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RISK MODELING AND SCREENING FOR BRCAI MUTATIONS AMONG FILIPINO BREAST CANCER PATIENTS

by

ALEJANDRO Q. NAT09 JR.

A Master's Thesis Submitted to the National Institute of Molecular Biology and Biotechnology College of Science University of the Philippines Diliman, Quezon City

As Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY

March 2003 In memory of my gelovedmother

Mrs. josefina Q -Vato who passedaway while waitingfor the accomplishment of this thesis...

Thankyouvery inuchfor aff the tremendous rove andsupport

during the beautifil'30yearstfiatyou were udth me...

Wom, you are he greatest!

I fi)ve you very much!

.And..

in memory of 4 collaborating 6reast cancerpatients

who passedaway during te course of this study ...

I e.Vress my deepest condolence to your (overtones...

Tou have my heartfeligratitude!

'This tesis is dedicatedtoa(the 37 cofla6oratingpatients who aftruisticaffyjbinedthisstudyfor te sake offuture generations... iii

This is to certify that this master's thesis entitled "Risk Modeling and Screening for BRCAI Mutations among Filipino Breast Cancer Patients" and submitted by Alejandro Q. Nato, Jr. to fulfill part of the requirements for the degree of Master of Science in Molecular Biology and Biotechnology was successfully defended and approved on 28 March 2003.

VIRGINIA D. M Ph.D. Thesis Ad

RIO SUSA B. TANAEL JR., M.Sc., M.D. Thesis Co-.A. r Thesis Reader

The National Institute of Molecular Biology and Biotechnology endorses acceptance of this master's thesis as partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology and Biotechnology.

,_all CYNTHI YDA, Ph.D. Director National Institute of Molecular Biology and Biotechnology

This master's thesis is hereby officially accepted as partial fulfillment of the requirements for the degree of Master of Science in Molecular Biology and Biotechnology.

RHODORA Ph.D. Dean, Ilege of Science iv

PLEASE POST PLEASE POST

THE NATIONAL INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY College of Science University of the Philippines Diliman, Quezon City

ANNOUNCEMENT OF THE MASTERAL EXAMINATION

Of

ALEJANDRO Q. NATO, JR.

In defense of his Masteral Thesis

"Screening by PTT, SSCP, DHPLC and Bayesian Modeling Techniques for,6RCAI Mutations Among Early-Onset and Familial Filipino Breast Cancer Cases"

For the degree of Master of Science in Molecular Biology and Biotechnology

9:00 A.M., Friday, 28 March 2003 Room 102, Albert Hall

THESIS ADVISER THESIS CO-ADVISER

VIRGINIA D. MONJE, Ph.D. APOLINARIO D. NAZAREA, Ph.D. Professor Professor National Institute of Molecular Biology Science and Society Program and Biotechnology College of Science College of Science University of the Philippines University of the Philippines Diliman, Quezon City Diliman, Quezon City

THESIS READER

SUSANO B. TANAEL, JR., M.Sc., M.D. Associate Professor Biology Department College of Science De La Salle University Taft Avenue, Manila

THESIS EXAMINER THESIS EXAMINER

AMEUIRFINA D. SANTOS, Ph.D. DR. CYNTHIA P. SALOMA Professor Associate Professor National Institute of Molecular Biology National Institute of Molecular Biology and Biotechnology and Biotechnology College of Science College of Science University of the Philippines University of the Philippines Diliman, Quezon City Diliman, Quezon City

ENDORSED BY: AUTHORIZED BY:

VIRGINIA D. RHODO ZA Ph.D. Director M* 1 Dean National Institute of Molecular Biology and College c ence Biotechnology University of the Philippines College o Science Diliman, Quezon City University of the Philippines Diliman, Quezon City V

BIOGRAPHICAL DATA Adwbiwh

Alejandro Q. Nato, Jr. Science Research Specialist 1 Health Physics Research Section, Atomic Research Division Philippine Nuclear Research Institute, Commonwealth Avenue, Difirrian I 01 Quezon City, Philippines aqnatopnri dost.gov.ph / aqnjpworld.nel.ph laqnjryalioo.coin Tel 632-9296011 loc. 235 Fax 632-9201646

Summary of Qualifications

Molecular Biology Molecular Genetics Radiation Biochemistry Performing (a) radioactive protein truncation test (using '5S) utilizing SDS-PAGE visualized by autoradiography and (b) single-sirand confiannation polymorphism analysis utilizing PAGE visualized by silver staining, in a pioneering endeavor to detect BRCAJ truncating mutations and/or putative polymorphisins among eaTly-onset and familial Filipino breast cancer patients Utilizing BRCAPRO, to determine apriori and aposterioriBRCA112 carr ier probabilities of breast cancer patients Performing DNA Analysis aer extracting genornic DNA from whole blood of familial and early-onset breast cancer patients Knowledgeable in the role of RRCA I gene mutation and the pathogenesis of breast cancer Utilized silver staining procedure for visualizing AFLP-PCR profile of radiation-inducedvariants and compared it with profile obtained sing `P Performing ALP-PCR Mutational Screening of "Co y radiation-induced variants of onamentals and foliage plants (e.g., Hurraya exotica and Dracaena sanderiona)after extracting plant genorruc DNA using ethanol precipitation or plant DNAzol as part of te IAEA TC Project PHI/5/027 Participated in reporting accomplishments for te IAEA TC Project on Mutation Breeding Studied te effect of different doses of Radiation on te proteins of Vigna ungulculaia Utilized 2-inelhyl DOPA as sbstrate to detect tyrosinase hrough production of dopachl'Orne found to have a peak at 484 nm Determined radioresistance of P. acruginosautilizing different doses of y-rays Drafted a synthesis of te recent trends in Hepatitis C Virus HCV) studies Performed a initial sudy on te tilization of AtB and AtC insect mosquito) cell lines in ile propagation of dengue virus and sbsequent detection usirig RT- PCR nd agarose gel clectrophoresis Determined CTT Tplex (CSFIPO, THOI, TPOX) STR loci fingerprint in a Filipino (Bicol) population utilizing PCR and PAGE vsualized hrough silver stainingof DNA bands Performed TXRF to detect trace elements present in isolated Gs-protein Performed gel electrophoresis (SDS-PAGE) as an initial sep in he isolation of Gs-protein of ile Oriental fruit fly (Bociroceraphilippinunsts) and sbsequent Iyophilization of isolated Gs-protein for electrophoretic-rerunning purposes Performed chromophore experiment and tyrosinase assay (using oxidized L-tyrosine as the substrate) for the Pupal homogenate of B. philippmenw to determine other possible effects of irradiation a 100 Gy Performed Bradford assay to determine aount of Gs-protein per pupa of B. philippinensis Performed human peripheral blood ymphocyte isolation and sbsequent trichloroacetic acid DNA extraction to determine the HGPRT mutation indices of Valerizuela and Las Pirlas residents followed by performing statistical calculations to analyze te database obtained

Health Pysics Environmental Participated as Pilippine Representative in te IAEA/RCA Poject Formulation Meeting on ]improving Regional Capacity for Assessment, Planning ad Responding to Aquatic Environmental Emergencies, 21-25 July 2003 Astralia Produced contour, wirefirarrie, and post aps of inarine radioactivity for te whole Asia-Pacific Region sing Surficr 7 for Windows s pail ofthe compilation of te Asia-Pacific Marine Radioactivity Database (ASPAMARD I and 2) Performed environmental and radiological surveillance in (a) former US Bases: Clark ad Sbic, (b) anufacturing corporation in the NCR, ad (c) oillerareas of interest in tile Pilippines (eg_ Palawan, Tanay, and Subic) sing the Berkeley Nucleonics Surveillance and Measurement Systems (BNC SAM935) coupled With QUantuin MCA Gold Soflware for quantitative radionuclide analysis and sared knowledge with colleagues Performed sediment sampling sediment core grab sampling) in Sbic Bay, murtian Bay Palawan), and Sulu Sea Performed initial study on radionuclide igration in Kara Sea sing tree-dimensional modeling (FLESCOT) as part of OJT on Dose Assessment in Battelle Pacific Northwest National Laboratory, Richland, WA, USA Environmental Srveillance within PNRI. at the perimeter of PNRI, and 25 km radius of PNR1 sing file igh pressure ionization camber and carborne y spectrometer (Exploranium 650) to easure abient y-radiation dose rate Participated as member of the Secretarial in the IAEA Project Formulation Meetings on ) Marine Coastal Environment and Its Pollution, 23-27 February 1998 Pilippines and 2) Reference Asian Man, Phase 11,June 1996 Pilippines Knowledgeable in the preparation of mixed radionuclide standards to be sed for HPGe detector

Advisorship Supervised and trained (year-, month-, or week-long) on-tlie-job-training and tesis students both local and international- graduate, undergrad. or igh school students) regarding vst range of laboratory echniques and topics

Computer Work Gathered PDF files offinportant scientific primary journal articles by downloading from te internet and interacting with the corresponding aLlihors Gathering ofhuge aount ofscientific data(i.e., binary files) and literature fro te Interne trough UNIX ad Windms-based systerns Made vrious templates aving appropriate formulae to aid computation of data for both environmental ad somatic mutation projects and lso for tile different administrative ad euipment request forms of te Health Pysics Research Section (HPRS) Gathered scientific research data through CD-ROM and University libraries (UPEPSF) Computed statistical antities concerning, srident needs at the national scale (UP OCG) Vi

Administrative Fine-tuned HPR procurement and onthly stock requisition from 1997-present by aking a computerized template Established diskette directory of te PRS Forniatted 2' announcement for final print out of programme for 2' Philippine Nuclear Congress (I 996) Taught computer software to officemaies Coordinated and spervised te forrialling and layouting of The Nucleus with te printing press and performing typographical ad grammatical corrections whenever necessary Acted as a Spply Officer in UP San Fernando -Diliman Office (which includes processing of requisition, payment and reimbursement of expenses concerning office equipment and spplies) ClericalICOMPLIter work includingjournal format layouting (UPEPSF)

Other Highlights Founding Member Constitutional Committee Chairperson I Membership Corrimiuee Assistant Chairperson (one of the steering cmmittee members who ensured srvival ofthe orpanization dring its first year), U.P. Green League (viectedos one afthe opi-lxunivxritty-bavede)rganizatiens, 199i awardedBusl Environmental Organization by DENR, 1998 awarded Presidential Youth Achievement Awardee in Evironmental Managemen Ctegoty. " PREYAA, Malacohang Palace, 2000, awordedastheBestOrganaoijon qUPDiliman. GowadChancellorAward, 2000) AY 1992-93 Corporate Secretary of te Ryal Cancer Research Fund-Philippines, Inc. (SEC A200009305)

Skills Familiar with IBM-PC Pentium IV and AMD-Athlon GHz or lower and proficient in Windows 95/98SE/ME1XP (Microsoft Oflice, Wordperfeci, rfanview, and oher variou%.%ofiware.%) Familiar with Three-Dimensional Modeling UNIX-based softwares like TEMPEST and FLESCOT

Career Highlights

2003 Philippine Representative during the IAEA/RCA Project Formulation Meeting on Improving Regional Capacity for Assessment, Planning, and Responding to Aquatic Environmental Emergencies, Australia (RAS/8/095) 2003 Lecturer on the search for heritable mutated breast cancer genes among- Filipinos by nuclear and molecular techniques: The BRCA I example 2002 Lecturer/Presentor on Bayesian probability mdeling for BRCAl mutation among early-onset and familial Filipino breast cancer cases 2002 Joint FAO/IAEA Fellow (FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria) 2001 Lecturer on applications of radioisotopes in molecular biology 2001 Member of the Philippine Nuclear Research Institute (PNRI) Website Development Committee 2001 Observer and Secretariat Member durin- te Re-ional Training Workshop on Modeling Water Quality and Validation usin- Radiotracer Techniques (Han-riful Algal Bloom Issues), Philippines, utilizing RMA software for 3D radiotracer modeling in the marine environment 2000-present Managing Editor of the PhilippinesNuclear Journal, the official pub] ication of the Philippine Nuclear Research Institute 2000-present Constitutional Committee Member of the UP Green League Alumni Association 2000 Corporate Secretary of the Royal Cancer Research Fund-Philippines, Inc. (SEC A200009305) 1999-present Project Co-Investigator and Bench Researcher of PNRI Research Pro.gram on Gene Technologiesfor Improving Cancer Monagenieni, Early Detection, and Prevention. Received financial assistance from the DOST-GIA, GATT-assisted projects, Philippine Charity Seepstakes Office PCSO), PCASTRD, and OVCRD 1999-present Study Leader and Bench Researcher of AEA- and DOST-funded PNRI Research Project on AFLP-PCR olecular Screening of "Co yRodiation-Induced Variants on Selected Ornamentaland Foliage Plants (PH1151027) 1999 Lecturer on radiation protection (dose assessment): environmental risk assessment and 3D modeling 1999 IAEA Fellow (Battelle Pacific Northwest National Laboratory, Richland, WA, USA) 1998 Lecturer/Presentor on BRCAI PTT as a definite eye opener for early detection of breast cancer 1998 Lecturer on evironmental radioactivity analyses in Japan 1998 Chairman of the Souvenir and Souvenir Program Committee during the Atomic Energy Week 1998 (AEW 1998) 1998 Secretariat Member of the Project Formulation Meeting on Marine Coastal Environment and Its Pollution, Philippines 1996-present Thesis/Research Adviser or Consultant 2002 BRCAl mutation and polymorphisms in breast cancer patients using PTT and SSCP (Gretchen Gayle S. Panganiban and Josephine Ann G. Santos from University of the Philippines - Manila) 2000 Putative radioresistant bacterial isolate from sewage ater (April A& Patricia Chua, Kristine Perez, April Rey, Kristel Rivor, Czarina San Pablo, and Enestine Santos from Miriam College High School) 1998 SDS-PAGE and its applications Mumi Indarwatmi, LAEA Fellow from Indonesia) 1997 Effects of y-radiation on Vigna unguiculata (Michelle A. Rosario.and JeSLIsa Brbara Gloria S Butor from Centro Escolar University) 1997 Interaction of radioprotective effects of reduced gutathione and butylated hydroxyanisole a known inducer of glutathione synthase in 3rd instar larvae of Bactrocera philippinensis (Dyan BuenaventUra, Samantha Marcelo and Elena Dacanay from Philippine Science High School): Finalist during the 49"' NCR Search for INTEL Outstanding Research, High School Division) 1996 Laboratory Techniques Sherwin Sayson fom Philippine Science High School) 1997 Nlanaging Editor of The Reactor, the official newsletter of the PNRI Employees' Union 1997 Chairman of the Technical Poster Committee (NukTeck 97) durin- the AEW 1997 1997 Invitation Committee Member of the Pilippine Association for Radiation Protection Biennial Convention 1996 JICA/JCAC Fellow Japan Chemical Analysis Center. Chiba, Japan) 1996 Co-Chairnian of the Technical E\hibits Committee durin- the AEW 1996 Vii

1996 Secretariat Member of the Project Formulation Meeting on Reference Asian Man, Phase 11,Philippines 1996 Technical Committee Member (Poster and Program) of the 2,d Philippine Nuclear Congress 1996 Tour Guide Committee Member during the AEW 1996 1995-1996 Editorial Staff (Editorial Asst.) of The Nucleus, the official publication of the Radioisotope Society of the Philippines 1995 Co-Chairman of the Tour Guide Committee during the AEW 1995 1995 Participant in the Hands-on Workshop on SDS Gel Electrophoresis, Flatbed soelectric Focusing, and Immunoblotting by the MBB Program (UP Diliman) 1994-present Bench Researcher and Field Work Staff for the following projects: 2002-present DOST-funded PNRI Research Program on Environmental Radioactivity Studies (Project Leader: Teresa Y. Nazarea) 200 -present CTBTO-funded Site Survey on ProposedSiresfor RN-.52 (Project Leader: Teresa Y. Nazarea) 1999-2001 IAEA-funded PNRI Project on Marine Radioactivity in South China Sea (Project Leader: Dr. Emerenciana B. Duran) 2000-2001 DOST-funded PNRl Research Project on Environmental and RadiologicalSurveillance in Former US Bases: Subic and Clark (Project Leader: Dr. Emerenciana B. Duran) using carbome spectrometer (Exploranium 650) 1999-2002 IAEA-funded compilation of the Asia-Pacific Marine Radioactivity Database (ASPAXMRD) (Project Leader: Dr. Emerenciana B. Dran) Specific responsibility.contour mapping of marine radioactivity in the whole Asia-Pacific Region using Surfcr 7 for Windows 1995-1999 DOST-funded PNRI Research Program on Evironmental Radioactivity Measurements in the Philippines (Program Leader: Dr. Emerenciana B. Duran) using high-pressure ionization chamber (HPIC) and carbome spectrometer (Exploranium 650) 1994-1998 DOST-funded research on Detection Methodfor irradiatedOriental Fruit Fly (Dacia dorsalis) quarantine purposes (Project Leader: Teresa Y. Nazarea) 1994-1995 PCHRD-funded research on Somatic Mutation in Peripheral Blood Lymphocytes mong Metro Manila Residents: ndicatorqfExposure o EnvironmentalPollution (Project Leader: Teresa Y. Nazarea) 1994 Participant in he AEA/PNRI/PCASTRD-sponsored I" National Training Course on Radiation Chemistry 1994 Participant in the Computerized Database Management Programming Course by PNRI 1994 Documentation and Proceedings Committee Member of te International Conference on Food Preservation and Sec6rity 1994 Tour Guide Committee Member during the AEW 1994 1993 Secretariat Member of te I" Global Youth Earth Saving Summit (Global YES)

Professional Background

Employment 2002-present Science Research Specialist II, Philippine Nuclear Research Institute 1994-2002 Science Research Specialist 1, Philippine Nuclear Research Institute 1993 University Research Associate I, UP Extension Program in San Fernando. Pampanga 1992-1993 Student Assistant, Officc of Counseling and Guidance, University of the Philippines-Diliman 1992 Interviewer, UPEHCO, University of the Philippines-Diliman 1990-1991 Student Assistant, Office of Counseling and Guidance, University of the Philippines-Diliman 1990 SWAP Trainee, Benguet Corporation

Education 2003 University of the Philippines - Diliman, M.Sc. in, Molecular Biology and Biotechnology Specialization: Molecular Genetics 1993 University of the Philippines - Diliman, B.Sc. in Molecular Biology and Biotechnology 1988 Philippine Science High School (Overall Top Ten Best in Research) 1984 Ednas School (Salutatorian/ Best in Math) 1976 Ednas School (Most OutstandingStudent)

Research Interests

M.S. Thesis Risk Modeling and Screening for BRCA I Mutations among Filipino Breast Cancer Patients

B.S. Thesis Design of Multivalent Synthetic Peptide Vaccines against Infectious Laryngotracheitis Virus and Equine lierpesviruses I and 4

Hi0 School Research Lime from Talaba (Crassostreavirginica) Shells as Block Additive (Winner of the 988 PSHS Science Fair) viii

Professional Awards, Scholarships, and Distinctions

Post-graduate 2003 Philippine Representative at the IAEA/RCA Project Formulation Meeting on Improving Regional Capacity for Assessment, Planning and Responding to Aquatic Environmental Emergencies (ANSTO, Lucas Heights, NSW, Australia) 2002 Joint FAO/IAEA Fellow at the AO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, Austria on Mutant Germplasm Characterization using Molecular Markers 2001-present DOST-Philippine Council for Advanced Science and Technology Research and Development (DOST- PCASTRD) Thesis Grant Recipient 2001-present UP-Office of the Vice-Chancellor for Research and Development (UP-OVCRD) Thesis Grant Recipient 1999 IAEA Fellow at the Battelle Pacific Northwest National Laboratory (Richland, WA, USA) on Radiation Protection (Dose Assessment): Environmental Risk Assessment and 3D Modeling 1998, 999 Nominated by PNRI (Dr. Alumanda M. dela Rosa) for the Philippine Talent Search for Young Scientists Award by DOST-NAST 1996 JICA/JCAC Fellow at the Japan Chemical Analysis Center (Chiba, Japan) on Environmental Radioactivity Analysis and Measurement 1995 I" Place, AEW Poster Presentation by PNRI for the study "Gs-protein: A tyrosinase enzyme from Bactroceraphilippinensis " 1994 2 dPlace, AEW Poster Presentation by PNRI for the study "Detection Method for Irradiated Oriental Fruit Fly (Dacus dorsalis) for Quarantine Purposes" 1994 V Place, Poster Presentation (Undergraduate Category) during the I h National Convention of Philippine Biochemistry and Molecular Biology (PSBMB) for the study, "Design of Multivalent Snthetic Peptide Vaccines Against Infectious Laryngotracheitis Virus and Equine Herpesviruses I and 4" 1994 1" Place, AEA/PNRIIPCASTRD-sponsored V National Training Course on Radiation Chemistry by PNRI 1994 3'd Place, 74'h Radioisotope Techniques Training Course (RTTC - Agriculture by PNRI 1993 Eligibility for Government Service (Career Service Professional Examination), July 8

Undergraduate 1990 University Scholar W.A.=1.403) by the University of the Philippines-Diliman 1988 University Scholar W.A.=1.218) by the University of te Philippines-Diliman 1988-1993 DOST-Science Education Institute (DOST-SEI) Scholarship Recipient 1988-1993 Jaime V. Ongpin Fund Scholarship Recipient by Benguet Corporation High School, Elementary, and Preparatory 1988 99+ Rating inthe National College Entrance Examination 1988 Best in Research of Batch 1988 (Recipient of the Mercury Drug Medalfor Excellence in Science) by the Philippine Science High School (PSHS) 1988 Tagumpay ng Isang Ina (Single Parent Award) Recipient by PSHS 1988 V Place in Research Poster by PSHS for the study "Lime from Talaba (Crassostrea virginica) Shells as Block Additive" 1984-1988 Consistent Director's Lister by PSHS (Highest rank obtained was 6 among 240 excellent students from a] I over the country) 1984-1988 PSHS Full Scholarship Recipient 1984-1988 Godparent Program Assistance Recipient 1984 I" Loyalty Award by Ednas School 1984 3rd Place in the National Quiz Bee. (Regional Level) by the Ministry of Education, Culture, and Sports (MECS) 1984 V Place in the National Quiz Bee (Division Level) by MECS 1981-1983 Half Scholarship Recipient due to Academic Excellence by Ednas School 1978-1984 Four (4)-time I"Place Winner of Declamation, Tula, or Vocal Solo by Ednas School 1978-1984 Seven (7)-time I" Place Winner of School Quiz Bees (Math, Science, and Spelling) by Ednas School 1976-1984 Consistent Academic Excellence by Ednas School (preparatory and elementary) ix

Publications

Technical Publications/Abstract 1. Custer C. Deocaris, Maria Cereza R. Velasco, Earl Louis Sempio Junie B. Billones and Aleiandro 0. Nato, Jr. 2002 An evolutionary perspective on the possible involvement of plasminogen in the pathology of bovine spongiform encelopathy. Phil J Vet Med Anim Sci. 28(2):58-68. 2. Aleiandro 0. Nato, Jr., Eliza B. Enriquez, Ariel T. Ortiz, and Custer C. Deocaris. 2001. Putative bioindicator Of 137CS in Perna viridis. Philipp Nucl J. 13:77-79. 3. Teresa Yulo-Nazarea, Ma. Lucia C. Cobar, Ale'andro 0. Nato, Jr., and Apolinario D. Nazarea. 2001. Somatic mutation in peripheral blood lymphocytes among Metro Manila residents: Indicator of exposure to environmental pollution. Philipp Nucl J 3:1 1 2 2. 4 Teresa Yulo-Nazarea and Ale6andro 0. Nato, Jr. 1996. Gs-protein: A tyrosinase enzyme from oriental fruit fly, Bactrocera philippinensis. The Nucleus. 32: 49. [Abstract only]

In Press 1. Custer C. Deocaris Agentina S. Casii)o, Maria Katrina Braulio, Gavino Rommel A. Cureg, and Alejandro Q. Nato. Jr. 2003. Anhydro-preserved bovine semen maintained at room temperature in trehalose extender. Phil J Yet MedAniin Sci (submitted for publication) 2. Custer C. Deocaris, Aleiandro 0. Nato Jr., and Apolinario D. Nazarea. 2003. Molecular design of a trivalent synthetic vaccine against anthrax. PhiIJVet MedAnim Si (submitted for publication)

International Non-Technical Publication I Aleiandro 0. Nato, Jr. 1997 A relatively impeccable first hand stint. Japan ChemicalAnalysis Center. 31:114-119.

International Technical Reports 1. Alejandro Q. Nato, Jr., Sheila C. Sajise, and Custer C. Deocads. 2002. Low incidence of germline mutation in BRCA1 exon II among early-onset and familial Filipino breast cancer patients. INIS PNRICNMO2002. 2. Ale4andro 0. Nato, Jr., Sheila C. Sajise, and Custer C. Deocaris. 2002. Radioactive PTT as part of screening protocol for prospecting radiation workers. INIS PNRICNMO2001. 3. Teresa Yulo-Nazarea and Ale'andro 0. Nato, Jr. 1994. Detection method for irradiated oriental fruit fly Dacus dorsalis) for quarantine purposes. INIS# PNRICAG94008. 4. Teresa Yulo-Nazarea, Ma. Lucia C. Cobar, Marla A. Endriga, Efren J. Sta. Maria, Aeiandro Q. Nato, Jr. et a. 1994. Somatic mutation in peripheral blood lymphocytes among Metro Manila residents: Indicator of exposure to environmental pollution. INIS# PNRICHP94007.

Proceedings 1. Ale*andro Q. Nato, Jr., Sheila C. Sajise, and Custer C. Deocaris. 2002. Radioactive PTT as prt of screening protocol for prospecting rdiation workers. Proceedings of the InternationalYouth Nuclear Congress 2002, Daejeon, KOREA. 6-20 April 2002. 2. Custer C. Deocaris and Aleiandro 0. Nato, Jr. 200 . Nuclear energy in the post-genomic era. Proceedings of the Korean Nuclear Society KNS) Autumn Meeting, Seoul, KOREA. 79-88.26 October 2001. 3. Custer C. Deocaris, Argentina S. Casino, Ma. Katrina G. Braullo, Joselito E. Muldera, Melvin F. Estonactoc. Gavino Rommel A. Cureg, and Aleiandro 0. Nato, Jr. 2001. Can bovine sperm be preserved at room temperature? A proof of concept. Proceedings of the PSAS 38"'Annual Convention. Manila, PHILIPPINES. 18-19 October 2001. p.21. 4. Custer C. Deocaris, Maria Cereza R. Velasco, Earl Louis Sempio Junie B. Billones and Ale'andro 0. Nato, Jr. 2001. Molecular evolution of the md cow's disease: Co-evolution theory of prions and prion-targets. Proceedings of the PSAS38'h Annual Convention. Manila, PHILIPPINES. 18-19 October 2001. p.1 12. 5. Emerenciana B. Duran, Eliza B. Enriquez, Cecilia M. De Vera, Carol B. Coloma, Alejandro Q. Nato, Jr., Teresa Yulo- Nazarea, and Ma. Lucia C. Cobar. 2001. Asia-Pacific marine radioactivity database (ASPAMARD). Proceedings of the 17'4 Philippine Chemistry Congress. Xavier Sports and Country Club, Cagayan de Oro City, PHILIPPINES. 23-25 May 2001. 6. Teresa Yulo-Nazarea, Alciandro 0. Nato, Jr., and Carol B. Coloma. 2001. AFLP-PCR fingerprints of 60CO radiation- induced variants of Murraya exotica and Dracaena sanderiana. Proceedings of the 17 M Philippine Cemistry Congress. Xavier Sports and Country Club, Cagayan de Oro City, PHILIPPINES. 23-25 May 2001. 7. Avelina G. Lapade*, Teresa Yulo-Nazarea, Ana Maria S. Veluz, Lucia J. Marbella, Alejandro 0. Nato, Jr., and Carol B. Coloma. 1999. Use of radiation-induced mutations and biotechnology for the development of new varieties of foliage and cutflower ornamentals. A011AEA Seminar on Mutation Techniques and Molecular Genetics for Tropical and Subtropical Plant mprovemen i Asia and the Pacific Region. ACCEED Conference Center, Makati City, PHILIPPINES. 11-15 October 1999. 8. Custer C. Deocaris*, Ae*andro . Nato, Jr., Apolinario D. Nazarea, and Angel B. Mateo. 997. Development of a rational approach in synthetic vaccine engineering subverting antigen rifts of the FMDV. Proceedings of the 64'b Annual X

Convention ofthe Phihvpine Veterinary Medicine Association VM4). Cebu Plaza Hotel, Cebu City, PHILIPPINES. 9-21 February 1997. 9. Deocaris CC*. Nato AJ , Nazarea AD and Mateo AB. 1996 'Winged' hypermutated epitopic mimetics of VP1 (135- 146) A novel molecular design for broad spectrum, multivalent synthetic vaccines for the foot-and-mouth disease virus serotype A. Recent Research Developments in Buffalo Production - Proceedings of he 2d Asian Buffalo Association Congress. Shangri-la Hotel, Makati City, PHILIPPINES. 912 October 1996. 181-193.

Presentations

Papers Presented - International (*Oral Presentor) 1. Ale*andro 0. Nato, Jr.* 2003. Country Report for the 1AEA/RCA Project Formulation Meeting on Improving Regional Capacity for Assessment, Planning and Responding to Aquatic Environmental Emergencies. Australian Nuclear Science and Technology Organization, Lucas Heights, NSW AUSTRALIA. 21-25 July 2003. 2. Teresa Yulo-Nazarea, Aleiandro 0. Nato, Jr.*, and Carol B. Coloma. 2002. AFLP-based characterization of ornamental crops and foliage plants with their radiation-induced variants. Short presentation during he FA017AEA Interregional Training Course on Mutant Germplasm Caracterization using Molecular Vfarkers 11 from 429 November 2002. FAO/IAEA Agriculture and Biotechnology Laboratory, Seibersdorf, AUSTRIA. 3. Alciandro 0. Nato, Jr., Sheila C. Sajise, and Custer C. Deocaris*. 2002. Radioactive PTT as part of screening protocol for prospecting radiation workers. During the International Youth Nclear Congress 2002, Daejeon, KOREA. 16-20 April 2002. 4. Custer C. Deocaris* and Aleiandro 0. Nato, Jr. 2001. Nuclear energy in the post-genomic age. During thedsian Young Generation orkshop (Theme: The Current and Future of Nuclear Energy in Asia) from 24-26 October 2001. Khunghee University, SuNvon, KOREA. 5. Avelina G. Lapade*, Teresa Yulo-Nazarea. Ana Maria S. Veluz, Lucia J. Marbella, Aleiandro 0. Nato, Jr., Carol B. Coloma, and Apolinar B. Asencion. 2001. Status of biotechnology with emphasis on molecular techniques for mutation breeding in the Philippines. During he Forumfor Nuclear Cooperation in Asia Workshopfor Plant Mutation Breeding: MolecularBiological Techniquesfor Mutation Breeding from 20-24 August 200 1. Bangkok, THAILAND. 6. Avelina G. Lapade*, Teresa Yulo-'Nazarea, Ana Maria S. Veluz, Lucia J. Marbella, Alejandro Q. Nato, Jr., and Carol B. Coloma. 999. Use of radiation-induced mutations and biotechnology for the development of new varieties of foliage and cutflower ornamentals. During the A011AEA Seminar on Mutation Techniques and Molecular Geneticsfor Tropical and Subtropical Plant Improvement in Asia and he Pacific Region from I I - October 999. ACCEED Conference Center, Makati City, PHILIPPINES. 7. Custer C. Deocaris*, Ale*andro 0. Natq_L, Apolinario D. Nazarea, and Angel B. Mateo. 1996 'Winged' hyper-mutated epitopic mimetics of VP1 135-146) A novel molecular deic n for broad spectrum, multivalent synthetic vaccines for the foot-and-mouth disease virus serotype A. Dring the 2 d Asian Buffalo Congress (ABA) from 912 October 1996. Shangri-La Hotel, Makati City, PHILIPPINES. 8. Ale'andro Q. Nato, Jr.* 1996. Country Report for the JICA/JCAC Group Training Course in Environmental Radioactivity Analysis and Measurement. Japan ChemicalAnalysis Center, Chiba, JAPAN. 7 September 1996.

