Cancer Linkage Consortium: Genetic linkage analysis in familial breast and ovarian cancer.” Am J Hum Genet 1993;52:678-701. Friedman, L.S. et al. “Confirmation of BRCAl by analysis o f germline mutations linked to breast and ovarian cancer in ten families.” Nat Genet 1994;8:399-404. Ford, D. et al. “Risks of cancer in BRCAl mutation carriers.” Lancet 1994; 343:692-695. Gao, Q. et al. “Prevalence of BRCAl and BRCA2 mutations among clinic-based African American families with breast cancer.” Am J Hum Genet 2000;107:186-191. Hall, J.M. et al. “Linkage of early-onset familial breast cancer to chromosome 17q.” Science 1990;250:1684-1689. Hsu, L.C. and R.L. White. “BRCAl is associated with the centrosome during mitosis.” Proc Natl Acad Sci USA 1998;95:12983-12988. Inoue, R. et al. “Germline mutation of BRCAl in Japanese breast cancer families.” Cancer Research 1995;55:3521-3524. Kolonel, L.N. et al. “A multiethnic Cohort in Hawaii and Los Angeles: Baseline Characteristics.” Am J Epidemiol 2000;151:346-357. Langston, A. A. et al. “BRCAl mutations in a population-based sample of young women with breast cancer.” N Engl J Med 1997;334:137-142. Martin, A.M. and B.L. Weber. “Genetics and hormonal risk factors in breast cancer.” J. Nat. Cancer Institute 2000; Vol.92 No. 14 1126-1135. Miki, Y. et al. “A strong candidate for the breast and ovarian cancer susceptibility gene BRCAl.” Science 1994;266:66-71. Narod, S.A. et al. “An evaluation of genetic heterogeneity in 145 breast-ovarian cancer families.” Am J Hum Genet 1995;56:254-264. Newman, B. et al. “Frequency of breast cancer attributable to BRCAl in a population- based series of American women.” JAMA. 1998;279:915-921. The New York Breast Cancer Study Collaborative Group. “Breast and ovarian cancer risks among women with BRCAl and BRCA2 mutations in the New York Breast Cancer Study (NYBCS).” Am J Hum Genet 2001;68:292. Nickerson, D. et al. “Phred and Phrap assembly program.” Nucleic Acids Research 1997 Jul 15; 25(14):2745-2751. 31 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Panguluri, R. et al. “BRCAl mutations in African Americans.” Am J Hum Genet 1999;105:28-31. Scully, R. et al. “Association of BRCAl with RadSl in mitotic and meiotic cells.” Cell 1997;88:265-275. Scully, R. et al. “BRCAl is a component of the RNA polymerase II holoenzyme.” Proc Natl Acad Sci USA 1997;94:5605-5610. Shen, D. et al. “Mutation analysis of BRCAl gene in African American patients with breast cancer.” J. Nat. Med. Assoc. 2000;92:29-35. Smith, S.A. et al. “Allele losses in the region 17ql2-21 in familial breast and ovarian cancer involve the wild-type chromosome.” Nat genet 1992;2:128-131. Szabo, C.I. and M.C. King. “Population genetics of BRCAl and BRCA2.” Am J Hum Genet 1997;60:1013-1020. Xu, X. et al. “Centrosome amplification and a defective G2 -M cell cycle checkpoint induce genetic instability in BRCAl exonl 1 iso form-deficient cells.” Mol Cell 1999;3:389-395. 32 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.

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Creator Xia, Lucy Yining (author)

Core Title BRCA1 mutations and polymorphisms in African American women with a family history of breast cancer identified through high throughput sequencing

School Graduate School

Degree Master of Science

Degree Program Applied Biostatistics and Epidemiology

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

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

Language English

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Advisor Henderson, Brian (committee chair), Coetzee, Gerhard (committee member), Ursin, Giske (committee member)

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