Papers Presented - National (*Oral Presentor) I. Alewandro 0. Nato. Jr.*, Sheila C. Sajise, and Custer C. Deocaris. 2002. Bayesian probability modeling for BRCAI mutation among early-onset and familial Filipino breast cancer cases. Dring he 29h PSBMB Annual Convention from 5-6 December 2002. SEARCA Auditorium, UP Los Baftos, College, Laguna, PHILIPPINES, 2. Avelina G. Lapade*, Teresa Yulo-Nazarea, Ana Maria S. Veluz, Lucia J. Marbella, Alejandro 0. Nato. Jr.. Carol B. Coloma, and Manny G. Rama. 2001. Mutation breeding in ornamentals. Duringthe Technical Sessions of the 29h Atomic Energy 1Veek on I I December 200 1. PNRI, Quezon City, PHILIPPINES. 3. Teresa Yulo-Nazarea*, Aleiandro 0. Nato, Jr.. and Carol B. Coloma. 2001. AFLP-PCR fingerprints of 6CO y radiation- induced variants of Murraya exotica and Dracaena sanderiana Dring the 17'h Philippine Chemistry Congress from 23- 25 May 2001. Xavier Sports and Country Club, Cagayan de Oro City, PHILIPPINES. 4. Emerenciana B. Duran*, Eliza B. Enriquez, Cecilia M. De Vera, Carol B. Co[oma, Alewandro 0. Nato. Jr., Teresa Yulo- Nazarea, and Ma. Lucia C. Cobar. 2001. Asia-Pacific marine radioactivity database (ASPAMARD) Dring he 17` Philippine Chemistry Congress from 23-25 May 2001. Xavier Sports and Country Club, Cagayan de Oro City, PHILIPPINES. 5. Custer C. Deocaris*, Argentina S Casino, Ma. Katrina G. Braullo, Joselito E. Muldera, Melvin F. Estonactoc. Gavino Rommel A. Cureg. and Ale6andro 0. Nato. Jr. 2001. Can bovine sperm be preserved at room temperature? A proof of concept. During he 38"'Annual Convention of the Philippine Society of Animal Science (PSAS) from 18-19 October 200 . Heritace Hotel. Metro Manila. PHILIPPINES. 6. Custer C. Deocaris*. Maria Cereza R. Velasco Erl ouis Sempio Junie B. Billones and Alejandro Q. Nato. Jr 2001. Molecular evolution of the mad cows disease: Co-evolution theory of prions and prion-targets. Proceedings of Me PSAS 38"' Annual Convention frorn 1-19 October 2001. Manila, PHILIPPINES. xi

7. Teresa Yulo-Nazarea*, Aleiandro 0. Nato, Jr., and Carol B. Coloma. 1999. AFLP-PCR molecular screening of 60C Y radiation-induced variants of selected ornamental and foliage plants (PH115/027). Report to 1A_A Expert (Shri Mohan Jain) on 23 November 1999. PNRI, Quezon City, PHILIPPINES. 8. Aleiandro 0. Nato, Jr.* 1998. BRCAI PTT A definite eye opener for early detection of breast cancer? During the " Philippine Breast Cancer Congress from 29-30 October 1998. Miriam College, Katipunan Avenue, Quezon City, PHILIPPINES. 9. Custer C. Deocaris*, Aleiandro 0. Nato, Jr., Apolinario D. Nazarea, and Angel B. Mateo. 1997. Development of a rational approach in synthetic vaccine engineering subverting antigen drifts of the FMDV. During the 64'h Annual Convention of the Philippine Veterinary Medicine ssociation (PYMA) from 921 February 1997. Cebu Plaza Hotel, Cebu City, PHILIPPINES.

Poster Papers (*abstracts are included in the program/proceedings) 1. Custer C. Deocaris, Alejandro 0. Nato, Jr., Diane M. Liu, Joaquin V. Moreno, and Apolinario D. Nazarea. 2003. Molecular design of multivalent synthetic vaccines against biological warfare (BW) agents. National Academy of Science Technology 25'6 Scientific Meeting from 910 July 2003. Manila Hotel, Manila, PHILIPPINES.* 2. Rolando Y. Reyes, Christina A. Petrache, Estrellita U. Tabora, Socorro P. Intoy, Teofilo, Y. Garcia, and Aleiandro 0. Nato, Jr. 2003. Gamma-ray surveys for geological studies and environmental monitoring: Experiences at the Philippine Nuclear Research Institute. National Academy of Science Technology 25'h Scientific Meeting from 910 July 2003. Manila Hotel, Manila, PHILIPPINES.* 3. Sheila C. Sajise, Alejandro Q. Nato, Jr., Custer C. Deocaris, and Cynthia P. Saloma. 2002. Mutation screening in exon 15 of the adenomatous polyposis coli (APC) gene in Filipino FAP patients. 4 HUGO Pacific Meeting Asia-Pacific Conference on Human Genetics from 21-30 October 2002. Chonburi, THAILAND.* 4. Sheila C. Sqjise, Alejandro 0. Nato, Jr., Custer C. Deocaris, and Cynthia P. Saloma. 2002. Mutation in codon 1309 and early-onset of colorectal cancer in two Filipinos with FAP. 4h HUGO Pacific Meeting 5h Asia-Pacific Conference on Human Genetics from 21-30 October 2002. Chonburi, THAILAND.* 5. Custer C. Deocaris, Peewee Marvin P. Gunay, Richmond R. Gregorio At Christian Y. Amurao, Mark Kim R. Zabala, Estrella B. Chico and Aleiandro Q. Nato, Jr. 2000. Isolation and purification of a-lactalbumin from selected dairy products: Prospecting for potential anti-cancer agents with semblance to human multimeric alpha-Lactalbumin (huMAL). Philippine Society of Biochemistry and Molecular Biology 32"h Annual Convention in December 2000. SEAFDEC Convention Center, Iloilo, PHILIPPINES.* 6. Custer C. Deocaris, Peewee Marvin P. Gunay, Richmond R. Gregorio, Art Christian Y. Amurao, Mark Kim R. Zabala, Estrella B. Chico and Aleeandro 0. Nato, Jr. 2000. Isolation and purification of a-lactalbumin from selected dairy products: Prospecting for potential anti-cancer agents with semblance to human multimeric alpha-lactalburnin (huMAL). Philippine Society of Animal Science 38h Annual Convention from 18-19 October 2000. The Heritage Hotel, Manila, PHILIPPINES.* 7. Teresa Yulo-Nazarea, Aeeandro 0. Nato, Jr., and Carol B. Coloma. DNA fingerprinting of 60CO y radiation-induced variants of foliage plants using AFLP-PCR. NationalAcademy of Science Technology 22 nd Scientific Meeting from 56 July 2000. Manila Hotel, Manila, PHILIPPINES.* 8. Emerenciana B. Duran, Teresa Yulo-Nazarea, Teofilo Y. Garcia, Cecilia M. De Vera, Rolando R. Reves. Aleiandro 0. Nato Jr., and Antonio A. Asada, Jr. Radiological assessment of former US bases: Clark Air Base. National cademy of Science Technology 22 nd Scientific Meeting from 56 July 2000. Manila Hotel, Manila, PHILIPPINES.* 9. Paul M. Leyson, Custer C. Deocaris, Carlos D. Hernandez, Aleiandro 0. Nato, Jr., and Gladys N. Punzalan. 1999. Rapid screening for tyrosinase-inhibitors in a microplate format. 15'h Philippine Chemistry Congress from 26-29 May 1999. Cebu International Convention Center, Waterfront Hotel, Lahug, Cebu City, PHILIPPINES.* 10. Teresa Yulo-Nazarea and Ale'andro Q. Nato, Jr. 1998 A chromogenic diagnostic kit for the radiosensitive tyrosinase enzyme in oriental fruit fly Bactroceraphilippinensis. 25hAtomic Energy Week Celehrationfrom 711 December 998. PNRI, Diliman, Quezon City, PHILIPPINES. It. Custer C. Deocaris, Aleiandro 0. Nato, Jr., Elena Dacanay, Samantha Marcelo, and Dyan Buenaventura. 1998. Design of a microcolorimetric assay for screening radioprotectors using the oriental fruit fly (Bactrocera philippinensis) model. National Academy of Science Technology 20'h Scientfc Meeting from 89 July 1998. Westin Philippine Plaza Hotel, Pasay City, PHILIPPINES.* 12. Custer C. Deocaris, Aeeandro 0. Najq_jL, Apolinario D. Nazarea, and Angel B. Mateo. 1997. Molecular engineering of winged-epitomorphic combipeptides mimicking FMDV serotype A VP1 polyprotein 135-146). 2"Annual Convention of the Philippine Societyfor Biochemistry and Molecular Biology (PSBMB) from 67 December 1997. CMW Auditorium, University of Santo Tomas. Espaha, Manila, PHILIPPINES.* 13. Teresa Yulo-Nazarea and Ale'andro Q. Nato, Jr. 1997 A chromogenic diagnostic kit for the radiosensitive tyrosinase enzyme in oriental fruit fly Bactroceraphilippinensis. ationalAcademy of Science Technologv 1'hAnnual Scientific .Veeling from 9- 10 July 1997. Westin Philippine Plaza Hotel, Pasay City, PHILIPPINES.* 14. Custer C. Deocaris, Alewandro 0. Nato. Jr., Apolinario D. Nazarea, and Angel B. Mateo. 1997. Epitomorphic combipeptides targetting te antigenic diversity of the VPI polyprotein 135-146) of the foot-and-mouth disease virus Xii

serotype A. National Academy of Science Technology 9"4 Annual Scientific Meeting from 90 Jul), 1997. Westin Philippine Plaza Hotel, Pasay City, PHILIPPINES.* 15. Teresa Yulo-Nazarea and Alejandro . Nato, Jr. A chromogenic diagnostic kit for tyrosinase in oriental fruit fly Bactroceraphilippinensis. 13'h Philippine Chemistry Congress from 28-31 May 1997. Palawan. PHILIPPINES.* 16. Teresa Yulo-Nazarea and Aleiandro 0. Nato, Jr. A chromogenic test for irradiated oriental fruit fly bactrocera philippinensis, as a quarantine diagnostic kit. 2',d Philippine Nuclear Congress from 10-12 December 1996. Manila Midtown Hotel, Manila, PHILIPPINES. 17. Teresa Yulo-Nazarea and Aleiandro 0. Nato, Jr. Gs-protein: A tyrosinase enzyme from Bactrocera philippinensis. Atomic Energy Week from 11-1 5 December 1995. PNRI, Diliman, Quezon City, PHILIPPINES. 18. Teresa Yulo-Nazarea and Aleiandro 0. Nato Jr. Gs-protein: A radiation sensitive enzyme in oriental fruit fly (Bactrocera sp.). 6h Asian Chemical Congress I Philippine Chemistry Congress from 22-23 May 1995. Philippine Village Hotel, Manila, PHILIPPINES.* 19. Aleiandro, 0. Nato. Jr and Apolinario D. Nazarea. Design of multivalent synthetic peptide vaccines against infectious laryngotracheitis virus and equine herpesviruses I and 4 19'6 National Convention of PSB,,vfB on 03 December 1994. MSI, UP Diliman, Quezon City, PHILIPPINES (undergraduatecategory).* 20. Teresa Yulo-Nazarea and Aegandro 0. Nato, Jr. Gs-protein: A radiation sensitive gene product of oriental fruit fly (Dacus dorsalis). 19'h National Convention of PSBMB on 03 December 1994. MSI, UP Diliman, Quezon City, PHILIPPINES (graduate category)* Atomic Energy Week from 12-16 December 1994. PNIRI, Diliman. Quezon City, PHILIPPINES.

Media Release (Television)

Genetic testing for breast cancer. 2001. The Global Filipino, The Filipino Channel, ABS-CBN (for international release).

Professional Affiliations Membership in Professional Organizations Philippine Association for Radiation Protection (PAJP), 1995-present Radioisotope Society of the Philippines (RSP), 1994-present Philippine Society for Biochemistry and Molecular Biology (PSBMB), 994-present PNRI Employees' Union (PNRIEU), 1994-present Membership in Professional Committees Member, PNRI Website Development Committee, 2001 Member, Constitutional Committee, U.P. Green League Alumni Association, 2000-present Chairman, Souvenir and Souvenir Program Committee, AEW 1998 Chairman, Technical Poster Committee (Nukteck 97), AEW 1997 Member, Invitation Committee, PAR-P Biennial Convention, 26-27 September 1997 Co-Chairman, Technical Exhibits Committee NucArtek'96), AEW 996 Member. Tour Guide Committee, AEW 1996 Co-Chairman, Tour Guide Committee, AEW 1995 Member, Tour Guide Committee, AW 1994 Xiii

Seminars Attended

9-1 Jul 2003 National Academy of Science Techriolo.-y (NAST) 25 11,Annual Scientific, Manila Hotel, Manila

I 0- I Jul 2002 National Academy of Science Technology (NAST) 24"' Annual Scientific, Manila Hotel, Manila

11-12 Jul 2001 National Academy of Science & Technology (NAST) 23d Annual Scientific Meeting and NAST 25"' Anniversary, Manila Hotel, Manila

2-7 April 2001 Regional Training Workshop on Modeling Water Quality and Validation using Radiotracer Techniques Harmful Algal Bloorn Issues), Island Cove, Cavite

5-6 Jul 2000 National Academy of Science Technology (NAST) 22"i Annual Scientific Meeting, Manila Hotel, Manila

8-9 May 2000 Conference on the Knowledge Economy: The Role of Information and Communications Technology, Manila Hotel, Manila

13 Jan 1999 Health Pysics Society - Columbia Chapter Meeting, Shilo Inn, Richland, WA, U.S.A.

29-31 Oct )998 I" Philippine Breast Cancer Congress, Miriam College, Quezon City

8-9 Jul 1998 National Academy of Science Technology (NAST) 20'h Annual Scientific Meeting, Westin Philippine Plaza Hotel, Pasay City

13 Au 1998 The Fture of Imaging (Philab Seminar), Sulo Hotel, Dihman, Quezon City

12 Mar 1998 Millipore Life Science Technology Seminar, Manila Diamond Hotel, Manila

30 Oct 1997 Recombinant Proteins Technology Symposium, Ateneo de Manila University, Quezon City

26-27 Sep 1997 7"' Biennial Convention of the Philippine Association for Radiation Protection (PARP), Subic Bay Metropolitan Authority, Olongapo City

14,16,21,23 Jul 1997 Biornolecular Modelling Lecture Series, Ateneo de Manila University, Quezon City

9-1 Jul 1997 NAST 19'4 Annual Scientific Meeting, Westin Phil. Plaza Hotel, Pasay City

19-21 Feb 1997 6" Anual Convention of te Philippine Veterinary Medical Association (PVMA), Cebu Plaza Hotel, Cebu City

10- 2 Dec 1996 2"" Philippine Nuclear Congress (Technical Committee member - Poster Pro-ram), Manila Midtown Hotel, Manila

6 Dec 1996 21' Annual Convention of the Philippine Society for Biochemistry and Molecular Biology (PSBMB) Uiversity of Sto. Tomas CME Auditorium, Manila

23-24 Au. 1996 Youth Forum on APEC National Conference, Ateneo de Manila University, Quezon CitN

16 Feb 1996 36"' Radioisotope Society of the Philippines (RSP) Convention, Sulo Hotel, Quezon Cil..

24-25 Ag 1995 Basic Operational Training on Macrophor DNA Sequencing, MBB Program, UP D iman Qezon City

28 Jul 1995 6'h Annual Convention of the PARP, DOH Auditorium, San Lazaro, Manila

24 Feb 1995 Philippine Society of Nuclear Medicine (PSNM)-RSP Joint Annual Convention, Manila Galleria Suites. Ortigas Complex, Manila

06-08 Jan 1995 Symposium on Molecular Approaches in Biology, STTC, Diliman. Quezon City

3 Dec 1994 19"' Annual Convention of the PSBMB, Marine Science Institute. UP Dilinian, Quezon City

08-11 Nov 1994 International Conference on Food Preservation and Security Documentation and Proceedings Committee member), EDSA Plaza, Shan-ri-la, Manila

17-22 Apr 1993 Global Youth Earth Saving Summit eniber of the Secretariat), Quezon City Circle and Batasan Complex, Quezon City Xiv

Extracurricular Activities

Post-graduate 1997-present Member (Bass), PNR[ Choir 1997-present Member, U.P. Green League Alumni Association 1997 I" Six, ARD Volleyball Team, Champion, PNRI Sportsfest 1996 Captainball/Coach/l" six, PNRI Volleyball Team for DOST Sportsfest 1994-1995 Member, PNRI Volleyball Team 1994 I" Six, ARD Volleyball Team, " Runner-up, PNRI Sportsfest 1994 Inter-School Debate Judge, Political Science 190 Class, Dept. of Political Science, U.P. Diliman, Au-, 23 1993-present Member, Earthsavers' Movement Undergraduate SY 1992-1993 I" Six, Narra Volleyball Team, I'l place, Alliance of Concerned Dormitories Sportsfest, UP Diliman Member, College of Science Vol leyball Team, 2dplace, U.P. Diliman Intramurals Finance Committee Associate Editor, Yearbook Committee, Agham 93 President/Executive Committee & Alumni Committee Chairperson, U.P. MBB Society Member, U.P. Subol Society (organization theme song composer) Founding Member Constitutional Committee Chairperson Membership Committee Assistant Chairperson, U.P. Green Lea-ue SY 1991-1992 Member, U.P. MBB Society SY 1990-1991 Workshop Committee Member, Philippine Guidance Personnel Association (PGPA) Annual Convention SY 988-1989 Trainee (Bass 2, U.P. Singing Ambassadors Elected Chairman, Kalayaan Residence Hall Council, UP Diliman High School, Elementary, and Preparatory SY 1987-1988 Press Relations Officer, Student Catholic Action (SCA) Librarian, Boys' Residence Hall Annex Library PSHS Senior Volleyball Varsity SY 986-1987 PSHS Junior Volleyball Varsity Member, SCA Member (Bass), PSHS Glee Club, I" place, Mixed Division, Meralco Musicfest and Overall 2 place, Meralco Musicfest, 1987 SY 1985-1986 PSHS Junior Volleyball Varsity - Sophomore Selection SY 1984-1985 INlember (Tenor), PSHS Glee Club Member, SCA SY 1983-1984 Class Secretary Platoon Scribe, Boy Scouts of the Philippines (Ednas School Division) SY 1982-1983 Class Secretary SY 1981-1982 Class President SY 1980-1981 Class President SY 1979-1980 Class President Prologue and Epilogue, Minsan May Isang Bata (about the life of the late former ]resident Ferdinand Marcos) Star Folk Dancer, Timawa Emcee, Elementary Commencement Exercises SY 1978-1979 Class President SY 1977-1978 Welcome Address, Preparatory Commencement Exercises Lead roles during elementary batch prcsentations/contest pieces in literary musical contests and sportsfests

Personal Information BirLhdate: 26 November 1971 Birthplace: JVFernandez Hospital, Dagupan City, Pan.-asinan, Philippines Hei-ht: 55" .eiaht: 135 lbs. Status: Sin-le XV

ACKNOWLEDGMENTS

Extreme gratitude is given to Mr. Custer C. Deocaris, whose DOST-GIA-funded research program on Gene Technologies for Improving Cancer Management, Diagnosis, and Prevention paved the way to put this study into a reality. Thank you very much, my dear close friend, for all the remarkable motivation you have instilled in me through all these years by giving me all the support that I need to achieve the long-awaited outcome of this research. This thesis would not have been also possible without te unselfish collaborating breast cancer patients, high risk individuals, and the low-risk individual who allowed me to access a specific part of their genorne, particularly BRCA L You can never imagine how much you have inspired me despite all the barricades that I have encountered along the way.

I astoundingly value the detailed cerebral action of my dear adviser, Dr. Virginia D Monje, Professor and former Director of the NMBB, on all the aspects pertaining to this thesis. Ma'arn Gie, thank you for understanding me through the ups and downs of this research. Your unique comprehension of life has influenced me to persevere until I have achieved acceptable results for this study. I also exceptionally express my heartfelt gratitude to my co-adviser, Dr. Apolinario D. Nazarea, Professor, Academician, and the first Director of the MBB Program (now known as the NMBB), for all the intellectual inputs and criticisms that you have imparted for this thesis. You have motivated me since day one in my undergraduate years. Your immeasurable knowledge in almost all the things that I know has constantly stirred my inner urge to continue pursuing a scientific career against all odds. Sir Nario, you know what I mean ..you alwaysdo...Q. lalsothankmyreader,thehighcaliberMedicalOncologistofUP-PGHandAssociateProfessorofDLSUDr.SusanoB.Tanae[,Jr.,foralithe significant inputs for this thesis. Integration of all your ideas regarding the incorporation of various risk models, as vefl as your noteworthy corrections and suggestions, resulted to a totally rehashed, intensely improved manuscript. I am also very grateful for all the references ou gave me.

With all the wonderful things you have taught me about life and with all the priceless love you have given me, losing both of you just a breath away intensely saddened me. I know that I have to move on... To the two most important women in my life, Mom and Lola, thank ou very much! At least, you are together now, happily watching over the whole family. You will always remain in my heart and in my memory. How I ish you could have stayed longer with us ...How I wish you could witness our successes in life ...Nevertheless, I assure you that all the hardships you have overcome for us will never be put to waste.

The contagious uplifting mood of rny thesis students, Ms. Gretchen Gayle S. Parganiban and Ms. Josephine Ann G Santos, along with their constant 'updates' regarding a special 'topic', have unconsciously maintained my sanity while performing SSCP during the last few months. Groo and Jo, congratulations and good luck in all your endeavors! I sincerely hope that both of you will be part of the precious rare breed of medical doctors someday... uhmm.. you also know what I mean. Thank you for your patience and friendship. I will be your D A. for good... 0. Of course, I can never forget my thesis 'partner', Ms. Sheila C. Sajise, who was able to superbly withstand all the pressures when we were in the latter stage of acquiring the materials and reagents needed for this type of research, as well as, during the template preparation and PTT days and nights ...and dawns ...Yup, at last we are able to finish each of our thesis.

My utmost recognition will always be for Dr Teresa M.U. Wagner and particularly to Ms. Daniela Mulur (Vienna, Austria) for extending their help in performing DHPLC and cycle sequencing in teir beautiful laboratory. I will never forget the time when I went to AKH I am also grateful for the assistance offered by Dr Haruhiko Sugimura and his laboratory staff in sequencing the same fragment. I wsh to thank Dr. Alex M. Garvin (Bureco, Switzerland) for all the necessary information about PTT, the negatives of your autoradiograms, and the mutation controls that you have given me. Aside from Dr. Garvin, scientists from several areas in the world have shared aliquots of their mutation controls. Unquestionably, I will never be able to perform both PTT and SSCP with high confidence without tese mutation controls. Therefore, I would like to thank Dr. Anna Jakubowska (Poland), Dr. James Fackenthal with Dr. Olofunmilayo Olopade (USA), Dr. Angela Sharp (Australia), Dr. Rosella Crucianelli (Italy), Dr. Gianfiranco Voglino (Italy), Dr. Peter Devilee (The Netherlands), and Dr. Stephen Abbs (UK) for the mutation controls and significant references that you have imparted to me. Dr. Mary-Claire King (USA) for all the significant suggestions regarding my thesis. I express my sincere appreciation for the help given by Dr. Jon Thor Bergthorsson, Dr. Igor Kourkine, and Dr. Piotr Kozlowski regarding all the details about SSCP. Dr. Peter Devilee UK) for sharing with me the fact that sample collection for breast cancer studies in other countries is also difficult. Thank you very much for the copies f the dissertations of your students, Dr. Renee Cornelis and Dr. Tamara Peelen I also acknowledge Dr. Anne-Lise Borresen Dale for introducing Custer Deocaris into the field of breast cancer mutation analysis. And, of course, I truthfully thank Dr. David Euhus and Ms. Catherine Elliott for the CancerGene software which contains the risk assessment (Gail and Claus) and prior probability (Couch, Shattuck-Eidens, Frank, and BRCAPRO mdels used in this thesis. I would like to thank the above-mentioned scientists for all the important references and PDF files that they have sent me.

Most of the collaborating patients..were crucially traced and convinced to join this study through the relentless supportive efforts provided by- (a) Dr. Samuel D Ang, Dr. Roe] Tolentino, Dr Pablo A. Candelario ad Ms. Charmaine Jaromay of SLMCCI and CGH, (b) Dr. Valoric Fulilon-Chan, Dr. Kalangitan R. Gutierrez, Dr Katherina Gutierrez-Querol, and Ms. Agnes P. Reyes of VMMC, (c) Dr. Ma. Victoria Abesarnis, Dr, Ramon C. Severino, Ms. May Calpatura, Ms. Julieta V. Cobangbang, and Ms Gilda D. Lucclo of EAMC, (d) Dr. Paul Bisnar of AFPMC, (e) Dr. Franklin Sable of Mapandan Hospital, and (O Dr. Emma L. Cancino, Ms. Lorie Pineda, and Ms. Cristy Aguilar of PNRI. Thank you very much to all of you for the remarkable help especially when you have to perform blood extraction yourself in a patient's house (ie., Dr. Bisnar).

Notably, most of my latest references were obtained from scientists that I have contacted during the course of this thesis I want thank all of them for sharing their articles with me. Here they are: Dr. Jose Rafael Blesa (Instituto de Investigaciones Citologicas, Spain), Dr. Richard E. Buller (Univ. of Iowa, U.S.A) Dr Kathleen Claes (University Hospital Gnt, Belgium), Dr. Richard A. Dicioccio (Roswell Park Cancer Institute, NY, USA), Dr. Barbara Weber (StellarChance Labs, PA, USA), Dr. Kate L. Nathanson (University of Pennsylvania, PA, USA), Dr. Maria J. Worsham (Henry Ford Hospital Molecular Oncology Laboratories, MI, USA), Dr Jean-Pierre Fricker (Centre Paul Strauss, France), Dr. Olufunmilayo I Olopade / Dr. Qing-Gao / Dr. James Fac6enthal University of Chicago, USA) Dr. Christopher Matthew (University of London, England), Dr. Donald Love and Dr. Madhuri Hegde (University of Auckland, New Zealand), Dr Douglas Macmillan (Nottingham, UK), Dr- Ui Soon Khoo (University of Hong Kong, Hong Kong), Dr Igor V. Kourkine Northwestern Univ, IL, USA), Dr. Sunil R. Lakhani (University College London, London, UK), Dr. Niklas Lornan (University Hospital, Lund, Sweden), Dr. Ake Borg, Dr. Ponic Puirnelaar and Dr. Jan .M Kijn (Erasmus Univ Medical Ctr, Rotterdam, The Netherlands), Dr. Pal Moller (The Norwegian Radium Hospital, Oslo, Norway), Dr. David F. Barker (University of Utah Health Sciences Center, Utah, USA), Dr A Perez-Valles (Hospital General Universitario, Valencia, Spain), Dr Manuela Santarosa (Centro di Riferimento Oncologico, Aviano, Italy), Dr. H 0zcelik Mount Sinai Hospital, Toronto, Ontario, Canada), Dr. Susan M Domchek (University of Pennsylvania, USA), and Dr. Barbara Pasini (Universita di Torino, Italy).

Financial assistance from various government agencies made it possible for us to acquire the materials and special reagents needed for this study. Department of ScienceandTeclinology-GraiitinAid(DOST-GIA)isthemainsourceoftherunds(-71%)andit%%ascoursedtlirOLighPNRI TheDCodeMutationDetection System was acquired through the PCSO (- 15%) Fnancial assistance was also augmented by the presence of funding from DOST for GAD-assisted projects (-9%). Some existing general lboratory cemicals of PNRI specifically from the (a) Radiation Biochemistry and Biotechnology Laboratory ofthe Health Physics Research Section and the (b) Cancer Research and Radiation Biology Laboratory of te Biomedical Research Section were ao tilized to enable s to Xvi

purchase the special chemicals needed (-3%). Financial support in the form of thesis grants (-I% each) from DOST-Philippine Council for Advanced Science and Technology Research and Development DOST-PCASTRD) and Office of the Vice Chancellor for Research and Development, UP Diliman (UP-OVCRD) [Grant #010104TSN] were also availed of to supplement a small portion of the expenses for this study. I express my sincerest recognition of the support given by these institutions for the success of this research

Funding and acquisition of materials and reagents were mainly coursed through PNR1. Although sometimes delayed by bureaucracy, the support I received from the Institute was truly heart-warming. Thank you very much to all of you for the sustenance that was given to me. Dr. Alumanda M. dela Rosa, Acting Director of PNRJ, thank you allowing us to have advanced deliveries of certain reagents so that we can perform the experiments on time. Dr. Emerenciana B. Duran, ARD Chief, for allowing bulk requisitions by coordinating with Dr. Graceta DL Cuevas to make the research move forward, and effectively stining me to finish my thesis. Thank you to oth of you. Ms. Teresa Nazarea, HPRS Head, thanks a lot for allowing me, my colleagues, and my students to work overtime and during weekends whenever we need to perforin more experiments. Ma'am I am very grateful for all the encouragement and intellectual discussions that I have received from you. I can never express how thankful I am for all the support you have given me even when I was still in UPSF Diliman Office. Ma'am Elvie, for the use of the electronic gadgets like cd-writer and laptop coupled by the inspiring words. Ma'am Gin and Ma'am Linda, for signing the requisitions important for the research. Ma'am Belen, for the never ending lunch treat most of the time we meet in the anteen. Ma'am Floops, for recently allowing me to finish my manuscript. Thanks a lot to all of you.

In performing the experiments, the computer and certain materials or chemicals are needed once in a while. For this, I sincerely thank my Health Physics Research family (Carol, Ate Liz, Ate Malu, Ate Pinky, Mong, Kuya Tony, and Kuya Phil) for giving way whenever I need to perform something on a time- bound basis. You also know what I mean ...0. Thank you for all the support you have shown through the years. Ate Cecile, for all the unwavering support since the time I have worked with you. You have instilled courage and perseverance in me. I am extremely thankful that you were part of the HIP family. Biomed (Ate Cena. and Ate Cel), for the pH meter and the supportive words. Ate Aida, Mang Pol, and Kuya Ed, for the use of the autoclave. AMR (Ate Sol, Ate Luchi, Kathy, Raymond, Jomike, Beng, and Analie), for the supply of minute amounts of liquid nitrogen and overflowing supply of milliQ H20- Chem (Ate Yen', Ate Maricel, Lucille) for the use of the ultra-low freezer. Training (Dr. Corazon Bemido, Dr. Star Relunia, and Kuya Nonong) for the red lamp and the binding machine. Cobalt-60 (Kuya Jun, Ate Haydee, and Ate Luvi) for the supply of cleionized H20. Ate Ellen for all the tips regarding computers. Boss Baby (Cancio), for processing most of the requisitions for this research. Info (Ate Rhoda, Ate Tina, Kuya Sim, Kuya Mar) for some impromptu support received. Property (Kuya Tony, Ate Odet, Ate Laura) for facilitating processing and delivery of the items needed. Thank you very much.

To the honorable dean of the College of Science, Dr. Rhodora V. Azanza, thank you very much for supporting me in my letter of reconsideration. It really meant a lot to me...

Developing BioMax MR I film after exposure to 35S was made easier by using the automated x-ray developing machine at the UP Infirmary Radiology ection in UP Diliman I would like to thank John Darriasco and Kuya Greg for all the assistance they have extended to me, Sheila, and my thesis students. In MBB, thank you very much, Ate Mariz, Ma'am Butch, and Ate Evelyn, for never giving up on me during the last few weeks before the oral defense date. Ate Ivy, Ate Precy, and Gigi of the CS Graduate office, thank you very much for all the help you have given me. You know what I mean... O. Karla, thank you, too, for the assistance whenever I needed it.

So many tedious tasks have to be performed during the course of this thesis. We have to transfer from one laboratory to another and we have to borrow some equipment from another laboratory once in a while. For this, I really thank the PNRI maintenance for their industriousness in helping us every now and then whenever we need their assistance. I also thank the PNRl security for keeping company wenever we go home late at night to ensure our safety. Thanks a lot for watching over the daily DCode overnight runs at room 219.

1 could not have survived without the cheerful disposition of my close friends (Efren, Clynt, Kuya Boyet, Kuya Rol Iy, Alex, Bernard, Ana, Go, Paulo, Russell, Richard, Jig-Jig, Mark, Rommel, Erwin, Ate Haydee, and Chat) who, in one way or another, lend a hand in the laboratory or in real life situations, crack ajoke, or share a deep prayer. I would like to thank all of you. Ate Bing2, thank you very much for being a sister to me and for taking care of Dave (my 'askal') whenever I have to perform experiments late at night. Kuya Rolly and Kuya Vic, thank you for keeping the car alive BS friends (Ate Joy, Ate Denia, Ate Berna, Ate Maricel, Ate Cory, and Ate Linda), thank you very much for your prayers. UP Green League, thank you very much for cheering me up. Ate Baby and Kuya Sonny, thank you for the sensible conversations. Arnie, Tess, Ruby, and Barbara, for the wonderful time in Vienna and Seibersdorf. Andy, Paul, Yasuo, Esther, and busmates in Richland, WA, thank you very much for sharing funny and memorable moments with me. To my friends and churchmates who were not mentionedhereyouknowwhoyouare wanttothankyousomuchforailthehelp.

To my dear dog, Dave, for guarding the house whenever I am away and for keeping me company especially when I am stressed or lonely.

The incessant support of my beloved bestfiriend, Mr. Ariel T. Ortiz, Associate Professor of MSU, will always be inherently remembered Thank you for all the times you have helped me in searching for data regarding demographics of breast cancer in the Philippines, as well as. in assisting me perform my laboratory work during holidays and weekends. You know how much you have affected my life through all these years.

Love begins within the family. I am absolutely grateful for my siblings (Kuya Jing, Kuya Joey, Achi Annie, and Kuya Joje), n-laws (Achi Gina, Achi Violy, Manong Adonis, and Achi Lida), uncles (Tio Eddie and Tio Pedro), aunt (Tia Ruth), cousins (Kuya Ped, Achi Bevy, Achi Cynthia, Kuya Boy, Achi Edwinna, Kuya Boyet, Kuya Glynn, Achi Hayddie, Achi Maine, Achi Judith, Jerry, Kareene, Laamie, and your husbands or wives), nephews (Aylwin, Jcjo, Jodael, Ulysis, Jonald, Joshua, Jericho, Junior, and Bryan), and nieces (Jinky, Jovy, Xyza, Leslyn, Anjoy, Jenny, Roxanne, An-An, Judy, Chivy, Vanessa, and Jozy). Thank you for always being there.

To you ...You know who you are ...Thank you very much for coming into my life.

Most importantly, I would like to thank You, my dearest Lord Jesus Christ, for being there during the lowest point in m% life. I give my highest praise to You.

Andrew Nato Manila, 2003 CONTENTS PAGE

Title Page i

Dedication ii

Certification Page iii

Public Announcement of M.Sc. Examination iv

Biographical Data v

Acknowledgments xv

List of Figures 4

List of Tables 5

List of Abbreviations 6

Abstract 7

I. Introduction 1.1 Burden of Illness 8 1.2 Objectives of the Study 8 1.3 Significance of the Study 9 1.4 Scope and Limitations 10

11. Literature Review 2.1 Clinical Categories of Breast Cancer I 2.2 Breast Cancer Susceptibility Genes I 2.3 Pathologic Manifestations of BRC,41 and BRC,42 12 2.4 Association of BRCA]12 %%ith FBC 13 2.5 Estimating Relative Risk and Lifetime Risk of BC 15 2.5.1 GailModel 2.5.2 Claus Model 2.6 Some Molecular Techniques and Mutation Studies on Detection of BRCAJ Mutation 6 2.7 High Frequency BRCA I Mutations and Their Corresponding Effects on Gene Function 22 2.8 Genetic Risk Assessment Based on Prior Probability Models 22 2. &I Couch Model 2.8.2 Shaltuck-Eidens Model (Myriad ) 2.&3 FrankModel(Myriadll) 2.8.4 RRCAPRO

111. Materials and Methods 3.1 Research Design 34 3.2 Recruitment of Patients 34 3.3 Inclusion Criteria 34 3.4 Exclusion Criterion 34 3.5 Pedigree Construction and Characterization of Families 34 3.6 Patient Information 35 3.7 Lifetime Risk Estimation 35 3.7.1 Estimation Using Gail Model 3.7.2 Estimation Using Claus Model 3.7.3 Estimation Using BRCAPRO 3.8 Screening Strategy 39 3.9 Template Preparation 39 3.9.1 Genomic DNA Extraction 3.9.2 Template Purity and ConcentrationDetermination 3.9.3 Pooled Genomic DVA Page 2 of 726

PAGE

3.10 BRCAJ Primers and Corresponding Lengths of Amplicons 43 3.11 Polymerase Chain Reaction 43 3.12 Single-Strand Conformation Polymorphism Analysis 44 3.13 Protein Truncation Test 44 3.14 Validation of Mutation Detected at Exon I I and Analysis of the DNA Sequence 45 3.15 Processing of Data Obtained from the Molecular Techniques Utilized 50 3.16 BRCAI Prior Carrier Probability Estimation for Familial Filipino BC Patients 50 3.161 Estimation Using Couch Model 3.162 stimation Using Shattuck-Eidens Model 3.163 EstimationUsingFrankModel(FrankandMyriadcom) 3.16 4 Estimation Using BRCAPRO 3.17 BRCA I Posterior Carrier Probability Estimation for Screened Patients 51 3.18 Unconditional Probability Estimation for Screened Unaffected High-Risk Individuals 5 1

IV. Results 4.1 Selection of Prospective Individuals at High-Risk for BRCA I Mutation 54 4.2 Characteristic Affliction of Relatives in Families 54 4.3 Clinicopathologicat Characteristics 54 4.3.1 ClinicopathologicalCharacteristics ofthe 36 BRCAJ Mutation High-Risk Individuals 4.3.2 ClinicopathologicalCharacteristics of the Familieswith Relatives Afflicted with BC 4.3.3 ClinicopathologicalCharacteristics ofthe Families with Relatives Afflicted with OC 4.3.4 ClinicopathologicalCharacteristics of the Familieswith Relatives Afflicted with Other Primary Cancers 4.4 Socio-Economic, Dietary, and Other Factors 55 4.4.1 Socio-Economic, Dietary, and Other Factors ofthe 36 BRCA I Mutation High-Risk Individuals 4.4.2 Socio-Economic, Dietary, and Other Factorsof the Families with Relatives Afflicted with BC 4.4.3 Socio-Economic, Dietary, and Other Factorsof the Families with Relatives Afflicted with OC 4.4.4 Socio-Economic, Dietary, and Other Factorsof the Families with Relatives Afflicted with Other PrimaryCancers 4.5 Relative and/or Lifetime Risk Estimates for Unaffected High-Risk Individuals 68 4.6 Working Genomic DNA Solutions 70 4.7 PCR Products 70 4.8 Single-Strand Conformation Polymorphism Analysis 70 4.9 Protein Truncation Test 7 4.9.1 Polyacrylamide Gel Electrophoresis(1401o) 4.9.2 GradientPolyacrylamide Get Electrophoresis (5-2016) 4.10 Denaturing High-Performance Liquid Chromatography (DHPLC) Analysis 72 4.11 Automated DNA Sequencing 73 4.12 BRCAJ Prior Probability Estimates for Familial Filipino BC Patients 73 4.13 BRCA I Posterior Probability Estimates for the Screened Patients 74 4.14 Unconditional Posterior Probability Estimates for Unaffected High-Risk Individuals 74 4.15 Patient Characteristics and Profiles 74

V. Discussion 5.1 The Only Mutation Detected at Exon I 99 5.2 Eight (8) Unique Putative Polymorphisms Detected at BRCA I 100 5.2.1 LowPrevalenceofPzitativePolymorphismsatBRCAI Exons 2 5, 17, and 22 May Indicate Probable Mutations 5.2.2 HighPrevalenceofPlitativePolymorphismsatBRCAI Exon 15 May Indicate Biological Insignificance 5.3 Breast Cancer Risk Estimation 102 5.3.1 RelativeRiskEstiniation 5.3.2 CnielativeLifelinzeRiskorUnconditionalProbabilityEsfin2ation Page 3 of 126

PAGE

5.4 Patterns in the Age at Disease Onset of Families with Cancer-Afflicted Relatives 103 5.4.1 FamilieswithRelalives.4fflictedwithBC 5.4.2 Families with Relatives Afflicted with OC 5.4.3 Families with Relatives Afflicted with Oer PrimaryCancers 5.5 Modeling and Screening Profiles of Families with Cancer-Afflicted Relatives 103 5.5.1 FamilieswithRelativesAffliciedwithBC 5.5.2 FamilieswithRelativesAffliciedwithOC 5.5.3 FamilieswithRelativesAfflictedwithOtherPrimaryCancers 5.6 Effect of Screening Results on BRCAPRO PosteriorCarrier and Unconditional Probabilities 105 5.7 5-20% Gradient vs. 14% Standard Polyacrylamide Gel for Separation of In Vitro Transcribed and Translated Protein Products 106 5.8 Cost-Effectiveness of Utilizing Prior Probability Models 107 Would Screening Only Exon I I Be ujjicientforBRCA I Genetic Testing in the Philippines? 5.9. Status of BRCAI Genetic Testing in the Philippines 108 5.9.1 The Irony ofSampling Collection 5.9.2 Low Socio-Economic Profile Demands Cheaper Genetic Testing 5.9.3 nformationObtainedRegardingPalhologicManifestationsandSocio-Economic, Dietary, and Other Factors: Still at a Very Early Stage

VI. Conclusions and Recommendations 6.1 Low Incidence of Germline Mutation in BRCA I Exon I I among Suspected Familial Filipino Breast Cancer Cases: The Only Mutation Detected is the Second Documented Fipino BC Case with BRC41 Mutation at Exon I I 110 6.2 Eight (8) Unique Putative Polymorphisms Detected at BRCA 1: Prevalence May Indicate Probable Mutation or Biological Insignificance 110 6.3 Probable Mutations at Exons 2 5, 17, and 22 May Suggest Initial Genotype-Phenotype Correlations in Filipino Breast Cancer Patients 110 6.4 Gail, Claus, and BRCAPRO Models can be Utilized for Estimating BC Risks of Unaffected Individuals III 6.5 Patterns in Age at Disease Onset Observed in Families ith Cancer-Afflicted Relatives HI 6.6 Most of the BRCAPRO and Myriad.com Prior Probability Estimates Coincide with the Presence of Mutation and/or Putative Polymorphisms in Suspected Familial Filipino Breast Cancer Patients II] 6.7 Screening Results Apparently Affect Posterior Probabilities IH 6.8 Cost-Effectiveness of using Gradient Gel Electrophoresis Coupled with Only One Primer Pair for Protein Truncation Test 112 6.9 BRCA I Genetic Testing in the Philippines Needs Nationwide Support from Various Sectors 112 6.10 Utilization of Prior Probability Models in the Philippines: A Pioneering Endeavor for Cost-Effective Pre-Genetic Test Screening 112

VII. Summary of Findings 113

Vill. Bibliography 114

IX. Appendices Appendix A. Bayes'Theorem Utilized in BRCAPRO 120 Appendix B. Informed Consent 121 Appendix C. BRCAJ Mutation Screening Program Questionnaire 122 Appendix D. Concentration and Purity of Isolated Genomic DNA 126 Page 4 of 126

PAGE List of Figures

Figure 1. Schematic diagram of the single-strand conformation polymorphism analysis. 19 Figure 2 Schematic diagram of the protein truncation test. 26 Figure 3 Protein encoding regions of BRC.41. 3 Figure 4 Estimation of the relative risk of a 40-year old woman (Ptl3) who had menarche at age 12 years using the Gail model. 36 Figure 5. Estimation of the relative risk of a 40-year old woman (Ptl3) who has a sister diagnosed of BC at age 40 years using the Claus model. 37 Figure 6 The BRCA-PRO model estimation of unconditional probability that the 40-year old woman (Pt 3 will develop BC. 38 Figure 7 Schematic diagram of the screening strategy and risk modeling used in this study. 42 Figure 8. Regions amplified for PTT and SSCP. 47 Figure 9 Estimation of the BRCAI prior carrier probability of a 46-year old woman (Pt25) using the Couch model. 52 Figure I . The BRCAPRO estimation of (a) BRCA I posterior carrier probability of a 40-year old woman (Pt 3 and (b) unconditional probability that she will develop BC. 53 Figure 11. Pedigrees of families of probands with first- or second-degree relatives afflicted with BC. 59 Figure 12. Pedigrees of families of probands with frst- or second-degree relatives afflicted with OC. 60 Figure 13. Pedigrees of families of probands with first- or second-degree relatives afflicted with other primary cancers. 61 Figure 14. Frequency distribution based on age at onset of disease of the 34 selected BRCA I mutation high-risk individuals. 64 Figure I S. Agarose gel electrophoresis (0.8%) of working genomic DNA solutions from suspected familial Filipino BC patients. 75 Figure 16. PCR products of pooled genomic DNA of the suspected familial Filipino BC patients at the smaller priority exons of BRCAJ. 76 Figure 17. PCR products (z-3468bp) of genomic DNA at exon I I from some of the suspected familial Filipino BC patients using BRCAI primers described elsewhere (Garvin, 998; Ottini et al., 2000). 77 Figure 18. SSCP analysis in the BRCAI gene. 78 Figure 19. SSCP analysis in the BRCA I gene in patients which probably have putative polymorphisms. 84 Figure 20. Protein products generated from exon I I PCR products of some of the familial Filipino BC patients through TNT Quick-Coupled in vitro transcription/translation system separated and visualized by 14% SDS-PAGE and autoradiography. 89 Figure 2 . Protein products generated from exon I I PCR products of the familial Filipino BC patients through TNT Quick-Coupled in vitro transcriptionltranslationsystem separated and visualized by 520% gradient SDS-PAGE and autoradiography. 9 Figure 22. DHPLC analysis of the segment containing the putative mutation of PtO4 at BRCA I exon I I (nt 1299-1758). 93 Figure 23. Sequence showing the precise mutational event (I 445 insGT) at BRCA I exon I I of PtO4. 94 Page of 126

PACE

List of Tables

Table 1. Pathology of BRCA1- and BRCA2-Associated BC Cases Compared to SBC Cases. 14 Table 2 Relative Risk Values of the Gail Model. 7 Table 3 Relative Risk Prediction Table Using Claus Model. 18 Table 4 1. BRCA I Mutations in Families with Hereditary Breast Cancer (HBQ Cases. 27 Table 42. BRCA I Mutations in Families with Hereditary Breast-Ovarian Cancer (HBOC) Cases. 27 Table 43. BRCA I Mutations in Families with Hereditary Ovarian Cancer (HOC) Cases. 27 Table 44. BRCAJ Mutations in Families with Hereditary Breast and/or Ovarian Cancer [HB(O)C] Cases. 28 Table 5. Frequent BRCAJ Mutations by Designation and their Corresponding Effects on Gene Function. 29 Table 6 Predicted Probability of a BRCA I Mutation Using Couch Model. 32 Table 7 1. Probability of a BRCA I or BRCA2 Mutation in a Woman with BC<50. 33 Table 72. Mutation Prevalence Table for Individuals of Non-Ashkenazi Jewish Acestry. 33 Table 8. BRCAJ Exons. 41 Table 9 BRCA I Primers. 46 Table 10. BRC I Mutation Controls from Different Countries. 48 Table I . The BRCA1 Mutation High-Risk Individuals from Various Institutions. 58 Table 12.1 Clinicopathological Characteristics of the 36 BRCA I Mutation High-Risk Individuals and their Corresponding Distribution. 62 Table 12.2 Clinicopathological Characteristics of the Families with Relatives Afflicted with BC. 63 Table 12.3 Clinicopathological Characteristics of tfie Families with Relatives Afflicted with OC. 63 Table 12.4 Clinicopathological Characteristics of the Families with Relatives Afflicted with Other Primary Cancers. 63 Table 13.1 Socio-Economic, Dietary, and Other Factors of the 36 36 BRCA I Mutation High-Risk Individuals and their Corresponding Distribution. 65 Table 13.2 Socio-Economic, Dietary, and Other Factors of Families with Relatives Afflicted with BC, OC, or Other Primary Cancers. 67 Table 14. 1. Relative and/or Lifetime Risk Estimates for 40-year old woman unaffected of BC (Pt 3). 69 Table 14.2. Relative and/or Lifetime Risk Estimates for 19-year old woman unaffected of BC (Pt34). 69 Table 15. Pooled SSCP Analysis in the BRCAJ Gene of Suspected Familial Filipino BC Patients. 83 Table 16. Patients with Putative Polymorphism Based on SSCP Analysis. 88 Table 17. Detectabifity of BRC,41 Mutation Controls at 14% Polyacrylamide Gel Electrophoresis. 90 Table 18. BRC,41 Mutation Controls Detected by 520% Gradient Polyacrylamide Gel Electrophoresis. 92 Table 19. BRCA112 Carrier Probabilities using Prior Probability Models. 95 Table 20. BRCAPRO Estimates of BRCA I Posterior Carrier Probabilities. 96 Table 2 .1 Unconditional Posterior Probability Estimates for Pfl 3. 97 Table 21.2 Unconditional Posterior Probability Estimates for Pt34. 97 Table 22. Profile of Individuals in Families of BC Patients. 98 Page 6 of 126

List of Abbreviations

ACN acelonitrile AROO number of alterations reported only once ASR age-standardized incidence rate ATFI cANT-dependent transcription factor I ATM ataxia telangeclasia mutated B(O)c breast and/or ovarian cancer BASC BRCAI-associated genome srveillance complex BIC Breast Cancer Information Core BOC breast-ovarian cancer Br or BC breast cancer BRCA I breast cancer ssceptibility gene, type I BRCA2 breast cancer ssceptibility gene, type 2 BRCA3 breast cancer susceptibility gene, type 3 BRCT BRCA I C terminal BSE breast self-examination cc colon cancer CDGE constant denaturant gel electrophoresis CDNA copyDNA CHK2 checkpoint kinase 2 CSGE conformation sensitive gel electrophoresis DGGE denaturing gradient gel clectrophoresis DHPLC denaturing high-performance liquid cromatography DIVTV number of distinct mutations, polymorphisms, and, variants DNA deoxyribonticleic acid E number ofentries FBC familial breast cancer gDNA genomic DNA HA lieteroduplex analysis HB(O)C mixture of hereditary breast cancer and ereditary breast-ovarian cancer HBC hereditary breast cancer HBOC hereditary breast-ovarian cancer het heterozygote HOC hereditary ovarian cancer HPN hypertension IDC infiltrating ductal carcinoma ILC infiltrating lobular carcinoma IR Ionizing radiation IVSP in vitro synthesized protein assay LR likelihood ratio mRNA messenger RNA mut mutant nt rincleotide position Ov or OC ovarian cancer PAGE po)yacry)ajnide gel electrophoresis PC prostate cancer PCR polymerase chain reaction PTT protein truncation test RNA ribonLICleic acid RT-PCR reverse transcriplase -polymerase cliam reaction SBC sporadic breast cancer SDS-PAGE sodium dodecyl sulfate -polyacrylamide gel clectrophoresis SSCA-HA combined single-strand conformation polymorphism aalysis and heterochiplex analysis SSCP or SSCA single-strind conformation polymorphism analysis ssDNA single-stranded DNA TPP tumor perimeter proportion wt wild-iype Page 7 of 126

ABSTRACT

Breast cancer susceptibility gene, type 1 (BRCAI) has been thought to be responsible for 45% of families with multiple breast carcinomas and for 80% of breast and ovarian cancer families. In this study, we investigated 34 familial Filipino breast cancer (BC) patients to: (a) estimate breast cancer risks and BCA112 mutation carrier probabilities using risk assessment and prior probability models, respectively; (b) screen for putative polymorphisms at selected smaller exons of BRCAI by single-strand conformation polymorphism (SSCP) analysis; (c) screen for truncated mutations at BCA] exon II by radioactive protein truncation test (PTT); and (d) estimate posterior probabilities upon incorporation of screening results. SSCP analysis revealed unique putative polymorphisms. Low prevalence of unique putative polymorphisms at exons 2, 5, 17, and 22 may indicate probable mutations. Contrastingly, high prevalence of unique putative polymorphisms at exons 13, 15, and 16 may suggest true polymorphisms which are biologically insignificant. PTT, DHPLC, and sequence analyses revealed a novel mutation in exon I I involving GT insertion that resulted to a stop codon which generated a 29.7 kDa truncated protein product. This is the second documented mutation in BCA I exon II in a Filipino BC patient since 1998. Initial genotype-phenotype correlations in Filipino BC patients may be elucidated based on screening tests performed. Our results corroborate the findings of a study on unselected incident Filipino BC cases where the reported prevalence of BRCAJ mutation is low. The higher prevalence of putative polymorphisms may be attributed to the increased stringency in patient prospecting. The Gail, Claus, and BRCAPRO models can be utilized to estimate BC risk in unaffected high-risk individuals but validation is needed. Most of the BRCAPRO and Myriad.com prior probability estimates coincide with the presence of BCAJ mutation and/or putative polymorphisms. This pioneering endeavor of utilizing these prior probability models as pre-genetic test screening tools has huge potential to minimize the expenses of relatives of BC patients who want to undergo genetic testing, as well as, to reduce unnecessary tests performed in genetic screening laboratories. The success of BCAI genetic testing depends on the collaboration among scientists, medical practitioners, and professionals in related institutions nationwide. Page of 126

I. INTRODUCTION

1.1 Burden of Illness

Cancer is the third leading cause of morbidity and mortality in the Philippines and a major problem worldwide, showing a 37% increase in number of cases from 1975 to 1990. Breast cancer (BQ ranks second as the leading malignancy among Filipino patients (Manila and Rizal registries) even if almost all cases occur in women. The 1980-1995 mean age-standardized incidence of BC (-44 in 100,000 for women and 0.7 in 100,000 for men) is the highest in Asia (Parkin et aL, 200 1; NBCC, 1998; Ngelangel et al., 1997).

Major risk factors for BC are age and a family history of breast and/or ovarian cancer B(O)C] especially at the first-degree relatives (DiMichele & Weber, 2000; Gayther & Ponder, 1997). The cumulative lifetime risk of developing BC by age 85 years is 12.5% while the estirnted lifetime risk of dying from BC for women is 36%.

Majority of BC cases are sporadic although familial and sporadic cases may have similar clinical presentation. About I out of cases of breast cancer is regarded as familial. Though clinically similar, 50% of these familial cases seem to be inherited as an autosomal dominant trait. BC susceptibility genes (BRC,41 and BRC,2) are probably responsible for 45% of multiple BC families and for >80% of B(O)C and early-onset BC families (Sorlie et al., 1998).

1.2 Objectives of the Study

The main objective of this study is to estimate BC risks and probabilities of carrying deleterious BRCAJ12 mutations with the detection of truncating mutations in BRCAI exon I I and putative polymorphisms in smaller BRCAI priority exons among familial Filipino BC patients. Specifically, this study aims to: (1) estimate breast cancer risks and BRCAY2 mutation carrier probabilities among selected patients using risk assessment and prior probability models, respectively; (2 sreen for putative polymorphisms at BRCAI exons 2 5, 13, 15, 16, 7, 18, 20, 22, and 24 among all familial Filipino BC patients by single-strand conformation polymorphism (SSCP) analysis; Page 9 of 126

(3) screen for truncated mutations at BRCA I exon I I in the same set of patients by radioactive protein truncation test (PTT); (4) estimate the apparent molecular weight MW) of the truncated protein product (5) approximate the possible location of the detected mutation by estimating the corresponding nucleotide position of the stop codon based on the apparent MW; (6) validate PTT results by denaturing high-performance liquid chromatography (DHPLC); (7) obtain te precise mutational event by cycle sequencing; and (8) estimate posterior probabilities upon incorporation of the PTT and SSCP results.

1.3 Significance of the Study

Due to the large size of BRCAI, sequencing the whole gene is very expensive and laborious. Several techniques used to screen for BRCAI germline mutations include single-strand conformation polymorphism. analysis (SSCP/SSCA), protein truncation test (PTT), denaturing high-performance liquid chromatography (DHPLC), among others. These techniques have varying degree of detection efficiency depending on the parameters and platforms used.

Single-strand conformation polymorphism analysis (SSCP) continues to be widely used due to its simplicity and cost-effectiveness (Orita et al., 1989a/1989b). SSCP detects base changes in single-stranded DNA when the folding of the single strand changes sufficiently to alter its clectrophoretic mobility in a non-denaturing gel. Although the sensitivity of this method is about 80% when single clectrophoresis conditions are employed, it is an appropriate technique in detecting mutations in the smaller exons of BRC,41 (Peelen, 2000; Vidal-Puig & Moller, 1994; Ravnik-Glavac, Glavac, & Dean, 1994;). Screening the whole BRCAI gene would be arduous and costly, entailing prioritization of exons that would be subjected to SSCP. In decreasing order, the most number of documented mutations are found in exons 11, 16, 20 2 18, 5, 13, 17, 15 and 24 (BIC, 2003). Since Matsuda et al. 2002) reported mutations in BRCA I exons 15, 22, and 24 among Filipino BC patients, exon 22 has been included for SSCP. On the other hand, protein truncation test (PTT) utilizes the principle of protein synthesis in vitro as encoded by a test sequence followed by the determination of its size. It has become a popular method as an efficient means of screening mutations in a coding sequence that lead to a truncated protein product. Propitiously, lot of genetic diseases result from inherited loss of function mutations that are usually due to premature truncation of the protein product (Garvin, 1998; Roest et al., 1993). Truncated proteins result from more than 61-75% of reported BRCA I mutations due to nonsense or frameshift mutations, 75% of which are found in exon II which comprises 6 % of the coding sequence (Gayther & Ponder, 1997; Borresen, 1996; Hogervorst et al, 1995). PTT is Page 10 of 126

therefore an appropriate screening method for this study since it detects these functional truncating mutations and provides the approximate location of the sequence causative alteration. Considering the high occurrence of truncating mutations and the reasonably large portion of coding sequence within one exon, screening of exon I I by PTT is a feasible, cost-effective, and suitable method, although incomplete, to evaluate presence of BRCAI mutations. Screening the I I exons mentioned through SSCP and PTT indicates detecting 80-90% of known BRCAJ mutations (BIC, 2003).

Various probability models for predicting carriers of deleterious BRC,41 or BRCA2 mutations in B(O)C families have been developed. These models essentially dictate whether mutation analysis has to be performed or not, for the cost-effectiveness of genetic screening. With the increasing incidence of BC in the Philippines, risk assessment ought to be carried out.

Corroborating information obtained from PTT and SSCP with risk assessment and prior probability models impacts strongly in understanding the disease.

1.4 Scope and Limitations

Blood samples come from in-patients rid out-patients in hospitals. A minimum of 4 BC patients was originally targeted as the source of samples. Choice of samples was based on exclusion/inclusion criteria distinguishing suspected familial from sporadic BC patients as indicated under Methods. Age of early-onset BC cases were only based on the age at disease diagnosis. BC risk assessment models were only applied to the unaffected high-risk individuals. Genornic DNA was the template used. Putative polymorphisms detected in some exons of BRCAI using SSCP were identified. Based on specificity and limitations of PTT, only the BRC,41 truncating mutation in exon I was identified in this study. Only the sample having truncated protein product based on PTT results was validated by DHPLC and subsequently sequenced. Prior probability models as well as the Myriad.com mutation prevalence table were utilized in estimating carrier probabilities of selected patients. Other mutation detection techniques, such as DGGE and CSGE, were not performed. Page 11 of 126

11. REVIEW OF RELATED LITERATURE

2.1 Clinical Categories of Breast Cancer

Sporadic breast cancer (SBC) occurs in 80-90% of all recorded breast cancer cases. One to two cases in a family scattered across 34 generations are estimated to occur randomly, unrelated to genetic predisposition. The definition of familial breast cancer (FBQ varies, but all require several cases of BC among near relatives in the same line of descent. The relative risk of BC in family members of an individual with BC compared to the risk in the general population is the usual measure of familial predisposition. Predisposition is site-specific, but there is variation in penetrance between and within families. Thus, FBC which is observed in 1-20% of all BC cases is clinically variable. In contrast, cases of hereditary breast cancer (HBQ are clinically similar despite genetically heterogeneous conditions occurring in approximately 50% of families. Several clinical characteristics which distinguish HBC from other types of BC include: (1) 10-20 years earlier age of onset, 2) bilateral BC, 3) increased incidence of second BC, 4) male BC, (5) occurrence of additional cancer diagnoses in a single individual or among close relatives, (6) occurrence of characteristic co-morbidities associated with known genetic syndromes, and (7) occurrence of multiple affected family members from one lineage, maternal or paternal (Scheuner, 1997). Presence of autosomal dominant inherited BC susceptibility genes (e.g., BRCAl and BRCA2) probably contributes to genetic heterogeneity despite homogenous conditions. Prevalence of clinically presented polymorphisms of BC susceptibility genes within various populations is currently poorly defined. This pattern found in HBC families overlap with characteristics of FBC (Gayther, Pharoah, & Ponder, 1998; Lynch et al., 1984).

2.2 Breast Cancer Susceptibility Genes

BReast CAncer susceptibility gene type I BRCAI), popularly called hereditary breast and ovarian cancer gene, is a large ene of 22 coding exons spanning >80 kb of genomic DNA (Smith et al, 1996). BRCAI is not a member of any known gene family. BRC,41 is located at l7q2I (Hall et al., 1990) and isolated by Miki et al. 1994) who reported the total coding sequence of 5592 nucleotides, 61 % of which is covered by exon IL The BRCAI mRNA has a size of 78 kb with a complex pattern of alternative splicing. Although expressed in numerous tissues like the breast, ovary, testis, spleen, kidney, brain cortex, colon, lymph node, lung, heart, esophagus, small intestine, pancreas, liver, thyroid, and thymus, BRCAl is found to be localized in ductal secretions of normal breast tissues (Bernard-Gallon et al., 1998; Xu et al., 1997). Page 12 of 126

The corresponding protein (Brcal) is composed of 1863 amino acids with a molecular weight of 220 kDa (Chen et al., 1996). Brcal is a tumor suppressor probably involved in DNA recombination, DNA repair, transcriptional regulation, and chromatin remodeling (Hedenfalk et al., 2002; Scully Livingston, 2000; Haile Parvin, 1999).

Like BRCAJ, BReast CAncer susceptibility gene type 2 (BRCA2) is a large gene of 26 coding exons spanning more than 70 kb of genomic DNA. BRCA2 is located at 13ql2-ql3 (Wooster et al., 1995) and isolated by Tavtigian et al. 1996) who reported the total coding sequence of 10, 257 nucleotides. The BRCA2 mRNA is 11-12 kb long. Similar to BRCAJ, it is also expressed in various tissues like the breast, thymus, testis, lung, ovary, and spleen (Tav-tigian et al., 1996).

The corresponding protein (Brea2) is composed of 3418 aino acids with a molecular weight of 384 kDa. Brca2 is also a tumor suppressor probably involved in DNA repair and transcriptional activation (Tavtigian et al., 1996).

2.3 Pathologic Manifestations of BRCAI and BRCA2

Pathologic manifestations of BRCAI- and BRCA2-associated BC cases, general and molecular, when compared with SBC are shown in Table I (Osin & Lakhani, 1999; Lakhani et al., 1997). More medullary and atypical medullary carcinomas are observed in BRCAI-associated BC compared to both SBC and BRCA2-associated BC. BCAI-associated cases have higher mitotic counts while BRCA2-associated cases have lower mitotic counts compared to SBC. The higher proliferation rate of BRCAI-associated BC suggests that the affected gne may encode a protein involved in cell proliferation. The lack of tubular carcinoma in the BRCA2-associated BC indicates a defective gene involved in cell differentiation or cellular interactions. BRCAJ carriers also showed deficiency of cancers with low rate of cell division and excess of cancers with high rate of cell division when compared to SBC. No such data is available for BRC,42-associated BC.

BRCAI-associated BC is usually estrogen- and progesterone-negative while SBC and BRCA2- associated BC are usually estrogen- and progesterone-positive.

BRCAI- and BRCA2-associated BCs exhibit similar pathologic manifestations in all the other characteristics. The higher overall pathological grade in both BRCAI- and BRCA2-associated BC cases indicates debatably poorer prognosis. Page 13 of 126

2.4 Association of BRCA112 with FBC

Association of both BRCAJ and CA2 with FBC has been established by the identification of germline mutations in affected individuals of families (Tavtigian et al, 1996; Miki et al., 1994). Linkage studies suggest that mutations in BRCAI and BRCA2 are responsible for 45 of families with multiple BC cases and 80% of families with B(O)C cases or multiple early-onset BC cases. BRCA112 mutations are associated with increased risk of cancers at other sites including the ovary, prostate, and pancreas (Sorlie et al., 1998; Gayther & Ponder, 1997; Easton et al., 1993).

More than 1200 sequence variations at the germline level have been reported for BRCA] (BIC, 2003; Szabo et al., 2000). Such germline mutations dispersed throughout the coding sequence of BRCA I predispose to B(O)C. The most number of documented mutations are found in exons 1 1, 16, 20 2 18, 5, 13, 17, 15 and 24 (BIC, 2003) A recent study among Filipino BC patients reported mutations in BRCA I exons 15, 22, and 24 (Matsuda et al., 2002).

Although majority of these variations in BRCA1 are unique, recurrent mutations like 185delAG and 5382insC are frequently observed and more than 75% of the sequence variants lead to a truncated protein (Theodor et al., 1998; Hogervorst et al., 1995). All mutations combined, penetrance at age 70 years is 56% to 87% in BC cases while that of ovarian cancer (OC) cases ranges from 16% to 63% (Birnbaum et al., 2000).

In the coding sequence of BRCA2, at least 1400 unique germline mutations have been reported and are dispersed throughout (BIC, 2003; Szabo et al., 2000). Recurrent mutations observed include 6174delT, 999de15, and 6503deITT. Majority of the mutations observed leads to a truncated protein.

BRCA] germline mutations are associated with B(O)C while BRCA2 mutations are associated with male BC although BRCA2 mutations also occur in female 13Q. Initially, the BC risk elucidated by BRCA2 mutations was considered equivalent to that of BRCA I, but in recent work based only on the recurrent BRCA2 999de15 mutation, estimated BC risk at age 70 years is 37%. The OC risk is lower than that of BRCAJ (Basharn et al., 2002; Lalloo et al., 1998; Thorlacius et al., 1998). Page 14 of 126

Jable 1. Pathology of BRCAI- and BRCA2-Associated BC Cases Compared to SBC Cases. Pathological Characteristic BRCAI-associated BC BRCA2-associated BC Medullary and atypical medullary carcinoma higher frequency of occurrence same frequency with SBC

Tubular carcinoma % occurrence in SBQ few few or none (defective -ene may be involved in cell differentiation or cellular interactions)

Proliferation rate Higher (affected ene may encode a protein involved in cell proliferation)

Mitotic count higher lower

Tumor perimeter proportion (TPP) with continuous pushing margin higher higher

Lymphocytic infiltration higher higher

Nuclear pleomorphism higher higher

Overall pathology grade Higher Higher (debatably poorer prognosis) (debatably poorer prognosis)

Other associated phenotypes in BC and epithelial ovarian cancer invasive invasive different similar (-estro-en receptor-negative) (-estrogen receptor-positive) different similar (-progesterone Teceptor-ne-ative) (-progesterone receptor-positive)

Frequency ofp53 mutation higher higher

p53 stabilization frequent frequent

HER2/c-erbB-2/neu overexpression less less

Immunoreactivity for steroid hon-nones absent absent

Somatic mutation rare rare

Cancer cells with low rate of cell division (I 4 per IO high power fields) deficient

Cancer cells with high rate of cell division (>10 per 10 high power fields) excessive Page 15 of 126

2.5 Estimating Relative Risk and Lifetime Risk of BC

In families with definite hereditary or familial tendencies, age of onset is relatively earlier. Risk management is affected by calculated risk at specific time intervals to estimate absolute lifetime BC risk. The Gail and Claus models are the most commonly used models. Both models are derived from datasets involving large population-based studies. Cumulative risks of BC calculated by these two models may be used in a clinical setting to: (a) identify women who are at increased risk, (b) discuss modified options for breast cancer screening (i. e., beginning mammography at a younger age or having more frequent clinical breast examinations, and (c) provide reassurance to many women who had previously overestimated their risk of breast cancer. Since these models cannot be applied to probands who had been diagnosed of BC, these can only be utilized on the unaffected high risk individuals included in this study.

2.5 Gail Model Variables considered in the model are: (a) age at menarche, (b) current age, (c) number of breast biopsies, (d) age at first live birth, and (e) family history of BC which is the number of first- degree relatives afflicted with BC (Gail et al., 1989). The equation used to predict lifetime risk is as follows:

f'A(u)RR(u;x)exp(-f' (A(v)RR(v;x) + h2(v))dv)du AR(ala2; X) f2 I (1) exp(- (v) RR (v; x) + h, (v)) dv) and RR(u;x) exp(B'X(u)) (2)

The absolute risk AR(al, a2;X) is the probability hat the woman with age al and risk factors x will be afflicted by BC by age a2. It is affected by the functions h](u), h2(u), and RR(u; x), wherein hu) is the age-specific incidence rate for an individual of age u without the incorporated risk factors, h2W is the mortality hazard from other death causes aside from BC, and RR(u; x) is the relative risk for BC for a woman with covariates x(u) at age u (or simply ratio of h2 (U) to hi u)).

Information to be obtained from this equation may be biased, ie., over- or underestimated An increase in the number of breast biopsies without atypical hyperplasia (particularly in women age <50 years) will also increase RR by almost twice to thrice relative risk value compared to those who had not undergone any biopsy resulting in inflated risk estimates. Since paternal family Page 16 of 126

history of BC is not incorporated in the Gail model, risk is underestimated by 50% in families with cancer in the paternal lineage. The Gail model is optimally used in calculating cumulative BC risk, i.e., adding the absolute risks at 10-year intervals until age 70 years to calculate the lifetime risk, even in the absence of family history in women. Relative risk (RR) values used to calculate summary RRs are shown in Table 2.

2 2 Claus Model Using Claus model, relative risk is estimated based exclusively on the family history of BC, maternal or paternal. The model is more applicable to women with family history of BC since it considers the age at BC diagnosis and evaluates the information from both first- and second- degree relatives (Claus et al., 1994) A prediction table has been devised with I 0-year increment of age of woman at time of diagnosis (Table 3.

2.6 Some Molecular Techniques and Mutation Studies on Detection of BRCAJ Mutation

BC susceptibility in early-onset families has been located in band 17q21 using linkage analysis. Claus et al. 1991) also presented evidence for the existence of a rare autosomal dominant allele (q = 00033) leading to increased susceptibility to BC.

Miki et al (I 994) also identified a strong candidate for the BRC,41 gene using positional cloning methods where predisposing mutations detected in five of eight kindreds are presumed to segregate BRCAI susceptibility alleles. Due to the large size of BRCAI 5592 bp) encoding a protein of 1863 amino acids, detection of predisposing mutations is very laborious and expensive if sequence analysis will be employed. Different types of mutation analysis are therefore utilized to screen for BRCA I mutations.

Single-strand conformation polymorphism. (SSCP or SSCA) analysis is an extensively used technique to detect mutations due to its simplicity. A schematic diagram of SSCP is shown in Figure 1. The region of interest in the genome or DNA is amplified by PCR, denatured to form single strands, and analyzed by nondenaturing polyacrylamide gel electrophoresis (Orita et al., 1989a). The two denatured strands PCR-arnplified DNA of the test sample adopt a stable conformation which may differ from the wild-type in the presence of a polymorphism or mutation. This results in an alteration of the electrophoretic mobility of the single-stranded DNA (ssDNA) bands (Glavac and Dean, 1993; Hayashi and Yandell, 1993). In an initial stud-, on BRCA] using SSCP on PCR-amplified genomic DNA of 50 probands with family history of B(O)C. 8 putative heterogeneous disease-causing alterations are found. Paoe 17 of 126

Table 2 Relative Risk Values of the Gail Model. Relative Risk Factor Relative Risk Relative Risk Factor Relative Risk Value Value

Main Risk Factor

Age at menai che (in years) >14 00 12-13 10 <11 21

Relative Risk '.Modifiers

Number of biopsies Biopsy with atypical hyperplasi 2 Not applicable or unknown I 0 Age <50years No 93 No biopsy I 00 Yes 1.82 1 biopsy 1 0 >2 biopsies 2.88 Mammographic density Not specified I 000 Age 501years 0% fatty 0 405 No biopsy I 00 I% to 24% fatty 0 636 1 biopsy 1 27 25% to 49% fatty 1.000 >2 biopsies 1 62 50% to 74% fatty 1.121 75% to I 0% fatty 1 761 Number of P relatives with BC LCJS on bwpsy2 Age afirst live birth <20years Not applicable I 0 No relative 1.00 B iopsy at ae 40 years 70 I relative 2 61 Biopsy at ace 40-44 years 6 0 >2 relatives 6 80 Biopsy at age 45-49 years 60 Biopsy at ae 50-54 years 4 0 Age atfirsf live birth 20-24 yea)s Biopsy at age >54 years 3.10 No relative 24 1 relative 2 68 !2 relatives 78

Age atfit st live birth 25-29 yeal s No relative 55 1 relative 2 76 >2 relatives 4.91

Age atfirst live birth 30years No relative 93 1 relative 2 83 >2 relatives 4 17 Page 18 of 126

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Figure 1. Schematic iagram of single-strand conformation polymorphism (SSCP) analysis. Wild-type (wt), heterozygote (het) ad mutant (MLIt) PCR products are shown at te top T dots on the DNA strands represent existing point Mutations. Darker single strands represent the sense strands while the lighter sngle strands represent te anti-sense strands. Sequence ariations between sense and anti-sense strands exhibit different folding patterns. Different single-stranded confoijnations lead to differential obility in te non-denatUring gel. Notice the presence of four single-strands i the heterozygote DNA aving aleles of both wild-type and rutant DNA. Source Grompe 1993 \ Gmet 111-117 Onta et a/ 1989a GenonncT 874-879, Orita et al 1989b Pro: \adAcad Sci U4 86 2766-1-770 Page 20 of 126

The large size of the gene and eterogeneity of mutations indicate technical hurdle for clinical applications. Genornic DNA from patients have been mixed together and subjected to PCR followed by SSCP to test the limits of pooling. SSCP of pooled DNA allows rapid detection of germline mutations in large genes like BRCA I. In pools composed of four to five DNA samples, fragments containing germline mutations could be easily detected (Kozlowski et al., 1996). However, it is safer to have pools composed of three DNA samples having exactly the same concentration and quality (KozlmNski, personal communication, 2002).

Since more than 75% of the reported BRCAJ mutations result to truncated proteins, the protein truncation test (PTT) by Hogervorst et al. 1995) was developed to screen for mutations in exon I 1. PTT, also referred as the in vitro synthesized protein assay (IVSP), exploits the principle of in vitro protein synthesis. The general strategy utilized in these studies entails cDNA to be used as PCR template for the 21 smaller exons containing 39% of the coding sequence, while exon I I comprising 61% of the coding sequence (Miki et al., 1994) is amplified from genornic DNA. In PTT, the coding region of a gene is screened for presence of translation terminating mutations using de novo protein synthesis from amplified copy. This procedure includes three important steps. First step involves isolation of genomic DNA and amplification of the target gene coding sequences using PCR, or alternatively, isolation of RNA and amplification of the target sequence using RT-PCR. The resulting PCR products are used as templates for in vitro synthesis of RNA, which are subsequently translated into proteins. Final step involves the SDS-PAGE of the synthesized proteins. Shorter protein products of mutated alleles are easily distinguished from full length protein products of normal alleles (Hogervorst, 1997) A schematic diagram of PTT is shown in Figure 2.

PTT was first utilized in analyzing BRCA mtations in 45 patients from B(O)C families in the Netherlands (Hogervorst et al, 1995). Six novel mutations 13%) are detected in this study which included two single nucleotide insertions, three small deletions (1-5 bp), and a nonsense mutation resent in two unrelated families. Aberrantly spliced products affecting exons 5 and 6 are also detected. This is followed by the study of De Benedetti et al. 1996) among 70 Italian B(O)C patients. BRCAJ mutations are identified in 9 of 29 patients 31%) with a family history of cancer and in 3)of 41 women 7%) with early-onset BC. Some of the studies that screened for BRCA112 mutations utilizing SSCP, PTT and other mutation detection methods are shown in Tables 41 to 44 (BIC, 2003). Truncating mutations are most frequent in hereditary breast- ovarian cancer (HBOQ families occurring in 47 8% of the families screened (Table 42) which is followed by hereditary ovarian cancer (HOC) families, HB(O)C families, and HBC families, occurring in 39.1%, 21 6, and-8 6 of the families screened, respectively (Tables 43 44, and Page 21 of 126

4.1). Truncating mutations least occurred in pure HBC families. Occurrence of missense mutations exhibited the same trend except that the mean occurrence in HOC cannot be calculated since only one of the three references specified the results (Table 43). The immense nmber of HB(O)C families screened definitely include HBOC, HOC, and HBC families indicating that the mean occurrence could be represented by HB(O)C families alone since the number of families screened for the other three is relatively few.

Studies identifying mutations in the BCAI gene of Filipino BC patients is quite limited. The first mutation detected is a Filipino BC patient in Henry Ford Hospital, Michigan, U.S.A. which revealed a point mutation in exon I I at nucleotide 2178 resulting in a C>T transition (2178C/T) that caused stop codon at aa687 (Worsham et al 1998). Another study screened exons II, 15, 22, and 24 and reported 3 mutations in exons 15 QI538X), 22 (5454deIC), and 24 (RI835X). The total estimated BRCAI mutation prevalence is 1.0% of 294 Filipino BC patients from Manila. The same study mentioned BRCAl exon 22 5454delC mutation detected in a Filipino BC patient from Hawaii (Matsuda et aL, 2002). Both studies utilized PTT at exon I .

More sensitive techniques requiring shorter time have been developed aside from SSCP and PTT. One of these methods is the denaturing high-performance liquid chromatography (DHPLC) that compares sequence based on heteroduplex detection (Wagner et al., 1999). Heterozygosity for a mutation or polymorphism in a sequence can be detected by the generation of heteroduplex molecules between the wild-type and the mutant sequences. Heteroduplex formation is induced by heating followed by slow cooling of the PCR product from that sequence. Partially denaturing conditions are used to separate homo- and heteroduplex strands. DNA applied binds to the hydrophobic column (e.g., DNASep column by Transgenomics) via the ion-pairing agent, triethylammonium acetate (TEAA). DNA is then eluted by an increasing gradient of acetonitrile (ACN). ACN disrupts the interaction between TEAA and DNA causing the DNA to be luted from the column and detected as a peak by D260nn monitoring of the eluate

At a non-denaturing temperature, the concentration of ACN required to remove any given fragment depends on the size and sequence of that fragment. In a linear radient of ACN, any DNA fraament therefore elutes at a characteristic point (retention time) When the column temperature is increased, elution takes place at progressively lower ACN concentrations as te DNA starts to denature. Heteroduplex and homoduplex DNA have different melting characteristics that under conditions of partial denaturation, heteroduplex DNA has a reduced retention time on the column. Therefore, DNA containing mismatches vill clute at an earlier time. Page 22 of 126

Multiple peaks (up to 4 are often seen representing differences in the retention times of 4 species 2 X homoduplexes and 2 X heteroduplexes). More often, less than 4 peaks are seen, and the presence of heteroduplexes is determined by comparing the lution profile obtained from a test sample with that for the wild-type sample.

Sensitivity has been shown to be 90-100%. Additional advantages of DHPLC include: (1) simplicity in primer design; 2 use of unlabelled primers (same primers can be used for sequence analysis of the same fragment); 3) inexpensive - once set up, high throughput analysis can be done due to 96-well format and -8min analysis time per sample (MRCPath, 2001/1999).

2.7 High Frequency BRCAI Mutations and Their Corresponding Effects on Gene Function

Among the 1200 sequence variations reported for BRCAI, the 20 most frequent mutations based on designation with their corresponding effects on gene function are shown in Table 5 (51C, 2003; Szabo et al., 2000; Neuhausen, 2000). The protein encoding regions of BRCAI are shown in Figure 3 BRCAI has several important functional domains (RING finger domain, p5) interaction domain, transcription activation domain, among others). Truncating mutations in families with high proportion of OC tehd to be located at the 5' end (exons 213), while families primarily exhibiting BC apparently have truncating mutations at the 3 end of BRCAJ (exons 13- 24) (Gayther et al., 1995; Shattuck-Eidens et al., 1995). This genotype-phenotype correlation is supported by cell line studies wherein near full-length truncated Brcal have been shown not to arrest 13C, but to inhibit OC cell growth (Holt et al., 1996). Furthermore, truncation of conserved terminal regions of Brcal protein appear to be associated with highly proliferating BC (Sobol et al., 1996).

2.8 Genetic Risk Assessment Based on Prior Probability Models

Making the decision, on whether or not an individual should undergo genetic testing and probably gain informative results, is facilitated by the estimated probability that an individual or family carries a mutation in BRCAI or BRCA2. Different centers have adapted the suggestion of the American Society of Clinical Oncology (ASCO) that consideration of BRCA gene mutation testing should be limited to individuals whose prior probability exceeds 10% (greater-than-10% mutation probability threshold) while other centers still consider >5% prior probability for testing. The widely used models for estimating prior probability are as follows: (a) Couch Model, Page 23 of 126

(b) Shattuck-Eidens Model, (c) Frank Model, and (d) BRCAPRO. These models do not assess actual cancer risk.

2 8.1 Couch Model This model estimates the probability of finding a BRCA I mutation within a family with 2 BC cases. Data utilized in generating this model is derived from heteroduplex analysis using conformation sensitive gel electrophoresis (CSGE) and logistic regression from a study population with an overall BRCA I mutation prevalence of 16%. The Couch model has now been modified to include both BRCA1 and BRCA2 mutation detection by full sequencing. Variables included in the modified model are the: (a) number of women diagnosed with BC before age 0 years, (b) breast-ovarian multiple primary cases, (c) ovarian and fallopian tube carcinomas, (d) male BC, and (e) Ashkenazi Jewish ancestry (Couch et al., 1997). The Couch model is best utilized for families with >4 B(O)C cases and is particularly useful in predicting BRCA1 mutation status among Ashkenazi Jews. However, it is not applicable to site-specific OC families. Since the study population is a relatively small sample size, confidence intervals. are wide, or could not be calculated, for some point estimates, such as families with BOC in a single individual. Its reliance on mean age at BC diagnosis could result in the same probability estimates (i e. a woman with one relative with BC at age 3 years and another woman with several relatives afflicted with BC but having a mean age of diagnosis of 3 years will have equal probability estimates).

Estimates of prior probability for unaffected individuals still need to be extrapolated from the closest affected relative without a built-in Bayesian adjustment for age. CancerGene calculates probabilities based on maternal and paternal lineages separately then the highest estimate is chosen. A prediction table has been devised with 5-year increment of average age of women at their time of diagnoses (Table 6.

2 2 Shattuck-Eidens Model Myriad ) This is the second logistic regression model intended to estimate the probability of finding a BRCAI mutation which is derived from a study population having overall BRCA1 mutation prevalence of 13%. It is the first of several models developed by Myriad Genetics (Salt Lake City, UT). Variables included in the model are: (a) younger age or bilateral BC at diagnosis, (b) OC in the tested patient, (c) relative with B(O)C, and (d) Ashkenazi Jewish ancestry Shattuck- Eidens et al., 1997). Estimated prior probabilities are presented as graphs for specific family histories. This model can be utilized in affected indi-, iduals v6thout any family history of B(O)C as well as patients with site-specific OC. Aside from the tested individual, only one affected Page 24 of 126

relative is used to determine the probability of identifying a mutation (e.g., a BC patient with I affected sister is assigned the same probability as that of another BC patient with 3 affected sisters). Shattuck-Eidens model is therefore useful for Ashkenazi Jewish families with small number of affected members since it underestimates risk with multiple affected members. Moreover, this model is not applicable to women diagnosed with BC before age 30 years and the graphs presented cannot be used for unaffected individuals (probabilities can only be derived or extrapolated from closest affected family member)

Prior probabilities are determined on an individual basis so probability can vary within a family (e.g., a woman with BC will have a different prior probability with her sister who has BOC). CancerGene uses Mendelian coefficients to calculate proband probability based on calculations from every affected relative and chooses the highest estimate.

2.8.3 Frank Model (Myriad 11) This model generated from the second multicenter study coordinated by the Myriad Genetics estimates prior probability for both BRC41 and CA2 mutations. Estimates are derived from study population having BRCAI12 mutation prevalence of 39% with the more stringent criteria used. Variables identified based on logistic regression analysis are: (a) BC diagnosis before age 40 years, (b) ovarian cancer, and (c) bilateral BC (Frank et al., 1998). Although 20% of the study population reported Ashkenazi Jewish ancestry. this is not considered as an independent predictor for this analysis of high-risk individuals. This model is best utilized in providing prior probabilities for both BRCAI and BRCA2 among families with multiple women (>l first or second-decree relative) diagnosed with BC before age 50 years or OC at any age. However, family history is not applicable to BC patients diagnosed after age 50 years.

Risk estimates are for individuals (not families). extrapolation is required for unaffected individuals, and, when compared to other models. higher estimates are often produced (i.e., lowest estimated probability is 025 which is still higher than probabilities enerated in a series of smaller, site-specific BC families) due to the stringent set of high-risk families (Peto et al., 1999) A prediction table devised by Frank et al. 1998) is shown in Table 71. Prevalence tables for BRCAI and BRCA2 mutations have been constructed using more than 10,000 individuals tested in Myriad Genetics in various clinical situations (Frank et al., 2002). These data (new Myriad 11) are empiric and not modeled yet. Unlike the original Frank model, the mutation prevalence values range from to 100% (Table 72). At least I BC case in a relative is needed for the Myriad.com data to be applied. The M- rad com mutation prevalence table for non- Page 25 of 126

Ashkenazi families can be utilized for estimating carrier probabilities of the probands in this study.

2.8.4 BRCAPRO This Bayesian model, developed by Parmigiani, Berry, Aguilar 1998), provides estimates for the likelihood of finding either a BRC I or CA2 mutation in a family. Variables included are: (a) published BRCAI and BRCA2 mutation frequencies, (b) cancer penetrance in mutation carriers, (c) cancer status (affected, unaffected, or unknown), (d) bilaterality, (e) age at diagnosis of disease, and (f) age of the first- and second-degree male and female relatives of the proband. The model is expressed through the equation,

P[BRCAIBRCA2jFamiIy History]= P(BRC,41]P[BRCA2]P[Family_HistorylBRCAIBRCA2] (5) P[Family History] wherein the carrier probability estimate is calculated using the maximum ikelihood method (Appendix A). The value of P[Family.. HistoryjBRCAlBRCA2] depends on the: (a) probability of the genetic status of offspring given that of their parents, (b) probability of the genetic status of parents given that of their offspring, and (c) penetrance based on published reports. Since the analysis is based on large, high-penetrance families, this model often produces higher estimates for clinic-based families compared to the Couch and Shattuck-EideDS models. BRCAPRO apparently performs well in a population at hicyh risk for BRCA112 mutations 71% of families had >3 B(O)C cases, 42% of families were of Ashkenazi Jewish descent) but it has to be taken into account that only 56% of the study population had BRCA112 mutations. Moreover, only first- and second-degree relatives are included in this model so the effect of occurrence of cancer in third- to fifth-degree relatives will not be included in the probabilities estimated.

Probabilities vary depending on which family member is used for analysis. Choosing the individual who best captures the core of affected relatives may provide the best estimate. CancerGene a publicly available software, provides prior probabilities from BRCAPRO as well as Couch, Shattuck-Eidens, and Myriad.com values (http:HwwW3.utsouthwestern.edu/ cancergene). BRCAPRO can also be used to estimate cumulative unconditional probability of BC before a particular age by obtaining the mean cumulative incidence probabilities for carriers and noncarriers. Page 26 of 126

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td ci 00 n m a, o Cl) It C CD V) cq CA

tj cc In (M C/) Table 44. BRCAJ Mutations in Families with Hereditary Breast and/or Ovarian Ca cer HB(O)C] Cases. Number of- Families Truncating Missense Selection Criteria Mutation Detection Methods Reference Screened Mutations Mutations 50 6 2 3' early-onset Br or 2 Br & I'Ov SSCP all exons Castilla 1994 (Nature Genet 387) 10 12 0 3 early-onset Br or 2Br & I' v DS entire coding region Simard 1994 (Nature Genet 392) 47 14 2 4 Br or 4' Br/Ov SSCP, ASO, PTT, entire coding region - Friedman 1994,1995 (Nat Genet 399, Am J Hum Genet 57 1284) 35 6 NS Prior probability BRCA 1> ITTentirec6dingregi ji 95 CNatGen 04 208) 60 32 0 4' Br <60, Ov any age 2 Hogervorst 9 SSCP/HA all exons Gayther 1995 (Nature Genet II 428) 47 1 14 1 3 Br or Ov, I' 50 gPTT exon I , SSCP/HA small ons Johannsson 1996 (Am Hum Genet 58 44 1) 162 1 34 1 4' Br or Ov 2 Ov SSCP/HA detecting -50% of mutations Gayther 1996 (Am J Hum Genet 58 45 1) 70 12 0 Ov or Br <50 and FH', Br<36 gPTT exon I I De-Benedetti 1996 (Oncogene 13 1353) 63 3 0 Bi<40, Br<40 &FH', Ov &FH' PTT entire coding region Garvin 1996 (J Med Genet 33 72 1) 20 3 0 3' Br, ay Ov SSCP entire coding region Jandng 1996 (Int J Cancer 68 188) ,6 6 2 1 n ly-onset Br, I 1', my Ov ASO for I intilations, DS 20 flls) Caligo 996 (OJ1L0gC11C 13 1483) 16 6 1 3Br or Ov any age gP17 exon I , SSCP entire coding region Montagna 1996 (Cancer Res 56 5466) 21, 5 0 3Br or Ov any age DS DDI'enurecodingreg:011 DUrochcr 1996 (J Med Genet 33 814) 220 91 NR 2Bi I' dagnosed <50 Screening for 185delAG, 188del II, 5382insC Tonin 1996 (Nature Med 2 1179) 49 6 0 3' Br or Ov any a SSCP entire coding region Xu 1997 (Genes Chrom Cancer 1 102) - 29 6 1 3' Br or Ov any ag PTT exon I , SSCP small exons Hamann 1997 (Genes Chrom Cancer 18 126) 45 3 1 3' Br or Ov, 2' under 60 PTT exon II, SSCP smal I exons Hamann 997 (J Med Genet 34 884) 160 38 0 FH' complex DGGE entire coding region Stoppa Lyorinct 1997 (Am J Hum Genet 60 02 1) 666 79 0 FIV complex, prior probability BRCA 1>0 I ---- PTT exo I , additional screening some samples Peelen_1,997(AmJHumGenet60 1041) 42 16 0 2' Br or Ov, any age CSGE exons 2, 20 (185delAG, 5382insC) Levy-Lahad 1997 (Am J Hum Genet 60 1059) 106 23 1 FH', complex, but majority 3' Br PTT exon I , SSCP small exons Hikansson 1997 (Am J Hum Genet 60 1068) 9 5 1 Prior probability BRCA I>0 20 DS entire coding region Gao 1997 (Am J Hum Genet 60 1233) 32 1 7 0 2' Br or Ov any age, Ist degree PTT exon I , SSCPIHA small exims Ramus 1997 (Am J Hum Gene 60 1242) 798 99 3 FH', complex DS all coding real ns &intron boundaries Shattuck-Eidens 1997 (JAMA 278 1242) 122 2 1 2' Br or Ov any age SSCP-HDX all exons Sobczak 1997 (Oncogene 15 1773) 100 10 0 3' Br or Ov any age PTT exon I , SSCP/HA small exons, DS Vchimmen 1997 (Hum Mot Genet 6 2309) 19 13 1 2' Ov, any Br PTT exon I , SSCP/HA small exons Gayther 1997 (Am J Hum Genet 60 1239) 86 6 NS'O Bre40,,-Br<40'&,FH1,, OV& FH' VTT entirito ding re '7 7 77 (J Med Genet 34, 990)',-,' 33 5 Hi risk fa lies' "PTTexon'l 1,- I, 99-7;(Eur J Cancer 33 23 170 3 NS, High nsk1hridies 774 25 1 6 0 4' Br (2 <55), 2' Br <60 and Ov any age, 2' Ov any age CDGE all exons Andersen 1998 (Hum Mutat 11 166) - 86 17 6 2' Br (<55) or Ov (any age), 3' Br (2 <60) and/or Ov (any age), I case Br <30 DHPLC all exons Wagner 1998 (Int J Cancer 77 354) 17 6 0 2' Br (<50) or Ov (any age) 9PTT exon I 1,SSCP all coding exons Lancaster 1998 (Br J Cancer 78 1417) 30 8 0 V Br <40), or I' Br (<45) and I' Ov (<60) SSCP-HD all exons Eccles 1998 (Br J Cancer 77 2199) 238 60 3 1 Br <50, or Ov any age, and 1+ Ist/2nd relative with either diagnosis DS all exons Frank 1998 (J Chn Oncol 16 2417) 61 17 1 2' Br <60, or Br cases FH' and diagnosed <50 SSCP all exons Dong 1998 (Hum Gcnct 103h ciri 14) -40 6 13 High risle flieg (some sporadic cases) 'Gi)eiiman-4999 Geni-eC I ,hbX21-24 46 7 1 High risk families P DS Wl'* e-1A`TK -',7--77777 18 1 2 1 Multiple Br, I' Br <40 SSCP all exons . "I Li 1999 (Hum Genet 104 201)8381) IT Alvin, 49 5 NS 2' Br or Ov in I st or 2nd degree PTT exon I , SSCP small ons Santarosa 1999 (Int J Cancer 83 15 3 NS PIT, CSGE, DS Bal6i'1999 (Eur J Cancer 35,, 70 ), ,, 618 120 12 Established genetic model (HBC, HBOC. HOC MHBC a present) SSCP,.CSGEDGGE, FAMA PTTDS Chanj-Claudi 1999'(LoiMarkers-'15'53)--,'C, 1V 49 5 3 3'BrorOv I'deg 2 Btor0v(l'- 'deg, <45), ilB, iTBOC PTT'ixon 83 5 3 High riskfamilles, PTTI eio-n 70 1 NS High nskfarnifies, 'PTT;DS' 37 14 NS 'Trick6i'2000'(Bull 28 1 I - I - I 11 ' High risk families PTr, SSCP. HA,-DS 27 3 0 3' Br, Br<50 PTT exon I , SSCP small exon Osono 2000 (Br J Cancer 92 1266) 66 27 7 3' Br or Ov, +<50 SSCP all exons Gorski 2000 (Am J Hum Genet 66 1963) 30 6 0 2' Br <50 ato Ov any age PTT exon I , DS smal I excins Konstantopoulou 20GO(Hum Mutat 359 on 53 8 I I High risk families, PTT, HA, DS' Yazi6i2000 (BrJ Cancer,83.737) 51 7 3 High risk families -CSGE, DS 119 5 3 Multifocal and bilateral BCfainflies PTT, P all coding exons" 86rgtBI&M 21. IH_MutatOnlirie'15381)I (j Med Ge= it 21 1 8 2 V Br and Ov in I I families, 9 BCs, and I Ov RNA-based DS Jakbbowska 2001 (Hum Mina 00k1j 100 25 NS I' BR and I' Ov same Imeage CSGE.HA.DS MartiW2001 (J Chn Oncol 19 2247)' H:gh ' fairulles PTT, DHPLC, DS Moller 2001 (Fur J Canter 37 1021) ISS g 082 51; 98 NS H HE families PTT, SSCP, ASO, DS Verhoog 2DOI (Eur J Cancer 37 2 1-i Legend: NR not relevant NS not specified in manuscript Source BIC, 2003 and some additional references indicated by shaded regions C., Page 29 of 126

Table 5. Frequent BRCAI Mutations by Designation and their Corresponding Effects on Gene Function. - Clinical Designation Nature of Location Codon Effect on BRCAI and Comment Type Mutation Its Function Hereditary 185de]AG Frameshift Exon 2 23 Disrupts the RrNG domain Increased lifetime risk of B(O)C, (truncating) and all domains after it, Families predominantly have OC Results inBRCAI cases, Founder mutation of inactivation Ashkenazi Jeviish population (Gayther et a 1995, StrUewing et at, 1995) C61G Missense Exon 5 61 Disrupts RNG binding Founder mutation of German and domain removin- effect on Latvian populations (Backe e a/, protein ubiquitination and 1999, Csokay e al , 999) transcriptional modulation of ATFI (Baer& Ludwig, in press, Houvras, 1999) 1135insA Frameshift Exon I] 339 Disrupts 3 interaction Families predominantly have OC (truncating) domain and all domains cases, Founder mutation of after it, Results in BRCA I Norwegian population (Borg et inactivation at, 1999, Dorum et al, 1999, Gayther e a 1995) Q356R Missense Exo I 356 Neutral to positive amino May be related to BC when acid (no reported apparent Simultaneously present with effect) S 521 (Hadjisavvas et at, 2002) R496H Missense Exon I 496 No reported apparent effect Rare variant 1675de]A Frameshift Exon I 319 Disrupts -AD51 binding Families predominantly have OC (truncating) domain and all domains cases, Founder mutation of after it. Results in BRCA I Nor-vi egia n population (Borg et inactivation a/, 1999, Dorum e al, 1999, Gayther et at, 1995) Q563X Nonsense Exon I 563 Defectne i stimulating Families predominantly have OC (truncating) transcription ad DNA cases, Founder mutatio of repair ad fail to induce Swedish population (Johrinnsson chromatin unfolding d t et l. 1996, Ga) ther et at, 1995) absence of COOH terminus of the Brea (Ye et at, 200 , J in et a, 2000) 2804de]AA Frameshift Exon I] 895 Disrupts RAD51 bnding Families predominantly ave OC (truncating) domain and all domains cases, Founder utation of utch after it Results in BRCAI population (Peelen et a, 1997, macti aion Gayther et a 1995) RI443X Nonsense Exon 3 1443 Disrupts cranscription Families predominantly ave (truncating) activation, domain and all B(O)C cases, Founder utatio of domains after it, Results in French-Canadian population BRC,41 inactivation (Sobol et /, 1996, Gayther e /, 1995, Simard e a 1994) 5382insC Frameshift Exon 20 1756 Disrupts ranscription Families predominantly have BC (truncating) activation, domain and all cases, Founder utation of domains after it Results in Ashkenazi JeiNish, German, BRCAJ inactivation Latvian, and Russian POPUlations (NeLfliausen, 2000, Sobol et a, 996, Gayther el at. 1995 Tonin et al, 1995) IVS21-36del5lO Frameshift Exon 22-36 1778 Disrupts transcription Families predornmantly have BC (truncating) activation, domai ad all cases, Founder mutation of Dutch domains after it, Results in population (Petrij-Bosch et al, BRC.41 inactivation 1997, Sobo e a 1996, Gayther el a/, 995) Source of Frequent MU1,111ons by DesiR11,A1011 BIC, 2003 Page 30 of 126

Table 5. Frequent BCAI Mutations by Designation ... (continued) Clinical Designation Nature of Location Codon Effect on BRCA I and Comment Type Mutation Its Function

Familial IVS5-1 IT>G Splice Exon 611 - No reported apparent effect Rare variant

R84 W & Missense Exon I 841 Positive to non-polar R84 I W is a rare polymorphism, M 10081 1008 hydrophobic amino acid, Increased risk of BC remains to be R841 W is likely to be an confirmed etiologically significant lesion with involvement to -1 %of all BOC in Barker el al (I 996) No reported apparent effect of M I 081

E125OX Nonsense Exon I 1250 Disrupts transcription Families predominantly have OC truncating activation, domain, minimal cases (Gayther el al, 995) transcription activation domain, p53 interaction domain 2 and CBP/p3OO binding domain, Results in BRCA1 inactivation

3875del4 & Frameshift Exon I 1252 Disrupts transcription Families predominantly have OC 4184de]4 (truncating) 1355 activation, domain and all cases (Gayther et al, 1995) domains after it, Results in BRCAJ inactivation

RI347G Missense Exon H 1347 Positive to non-polar Rare variant, Weak association hydrophobic amino acid, No with BC risk reported apparent effect

S 15121 Missense Exon 15 1512 Polar hydrophilic to o May be related to BC when polar h)drophobic aino simultaneously present %vith acid Q356R (Hadjisavvas el al, 2002)

M 1628T Missense Exon 6 1628 Non-polar to polar aino Beni.-n polymorphism acid, No reported apparent effect Source of Frequent Mutations by Designation BIC, 2003 Main Breast Cancer Region 0 0 0 0 0 0 0 0 * Main Ovarian Cncer Region

1711-1-

2 3 5 6 7 8 9 10 it 12 13 14 15 16 17 18 19 20 21 22 23 24

RING domain NLS I nd 2 RAD51 binding domain gr.n.n homology Tanscrpt.on act.aoon domain (.a. 24-64) (aa 501-507 ad 607-614) (.a 58-1064) (na 1214-1223) (im 1560-1863)

RARDI binding p53.meacoon doma I Leucme zippei Minimal ranscription (aa 1-101) 224-500) (aa 1209-1230) acin,aton domain (an 1760-1863)

E2F cycl n, CDK URCT domains I and 1 i:,lerac'iwn (aa 1650-1855) (an 2-64)

RIIA binding O 6-1800)

163 miierilchon domai 2 (Ra 1760-1863)

CBP/1)300 binding (an 1760-1863)

Figure 3 Protein encoding regions of BCAL Approximate locations of the functional domains are presented with black lines below tile ,aene. Te genotype-phenotype correlations are shown with square dots (OC region) and round dots (13C region) above the gene. Exon diagr,,ims are drawn to scale bsed on te number of nucleotides (bp).

Source Huusko ( 999), Gayther ei al (I 995), and Shattuck-EidCIIS el 6if (I 995) Page 32 of 126

Table 6 Predicted Probability of a BRCAI Mutation Using Couch Model.

Average Age at Diagnosis BRCAI Mutation Carrier Probability of Breast Cancer BC Only Family* B(O)C Fmily*

<35 years old 0.174 0 550

3 539 Nears old 0.117 0.435

40-44 years old 0 077 0 327

45-49 years old 0.050 0 234

50-54 years old 0 032 0 162

55-59 ears old 0 021 0 108

>59 Nears old 0 013 0.071

*Avera-e of'3 5 camers/family Source 'ouch ei d 1997 Eng J Ied 336:1409-141 Page 33 of 126

Table 71. Probability of a BRCA1 or BRCA2 Mutation in a Woman with BC<50. Any Relative with BC<50? Any Relative with OC? Proband with Bilateral BC Probability or OC?

25 35 0 51

71 Source Frank eta/ 1998 J Clin Onc 16:2417-2425

Table 72. Mutation Prevalence Table for Individuals of Non-Ashkenazi Jewish Ancestry. Family History (Includes at least one first- or second-degree relative)

BC<50 in BC<50 in OC at any OC in more Patient's History No BC<50, one relative; more than age in one than one BC<50 and or OC in no OC in one relative; relative; no relative; no OC at any any relativef any, relative no OC in BC<50 in BC<50 in ageti anN relative anv relative any relative

No BC or OC at any age 0 029 0 042 0 098 0 058 0 087 0 67

13C:50 0 032 0 083 ij 14 0 074 0 098 0 98

BC<50 0 078 0 178 0 316 0 67 0 3 12 0 445

Male BC 0 204 0 238 0 500* 0 000* None Tested I 000*

OC at any age, no BC 0 1119 0 293 O388 0 247 0 322 0 514

BC>50 and OC at any age 0 176 0 21 1* o 43811 0 182* 0 444* 0 500*

BC<50 and OC at any age 0 320 0 567 ( 722* 0 588* 0 625* 0 8 3

May include families wh BC>50 (in women or men) Number of observations is 10231 ,+Includes aily members with either or bot dagnoses *N<20 Note Te ethod sed to develop tis mutation prevalence table of the MN r ad Genetic Laboratories as recemlN been published Frank et l 2002 J Chn Onc 20:1480-1490 Page 34 of 126

111. MATERIALS AND METHODS

3.1 Research Design

The case series of Filipino families with genetic susceptibility to breast cancer was undertaken in this study. Truncating mutations and putative polymorphisms in the BRCAI gene of the Filipino families were defined.

3.2 Recruitment of Patients

Probands and immediate family were recruited from the following participating hospitals or institutions: St. Luke's Medical Center Cancer Institute, Chinese General Hospital, East Avenue Medical Center, Veterans' Memorial Medical Center, Armed Forces of the Philippines-Medical Center, Mapandan Hospital, and Philippine Nuclear Research Institute Medical Clinic. A seminar with medical practitioners was conducted to screen and identify prospective participants. A letter of informed consent (Appendix B) was accomplished explaining the objectives of the research. Guided by the investigators, the participants also answered the structured questionnaires (Appendix Q.

3.3 Inclusion Criteria

Patients with sspected FBC were included. The characteristics that indicate possible familial tendency include at least one of the following: (a) early-onset BC patients (i e , _<40 years old at disease diagnosis), (b) BC patients with OC, () BC patients who have first-degree relatives diagnosed with breast and/or ovarian cancer, and (d) bilateral BC patients. Two unaffected high- risk individuals for BRCAI mutation were also included.

3.4 Exclusion Criterion

BC patients who have no relative with B(O)C diagnosed at age >40 years were excluded from the studv.

3.5 Pedigree Construction and Characterization of Families

Pedigrees of patients (probands) were drawn using CancerGene software. Current age. ape at diagnosis or death, and type of cancer were indicated. Only first- and second-degree relatives Page 35 of 126

were included in the constructed pedigree. Families of BC patients were grouped according to characteristic affliction of their corresponding relatives.

3.6 Patient Information

When available, clinicopathological characteristics of patients and their respective information on hormonal, socioeconomic, environmental, and dietary factors were summarized.

3.7 Lifetime Risk Estimation

3 7 Estimation Using Gail Model Figure 4A is a pedigree of a patient included in this study. The proband is a 40-year old woman (Pt 3 with a relative risk (RR) value of 1 I (Table 2 given age at first menarche is 12 years. Using Table 2 RR of risk modifiers are obtained (Figure 413). By multiplying the values of the different risk modifiers, the summary RR value at her current age is computed at 31 1. Notethat family history is defined by the number of first-degree relatives affected with BC. Presence of OC in relatives will not affect the estimation of RR. Using CancerGene, the summary RR value (Figure 4Q based on three RR values given by variables a (Age Men), b and (Age Bx), and d and e (FLB FHx) indicated in the R6iew of Related Literature (RRL) is 282, which almost approximates the average of her summary RR < 0years old. Her RR values at various ages can be calculated by dividing her risk (subject) by the risk of "no risk" individuals. Her lifetime BC risk is 19.6% (Figure 4Q.

3.7 2 Estimation Using Claus Model Based on the Claus prediction table (Table 3 the relative risk (RR) for the same 40-year old woman (Ptl3) who has a primary relative (sister) diagnosed of BC at age 40 years (Figure 4 is 0.032 or 32%. This value, is similar to the RR value obtained using the Gail model (e.g., 31 1). Usin- CancerGene. the summary RR value is also 32 Figure 5). Z:1 in

3.7 3 Estimation Using BRCAPRO Although primarily a prior probability model, BRCAPRO can also estimate unconditional probability of developing BC before a particular age including the cumulative lifetime BC risk at age 85 years. Using CancerGene, the unconditional probability value for Ptl3 (Figure 4 at age 45 is 28% and her cumulative lifetime BC risk at age 85 years is 19.9% (Figure 6. Page 36 of 126

(A) 56 Br 52

74 72 71 69 75 76 72 Br 62 Pro 62

49 48 47 41 38 40 42 45 44 k 40 O 41 Relative Risk Factor/Modifier Relative risk value (B) Age at rnenarche: 12 1.10 Number of biopsies: and Ae: 40 1.00 Age at first live birth: N/A and Family history: sister 2.83 Atypical ductal hyperplasia (ADH): No 1.00 Lobular carcinoma in situ (LCIS) No and LCIS o biopsy at age (AgeQ LCIS): 0 1 00

Summary Relative Risk (<50 since she is 40 years old): 3.11

(C) 1111111 .. Fli

613012003 Gail General Risk Assessment Model PT13-A-PRIORI IIISPA141C ADW H Family Hstory 1131 Age 40 LCISi710 SISTER Age at Menarche 12 Age LCIS 0 Number of Biopsies Age First Birth

ProbablUty of Developing Breast Cancer by Ae so RR I I I 2

40 I

0 Chent Age Age FLB 00 30 IM Popi3lation Men Eix FH:O)

20 r_1 No 16sk Relative Risk (, Relative Risk (>50) Age Subiect Population No Risk 10 YCW 4, 0 3 Do 7 0 4 6051) ,02 ,6 25 1200 49 0 _JO_ nm, 2 O' ' 13 Life 6 13 5 Aqye Copyr i9 hi The Un ive is II Y of Texas 1997 All rights reserved OUfDUt 5 M.del /\ BRCkPPO /\ EIRCAProbs /-\ G

Fi-ure 4 Estimation of the relative risk of a 40 year-old woman (Pt13) who hd menarche at age 12 years using the Gail model. The pedigree (A) and te details regarding te relative risk modifiers with teir corresponding values (13) are shown Rsults of CancerGene (C) are also shown RR value and lifetime risk are pointed by arrows ad enclosed i a circle ad in a damond, respectively Family istory is defined by te number of ffist-degreet relatives afflicted wt BC Page 3 7 of 126

File 7/2/2003 Claus amily History Model PT 3 APRIORI 77ie Ckzus table used in tis calculation is: Oe 1131 first-ekqree riekiiive Remaining Risk 29 3 9 59 69 '79 Age % 03 L2 6A 10.1 13.2 Probability of Developing Breast ance ge 45 1.01 50 2.15

50 55 3.77 60 5.45 40 65 7.32 70 9.14 30 75 10.71

20

10

0 To Aae 79: 11.97 45 50 55 60 65 70 75 80 85 Aae t, Copyright The Universityof Texos, 1998 All rights reserved Claus Model \_EjA P odel / -b, G.fllllode

Figure 5. Estimation of te relative risk of a 40 year-old woman (Pt.13) who has a sister diagnosed of BC at age 40 years using the Claus model. Results of CancerGene are sown. RR value is enclosed in a circle and pointed by the arrow. Remaining risk is also indicated. Page38 of 126

File

7/3/2003 BRCAPRO: The Duke University Model PT13 A PRIORI 1131 CarrierProbabilities BRCAI: 0.138 BRCA2: 0,011 BRCA I or 2 0.148

Probability of Breast or Ovaiian Cancer by Ag 0.5 Age gegsf '0var!= 45, 0027789 0005640 .50 D589176' 0015581 0.4 0.08t8O 6.02808 "60"0 111944 0036255 65 o 1332-7o--'6.04320-9 0.3 11,70 75 0 170319,',0,05i'65 0.2 "80 188665 604888 R5 d 1§994',b 056801

0.1 0.0 L 45 50 55 60 65 70 75 80 85 Age Copyright The University of Texas, 1998 All rights reserved I

Figure 6 The BRCAPRO model estimation of unconditional probability that the 40-year-old woman (Pt13) will develop BC. Results of CancerGene indicating the unconditional probabilities at various acres including the cumulative lifetime BC risk at ae 85 years, Page 39 of 26

3.8 Screening Strategy

Blood samples (five 4ml tubes: 20 ml) were collected from the patient by a medical technologist or nurse of the hospital through a vacutainer having EDTA as anticoagulant. BRCA I is a large gene, composed of 22 coding exons which generate a protein of 1863 amino acids. Genomic DNA (gDNA) is the template utilized for SSCP and PTT. SSCP was performed on exons 2 5, 13, 15, 17, 18, 20, 22, and 24 encoding approximate]y 23.3% of the entire gene. PTT was performed on exon I which encodes 61% of the entire gene and contains about 75% of the truncating mutations detected in BRCAL Exon I I was obtained in the form of in vitro transcription/translation product. DHPLC and cycle sequencing were employed for the sample which revealed a truncating mutation. Exon I I together with the exons studied using SSCP encode 84.6% of the entire gene and contain about 80-90% of all the mutations detected in BRCAI (BIC, 2003). The location of BRCAI exons with their respective number of base pairs, codon, number of amino acids, and portion of BRCAI encoded are shown in Table 8. Pooled genomic DNA 3 patients per tube) was initially used as template for SSCP before detecting polymorphisms for individual patients. Schematic diagram of the screening strategy is shown in Figure 7.

3.9 Template Preparation

3 9 Genonfic DNA Extraction Genomic DNA was isolated from whole blood using salting-out method from 4ml EDTA-treated blood (Miller, Dykes, Polesky, 1988; Helms, 1998; Garvin. 1998). Extra 4ml tubes were stored at 70'C. All of the reagents and materials used in this study were autoclaved. The tube was inverted several times to get a homogenous solution and transferred to a labeled 50ml graduated conical polypropylene tube. Vacutainer tube was immediately rinsed with cold IX blood lysis buffer or BLB (155mM NH4Cl, ImM KHCO3, 1mM EDTA, pH 74) and poured into the Falcon tube. Cold IX BLB was added well to a final volume of 50ml followed by removal of remaining blood clots with a sterile Pasteur pipet. Resulting solution was transferred equally into two 32ml polypropylene tubes, incubated on ice 'C, 45-60min) to lyse the red blood cells, then centrifuged (1,800rpm, 10min, 4C). Supernatant was discarded. Pellet was rinsed 34 times by resuspending in 15ml cold IX BLB, inverting several times to obtain a homogenous solution, centrifugation (1,800rpm, 10min, 4Q ad discarding the supernatant. Resulting pellet should be clean, white, and without blot clots. Into the pellet. 3ml nucleus lysis buffer or NLB 10 mM Tris-HCI, pH 8.0 04 M NaCl 2 mM EDTA, pH 2 was gently added then immediately resuspended with a vortex for 15s and transferred to a 5ml polypropylene Page 40 of 126

tube. This was followed by the addition of I 0tl of 20mg/ml pronase E and 3 00[d of I % SDS to achieve a clear viscous solution. This was incubated (room temp., overnight) on an orbital shaker. On the second day, solution was transferred to 32ml polypropylene tube. Saturated NaCl stock (-6M) was re-mixed prior to addition of Iml aliquot into each tube then agitated vigorously for 15s followed by centrifugation (3,000rpm, 15min). Supernatant was poured into another 32ml polypropylene tube and centrifuged (3,000rpm, 15min) to obtain a clearer supernatant. Absolute EtOH (8ml) was gently added to the supernatant followed by several slow tube inversions to precipitate the DNA. Precipitated DNA was fished out using a sealed Pasteur pipet then transferred to a 1.5-ml microfuge tube and washed (14,000rpm I min) with 500tl cold 70% EtOH. The supernatant was decanted and the pellet was air-dried for I Ornin followed by the addition of 100-500tl IX TE (10mM Tris-HCI, .ImM EDTA pH 8.0) depending on the size of the pellet. Genomic DNA stock solution was incubated (65'C, 30min) to get rid of any DNase contamination.

3.9.2 Template Purity and ConcentrationDetermination Purity and concentration of gDNA were determined spectrophotometrically (Bio-Rad SmartSpec 3000) and electrophoretically (Mupid 07% agarose gel electrophoresis and Hoefer UV Transilluminator). Ethidium bromide (EtBr)-stained agarose gels were documented using Polaroid 667 Film. Stock gDNA was diluted accordingly with I X TE to obtain working solutions within the range 25-100pg/ml.

3 9 3 Pooled Genomic DNA Working solutions of the genomic DNA from the 34 collaborating patients and 2 high-risk individuals were pooled as follows: GI (PtOI-N03), G2 (PtO4-PtO6), G (PtO7-PtO9), and so on until G12. The corresponding concentration of each genomic DNA was 20 tg/ml to a final concentration of 60 tg/ml 3 genomic DNA per pooled sample). In the presence of putative polymorphisms detected in the pooled samples, the ) corresponding individual genomic DNA, were utilized as templates. Page 41 of 26

Table 8. BRCAI exons.

Exon Location Number of Codon Number of % of BRCAI base pairs amino acids gene I I- 100 (100) 2 101- 199 99 (80) I- 26 26 77

3 200- 253 54 27- 44 8

5 254- 331 78 45- 70 26 1.39 6 332- 420 89 71- 100 30 7 421- 560 140 101- 146 46 8 561- 665 105 147- 181 35 9 666- 712 47 182- 197 16 10 713- 788 76 198- 222 25 1 789-4215 3427 224-1366 1143 61 28 12 4216-4302 87 1366-1394 29 13 4303-4476 174 1395-1452 58 3 It 14 4477-4603 127 1453-1494 42 15 4604-4794 191 1495-1558 64 3 42 16 4795-5105 311 1559-1661 103 5.56 17 5 106-5193 88 1662-1691 30 57 18 5 194-5273 80 1692-1717 26 43

1 9 5274-5310 37 1718-1730 1 3 20 5311-5396 86 1731-1758 28 54

2 1 5397-5451 55 1759-1777 19

22 5452-5526 75 1778-1802 25 34

23 5527-5586 60 1803-1822 20

24 5587-5711 125 1823-1864 4 2 23

Total 5711 1863 84.64 Note Start codon (AUG) is at nt 120 Total number of base pairs used for encoding Brcal=5592 nt Location is based on Genbank accession number U14680 Filipino BC or B(O)C patient Inclusion and exclusion criteria Suspected Familial Filipino BC patient Letter of Informed Consent One-on-one interview to answer the sructured estionnaire

Blood Sample Collection Evaluation of amily History

Genomic DNA Extraction Breast cancer patients, were included if first- or second-degree relatives have dsDNA I) I breast cancer case 2) I ovarian ea cer case OD2(Ai/2x(,ratio 3)other primary cancer cases agarose gel clectrophoresis High-ml, individuals were also included

Touchdown PCR for BRCA I PC R for BRCA I exon 1 1 exons 2, 5, 13, 15, 16, 17, 18, 20, 22, and 24 Pedigrees of PtOl, PtO2, PM, Ptl3, PE14, Ptl5, Pt22, Pt25, Pt26, dsDNA - dsDNA Pt27, Pt28, PGO, Pt33, and Pt34

rose Gel Electrophoresis of PCR products Agarose Gel Electrophoresis of PCR products ancerGene cancer risk and prior probability models ATG Pedigrees should contain - dsDNA dsDNA 1)speafic cancers orrelatves

T7 In into Transcription 3)2) ages ofdiscascand sexes ofdiagnosis relatives and I ranslation 4) age at death (ifdeceased) RNA AUG Denature at 95'C with forinamide Perform CancerGene analysis Protein snap-cool dsDNA Obtain cancer risks ad/or prior carrier probability values

SDS IIAGL and ssDNA Incorporate results ofP 7 and SSCP Autoradiography (BRCAI truncating mutation and putative polymorplusins) Determine tuncated protein produFts Non-denaturing polyacrylamide gel electroplioresis DHPLC (DNA of patient with truncated Silver Staining Obtain posterior caff ter probability values protein product through PTT at exon I ) Detect aberrant mgration patterns by BRCAPRO

DNA Sequencing

Data Aalysis Data Aalysis Data Analysis

Figure 7 Schematic diagram of the screening strategy and risk modeling used in this study. Page 43 of 126

3.10 ERCA] Primers and Corresponding Lengths of Amplicons

Twenty-two 22) primers were utilized in this study (Table 9 All of the primers were based on Genbank accession numbers U14680 (exon 1 1) and L78833 (exons 2 5, 13, 15, 16, 17, 18, 20, 22, and 24) and on published primer sequences. Primers for exon I I are within the exon while primers for the smaller exons are intronic sequences (Ottini et al, 2000; Garvin, 1998; BIC, 2003). However, exon 2 primer pair was designed using Web Primer (http://genoi-ne- www2.stanford.edu/cgi-bin/SGD/web-primer). The regions that were amplified in preparation for PTT and SSCP are shown in Figure .

3.11 Polymerase Chain Reaction

Lyophilized primers were resuspended in IX TE (lOmM Tris-HCI, pH 75; 1.0 M EDTA pH 8.0) to obtain 40 VtM working solution. Final volume for each of the PCR reactions was 20 [11 having -I 00 ng of template, 200 nM of each primer, and 200 tM of each dNTP.

To amplify pooled or individual genomic DNA for SSCP, lU of Taq polymerase was added to each of the amplification reactions. PCR was performed in the Mastercycler Gradient 5331 after series of optimizations. Initial denaturation was performed at 94'C for 2min followed by 35 cycles at 94'C for 30s, annealing temperature specified in Table 9 'C) for 3)Os, 72'C for Imin. This was followed by a final elongation at 72'C for 10min. PCR products were electrophoretically 2.0% agarose) evaluated based on purity and expected molecular weight (Table 9 and Figure 8).

To obtain PCR products at BRC I exon I I for PTT a mixture of forty 40) parts Taq polymerase and one (1) part proofreader Pwo polymerase was prepared. An aliquot of lU (0.976U:0.024U) from this mixture was added to 2 MM MgC12 for each of the amplification reactions. Touchdown PCR was performed in the Eppendorf Mastercycler Gradient 5331 after series of optimizations. Initial denaturation was performed at 94'C for 2min followed by 36 cycles at 94'C for 30s, 65'C for 30s, 68'C for 2min having I C annealing temperature reduction for the first I I cycles until 55'C has been attained and carried out for the succeeding 25 cycles. This was followed by 14 additional cycles at 940C for 30 s, 50'C for 3 s, 6'C for 2 min and final elongation at 72'C for 10min. PCR products were electrophoretically 07% agarose) evaluated based on purity and expected molecular weight 468bp). Page 44 of 126

Genornic DNA of the wild-type low-risk individual (Pt37) as well as mutation controls (positive controls) obtained from various scientists abroad were also amplified at the different exons of BRCA1 for PTT and SSCP (Garvin, 1998; Jakubowska et al, 2001). Mutation controls from Australia, Italy, The Netherlands, Poland, Switzerland, UK, and USA were given by Dr. Angela Sharp (Molecular Genetics Laboratory, Royal Brisbane Hospital, Herston, Queensland, Australia), Dr. Rosella Crucianelli (irc Institute of Molecular Oncology, Diagnostics of Hereditary Tumors Laboratory Building n 9 via Adamello, Milano, Italy), Dr. Gianfranco Voglino (Pathology Department, 0IRM S. Anna, Italy), Dr. Peter Devilee (Departments of Pathology and Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands), Dr. Anna Jakubowska (Department of Genetics and Pathology, Pomeranian Academy of Medicine, Poland), Dr. Alex M. Garvin (Bureco, AG, Switzerland), Dr. Stephen Abbs (Guy's and St. Thomas Hospital Trust, Genetics Centre, 8h Ir Guy's Tower, Guy's Hospital, St. Thomas St., London, UK), and Dr. James Fackenthal with Dr. Olufunmilayo 1. Olopade (University of Chicago Medical Center, Chicago, Illinois, USA), respectively. Most of the controls that were received from them were used in this study (Table 10).

3.12 Single-Strand Conformation Polymorphism Analysis

Amplified samples (pooled) were diluted 1 I in formarnide buffer 95% formamide, 20 mM EDTA, pH 8.0, 0.05% bromophenol blue, 0.05% xylene cyanol), heated to 95'C for 10 min and chilled on ice for min. Twenty 20) microliters of this mixture was loaded onto vertical non- denaturing 7 or IO% polyacrylamide gels (with 5% or IO% glycerol) depending on the product size and run at 40-IOOV for 20 h in IX TBE depending on size of PCR product. Variant bands were detected by silver staining. Exons 2 5, 13, 15, 17, 18, 20, 22, and 24 were analyzed by SSCP analysis to further detect the presence of additional mutations. Corresponding individual genornic DNA based on pooled DNA samples exhibiting aberrant mobility patterns were amplified and analyzed by SSCP to determine the patient which has the polymorphism.

3.13 Protein Truncation Test

PCR products were transcribed into BRCAl rnRNA and translated into protein simultaneously using down-scaled TNT-"' Quick Coupled Transcription Translation System (Promega LI 170). Redivue 35S-methionine from Amersham Biosciences (0.2tl), T7 TNTO PCR Enhancer (O.I Vd), and Nuclease-free H20 (0.2VLI) were mixed with TNTO Quick Master Mix (4tl) from Prornega. To these fr components, 0.75tl of the PCR product was added and ncubated at 30'C for 90min. Resulting translation products were fractionated using standard procedures of SDS- Page 45 of 126

PAGE using the Bio-Rad DCode Universal Mutation Detection System in a 0cm x 20cm fixed (14%) or Bio-Rad Model 473 Gradient Delivery System-casted gradient 5-20%) acrylamide gel. Benchmark Protein Ladder (Invitrogen) was used to estimate apparent molecular weights of the translation products. Marker portion of the gels were stained with 0. 1% Coomassie Brilliant Blue R250 in 7 acetic acid. Gels were placed in a plastic box and covered with fixing solution (50% methanol, 10% acetic acid) for 30 min, drained then followed by soaking solution 7% acetic acid, 7 methanol, 1% glycerol) for min. Gels were finally dried for 40min at 80'C usinID Bio-Rad Model 583 and Hydrotech Gel Drying System set at drying cycle for gradient gels. Autoradiography was perfon-ned by exposing the gel in Kodak Biomax MR I film for 10-16 h. The film was developed using automated Kodak Developing System.

3.14 Validation of Mutation Detected at Exon 1 1 and Analysis of the DNA Sequence

The proband that revealed a truncated protein product with PTT at BCA I exon I I was selected for DHPLC analysis and DNA sequencing with Ms. Daniela Muhr in the laboratory of Dr. Teresa M.U. Wagner at the Division of Senology. Department of Obstetrics and Gynecology, University of Vienna, Vienna, Austria. The amplified fragments were analyzed first using denaturing high-performance liquid chromatography (DHPLC). PCR products were denatured at 95'C for 3 min and allowed to renature over 30 min by decreasin te temperature from 95'C to

65'C then stored at 4C until analyzed on the WAVE system equipped with a DNASep TM column, both purchased from Transgenomic with the Gradient (Eluant A: 0.1 M triethylammonium acetate, pH 70; Eluant B: 0 I M triethylammonium acetate, pH 7 . 25% acetonitrile) 50-59%B in 0.5 min / 59-66%B in 35 in at 56'C. After DHPLC analysis, the PCR products were purified with the QIAquick 8 PCR Purification Kit (Qiagen) according to the protocol. Cycle Sequencing was performed using the DNA Sequencing Kit Bia Dye Terminator V3.0 (Applied Biosystems) according to the protocol. The sequencing reaction is then purified on Multi Screen-HV Clear Plates (Millipore) filled with Sephadex G50 Fine (Pharmacia) and then analyzed on the ABI 3 OOTM 16 capillaries sequencer (Applied Biosystems). The results were evaluated by using the SeqScape Software (Applied Biosystems). Table 9 BRCAJ Primers.

S'Ohgo 3' Oligo 5' Oligo: 3' Oligo. Exon Amplicon Annealing Method Exon 3' Length Length hp from hp from Size Length Temp. Used (lip) OP) e\on exon (bp) (lip) (IC)

2 GAA I I il C A I I I I A IAA AC (457 4S77) Tc r TCC C A GTA rGT AAG GT A 4807-4786) 2 22 42 67 99 251 53 SSCP

I I I I AGG (. A I I I M 22092 2211 1) AI GI I 1 A IA(, (,AA I AI (, 22161)221 1) 20 22 99 69 7\ 278 Ss "V V

I I 66A IC 17 I I AA r I I r AG AC 792-91 1) FAA Gi r I GA A I C CA I GCl n G 4214-4193) S7 2 - - 3427 3468 Ss PTT

I I AA I GGA AAG TT CT( AAA GTA (6100-46120) AlG TTG GAG CTA GGT CCT TAC (46419-46399J 2 2 35 7 172 320 55 SSCP

I ( .A( I I I A(.0 I I I I A IN WS I 107 1) I , I I I I I A A I A A(, M, (S4414-54414) 2 2 133 2 Of 378 55 SKI,

P, AA I I I I AA , A(,A ( A AA (S74 I -S7411)) AAA A( I I I I ( AOA A I 1-1 I S7867-S7846) 22 22 S4 1 III 4SO 55 SSCII

7 ACC TGT GTG CTA GAG GTA ACT C 60986-61007) GTG GTT TA TGC AGC AGA TG 61175-61156) 22 20 30 30 89 190 55 SSCP

I (.(, I I A 6C I I C I AG (,A( (64704 64724) I AA C I AGC Al C AGC (64962 6494i) 2 18 S7 RS 78 2S9 S9 SSCP

20 I A I A C I I C I A I C( AC 71518-71537) AGI CTT ACA AAA rGA AGC GG 71776-71757) 20 20 60 75 84 259 56 SSCP

22 TCC CAT TGA GAG GTC TTG C 79433-79452) GAG AAG ACT TCT GAG GCT AC 79729-79710) 20 20 90 93 74 297 59 SSCP

24 ATG AAT TGA CAC TAA TCT CTG C (S2995-92916) GTA GCC AGG ACA GTA GAA GGA 83174-83154) 22 2 9 (93) (125) 280 58 SSCP The ntimbei s In parentheses i cfct [lie psit ions according to Gcnbank accession numbers 'U 14680 ad 178831 T7 refei s to 7 polynicrasc Kodak consensus sequence TAATACGACTCACTA rAGGG AGACCACCATG I lip) A GC loop %N.iNa(led to u(l of Ilie lojiva d p miw for e\o I I 3468 bp

,MMT IMTPFA77 7, M000n, Dr,42 ZI 0 00 0 D 2 3 6 7 8 9 10 it 12 13 14 Is 16 17 18 19 20 21 22 23 24

320 217 b,

2 bp 378 bp 278 bp 259 bp 280 b,

450 bp 259 bp

Figure S. Regions amplified for PTT and SSCP. Exon diagrarns are drawn to scale based on the number of nucleotides (bp). Arnplicon size above te exon diagrarns idicates region amplified for PTT wile arnplicon sizes below te exon diagrarns are te regions amplified for SSCP. Table 10. BRCAJ Mutation Controls from Different Countries. Base Mutation Est. MW, if Country Code, if from n Times Recorded Exon Codon Designation Change AA Change Type Protein Product is (Tube it) mutant cell line (Rank) (BIC, 2003) Truncated (kDa) 2 --- 7 139de19 de19 11(I)-RAD4 2 23- 183ins 1-1 111STI, A 2 23 185delAG de1AG Stop 39 A 490 (1) 2 23 185de]AG delAG Stop 39 F USA (1) 490 (1) 3 3 212CG 13 M) C to Ile to Met 11(2)-SODI 5 61 C61 G T to Cys to Gly M H 3 - CRI 1 67 (1) 5 61 C61 G F to Cys to Gly M P 3) 67 (1) 5 64 ('64 Y Cys to 1'yr USA 2) 7 IVS7+3A>G UK (1) 7 IVS6-IG>l UK 2) 8 IVS7-34C'> I' 12 3) 8 IVS8+21A 11(4)-DEL9 3 9 IVS8-17G USA 3) Ila 225 794delT delT Stop233 F 0 41 P (8) 1 ]let 266 (17de I derri, Stop 285 1 11(5) -'I'AN I Ila 327 11 00del I delAT Stop 329 F 11 85 A 2 Ila 392 1294del4O del4O Stop 397 F 11 93 A 21 3) llb 672 2135delA delA F 50 07 S 709) GMI3709 111) 681 2163 m I Insl 1 51 1 I, (I ) Hb 720 2279in,,A insA F 55 41 S 712) GM 3712 I 891 271Md(JI dcl6AAA Stop 99 1 74 63 A 3 I iZ- 95 6- 2982de[5 or 2985dcl5 dcl5bp or Stop 968 F 81 56 P(9)exl I 12 1 or 2 delTCTCA lid ex II l5de15 de15 P (Io) lid IIH 3450dcl4 delCAAG -5toL 15 F 98 78 A 9 12) lid 1234 3819de[5 delGTAAA Stop 1242 F 112 44 P (5) 16 9) lid 12O 1-125OX Gto T Glu to Stop N 114 22 S 713) GM 13713 23 (5 5) lid 1252 3875de]4 dc[GI'C I' Stop 1262 F 114 52 A 35(3) lid 1252 3875de14 delGTC'r stop 1262 114 52 S 705) GM13705 35(3) lid 1345 4153delA delA Stop 1365 124 82 P 2) lid 1347 R1347G 3751-4190) Arg to Gly USA 4) lid 1355 4184del4 delTCAA Stop 1364 F 125 96 A 38 or 39 2) 12 1373 4236GT (El 373X) Gto T Glu to Stop 11(6)-MAR1 2 12 1387 4280del C dcl iC USA (5) 13 1405 11405V Ile to Val USA 6) 13 - exonl3dup exon 13 stop 1460 F A I 4603GT (RI 495M) duplication 14 1495 GtoT Arg to Met 11 7 - GRI2 15 1512 S15121 12 (1) Table 10. BRCAI Mutation Controls (continued Base Mutation Est. MW, if Country Code, if from 9 Times Recorded Exon codon Designation Change AA Change Type Protein Product is (Tube N) mutant cell line (Rank) (BIC, 2003) Truncated (kDa) 16 1563 YI 563X I'yr to Stop USA 7) 7 1665 V1665M eICTAA Val to Met USA (8) 7 1677 5149del4 d Stop 1678 11(8 - VOL 6 or 7 1) 17 1677 5149de14 delCTAA Stop 1678 F P 7) 6 or 7 1) I 170i 1 17051, 12 2) 18 IVS18+65(j/A 12 4) 19 1726 5296de]4 de14 USA 9) 20 1751 R175 X C to'l, Arg to Stop N P 6) 20 1756 5382insC insC Stop 1829 F A 268 (1) 20 1756 5382insC msC Stop 1829 F 11(9)-MAD3 268 (1) 20 1756 5382msC insC Stop 1829 F P () 268 (1) 20 1756 5382msC msC Stop 1829 F S 715) 268 (1) 20 1756 5382msC msC Stop 1829 F USA (IO) 268 (1) 21 1773 5438msC insc stop 1829 F S 714) 1 2 4) 2 1778 IVS21-36de]5 I del 10 Stop 1805 F P 4) 39 1) 24 1835 RI835X Arg to Stop USA (I ) 24 1837 5628TC (WI 837R) T to C Trp to Arg, 11 10)-BACI

Scientists who sent aliquots of teir mutation controls (total = 54): Australia (A) Dr Angela Sharp, Molecular Genetics Laboratory, Royal Brisbane ospital, Herston Q 4029, Australia IOcontrols Italy (I1) Dr Rosella Crucianelli, Fire Institute of Molecular Oncology, Diagnostics of Hereditary Tumors Laboratory Building n 9 via Adarriello, 16 20139 Milano, Italy I controls Italy 2 12) Dr Gianfranco Voglino, Italy 4 controls The Ntherlands (N) Dr Peter Devilee --- exons 13 and 22 2 controls Poland (P) Dr Anna Jakubowska II controls Swit/crIand (S) Dr Alex Garvin 6 controls UK (UK) Dr Stephen Abbs, Guy's ad St Tomas Hospital Trust, Genetics Centre, ' FIr Guy's Tower, Guy's Hospital, St Tomas St London SE I 9RT, UK 2 controls USA (USA) Dr James Fackenthal, University of Chicago Medical Center, 5841 Maryland Ave, MC 2115, Chicago, IL60637-1470, USA II controls Page 50 of 126

3.15 Processing of Data Obtained from the Molecular Techniques Utilized

Presence of aberrantly migrating single strands in the SSCP analysis of selected exons of BRCA I indicated putative polymorphisms. Low prevalence (i.e., I or 2 patients in 34 samples) was noted for the possibility that the putative polymorphism detected is actually a mutation in that particular exon.

Lower molecular weight of sample transcription-translation product when compared to control PTT product indicated a detectable truncating mutation. Possible location of detected mutation was estimated by estimating the corresponding nucleotide position of the stop codon based on the estimated apparent molecular weight of the truncated protein product. Estimated nucleotide position of the stop codon was the basis for designing the sequencing primers. DHPLC profile of sample was compared to a wild-type (control) sample to confirm the mutation detected through PTT. Sequencing data of sample manifesting a truncating mutation was compared with the control (known) sequence to determine the precise mutational event.

3.16 BRCAI Prior Carrier Probability Estimation for Familial Filipino BC Patients

3.16.1 Estimation Using Couch Model Based on the Couch prediction table (Table 6, the carrier probability for the 46-year old woman below (Pt25) would be 0. 17 (Figure 9. This means that among I 0 individuals from 'BC only' families with the mean of diagnosis belonging to the same range as Pt25, 11-12 are BRCAI mutation carriers [BRCAI(+)]. The reported value for CancerGene (Figure 9 jibes with the Couch prediction table (Table 6 Couch model is particularly useful for Ashkenazi Jewish families with 4 B(O)C cases. Although N25 has >4 BC cases, this model cannot be utilized since she is not of Ashkenazi Jewish descent. Since all of the patients in this study are not of Ashkenazi Jewish descent, this model cannot be utilized.

3.16 2 Estimation Using Shattuck-Eidens Model Although the family of Pt25 has no OC case, the Shattuck-Eidens model reported through CancerGene a carrier probability value of 0172 which is higher but almost similar to that of the Couch model (Figure 9 Shattuck-Eidens model is particularly useful for Ashkenazi Jewish families with small number of affected members. This model needs only one relative afflicted of BC to estimate the carrier probability value. Since Pt25 has multiple affected relatives. the carrier probability would be underestimated. She is also not of Ashkenazi Jewish descent, therefore, this Page 51 of 126

model cannot be used for this type of case. Since all of the patients in this study are not of Ashkenazi Jewish descent, this model cannot be utilized.

3 6 3 Estimation Using Frank Model (Frankand Myriad com) Based on the Frank model (Table 71) and Myriad.com mutation prevalence table (Table 72), the carrier probability values of carrying a BRCAY2 mutation for Pt25 are 0250 and 0.316, respectively. CancerGene, owever, reported a value of 383 (Figure 9 Since the Myriad.com mutation prevalence table (Table 72) has the latest version (updated August 2002), the value obtained from this table was used as the acceptable estimate.

2 6.4 Estimation Using BRCAPRO CancerGene reported BRCAPRO BRCAI prior carrier probability of 0939 for N25 (Figure 9) based on the presence of numerous first- and second-degree relatives with BC, two of which are early onset cases. This shows that there is only a 6 chance that Pt25 will not have a BRC I mutation. This is also an acceptable estimate due to the incorporation of important variables. This model can be utilized in this study together with the updated Myriad.com mutation prevalence table.

3.17 BRCA1 Posterior Carrier Probability Estimation for Screened Patients

The BRCAPRO Model was Utili7ed to estimate posterior BRCAJ carrier probability values by incorporating the results of PTT and SSCP. Two posterior probability values were estimated for patients with putative polyrnorphisms (based on SSCP results) since the aberrant band may just be a polymorphism [BRCA](-)] or may actually be a mutation [BRCAI(+)]. In the case Ptl3 (Figure 4A) who has putative polymorphisms at exons 13, 15, and 16, her posterior carrier probability value is 0.0 6 if these are just polymorphisms making her a noncarrier (Figure OA), but if the aberrant band in at least one of these exons is a mutation making her a carrier, it will automatically be 1.000 (Figure 1013).

3.18 Unconditional Probability Estimation for Screened Unaffected High-Risk Individuals

The BRCAPRO was also utilized to estimate the unconditional probability of developing BC before various particular ages for the screened unaffected high-risk individuals in this study. Three probability values were obtained for Ptl3 (Figure 4A). CancerGene estimated the following various unconditional probabilities: (a) if she is a noncarrier (Figure 10A). (b) if she is carrier Figure 1013). and (c) before incorporation of PTT and SSCP results (Figure 6. Page 52 of 126

(A) 45

00 6- 69 71 618 44 45 so 29 46 38 Br 42 Br 42 Br 27 Br 35

44 4 3 36 -T Br 39 8 12 13 9 5 3 1 9 8 III T- 22 19 15 12

(B) --isW nE File

7/2/2003 BRCA Mutation Probability Models PT26 A PRIORI 1261 Pedigree Information Ashkenazi family NO BR 1 Number of family members 35 Number with breast cancer only Couch (U. Penn 0.117 Number with ovarian cancer only Shattuck-Eiden (Myriad 1) 0,172 Number both breast ad ovarian cancer BRCAPRO 0.939 Number with bilateral breast cancer

BRCA2 BRCAPRO 0.013 Values expressed as BRCAI or 2 probabilities, not percents

NCI CART ..none" means no calculation

Myriad.com (Myriad1l) possible C&acnr6cfleVersion 33 BRCAPRQ Myriad.com table 0123/2000

CopyrightOTheUniversityofTexos,1998-2000 Arigh1sreserved

S

Figure 9 Estimation of the BRCA1 prior carrier probability of a 46-year-old woman (Pt25) using te Couch model. Te pedigree0 wt teir corresponding aes0 at diagnosis is shown Results of CancerGel7e for all the prior probability models are also sown Note tat bot te Couch prediction table and CancerGene estimated te sarne carrier probability valUe of 01 7, Even if tere ae >3 BC cases in te farnily of Pt25, Couch rnodel cannot be tilized since se is not of Ashkenazi Jevish descent Page 53 of 126

(A) File

7/4/2003 BRCAPRO: The Duke University Model PT13 A POSTO 1131T CarrierProbabilities TlRckl: OB16 BRC,%2. 0,01-1 BRCA I or 2 01)28

Probabih!y of Breast or ovarian Cancer by Ag

0.5 &-_e reast van 45 0010071 OX01419 50 0 02292 1 003655 0.4 55 0 03-750 006401' 60 03?91 6609272 65 0 070966-10 2Q68 0.3 70 08669,0 01 4652 75 0,106i3i O 016874 80 0 12212, b.bl'?,574 0.2 85 0 1348E, 0 C68§9

0.1 (.0 L L L L 45 50 55 60 65 70 75 80 85 Age Copyright'DTheUniversifyofTexos,1998 Allrightsreservedi Outputblaxager/\ Claushlodel BRCAPRO

(B) He

714/2003 BRCAPRO: The Duke University Model PT13 A POST(+) 1131j' CarrierProbabilities BRCAL 11100 BRCA2: 01100 DR CA 2: g1000

Probaby of Breast a c 0m III Cancer by Age 0.5 A.C Breast 45 13142: 035503 50 0 31031 0099961 04 55 0 43922S 11195 60 0 523321 27172 65 0 574090 0 2630 0.3 70 0 602542 25031 75 0 623741 0 00243 80 0 642327 0311818 0.2 85 0 66645 0 19377

0 1

0.0 45 50 35 60 65 70 73 80 85 Age Copyrigh 0 The University of Texas 1998 All rights reserved I Output Manage Claus Model 7\ BRCAPRO Probs, 11I.&I

Ficure 10A-10B. The BRCAPRO estimation of (a) BRCA1 posterior carrier probability of a 40- year-old woman 03) and (b) unconditional probability that she ill develop BC. ReSLI]tS of CancerGene are sho,,,m. (A) if she is a noricarrier and (B) if she is a carrier. Page 54 of 126

IV.RESULTS

4.1 Selection of Prospective Individuals at High-Risk for BRCAJ Mutation

Thirty-six 36) BCAJ mutation high-risk individuals out of 750 BC patients from 7 institutions were recruited, 34 of which are BC patients and the remainin 2 are unaffected individuals who have high family history of BC (Table 11). Patient lDs (Pt#) have been assigned to these individuals during the course of the study, i.e., PtOl up to Pt36, PtOl being the first patient who had her blood extracted and Pt36 being the last. Ptl3 and P04 are the unaffected high-risk individuals, Pt37 is a wild-type low-risk healthy individual (normal) without family history of any type of cancer.

4.2 Characteristic Affliction of Relatives in Families

Participants were grouped according to characteristic affliction of relatives in families: (a) families of BC patients with first- or second-degree relatives afflicted with BC (Figure 11), (b) families of BC patients with first- or second-degree relatives afflicted with OC (Figure 12), and (c) families of BC patients with first- or second-degree relatives afflicted with other primary cancers (Figure 13). Observed patterns for each type of affliction were taken into account.

4.3. Clinicopathological Characteristics

4 3 ClinicopathologicalCharacteristics of the 36 BRCA I Mutation High-Risk ndividuals The clinicopathological characteristics of 36 BCAI mutation high-risk individuals from 7 institutions are summarized in Table 12.1. The mean age of diagnosis was 39.1±7.2 years (range: 30-71 years). Ages of Ptl3, Pt34, and Pt37 are 40, 19, and 47 years, respectively. Frequency distribution of age at BC diagnosis of patients is shown in Figure 14. Twenty-six patients 76.4%) are early-onset BC cases (diagnosed 40 years old), 14 41.2%; 53.8% of early- onset cases) of which have familiarity of the disease. Among the patients that have been diagnosed above 40 years old, 7 20.6%; 87.5% of regular-onset cases) have family history of BC. One patient who was diagnosed at age 42 years, who have 2 first-degree relatives with other cancers but had no history of B(O)C, was included to note any possible difference. Classification of the patients with respect to family history was based on the nearest relative with B(O)C or other cancers. Ptl3 has ') relatives with B(O)C and I maternal uncle with prostate cancer while Pt34 has 2 maternal relatives afflicted with BC and I paternal uncle with lung cancer. Eighty- Page 55 of 126

five percent (85%) of the 20 patients with known histology type had invasive ductal carcinoma (IDC) while the remainin 3 patients (15%) had papillary or mucinous carcinoma. Only 14 (41.1%) and 12 35.3%) patients had their estrogen receptors (ER) and progesterone receptors (PR) assayed, respectively, approximately 40% of which are ER- 35.7%) and PR- 41.7%).

4 32 ChnicopathologicalCharacteristics of the Families with Relatives Afflicted with BC The clinicopathological characteristics of families with relatives afflicted with BC are shown Table 12.2. The mean age of diagnosis was 42 years (range: 38-46 years). All of the probands have family history of BC and the relatives afflicted with the disease are from the maternal lineage. Two probands (50%) are early-onset BC cases. The 2 patients with known histology type had IDC. Pt 25 is ER- and PR- while Pt27 is ER+ and PR+.

4.3.3 ClinicopathologicalCharacteristics of the Families with Relatives Afflicted with OC The linicopathological characteristics of families with relatives afflicted with OC are shown Table 12.3. The mean age of diagnosis was 31.5 years (range: 3)0-3 )3 years). All of the probands have family history of B(O)C. Two probands (I 0%) are early-onset BC cases while Pt I 3) is an unaffected individual. Existence of relatives afflicted with BC from the paternal lineage is observed. The histology type of both PtOl and PtO4 is IDC. No patient had her ER and PR assayed.

4.3.4 Clinicopathological Characteristics of the Families with Relatives Afflicted with Other PrimaryCancers The linicopathological characteristics of families with relatives afflicted with other primary cancers are shown Table 12.4. The mean age of diagnosis was 40.5 years (range: 34-51 years). All of the probands have family history of BC, majority of which is from the maternal lineage but are usually regular-onset BC cases. Five probands 83%) are early-onset BC cases while Pt34 is an unaffected individual. Five probands 83%) with known histology type had IDC. PtI is ER- and PR- while PtO2 is ER- and PR+. Pt is ER+.

4.4 Socio-Econornic, Dietary, and Other Factors

4 41 Socio-Economic, Dietary, and Other Factors of the 36 High-Risk Individuals Patient information on various factors which may or may not affect the risk of developing cancer is summarized in Table 13.1 Almost half 47%) of the BC patients had their first menarche at an early age 12 years) and about 41% were nulliparous or were >')I years old when their first child was born. Early menarche directly increases lifetime exposure of the breast tissues to Page 56 of 126 circulating honnones. The other factors included herein were intended for documenting other characteristics of the patients for probable future correlations. Age at first sexual contact was highest 44.1%) at the midrange (20P40,000/month) in this study is the same as the lowest-income bracket families. Both of them comprise 20.6% of the number of individuals each. At least one-third of the BC cases 35.3% in this study come from Quezon City, 14.7% come from outside Metro Manila, and the rest are within the Metro Manila area. More than half 55.9%) have been exposed to cigarette smoke whether active, passive, or both types of exposure. On the other hand, most of the patients (61.8%) have no alcohol intake while only 14.7% have no caffeine intake. Although 64.7 of the subjects generally used vegetable oil for cooking, 70.6% of the 34 BC patients re-used their cooking oil.

4 42 Socio-Economic, Dietary, and Other Factors ofFamilies with RelativesAfflicted with BC Proband information on various factors is shown in Table 13.2. Majority 75%) of the BC patients had their first menarche at age >12 years and all of them were parous, latest of which was 28 years old when the first child was born. Age at first sexual contact was relatively early (age 17-28 years). Majority 75%) of the patients had experienced breast feeding and Pt25 admitted experiencing personally-induced or "quack doctor" abortion. All of them were married and 3 patients 75%) were Roman Catholics. Half of the patients (50%) have an undergraduate degree or higher level of education but 3 BC patients 75%) have a net monthly income below P15,000. Majority 75%) have been exposed to cigarette smoke, have no alcohol intake, have regular caffeine intake, used vegetable oil for cooking, and re-used their cooking oil.

4.4 3 Socio-Economic, Dietary, and Other Factors of Families with Relatives Afflicted with OC Proband information on various factors is shown in Table 13.2. Most of the high-risk individuals had early menarche and were nulliparous. Age at first sexual contact was relatively late (ages 26 and 48 years). PtO4 had experienced breast feeding and no one admitted experiencing personally- induced or "quack doctor" abortion. Two of them 67%) were married and were Roman Catholics. All of them have an undergraduate degree or higher level of education but the 2 BC Page 57 of 126 patients have a net monthly income below PI 5,000. Majority 67-100%) are non-smokers, have no alcohol intake, have regular caffeine intake, used vegetable oil for cooking, and re-used their cooking oil.

4.4.4 Socio-Economic, Dietary, and Other Factors of Families with Relatives Afflicted with Other Primary Cancers Proband information on various factors is shown in Table 13.2. Majority 71%) of the BC patients had early menarche at age >12 years and half of them were parous, latest of which was 33 years old when the first child was born. Age at first sexual contact was diverse (ages 18-35 years), almost half of which never had any sexual contact. Majority 75%) of the patients had not experienced breast feeding and Pt28 admitted experiencing personally-induced or "quack doctor" abortion. Less than half 43%) of them were married and 6 patients 86%) were Roman Catholics. More than half of the patients 71%) have an undergraduate degree or higher level of education but half of the BC patients have a net monthly income below P25,000. Majority have been exposed to cigarette smoke, have very occasional alcohol intake, have regular caffeine intake, used vegetable oil for cooking, and re-used their cooking oil. Page 58 of 126

Table 11. The BRCA1 Mutation High-Risk Individuals from Various Institutions.

Institution Number of Collaborating Patients

SLMCCI/CGH 19

EAMC 8

vMMc 4

AFPMC 3

MH I

PNRl I

Total 36 Page 59 of 126

(A) Pt25

60 65 69 71 68 50 29 46 38 B, 43 Ik 42 RI 27 Br 35

__TT_44 IE 39 8 12 13 9 9

32 19 is 13 (B) Pt26

64 65 67 74 71

36 37 49 49 43 42 40 4, B, 40

30 29 28 (C) Pt27 Alex 1 . a, 76

38 39 44 43 41 38

16 Is

(D) POO Uk 1' 4

k 4

se 60 6) 64 41 f6

Figure IIA-IID. Pedigrees of families of probands with first- or second-degree relatives afflicted with BC. Mean age at BC onset of probands is 42 years (range: 38-46 years). All of the probands have family history of BC. All of the relatives afflicted with BC are from the maternal lineage of the immediately preceding generation. The observed difference in the a-e of onset between proband-mother, proband-sibling, and proband relative ranges from I to 6 years except for 111-5 - 11-3 of Family C 38 years) and the lowest difference is observed between proband- mother in families A and D The observed earlier aYe of onset of BC in the probands and their relatives is a distinctive characteristic of inherited form of cancer as indicated by the Knudson's 2-hit model of carcino-enesis (A) Ace of disease onset difference between proband N25 111-3) and mother 11-6) is I year Majority 7 1%) of the afflicted members of this family had early-onset BC which includes both proband and her mother. Mean ae of BC onset amon- second-degree relatives (I-8, 11-9, 11-12, and 11-13) is 36.5 years. (B) Proband Pt26 11-4) age of disease onset difference with sister (II-7) is 6 years. Her sister 11-7) had early-onset BC. (C) Age of disease onset difference between proband Pt27 (111-5) and mother 11-3) is 38 years Pt27 (111-5) had early-onset BC while her mother 11-3) had late-onset BC (D) Proband POO (11-8 ae of disease onset difference with mother 1-2) is 3 years Both had recrular-onsetz:I BC. POO has only oe sister 11-9) and 7 brothers (11-1 to 11-7) Page 60 of 126

(A) PtOl

92 65 01 50

4 63 65 68 70 k 33 72 69 Ik 59

(B) PtO4

92- 70' 78

52 so 54 55 57 So 59 1 34 5 1 48 47 4 5

4 31 DI 43

30 28

5 1

(C) P03

56 Er 52

74 72 1 6 72 B, 62 R10 62

49 48 47 41 38 40 42 44 45 el 40 0 41

Fi-uret' 12A-12C. Pedigrees of families of probands with first- or second-degree relatives afflicted with OC. Mean a-et, at disease onset of the two probands (Pt I and 4) is 31.5 years 30 and 33 years) which are definitely early-onset C cases while PtI3 is an unaffected individual. All of the probands have family history of B(O)C Relatives afflicted with OC are from the maternal lineage while appearance of C from the paternal lineage is observed indicatingZI linkage of expression of C in the proband from both lineagest, (A) Proband PtOl (11-5) had early-onset C (IDC, left) followed by a second C dagnosisb 26 years after (IDC, right) indicating bilaterality She had breast cysts thrice before disease onset at ages 16 years (I cyst), 22 years 2 cysts, left), and 25 years 2 cysts, right). Her mother 1-2) had OC at age 50 years. (B) Proband PtO4 (II IO) had early-onset C. Her mother had OC at age 45 years (breast cyst during her teenage years) She has a paternal aunt (II-6) with early-onset DC. Two of her maternal cousins have breast cysts (not shown). (C) Proband PtI3 (III-6) is an unaffected 40-year old high-risk individual who had breast cyst and had undergone umpectomy twice age 36 and 37 years). All of her sisters 11-7 to III-10) also had breast cysts One of them (111-10) had OC Her maternal randmother 1-3) had recrular-onset DC while one of her two maternal uncles (11-8) had PC. One of her two paternal aunts I-4) ad late-onset DC Pa,-,e 61 of 126

A: C3a (PtO2) E: C3e (Pt28)

78 7. 73 71

iu '2 i!,

39 13 '5

B: C3b (PtA) F: Of (Pt33)

7 1 C.a, 0- W 33 51 El 4

33 3 37 .1 10 35 38

C: O (PtI5) G: C3g (Pt34)

it 69 68 61 39 53 52 50 L 5 L_ 51 51

IF O 23 21 P9

12 2. 27 26 23 21

15 D: C3d (Pt22)

C_ so k

0 45 41 48

Figure 13A-13G. Pedigrees of families of probands with First- or second-degree relatives afflicted with other primary cancers. Mean age at disease onset of probands of families A to F is 40 years range 34-51 years) Five 83%) probands (families A to E) are early-onset BC cases while P03 (family F) ad reo lar-onset BC and Pt34 (family G) is an unaffected individual All of the probands (100%) have family istory of BC from the maternal lineage All of the relatives afflicted with BC are regular- or late-onset cases while te probands have eaTly-onset BC in families A, B, C, and E (A) Proband PO2 11-4) had early-onset BC while her mother 11-4) and aternal grandmother (I- 3) have late- and regular-onset BC, respectively The ae at disease onset differences are 38 years and 26 years for proband-mother and proband- maternal -randmother, respectively Her father 11-3) has prostate cancer (PC) She also has a maternal half Cusin with PC at ae 60 years (not shown) (B) Proband Ptl4 (111-5) had early-onset BC while 2 of her maternal aunts, 1-4 and 11-7, have regular-onset BC with age at disease onset differences of 22 years and 15 years with the proband, respectively Her mother 11-2) had metastatic lipoma while her father (11-1) had pancreatic cancer (C) Proband N5 111-3) ad early-onset BC while I of her 2 sisters II14) and her mother 1-8) have regular-onset BC with age of onset differences with the proband of years and 15 years, respectively She has a paternal aunt 11-2) who had lung cancer (D) Proband Pt22 (11-5) had regaular-onset BC while one of her maternal aunts (1-5) had early-onset BC followed by hng cancer that metastasized to the brain 13 years later Another maternal ant 11-3) died of astric cancer while her maternal grandmother (1-1) died of throat cancer that metastasized to the lung (E) Proband Pt28 111-4) had early-onset BC and relatives afflicted with BC from both maternal and paternal lineages Both of her maternal and paternal grandmothers 1-3 and 1-1) had re-ular-onset BC Both of them have 21-year age at disease onset difference with te proband Se has a maternal aunt (11-1 5) with cervical cancer Also from te aternal lineage, she as a grandaunt %Nith gastric and colon cancer. a randCOLISIll who had uterine cancer, and a 2 d cousin wth brain cancer (not shown) From the paternal I neage, she has a grandaunt with regular-onset BC ad a randuncle wth oral cancer (not shown) (F) Proband N33 11-2) and one of er sisters 11-3) ad regular-onset BC Her maternal ant 1-4) has colon cancer (CC) Se has maternal female cousin with CC, maternal half-uncle wh PC, maternal half-cousin with OC, and another maternal half-cousin wth pancreatic cancer (not shown) (G) Proband Pt34 11-3) is the naffected I 9-year old niece of Pt33 who is a hi-h-risk individual Both of her mother 11-6) and maternal aunt (11-8) had regular-onset BC She has a paternal uncle 13) with king Te other affected relatives are the sme as Pt33 except tat P04 belongs to the -generation succeeding P33 (not shown) Table 12.1. Cinicopathological Characteristics of te 36 BRCA1 Mutation High-Risk Individuals and their Corresponding Distribution. Distribution High-Risk Individuals Characteristics Wild-Type Low-Risk Age at Onset of BC (years)' Unaffected' Individual <40 >40 (normal) 'S"ex Female 25 73.5) 8 23 2 1 Male 1 29) 0 Family history rainilies wth B(O)C cases 14 41.2) 7 20.6) PtI3, Pt34 E'a:n:;:cs w t 0 0 P03(l) 1,:iat leas I BOC case i t first degree relatives ra n es wi at least 2 BC cases i te first-degree relatives 1 2 9 0 Families wit I BC case in te first-degree relatives 4 (11 8) 5 14 7 Pt34(l) Families wth at least I OV case in the first-degree relatives 2 ( 9 0 Families with at least I B(O)C case in second-degree relatives 6 20 6 2 ( 9 Pt 32), Pt34(l) Families wth at least I B(O)C case in third-clegree relatives 1 2 9 0 Families without B(O)C case 12 35.3) 1 (2.9) Pt37 Families with other ypes of cancer first-clegree relatives 2 ( 9 i (2 9) Families with other types of cancer in second- or third-clegree relatives 3 (S 8) 0 Ptl3(l), Pt34(5) No family istory ofany type ofcancer 7 20 6 0 Pt37 Histopath of BC of proband 3 Invasive ductal 15 75.0) 2 (10.0) Invasive lobular 0 0 Papillary 1 (5.0) 0 MUCinous 2 (10.0) 0 Unknown 9 5 Not applicable 0 0 PtI3, Pt34 Pt37 EStj ogen receptor3 Negative 4 28.6) 1 (7.1) Positive 9 64.3) 0 Unknown 12 7 Pt13, Pt34 Pt37 Not applicable I 0 Progesterone receptors Negative 4 33.3) 1 (8.3) Positive 7 58.3) 0 Unknown 14 7 PtI3, P04 P137 Not picable I 0 'enclosed iii parentheses are the Witesponding percentages n 34 ptients 2criclosed n parentheses are the number of cancer cases percentages were calculated from the cases were information was available ad applicable Table 12.2. Clinicopathological Characteristics of the Families with Relatives Afflicted with BC. Age at 13C Number of First- or Sccond-De,,ree Age Mean Age) of BC Onset of Frst- Age (Mean Age) of BC Histopath Patient Onset of Relatives Aflicted with BIC' Degree Relatives (years) Onset of Second-Degrcc of BC of Estrogen Progesterone 11) l1roband Relatives yelrs) Proband Receptor Receptor (years) Matei nal Paternal Sibling Matei nal Paternal Sibling Maternal Paternal Pt25 39 5 0 0 38 - 36 IDC Negative Ne-ative Pt26 46 0 0 1 - 40 Pt27 38 1 0 0 - - 76 IDC Positive Positive 1100 45 1 0 0 42 42

Table 12.3. Clinicopathological Characteristics of Families with Relatives Afflicted with OC. Age at BC Age at OC Number of First- or Second- Age (Mean Age) of Patient Onset of Relative Onset Degre Ierative!, Afflicted Age Mean Age) of 1C Oset of BC Oset of Second- Histopath Estrogen Progestercine ID Proband Afflicted Relative with BC First-Degrec Relatives yeais) Degree Relatives of BC of Receptor Receptor (years) with OC (years) (yea rs) Proband Maternal Paternal Sibling Maternal Paternal Sibling Maternal Paternal ptol 33, 59 Mother 50 0 0 0 - IDC Pt04 3 0 Mother 45 0 1 0 - - 3 IDC Ptl3l N/A Sister (BOC)-* 4 1 1 1* 40** 52 62 N/A N/A N/A *unafffected individual I*sanic individual

Table 12.4. Clinicopathological Characteristics of Families With Relatives Afflicted With Other Primary Cancers. Age at BC Other Primary Number of First- or Second- Age (Mean Age) of Patient Onset of Cancers of First- or Degree Relatives Afflicted Age (Mean Age) of BC Onset of BC Onset of Second- Histopath Estrogen Progestercine ID Proband Second-Degree with BC First-Degree Relatives (years) Degree Relatives of BC of Receptor Receptor (years) Relatives (ye rs) Proband Maternal Paternal Maternal Paternal Sibling Maternal Paternal Sibling Maternal Paternal l1t02 34 Prostate 2 0 0 72 60 IDC Ncgative Positive Ptl4 38 Metastatic Pancreas, 2 0 0 - - 56 IDC Negative Negative liporna islet Ptl.) 40 Lun. I 0 1 55 45 - IDC Positive Pt22 41 Lung 1** 0 0 - - 38** IDC with Throat, lobular Gastric, component Brain Lung mclastasis- Pt28 39 Cervical Hodgkin's I I 0 - 60 60 Disease Pt33 5 1 Colon 0 0 1 44 - - IDC l't34* N/A Lung 2 0 0 51 N/A N/A N/A lunafffected idividual "sani idividual Page 64 of 126

In 20 - co :2 18

16 - -he Cn 14 -

12 - 0

8

co 6 4 -

U) 2 0 0 FN 2 90 29-34 35-40 41-46 47-52 53-58 59-64 65-70 71-76 Age at Onset of Disease (years)

Figure 14. Frequency distribution based on age at onset of disease of the 34 selected BRCAI mutation high-risk individuals. Most of the patients have been diagnosed of BC before reaching age 41 years. The patient diagnosed at 71 years as a sister afflicted with BC. Page 65 of 126

Table 13.1. Socio-Economic, Dietary, and Other Factors of the 36 High-Risk Individuals and their Corresponding Distribution. Distribution High-Risk Individuals . . Wild-Type Socio-Econontic, Dietary, and Other Factors Age at Onset of BC Low-Risk (years)' Unaffected2 Individual :540 >40 (normal) Age at first menarche (years) :5i2 10(29.4) 6(17.6) Pt37 >12 12(353) 2 59) P03, Pt34 Unknown 3 ( 8) 0 I-lot applicable 1 29) Age at first sexual contact (years) <20 4(11.8) 1 29) >20 o 30 11(32.4) 4 11.8) Pt37 ?30 3 (8.8) 2 59) Unknown 2 59) 0 Never had any sexual contact 4 (11.8) 2 59) Ptl3, Pt34 Not applicable 1 29) 0 Parity Niffliparous 7 20.6) 4 (11.8) Ptl3 Parous 17 (50.0) 4 (11.8) Pt3? Age at first cild 30 years old 14 412) 4 (118) Pt37 Age at first cild 30 years old 3 (88) 0 Unknown 1 2 9 0 Not applicable 1 29) 0 Pt34 Breast Feeding Yes 10 29.4) 3 (8.8) Pt37 No 10 29.4) 5 14.7) Ptl3 Unknown 5 14.7) 0 Not applicable 1 29) 0 Pt34 Personally-induced or "quack" doctor abortion Yes 2 ( 9 0 No 22 64.7) 8 23.5) Pt13 Pt37 Unknown 1 29) 0 Not applicable 1 2 9 0 Pt34 Civil Status Single 7 20 6 2 59) Ptl3, Pt34 Married 19 (5 9 6 17 6 Pt37 Religion Buddhism 2 (5.9) 0 lglesia Nt Cristo 0 1 29) Prolestantisinl0ther Cristian GrOLIPS 5 14.7) 1 2 9 Pt34 Roman Catholicism 19 55.9) 6 17.6) Pt13 Pt37 Educational Attainment Flementary/High-School 2 (5.9) 2 ( 9) Undergraduate (B S /BA /A 17 (50.0) 4 (118) Pt34 Graduate School (MS Ph D Medic ine/Law/ete 5 14.7) 2 (5.9) Ptl3 Pt37 Unknown 2 ( 9 0 Net Monthly Family Income

Table 13.1. Socio-Economic, Dietary, and Other Factors of the 36 High-Risk Individuals and their Corresponding Distribution ... (continued) Distribution High-Risk Individuals Wild-Type Socio-Economic, Dietary, and Other Factors Age at Onset of BC Low-Risk (years)' Unaffected 2 Individual :540 >40 (normal) Smoking Active and passive 1 29) 0 Active 3 ( 8) 2 (5.9) Passive 10(29.4) 3 (8.8) Pt34 Non-Smoker 10(29.4) 3 (8.8) PtI3 Pt37 Unknown 2 59) 0 Alcoholism Regular >3 lasses per week) 0 0 Very Occasional <3 glasses per eek and only dring occasions) 9 26.5) 2 ( 9 Pt34 No intake 15 44.1) 6 17 6 Ptl3 Pt37 Unknown 2 59) 0 Caffeine Intake Relatively Elevated 2 (5.9) 2 59) Pt37 Regular 18 52.9) 5 14 7 Ptl3 No intake 4 (11.8) 1 2 9 Pt34 Unknown 2 59) 0 Cooking Oil Used Animal 7 20.6) 2 59) Vegetable 17 (50.0) 5 14.7) Pt13, Pt34 Pt37 Canola 2 ( 9 0 Corn 9 26 5) 2 ( 9 PM Pt37 Olive 1(2 9 0 Soya 1(2 9 2 ( 9 Pt34 Regular 1(2 9 0 Corn and Olive 3(8 8) 1 (2 9) Animal and Vegetable 0 1 (2.9) Unknown 2(5.9) 0 Re-usage of Cooking Oil 2:2 X I (32.4) 2 (5.9) 1 x 7 20.6) 4 (11.8) Ptl3 Pt37 Never re-used 4 (11 8) 2 ( 9 Pt34 Unknown 4 (11 8) 0 'enclosed in parentheses are te corresponding percentages in 34 patients 'enclosed in parentheses are te number of cancer cases Ipercentages erc alculated from te cases here inforination was available and applicable Page 6 7 of 126

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4.5 Relative and/or Lifetime Risk Estimates for Unaffected High-Risk Individuals

Estimates of relative and/or lifetime risks for NH (Figure 4 and 12C) and Pt34 (Figure 13G) using Gail, Claus, and BRCAPRO Models are shown in Tables 14.1 and 14.2. Relative risk (RR) values given by the Gail model indicate -3X risk of "no-risk" individuals for both P03 and Pt34. Although the RR is higher or 10 years from the current age (except for age 29 of P04 due to 0.0 risk value of "no risk" population), the RRs for these individuals are actually almost similar during their lifetime. While the Gail model estimates cumulative lifetime risk based on risk modifiers and Claus model incorporates family history, BRCAPRO incorporates carrier probabilities to estimate unconditional probability. The Gail model and BRCAPRO gave similar lifetime risk estimates for Ptl3 but not for Pt34. This is due to the effect of family history of P04. Claus estimated higher remaining risk for P014 because she is only 19 years old with 2 relatives afflicted with BC. The Gail model is particularly useful in estimating relative and lifetime risks for individuals with early menarche regardless of their family history. The Claus and BRCAPRO models are quite useful in estimating cumulative lifetime BC risks for individuals with family history of BC and have high-risk of being BCAI mutation carriers, respectively. Page 69 of 126

Table 14.1. Relative and/or Lifetime Risk Estimates for 40-year old woman unaffected of BC (Ptl3). Relative Risk Cumulative (Remaining) Risk or Unconditional Probability

Age Unconditional Gail Model Claus Model* Gail Model Claus Model Probability

40 3 11 3 20 - - -

45 3 25 - 0 0130 0 0101 0 0278

50 2 89 0 0260 0 0215 0.0589 55 - - 0.0377 0.0874

60 2 79 0.0670 0 0545 0 119 65 - - 0 0732 0 1333

70 2 79 01200 0.0914 0 1523 75 - - 0.1071 0 1703

80 - 0 1866 85 - - - 0 1995

Lifetime 2 61 01960 0 197* 0 995** *from CancerGene and Table 3 **to age 79 years **to age 85 years

Table 14.2. Relative and/or Lifetime Risk Estimates for 19-year old woman unaffected of BC (Pt34). Relative Risk Cumulative (Remaining) Risk or Unconditional Probability Age Unconditional Gail Model Claus Model* Gail Model Claus Model Probability

19 3 11 I 10 at 29 - - - 24 - 0 0000 0 0055 0 0001 29 0 Vs 0 0001 0.0191 0 0008

34 - - 0 0327 0 0003

39 3 50 0 0007 0 0598 0 0077

44 - - 0 0870 059

49 3 00 0 0300 0 1247 0 0266 54 - - - 0 1624 0 0392

59 - - - 0 991 0 0534

64 - - - 0 2358 0 0689

69 - - - 0 2604 0 0852

74 - - - 0 2851 0 018

79 - - - 0 1172

84 - - - 0.1300

Lifennie 2 58 - 01990 0 2851 0 1300**

'trom unc, i,en oly "to age 79 eals *'*to age 84 yeais Page 70 of 126

4.6 Working Genomic DNA Solutions

Characteristics of stock and working genornic DNA solutions are shown in Appendix D. Agarose gel electrophoresis results of working genomic DNA solutions (Figure 15) corroborated the concentration and purity of isolated genornic DNA. Concentration was calculated using the spectrophotometric conversion for double-stranded DNA D260=1 for a 50tg/rnl solution). Purity was calculated using D260/OD2gO ratio (Freifelder, 1982; Warburg Christian, 1942).

4.7 PCR Products

PCR products for SSCP at BRCA1 exons 2 5, 13, 15, 17, 18, 20, 22, and 24 from (a) pooled or (b) individual genornic DNA of suspected familial BC patients were successfully generated through PCR. Based on EtBr-stained agarose gel electrophoresis and Polaroid manual documentation system, the fragments apparently exhibited the expected sizes (Table 9 Figure 8). Pooled genomic DNA were as follows: GI (PtOl-PtO3), G2 (PtO4-PtO6), G3 (PtO7-PtO9), G4 (PtIO-Ptl2), G5 (PO3-1`05), G6 (PtI6-Ptl8), G7 (Ptl9-Pt2l), G8 (Pt22-Pt24), G9 (Pt25-Pt27), GIO (Pt28-POO), GI PO I-Pt33), and G12 (1`04-PO6). Mutation controls (lane 16) used were as follows: exon 2 185delAG, American), exon 5 (C64Y, American), exon 13 (II405V, American), exon 15 (SI5121, Italian), exon 16 (YI563X, American), exon 17 (VI665M, American), exon 18 (LI705L, Italian), exon 20 (5382insC, Australian), exon 22 (IVS21- 6del5lO, Polish), and exon 24 (WI 837R, Italian) Figure 16).

PCR products at BRC,41 exon I I for PTT from genomic DNA of suspected familial BC patients were successfully generated through touchdown PCR. Based on EtBr-stained agarose gel electrophoresis and Polaroid manual documentation system, the fragments apparently exhibited the expected size of z' )468bp. Lanes 34 615, 19-26, and 28-31 correspond to PCR products at exon 11 of PtOl-PtO2, PtO4-Ptl3, Ptl4-Pt2l, and Pt23-Pt26, respectively (Figure 7). Lanes and 27 correspond to PtO_3) and Pt22, respectively. PCR products from the genornic DNA of these patients, as well as Pt27-Pt' )7, were later successfully generated (data not shown).

4.8 Single-Strand Conformation Polymorphism Analysis

Formamide- and heat-denatured, snap-chilled PCR products were loaded and run in non- denaturing polyacrylarnide gel electrophoresis at I OOV for 20h then visualized by silver staining. SSCP profiles of pooled enomic DNA at exons 2 5, 13, 15, 16, 17, 18, 20, 22, and 24 are shown in the succeeding pagesI., FigureIm 18). Page 71 of 126

Results of SSCP analysis of PCR-amplified pooled DNA of suspected familial Filipino BC patients are shown below (Table 15). Genomic DNA of patients that could possibly have the putative polymorphisms based on the pooled SSCP analysis were amplified and subjected to SSCP analysis individually (Figure 19).

SSCP analysis of pooled and individual samples was able to pinpoint unique putative polymorphisms among 28 collaborating patients 26 BC patients and 2 high-risk individuals) at BRCAI exons 2 5, 13, 15, 16, 17, and 22 (Table 16). This makes the prevalence of BCAJ putative polymorphisms for this study to be approximately 76.5% 26/34). Low prevalence (

4.9 Protein Truncation Test

4.9.1 Polyacrylamide Gel Electrophores'is(14%) Protein products generated from in vitro transcription and translation of PCR products at BCA I exon I I were separated by SDS-PAGE 14%) and visualized through autoradiography generally exhibited the expected size of the wild-type brcal protein (427kl)a) and the banding pattern of the reticulolysate system used (Figure 20). Lower molecular weight truncated protein products (12kDa, 55kDa, and 75kDa) were detected using 14% SDS polyacrylamide gel. Higher molecular weight truncated protein products (1 12 kDa, II 5kDa, 125kDa, and 126kDa) were not clearly separated from the wild-type brcal protein (Table 17). Nationality and details of these mutation controls are shown in Table 10 (Methods). Negative controls were as follows: loading buffer (lanes I and 12), PCR negative control subjected to PTT (lane I ), and PTT negative control (lanes 24 and 26). All the negative controls exhibited no banding patterns except for the presence of unincorporated 35 S-methionine in the PCR and PTT negative controls (lanes 11, 24, and 26). Lanes 28, 13-20, and 28-38 correspond to Filipino BC patients PtOl-PtO7, PtO8-PtI5, and PtI6-26, respectively. Lanes 23 and 27 correspond to Pt37 (wild-type, low-risk individual). A truncated protein product (-29.7kDa) was observed at lane corresponding to PtO4. This protein product has almost of the same apparent molecular weight (MW) as the lower MW protein product of mutation control 2279insA. However, the expected protein product of 2279insA is a 5 5kDa band which is actually the darker higher MW band at lane 2 . Page 72 of 126

4.9.2 GradientPolyacrylamide Gel Electrophoresis(5-20%) To remove the possibility of not detecting mutations resulting into higher molecular weight truncated protein products (i.e., above lOOkDa) as exhibited by the mutation controls in Figure 20, and to also increase the resolution of separation for any ultra-low molecular weight protein product, gradient SDS-PAGE 5-20%) was performed then visualized by autoradiography using the same PCR products that were transcribed and translated in vitro (Figure 21).

The expected size of the wild-type brcal protein (;z:127 kDa) and the general banding pattern of the reticulolysate system were manifested by the protein products of most of the samples. In fact, the resolution of the bands was more pronounced. Lanes 36 911, 13-14, 21-31, 38-48, 56-58, 59, and 60-61 correspond to protein products of in vitro transcribed and translated exon I PCR products of Filipino patients PtOl-PtO4, PtO7-09, Ptll-PtI2, Ptl3-Pt23, Pt24-PO4, PtO4-PtO6, PtJO, and P05-PO6. Lanes 2 20, 37, and 55 contain the protein products of the low-risk individual (Pt37). Mutation controls were now clearly separated even for the higher molecular weight truncated protein products (Table 18). Ex I 1. 15delA exhibited the same apparent MW as E 125OX 14 kDa) while controls 4153delA and 4184del4 only have 12 kDa difference with the wild-type protein, Negative controls were as follows: loading buffer (lanes 7 and 53) and PTT negative control (lanes 1, 8, 19, 36, and 54. The negative controls had no detectable banding patterns except for the presence of unincorporated 35 S-methionine (lanes 1 7 8, 19, 36, 3, and 54). Majority of the bands of luciferase DNA (lane 65) was about 61 kDa Z90%). The banding pattern of control 2279insA (lane 16) was much better when a 520% gradient gel was used, However, the low MW band at lane 16 is still distinguishable and appears to be smaller than the truncated protein product of PtO4 (lanes 6 and 56).

4.10 Denaturing High-Performance Liquid Chromatography (DHPLC) Analysis

Using standard calculations the apparent molecular weight of the truncated protein product of the in vitro transcribed and translated PCR product at BRC,41 exon I I of PtO4, was estimated to be approximately 29.7 kDa 29,664 Da). Since I kbp DNA 37 kDa Z; 333 amino acids, the estimated location of the stop codon due to the unknown mutation is approximately at nt 802. The approximate nucleotide position of these sequencing primers in the whole BRCAJ gene 'Nas estimated by adding 789bp to the position in exon 11 resulting to nt 1591. Sequencing primers flanking the approximate location of the stop codon (nt 1591) were, therefore, chosen in te laboratory of Dr. Teresa M.U. Wagner (Division of Senology, Department of Obstretrics ad Gynecology, University of Vienna, Vienna, Austria). Forward sequencing primer (GGT TCT GAT GAC TCA CAT GAT GGG) used, designated as EXI I EF, contains 5' nucleotides 1299- Page 73 of 126

1322 of BRCAJ. The reverse sequencing primer (TCA TCA CTT GAC CAT TCT GCT CC) used, designated as EX11ER contains 3 nucleotide positions 1758-1736. Amplicon size is 460bp and annealing temperature is 65'C-58'C.

Touchdown PCR was performed by using Ix Optimase buffer, 200LM of each dNTP, 12.5pmol

(625 nM) of each primer, 1.5mM MgSO4, and 2.5U Optimase TM (Transgenomic) in the Applied Biosystems 9700 Thermal Cycler. Initial denaturation was performed at 95'C for min followed by 34 cycles at 95'C for 30s, 65'C for 30s, 72'C for Imin having 0.5'C annealing temperature reduction for the first 14 cycles until 580C has been attained and carried out for the succeeding 20 cycles. Final elongation step was performed at 72'C for 5min. PCR products were processed for DHPLC analysis and amplicons were eluted with the Gradient (Eluant A: 0.1 M triethylammonium acetate, pH 70; Eluant B: 0.1 M triethylammonium acetate, pH 70, 25% acetonitrile) 50-59%B in 0.5 min 59-66% in 35 min at 56'C. The presence of heteroduplexes is deten-nined by comparing the elution profiles obtained from the test sample with that of the wild-type sample. Under partially denaturing conditions, heteroduplex DNA has reduced retention time compared to homoduplex DNA and is therefore eluted at an earlier time. In PtO4, the mutation is indicated by the presence of a small peak at 4.4 min in the test sample that is absent in the homozygous control thereby validating the PTT result (Figure 22).

4.11 Automated DNA Sequencing

PCR products (460bp) were purified and cycle sequencing was performed. Sequence analysis indicated the presence of the insertion of "GT" at nucleotide position 1445 resulting in a frameshift mutation causing a stop codon at amino acid 452 and expressed as a truncated protein product of 29.7 kDa when transcribed and translated in vitro (Figure 23). This is the second documented mutation detected in BRCAI exon I I among Filipino breast cancer patients, first of which was in a case report in the United States (Worsham et al., 1998). This mutation (1445insGT) has not been detected in any other individual worldwide based on the BRCAJ mutation database at the BIC website (BIC, 2003).

4.12 BRCAI Prior Probability Estimates for Familial Filipino BC Patients

Estimates of BRCAJ prior carrier probabilities for families characterized by cancer-afflicted relatives (Figures 11 to 13) appropriately using Myriad.com Prevalence Table (Table 72) and

BRCAPRO Figure6 9 depending on the variables included are shown in Table 19. Page 74 of 26

4.13 BRCAJ Posterior Probability Estimates for Screened Patients

BRCAPRO estimates of BRCA1 Posterior carrier probabilities for families characterized by cancer-afflicted relatives (Figures 11 to 13) based on screening results are shown in Table 20. Prior probabilities are shown for comparison.

4.14 Unconditional Posterior Probability Estimates for Unaffected High-Risk Individuals

BRCAPRO unconditional probability estimates for Ptl 3 (Figures 4 and 2C) and Pt34 (Figure 13G) are shown in Tables 21.1 and 21.2. Since both Ptl' and Pt34 exhibited putative polymorphisms (Figure 18), two unconditional posterior probability estimates were obtained for each patient. Unconditional prior probabilities are also shown for comparison.

4.15 Patient Characteristics and Profiles

Main characteristics of the Filipino BC patients and their respective risk modeling and screening profiles after performing SSCP at exons 2 5, 13, 15, 16, 17, 18, 20, 22, and 24 and PTT at BRCAI exon I I and are shown in Table 22. Te clinical and family histories of each of the collaborating patients with their corresponding ethnic origins and screening profiles based on PTT and SSCP results are presented. The BRCAPRO prior and posterior carrier probabilities of the selected patients are also included. Page 75 of 126

6ISRP

2000bp-

10000pRR- - 2000bp-

2000bp:

Figure 15. Agarose gel electrophoresis (0.8%) of working genomic DNA solutions from suspected familial Filipino BC patients. Lanes 415, 19-30, and 34-45 working genornic DNA solutions of PtOl-PtI2, Pt]3-Pt24, and Pt25-Pt36, respectively); lanes 1, 16, and 31 (Invitrogen High DNA Mass and Concentration Ladder 110kb); lanes 2 17, and 2 loading buffer-negative control); lanes 3 18 ad 33 workingI t 0genornic DNA solution of wild-t pe ov, risk individual, Pt37). Page 76 of 126

2""Oft,- - ..... P- 350bp- Z- 150bp- 35-Obp- 50bp- if; Obp-

Exon 2 amplicon: 251 bp 50bp- Exon 16 amplicon: 450 bp 1111b,- 16 P_ Wbp P bp 150bp- 150bp- 50bp- 50bp-

Exon 5 arnplicon: 278 bp Exon 17 aplicon: 190 bp

2612 'P- 2652bp

ftp- 800bp 350bp- 350bp 150bp- 150bp 50bp- 50bp Exon 13 amplicon: 320 bp Exon 20 amplicon: 259 bp

2612bp- 2612bp-

"'P_ bp_

350bp- 350bp 150bp- 150bp- 50bp- 50bp-

Exon 15 amplicon: 378 bp Exon 22 amplicon: 297 bp

Figure 16. PCR products of pooled genomic DNA of the suspected familial Filipino BC patients at the smaller priority exons of BRCAI. Lanes 315 (PCR products of GI-GI2); lane 1 (50 bp ladder); lane 2 (negative control), and ane 16 (mutation control). PCR products of exons 18 (259bp) and 24 280

bp) were also r>generated (data not shown). Page 77 of 126

JIMIMI.

.1101......

Figure 17. PCR products --3468bp) U genomic DNA at exon 11 from some of the suspected familial Filipino BC patients using BRCAI primers described elsewhere (Garvin, 1998; Ottini et a, 2000). Lanes 34 615, 19-26, and 28-31 (PCR products of PtOl-PtO2, PtO4-Ptl3, Ptl4-Pt2l, and Pt23- Pt26, respectively); lanes I and 17 500bp ladder); lanes 2 and 18 (loading buffer); lanes 16 and 32 (negative controls). Lanes and 27 correspond to PtO3 and Pt22, respectively, which were later successfully generated (data not sown). Page 78 of 126

(A) Exon 2

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 19 2,652 bp-

800 bp- 700 bp- 600 bp- 500 bp-

350 bp-

250 bp-

(B) Exon 5 1 2 3 4 5 6 7 9 10 11 12 13 14 15 16 17 2,652 bp-

800 bp- A 700 bp- 600 p-,4,

500 b P_ m "'A

350 bp-*

Figure 18A-18B. SSCP analysis in the BCAI gene. Lane 1 (50 bp ladder), lane 2 (formamide buffer), and lane 3 (wild-type, P07). Closed and open arrows indicate aberrantly migrating bands of mutation controls and pooled samples, respectively (A) Exon 2 lane 4 negative control), lanes 516 (pooled samples GI G 12) Note 2 putative polymorphisms at G8 and GI I], lane 17 (185delAG, Australian, lane 18 (185delAG, American), and lane 19 (183insTT, Australian). (B) Exon 5 lanes 415 (pooled samples GI-GI2) [Note: I putative polymorphism at G121, lane 16 negative control), and lane 17 (C64Y American) Page 79 of 126

(C) Exon 13 2 3 4 6 9 12 13 14 15 16 17 2,652 bp-

800 bp- 700 bp- 600 p-

500 bp-

350 bp-

250 b-

(D) Exon 15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

0- 2,652 bp-

800 bp- 700 bp- 600 bp-

500 bp-

350 bp-

250 bp-

Figure 18C-18D. SSCP analysis in the BRCAI gene. Lane 1 (50 bp ladder), lane 2 (formamide buffer), and lane 3 (wild-type, Pt37) Closed and open arrows indicate aberrantly migrating bands of mutation controls and pooled samples, respectively. (C) Exon 13: lane 4 (negative control), lanes 516 pooled samples GI-G12) [Note: 4 putative polymorphisms at G, G5, G7, and G91, lane 17 (negative control); and lane 18 (11405V, American). (D) Exon 15: lanes 415 (pooled samples G-G12) rNote: 10 putative polymorphisms in almost all of the Ro1ed samples except G2 and G8], lane 16 (SI 5121, Italian, notice the high intensity of the slow migrating band), and lane 7 negative control) Page 80 of 126

(E) Exon 16 1 2 3 4 6 7 9 0 11 12 13 14 15 16 17 2,652 bp-

800 p- 700 bp-

600 bp-

500 bp-

350 bp-

(F) Exon 17 1 2 3 4 6 7 9 11 12 13 14 16 17 800 bp- 700 bp- 600 bp-

500 bp-

350 bp-

250 p-

150 bp-

Figure 18E-18F. SSCP analysis in the BRCAJ gene. Closed and open arrows indicate aberrantly migrating bands of mutation controls and pooled samples, respectively. (E) Exon 16: lane 1 (50 bp ladder), lane 2 formarnide buffer), ane 3 negative control), lane 4 (wild-type, P67), lanes 516 (pooled samples GI G 12) [Note: 4 putative polymorphisms at G 1, G2, G5 and G I I , lane 17 (negative control), and lane 18 (Y 1563X American). (F) Exon 17: lane 1 (50 bp ladder), lane 2 formamide buffer), lane 3 (wild-type, P07), lane 4 negative control), lanes 516 (pooled samples G I G 12) Note I putative 12olymo[phism at G41, lane 17 (negative control), and lane 18 (V I 66M, American). Notice the absence of aberrantly migrating band in the mutation control for this particular exon. Another mutation control (5 149de]4, Italian) showed clearer difference in the migration of sin-le strands (data not shown). Page 81 of 126

(G) Exon 18 1 2 3 4 6 7 9 0 11 12 13 14 15 16 7

800 bp- 700 bp-

600 bp-

500 bp- DA

350 bp-

(H) Exon 20 1 2 3 4 6 7 9 0 11 12 13 14 15 16 17 1 19

800 bp- 700 bp- 600 bp- 500 bp-

350 bp- N,

250 bp-

150 bp-

Figure 18G-18H. SSCP analysis in the BRCA1 gene. Lane 1 (50 bp ladder), lane 2 formamide buffer), and lane 3 (wild-type, Pt37). Closed arrows indicate aberrantly migrating bands of mutation controls. (G) Exon 18: lanes 415 (pooled samples Gl-G12) [Note: no putative polymorphism detected], lane 16 negative control), and lane 17 (JVS I 8+65G/A, Italian). (H) Exon 20: lanes 4 5 716 (pooled samples G I G 12) [Note: no putative polymorphism detected], lane 6 negative control), lane 17 53 82insC, Australian), lane 18 (R 175 X, Polish), and lane 19 (negative control). Page 82 of 126

(1) Exon 22 1 2 3 4 6 7 9 0 11 12 13 14 15 16 17 2,652 bp-

800 bp- 700 bp- 600 bp- 500 p-

350 bp-

250 bp-

150 bp-

(J) Exon 24 1 2 3 4 6 7 9 11 12 13 14 15 16 17 2,652 bp-

800 bp- 700 bp-

600 bp-

500 bp-

350 bp-

250 bp-

Figure 181-18J. SSCP analysis in the BRCAI gene. Lane 1 (50 bp ladder), lane 2 formamide buffer), and lane 3 (wild-type, P67). Closed and open arrows indicate aberrantly migrating bands of mutation controls and pooled samples, respectively (1) Exon 22: lanes 415 (pooled samples GI-G12) [Note: 3 putative polymorphisms at G7, G8, and G91, lane 16 (negative control) and lane 17 (IVS21-36de]510, Polish). (J) Exon 24: lanes 415 (pooled samples 01-GI2) [Note. no putative polymorphism detected), lane 16 (RI835, American), lane 17 (WI837R, Italian); and lane 18 negative control). Page 83 of 126

Table 15. Pooled SSCP Analysis in the BRCA.1 Gene of Suspected Familial Filipino BC patients.

Exon Pool in Which Putative Polymorphism was Possible Patients with Putative Polymorphism Detected

2 G8 and GI I Pt22-Pt24, Pt I Pt33

5 G12 Pt34-Pt36

13 GI, G5, G7, and G9 PtOl- PtO3, Ptl3-Ptl5, PtI9-Pt2I, Pt25-Pt27

15 All except G2 and G8 All except PtO4-PtO6, Pt22-Pt24

6 GI, G2, G5, and GI Pt I PtO6, Pt 3 -Pt 5, PO 1-Pt3 3

7 G4 PtIO-PtI2

8 Negative

2 0 Negative

22 G7, G8, and G9 PtI9-Pt27

24 Negative Page 84 of 126

(A) Exon 2 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2

800 bp-

700 bp-

600 bp

500 bp-

350 bp

(B) Exon 5

1 2 3 4 5 6 7 8

800 bp- 700 bp- 600 bp-

500 bp-

350 bp-

Figure 19A-19B. SSCP analysis in the BCAI gene in patients which probably have putative polymorphisms. Lane I 50bp ladder), lane 2 formarnide buffer), and lane 3 (wild-type, Pt37). Closed and open arrows indicate aberrantly migrating bands of mutation controls and individual samples, respectively. (A) Exon 2 Lanes 4 67 (PM-Pt24), lane 5 negative control), lanes -10 (Pt3l-Pt33) (Note: Pt33 has a unique putative polyrnoEphism which could orobably be a mutation as indicated by its low prevalence], lane I I (negative control), and lane 12 (185de]AG, Australian). No putative polyrnorphism was detected among Pt22-Pt24 (G8). (B) Exon 5: lanes 46 (Pt34-Pt36) [Note: Pt36 has a unique putative polymorphism which could probably be a mutation as indicated by its low prevalence], lane 7 negative control), and lane 8 (C64Y American). Page 85 of 126

(C) Exon 13

21652 bp_ 1 2 3 4 6 7 9 10 11 12 13 14 15 16 17

800 bp- 700 bp- 600 bp- 500 bp-

350 bp-

250 bp-

(E) Exon 16 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 2,652 bp-

800 bp- 700 bp- 600 bp-

500 bp-I

350 bp-

Figure 19C and 19E. SSCP analysis in the BRCAI gene in patients which probably have putative polymorphisms. Lane I 50bp ladder), lane 2 (formarnide buffer), and lane 3 (wild-type, Pt37) Closed and open arrows indicate aberrantly migrating bands of mutation controls and individual samples, respectively. (C) Exon 13 lanes 46 (PtOl-PtO3), lanes 79 (Pt]3-Ptl5), lanes 10-12 (Pt]9-Pt2l), lanes 13-15 (N25-N27) [Note: PtO3, Ptl3 Ptl4, PH5, Ptl9, MO, Pt2l, and Pt26 exhibit one (1) unique putative polymorphism], lane 16 (negative control), and lane 17 (11405V, American). (E) Exon 16: lanes 49 (NOI-PtO6), lanes 10-12 (Pt]3-Pt]5), lanes 13-15 (Pt3l- Pt33) Note- PtO2, PtO4, PtO5 PM, Ptl4, P05, and Pt3l exhibit one (1) unique putative polymorphism], lane 16 (negative control), and lane 17 (Y I 563X American) Page 86 of 126

(D) Exon 15

1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

2,652 bp-

800 bp 700 bp 600 bp

500 bp

350 bp

Figure 19D. SSCP analysis in the BRCA1 gene in patients which probably have putative polymorphisms. (D) Exon 15: Small solid boxes indicate the patients having same allele as the wild-type individual. Small dashed boxes correspond to the homozygous polymorphism alele carriers while the large boxes indicate the heterozygotes. Closed arrows indicate aberrantly migrating bands of mutation control which appears to be a heterozygote having the same migration pattern as the heterozygous patients. Lane I 50bp ladder), lane 2 formamide buffer), lane 3 wild- type, N37), lanes 46 (PtOI-PM), lanes 718 (PtV-P08), lane 19 (S15121, Italian), lane 20 (forniamide buffer), lane 21 50bp ladder), lane 22 (wild-type, Pt37), lanes 23-25 (Ptl9-21), lanes 26-37 (Pt25-Pt36), lane 38 (negative control but with leaks from lane 39 (S15121, Italian). [Nqte- Almost all of the collaborating patients except for 10 of them tO4-PtO6, Ptl0, PtI7, Pt2O'Pt22-Pt24 PM, Pt3O, Pt32, and Pt35) either have the homozygous putative polymofphism or the heterozygous polymoMhism resulting from the presence of the wild-!Xpe allele as well as the homozygous polymorphism allele in an individual Out of 37 collaborating patients, 15 40.54%) have wild-type allele, 15 40.54%) are heterozygotes, and 7 18.92%) have the homozygous putative polymorphism allele. In this exon, pooled SSCP cannot be employed. However, efficiency of pooling was validated by the migration patterns of the individual samples would corroborate the mobility observed in the pooled samples. Page 7 of 126

(F) Exon 17

1 2 3 4 5 6 7 8 800 bp- 700 bp-

600 bp-

500 bp-

350 bp-

250 bp-

(G) Exon 22 1 2 3 4 5 6 7 8 9 10 I 12 13 14 2,652 bp-

800 bp- 700 bp- 600 bp- 500 bp-

350 bp-

250 bp-

Figure 19F-19G. SSCP analysis in the BRCAI gene in patients which probably have putative polymorphisms. Lane I 50bp ladder), lane 2 (formamide buffer), and lane 3 (wild-type, P07). Closed and open arrows indicate aberrantly migrating bands of mutation controls and individual samples, respectively. (F) Exon 17: lanes 46 (PtIO- P02) Note- PO I and Ptl2 exhibit one (1) unique putative polymorphism which could probably be a mutation as indicated by its low prevalence], lane 7 (negative control), and lane 8 (VI665M, American). (G) Exon 22: lanes 4- 12 (Ptl9-Pt27) [Note: Pt22 and Pt26 have two 2 putative polymorphisms each of which could probably be a mutation as individually indicated by their low prevalenc ], lane 13 (IVS21-36de]510, Polish), and lane 14 negative control). Page 88 of 126

Table 16. Patients with Putative Polymorphisms Based on SSCP Analysis.

Pool in which #of Patie ts ft of Unique Exon putative Patients with Putative with Putative Prevalence Putative Polymorphism was Polymorphism Polymorphism (%)" Polymorphisms Detected

2 G8 and GI I P03 1 2.9 b

5 G12 Pt36 1 2 9 b

13 GI, G5, G7, and G9 PtO3, PtO4, Pt] 4, Pt 5, 8 7) 20 6 1 Pt] 9 MO, Pt2 1, Pt26

15 All except G2 and G8 PtOl, PtO2, PtO3, NO, 22 20) 58 8 1 PM, PtO9, Pt I 1, Pt 3, Ptl4, Ptl5, PtI6, PtI8, PtI9, Pt2l, Pt25, Pt26, PM, Pt29, Pt3 1, Pt3 3, P04, Pt36

16 GI, G2, G5, and GI I PtO2, PtO4, PtO5, PtI3, 6 (5) 14 7 1 PtI4, Pt3l

17 G4 PO 1, P02 2 5.9 b

22 07, G8, and G9 Pt22, Pt26 2 9 b

'Denominator used is 34 bThese unique putative polymorphisms could probably be mutations due to the low prevalence in these patients Note Numbers in parentheses indicate the number of patients included in the calculation ofprevalence Total number of patients ith putative polymorphisms is 26 so the prevalence in 34 patients is 76 % Page 89 of 126

1 2 3 4 5 6 7 8 9 10 I 12 13 14 15 16 17 18 19 20 21 22 23 24 25 132 kD - 132 kDa i'-; 66 kDa - 66 Da 45 kDa - 45 kDa i

20 kD - 20 kDa

14 kD - 14 kDa

26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 132 kDa -

K44 Plot 45 kDa -

20 kDa -

14 kD -

Figure 20. Protein products generated from exon 11 PCR products of some of the familial Filipino BC patients through TNT Quick-Coupled in vitro transcription/translation system separated and visualized by 14% SDS-PAGE and autoradiography. Coomassie-stained homernade markers composed of BSA (dimer and monomer), ovalbumin, trypsin inhibitor, and lysozyme were used to estimate apparent molecular weights. Lanes 28, 13-20, and 28-38 correspond to Filipino BC patients PtOl-PtO7, PtO8-PtI5, and Ptl6-26, respectively. Protein products of wild-We, low-risk individual (Pt37) were at lanes 23 and 27 (Pt37). Mutation controls were as follows: lanes 9 and 39 (1100delAT, Australian); lane 10 (2798del4, Australian); lane 21 (2279insA, GM13712, Swiss); lane 22 (3875de]4, GM13705, Swiss); lane 40 (3819del5, Polish); lane 41 (3875del4, Australian); lane 42 (4153delA, Polish), lane 43 (4184del4, Australian) (Table 17). Nationality and details of these mutation controls are shown in Table 10 (Methods). Negative controls were as follows: lanes I and 12 loading buffer); lane I I (PCR negative control subjected to PTT); lanes 24 and 26 (PTT negative control); and lane 25 (Iticiferase control DNA 90% z6lkDa band). Note the truncated protein product Z29.7kDa) at lane corresponding to PtO4. Page 90 of 126

Table 17. Detectability of BRCA1 Mutation Controls at 14% Polyacrylamide Gel Electrophoresis.

Truncated Protein Lane Positive Mutation Control Molecular Weight Remarks Products (Nationality) (kDa)

9 1100delAT (Australian) 12 Detectable

10 2798de14 (Australian) 75 Detectable Lower Molecular Weight 21 2279insA (GM13712, Swiss) 55 Detectable

39 1100deIAT (Australian) 12 Detectable

22 3875de]4 (GM] 3705, Swiss) 115 Not clearly separated

40 3819de]5 (Polish) 112 Not clearly separated

Higher Molecular Weight 41 3875de]4 (Australian) 115 Not clearly separated

42 4153delA (Polish) 125 Not clearly separated

43 4153de14 (Australian) 126 Not clearly separated Page 91 of 126

1 2 3 4 5 6 7 9 9 10 11 2 13 14 16 17 1 8 19 20 2 22 23 24 2S 26 27 28 29 30 31 32 33 34 35 Y

V A -220 kDa - 220 W - 60 kDa - '20 II)a - 160 kDa - 90 kDa - 120 kD - 70 kDa - 90 kDa - :50 kl) - 50 kDa -

4 30 kDa - 30 kDa -

20 kD. - 20 kDa -

10 kDa - 10 kDa

36 37 33 39 40 41 42 43 44 45 46 47 49 49 50 51 52 53 54 5 56 57 58 59 60 6 1 62 63 64 65

220 kDa - A 22 kDa - 160 kDa - 160 kDa - 1211 W. - [20 kDa - 90 kDa - 90 kDa - 70 kDa - 70 kDa - 5 G k Da - 50 kDa -

30 kDa - 30 kDa -

20 kDa - 20 kDn -

10 1,D,, - 10 kDa -

Figure 21. Protein products generated from exon 11 PCR products of the familial Filipino BC patients through TNT Quick-Coupled in vitro transcription/translation system separated and visualized by 520% gradient SDS-PAGE and autoradiography. Coomassie-stained Invitrogen BenchMark Protein Ladder consisting of 15 engineered proteins from 10 to 220 kDa was used to estimate apparent molecular weights. Lanes 36 911, 13-14, 21-31, 38-48, 56-58, 59, and 60-61 correspond to protein products of in vitro transcribed and translated exon I I PCR products of Filipino BC patients Pt I - PtO4, PtO7-09, Ptl I-PtI2, Ptl3-Pt23, Pt24-Pt34, PtO4-PtO6, PtIO, and Pt35-Pt36. Lanes 2 20, 37, and 5 contain the protein products of the low-risk individual (Pt37). Mutation controls were as follows: lane 5 (I 100delAT, Australian); lane 16 (2279insA, GM13712, Swiss); lane 17 (3450del4, Australian); lane 8 (3875de]4, GM13705, Swiss), lane 32 (1294del4O, Australian); lane 33 (2135de]A, GM13709, Swiss); lane 34 (2982del5, Polish); lane 35 (3875del4, Australian); lane 49 (2163insT, Polish); lane 50 (2798del4, Australian); lane 51 (38]9del5, Polish); lane 52 (4153de]A, Polish); lane 62 (EI25OX, GM13713, Swiss); lane 63 (4184del4, Australian); and lane 64 (exI 1.15del5, Polish) (Table 18). Negative controls were as follows: lanes 7 and 53 loading buffer) and lanes 1, 8, 19, 36, and 54 (PTT negative control). Lane 65 (luciferase control DNA 90% -61kDa band). Note the truncated protein product (z29.71

Table 1. BRCAI Mutation Controls Detected by 520% Gradient Polyacrylamide Get Electrophoresis

Truncated Protein Lane Positive Mutation Control (Nationality) Molecular Weight Products (kDa)

15 1 100deIAT (Australian) 12

16 2279insA (GM] 3712, Swiss) 55

17 3450deI4 (Australian) 99

32 1294del4O (Australian) 19 Lower Molecular Weight 33 2135delA (GM13709, Swiss) 50

34 2982de15 (Polish) 82

49 2163insT (Polish) 51

50 2798deI4 (Australian) 75

8 3875del4 (GM 13705, Swiss) 115

35 3875del4 (Australian) 115

51 3819de]5 (Polish) 112

Higher Molecular Weight 52 4153delA (Polish) 125

62 E125OX (GM13713 Siss) 114

63 4184del4 (Australian) 126

64 ex] I 15delA Page 93 of 126

2.0

1.5

1.0

0.0

0 1 2 3 4 i 6 7

Retantion Time Wu).

(b) 5

4

3

2

1

0

0 1 2 3 4 5 6 7

4

3

2

...... 0

0 1 2 3 4 5 6 7

Retention Time (min)

Figure 22. DHPLC analysis of the segment containing the putative mutation of PtO4 at BRCAI exon 11 (nt 1299-1758). (a) wild-type hornozygous control, (b) PtO4 --- note the small peak at 4.43i-nin that is not present in the control sample, and ) diluted solution of PtO4. The presence of heteroduplexes is determined by comparing the eution profiles obtained from the test sample (b&c) with that of the wild- type sample (a). Since heteroduplex DNA has reduced retention time (compared to homoduplex DNA) under partially denaturing conditions, it is therefore euted at an earlier time as exhibited by the clear presence of a smaller peak at -4.4min (b&c) even under diluted conditions. The smaller peak is distinctly absent in the control (a). Page 94 of 126

Summay CTTACTGGCCAGTGATCCTCATGAGGCTTTAKTATGT hAGTG)IAAGAGTTC&CTCCRAkTC),GTAGkOA Vadants . . . .i ...... Index 410 141 1420 142S 1430 143S 1440 144b 1450 1455 1460 1465 1470 .75 14( Rererence CTTACTOOCCAGTGATCCTCATGAGOCTTTAATATOT AAGTGkAAGAGTTCkCTCCAAATCAGTAGAGA, Referenm,t ...... D L L A S D P H E A L I C K 3 Z JR V H 3 X S V X WT11- CTTACTGGCIAGTGATCCTCATGA TGAAA:AGTTCACTCCAAATCAGTAGAG GGC ,,,,::CTTTAATATGTkkkAG C TT A C CAOTGATCCT CTTTAATATGT . A A TGkAA AGTTCACTCCAAATCAGTAGLGA

1_FO5_VVT1IBR1_EX11EIj 'TV

CTTA C TG G C CA 7 A C C CA TGA 0 C TTTA A TA TG T I qA A G GA AA G GTT CA C C CA AA TC A G A G. G BRCAI

1_FO6_WT11_BR1_EXI F-2 I wild-type 6v AWLAA, control PaL4_ CTTACTGGCCAGTGATCCTCkTOAGGCTTTAATATGTk AAGTGAAAGAOTTCACTCCYAATCkGTAGAD), C TTA CT G G C C A GTG JLTC C TC AT GA G CTTTA IT AT GT G T N- I.p c.d.. d. I. I 005 aL4 SRI CXIIEj f . .. WI t.t...

7 1 C T t t d A T G'T, 4 'ZCACZ'CCbAATCA G BRCAI I-G06_PaL4_SRIEX IF 1445insGT Stop452

Figure 23. Sequence showing the precise mutational event (1445insGT) at BRCAI exon 11 of PtO4. The insertion of GT at nt 1445 (indicated by the circle) is a frarneshift mutation resulting in a stop codon at amino acid 452 (indicated by the rectangle). Page 95 of 126

Table 19. BRCA112 Carrier Probabilities using Prior Probability Models.

BRCA1 BRCA1 or BRCA2

Patient ID BRCAPRO Myriad.com* BRCAPRO (Myriad 11)

Families with RelanvesAfflicted with C

Pt25 0 939 0 316 0 952

Pt26 0 017 0178 0 021

Pt27 0 013 0 078 0 015

Pt3O 0 023 0 178 0.027

Families with Relatives Afflicted with OC

Pt I 52 0 167 0 560

PtO4 0 206 0.445 0 222

Pt13 0138 0 167 0 148

Familieswith Relatives Afflicted with Other Primary Cancers

PtO2 0 009 0.078 04 Pt14 0 05 0.078 0 021

Pt] 0 226 0 178 0 272

Pt22 0 023 0 178 0 026

Pt28 0 016 0 078 0 020

Pt33 0 024 0 083 0 Oil

Pt34 0.014 0 042 0 006 *Values are obtained from the mutation prevalence table of the Myriad Genetic Laboratories which is developed based on the recently published method of Frank et al 2002 J Chn One 20:1480-1490 Page 96 of 126

Table 20. BRCAPRO Estimates of BRCA1 Posterior Carrier Probabilities. BRCAPRO Screening Profile BRCAPRO BRCA I Patient ID BRCAI Prior SSCP PTT Posterior Probability Probability

Families with RelativesAfflicted with BC

Pt25 0 939 Exon 15 0.654 1.000*)

N26 0 017 Exon 13, Exon 15 het, Exon 22 0.002 1.000**)

Pt27 0 013 Exon 15 0 001 (1.000*)

POO 0 023 H 0 002

Families with Relatives Afflicted with OC

Pt I 0 512 Exon 15 het H 0 095 1.000*)

PtO4 0 206 Exon 16 Exon I I - 1445insGT*** I 000

Ptl3 0 138 Exon 13, Exon 15, Exon 16 0 0 15 1.000*)

Families with Relatives Afflicted with Other Primary Cancers

PtO2 0 009 Exon 15 het, Exon 16 0.001 (I 000*)

P04 0 015 Exon 13, Exon 15, Exon 16 H 0 002 (I 000*)

P05 0 226 Exon 3, Exon 15 het 0 28 ( 000*)

Pt22 0 023 Exon 22 H 0 002 (I OOO-)

Pt28 0 016 H 0 002

Pt33 0 024 Exon 2 Exon 15 het H 0 003 (I 000**)

Pt34 0.014 Exon 15 het 0 001 (1.000*) Le.-end het = heterozygous, (-) negative *This is the BRCAJ posterior carrier probability if the putative polymorphism is found to be a BRCAJ mutation "Greater chance of having a BRCAI mutation de to low prevalence 17PUtative polymorphism observed in a certain e\on ***This the second documented mutation in BRCAJ exon I I in a Filipino BC patient since 1998 Page 97 of 126

Table 21.1. Unconditional Posterior Probability Estimates for NO.

Unconditional Prior Unconditional Posterior Probability Age Probability If BRCAI(-) (noncarrier) If BRCA P) (carrier)

45 0 0278 0 001 0 1531 50 0 0589 0 0229 0 3140 55 0 0874 0 0376 0 4392 60 0.1119 0 0538 0.5233 65 0 1333 0 0710 0 5741 70 0.1523 0.0887 0 6025 75 0.1703 0.1062 0 6237 80 0 1866 0.1222 0 6423 85 0 1995 0 1349 0 6566 Lifetime 0 1995* 0 1349* 0.6566* *to ae 85 years

Table 21.2. Unconditional Posterior Probability Estimates for Pt34. Unconditional Posterior Probability Age Unconditional Prior Probability If BRCAI(-) (nonearrier) If BRCAP) (carrier)

24 0 0001 0.0001 0 0048 29 0 0008 0.0005 0 0256 34 0 0003 0.0020 0 0818 39 0 0077 0 0055 0 1827 44 0 0159 0 0121 0 3 130 49 0 0266 0 0212 0 4420 54 0.0392 0 0327 0 5460 59 0.0534 0 0462 0 6176 64 0.0689 0.0613 0 6617 69 0 0852 0.0775 0 6867 74 0 118 0 0941 0 7035 79 0 1172 0 1095 0 7185 84 0 1300 0 1223 0 7304 Lifetime 0 1300* 0 1223 0 7304* *to age 84 years Page 98 of 126

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V. DISCUSSION

5.1 The Only Mutation Detected at Exon 11

At least 426 distinct alterations (DMPV) and 235 alterations reported only once (AROO) that are either frameshift or nonsense mutations in BRCAJ exon 1 1 alone have been reported worldwide. A total of 1776 entries of mutations have been documented in the Breast Cancer Information Core Database. These mutations which spread over a span of 3427bp would have a probability of I mutation per -5.2bp indicating high prevalence of BRCAJ mutations in B(O)C patients from the countries whose researchers and health professionals have submitted their respective mutation databases to this established central database which is continuously monitored by a core group of scientists (BIC, 2003).

Few BRCAI mutations have been identified in Filipino BC patients. The first mutation detected is a point mutation in exon I at nucleotide 2178 (2178C/T) causing a stop codon at amino acid 687. This particular patient has a 38-year-old sister with OC. Paternal grandcousins of the proband also had breast and ovarian cancer, respectively (Worsham et al., 1998). The second study screened exons 11, 15, 22, and 24 in 294 unselected incident Filipino BC patients from Manila, wherein seven cases with 2 relatives with BC<50 years old or OC were selected for screening by direct sequencing. The study recently reported 3 mutations in exons 15 (Q 53 8X), 22 (5454deIC), and 24 (RI835X) with a total estimated prevalence of 1.0%. There was no mutation detected at exon 1 1 (Matsuda et al., 2002). Both studies utilized PTT at exon I .

Furthermore, Matsuda et al 2002) reveals that only 6 out of the 294 unselected patients have family history of B(O)C which included 19 patients classified as BC<50 years old. In our study, we opted to increase the stringency to age 40 years at disease diagnosis and classified these cases as early-onset BC cases 26 patients 76.5%). More than half of these early-onset cases 14 patients out of 26 early-onset cases - 53.8%) have family history of the disease and actually cover a big portion of the total number of patients in this study (I 434 or 41.2%) (Table 12. 1).

In this study, the one and only mutation detected at exon 1 1 29.7 kDa truncated protein product) from among 34 suspected familial Filipino BC cases indicates an approximate prevalence rate of 2.9% (Figure 20 lane 5, Figure 21 lanes 6 and 56). The mean age of diagnosis of these patients is 39.1 years, which is -5 years younger compared with the patients in the study of Matsuda et al 2002). Subsequent direct sequencing of the appropriate coding region revealed a frameshift Page I 0 of 126

mutation in exon I I at nucleotide 1445 due to a GT insertion, resulting in a ten-nination (stop codon) in amino acid 452 and is now designated as 1445insGT (Figure 23). This is therefore the second documented mutation in BRCAI exon I I in a Filipino BC patient since 1998.

Medical information about the patient (PtO4 or C2b) is limited due to the few number of tests performed to profile her case due to economic reasons. She was diagnosed of IDC (stage 2 at age 30 years old. Mastectomy of the left breast was performed 7 months after diagnosis. Seven (7) out of 15 lymph nodes examined, as well as the skin and the nipple, were free of tumor cells. However, a year after mastectomy, the disease immediately progressed to stage 4 The patient is now experiencing bone metastasis. The aggressiveness of her type of cancer may be attributed, in part, to the presence of a mutation in BRCAI exon I . The mother of this patient had OC at age 45 years, aside from having breast cyst during her teens, and died when she was 51 years old. She also has a paternal aunt who had BC at age 31 years and is now 58 years old. Her maternal grandmother died due to a kidney problem at age 65 years. Mutations in exon 1 1 of BRCAI are generally covered within the main ovarian cancer domain (Huusko, 1999) and this is also evident from pedigree drafted for PtO4 (Figure 12B, Table 22).

Testing positive as a carrier of BRCAI mutation entails several approaches for appropriate management of the patient. This would include surveillance, prophylactic surgery, risk avoidance, and chemoprevention. However, the BRCAI-mutation(+) patient in this study already died after 6 months with metastatic bone cancer. The only procedure available for this case in the Philippines is 6OCo-therapy.

5.2 Eight (8) Unique Putative Polymorphisms Detected at BRCAI

SSCP analysis in this study revealed unique putative polymorphisms at BRCA I exons 2 5, 13, 15, 16, 17, and 22 (Table 16). The fact that several polymorphisms have been detected in 7 of the 10 smaller exons studied indicates that studies on BRCA.112 mutation in the Philippines should be improved and supported to further the knowledge obtained in this study as well as that of a recently reported research stating that prevalence of BRCA I mutation is relatively low in the country (Matsuda et al., 2002). With this new development obtained through SSCP a high BRCAI putative polymorphism prevalence of about 76.5% is observed. It should be noted, however, that these are predominated by putative polymorphisms at this stage of the research. Thus, cycle sequencing is needed to obtain the precise mutational event. Other exons should also be screened for putative polymorphisms to cover all the coding exons of BRCA 1. Page 101 of 126

5.2 1 Low Prevalence of Putative Polymorphisms at BRC I Exons 2 17, and 22 May Indicate Probable Mutations

Low prevalence <10%) of unique putative polymorpbisms at exons 2 5, 17, and 22 may indicate that these are probable mutations (Table 16). Assuming that these unique putative polymorphisms are actually BRCAJ mutations and integrating the mutation detected in exon I , the prevalence would therefore be 17.6% which is about 17-folds higher than previously observed (Matsuda et al., 2002). This may be due to the higher stringency used in patient prospecting for this study.

5.2.2 High Prevalence of Putative Polymorphisms at BRCAI Exon 5 May Indicate Biological Insignificance

The high putative polymorphism prevalence observed at exon 15 (58.8%) may indicate that it could probably exist in the general population and cannot be correlated with predisposition to BC. If we assume that the wild-type low-risk individual (Pt37) represents the hornozygous unaffected allele AA and consider the second group of homozygous allele aa to be the polymorphic allele, then the heterozygotes would have allele Aa. Based on the SSCP results, 14 BC patients have wild-type allele AA 14/34=0.412) (Figure 18D lanes and 11, Figure 19D lanes 10, 12, 17, 24, 29, 31, 33, and 36) and 6 BC patients have hornozygous polymorphic aele aa 6/34=0.176) (Figure 19D lanes 6 14, 25, 26, 28, and 30). The rest 14 BC patients) are heterozygotes Aa 14/34=0.412) (Figure 19D lanes 4 5 79, 11, 15, 6, 18, 23, 27, 32, 34, and

37). Using frequency of allele aa (0.176) as q 2 in the Hardy-Weinberg distribution equation (P2 +2pq+q 2=1), q=0.420 (Strachan & Read, 1996). Since p=l-q, then p=0.058 ,p2=0.336, and 2pq=0.487. By comparing the observed frequencies with the expected frequencies below,

Genotypes: AA Aa aa Frequencies: P2 2pq q2 Expected frequencies: 0.336 0.487 0.176 Observed frequencies: 0.412 0.412 0.176 less than 10% difference could be noted. This shows that despite the small sample size and the biases caused by the highly stringent inclusion criteria, Hardy-Weinberg distribution is manifested which could indicate that the putative polymorphism detected in BRCAJ exon 15 rnav not be biologically significant (Figure 19D). Pooling of samples for this particular exon is Page 102 of 126

not recommended. However, the results manifest the effectiveness and sensitivity of SSCP when using pooled samples to perform genetic screening cost-effectively.

Intermediate prevalence values of 20.6% and 14.7% are observed in exons 13 and 16, respectively. Unlike exon 15, presence of extra single strand conformers in exons 13 and 16 indicated these putative polymorphisms (Figure 19C lanes 612 and 14, Figure 9E lanes 7, 8, 10, 1, and 13).

5.3 Breast Cancer Risk Estimation

Risk assessment models are applicable to the two unaffected high-risk individuals, Pt 3 and Pt' )4 (Tables 14.1 and 14.2). Gail model proved to be useful for unaffected individuals regardless of family history of the disease as long as data on the relative risk modifiers is present. Claus model exhibited usefulness in estimating remaining risk as affected by age and family history of proband. BRCAPRO is definitely useful when more sufficient data on unaffected individuals and their relatives have been obtained to achieve a more comprehensive estimate of the lifetime risk (unconditonal probability).

5.3. 1 Relative Risk Estimation Under the Gail model, both have current summary RR value of 3) I I given the same RR value of 1.10 (Table 2 based on their ages at first menarche. Their lifetime RR are also similar. This indicates that based on the relative risk factors, both Ptl3 and Pt34 have same lifetime RR regardless of family history. For Claus model, however, only the current age relative risk is estimated. RR value for Pt13 320) jibes with the Gail model estimate while that of P64 if extrapolated, could be an underestimate of the supposed RR due to her young age.

5.3. 1 Cumulative Lifetime Risk or UnconditionalProbability Estimation Cumulative lifetime risk estimates of Gail model for both Ptl3 and Pt34 are also similar based on the risk modifiers included. For Claus model, however, Pt34 has a higher remaining risk than Pt13 due to her young age (Tables 14.1 and 14.2). Unlike the other prior probability models which only predict the presence of BRCAI or BRCA2 mutation, BRCAPRO can also indicate the risk of developing BC with the incorporation of family history of BC up to the second-degree relatives. Ptl3 has greater unconditional probability than N34 since the existence of the sibling of Ptl3 with early-onset BOC was already incorporated in the estimation. The mother and maternal aunt of Pt34 are both afflicted with regular-onset BC elucidating a lower unconditional probability compared to that of Pt34. Page 103 of 126

5.4 Patterns in the Age at Disease Onset of Families with Cancer-Afflicted Relatives

5.4. 1. Families with Relatives Afflicted with BC Minute differences observed between the ages of onset of proband and first- or second-degree relatives, except for Pt27, could indicate increased possibility that BC cases of future generations will have onset of the disease within the same age range (Figure 11). All of the relatives afflicted with BC are from the maternal lineage suggesting the possible existence of one mutated allele from this lineage l" hit) and the 2 d hit (from Knudson's two hit model hypothesis) induced by environmental factors resulted in the relatively early age at disease onset of the probands (Feuer et al., 1999).

5.4.2. Families with Relatives Afflicted with OC Mean age of onset of disease is much earlier in these families. Relatives afflicted with OC are generally from the maternal lineage coupled with the appearance of BC from the paternal lineage (Figure 12). Development of BC at a much earlier age in the generation of the proband may be induced the linked expression of BC by the OC allele from the maternal lineage and the BC allele from the paternal lineage.

5 43. Families with Relatives Afflicted with other Primary Cancers All of the relatives afflicted with BC in these families are either regular- or late-onset cases but 83% of the afflicted probands are distinctly early-onset cases Figure 13). Further studies are necessary to elucidate whether the interplay of genes linked to these primary cancers has a tangible effect on the age at disease onset in future generations.

5.5 Modeling and Screening Profiles of Families with Cancer-Afflicted Relatives

The Myriad.com mutation prevalence table (Myriad 11) is utilizable with BRCAPRO for this study population of BC patients selected using high stringency. Not included in the fairnifies discussed in the succeeding pages are the putative polyrnorphisms at exon 17 (PtI I and Ptl2) and exon 5 (Pt36) which occurred at a low prevalence rates and thus may be probable mutations (Figure 19F lanes and 6 Figure 19B lane 6 Ptl I and PtI2, who probably have the same mutation at exon 17, have been diagnosed of BC above age 40 years but both have family history of BC. On the other hand, P06 has a maternal aunt with BC and another maternal aunt with unknown type of cancer. Further data that will be obtained in future studies on BRCA in Filipinos would be needed to facilitate genotype-phenotype correlation in Filipinos. At this stage, carrier probability values and ptative polymorphisms observed among selected Filipino BC Page 104 of 126

patients are indicated in the families with characteristic affliction of relatives. The only way to elucidate more probable genotype-phenotype correlations in these putative polymorphisms is by sequence analysis in future studies.

5.5. 1 Families with Relatives Afflicted with BC (Figure II) The high prior probability estimates of BRCAPRO 0.939) and Myriad.com 0.316) for Pt2 is attributable to the presence of BC cases in the first- and second-degree relatives. BRCAPRO has been observed to be a very effective predictor for families with >3 BC cases. Both BRCAPRO and Myriad.com suggest presence of deleterious mutation of Pt25 in BRCA1. Carrier probability values of Pt26 PM, and POO are quite low in the presence of only one relative afflicted with BC. However, their Myriad.com values are relatively higher (Table 19). This corroborates with the lowest occurrence of truncating and missense mutations in purely HBC families (Table 41) but the proportion of pure HBC families included in the studies on HB(O)C families (Table 44) should not be disregarded.

Both Pt25 and Pt27 exhibited putative polymorphism at exon 15 which may be biologically insignificant. Posterior probability of Pt25 is still quite high 0654) even if the putative polymorphism turns out to be a polymorphism but for PM, it becomes almost zero. Pt26 exhibited putative polymorphisms in exons 13, 15, and 22. The rare putative polymorphism of N26 at exon 22 indicates a higher probability that it is a mutation with a posterior probability of 1.000. The same value (1.000) is expected with Pt25 and Pt27 if their putative polymorphism turns out to be a deleterious mutation. POO posterior probability of PGO remains at 0002 since it is negative in all the exons studied (Table 20).

Presence of putative polymorphisins in 3 out of 4 families in this group (one of which is a highly probable mutation) may indicate existence of BRCAI mutation in Filipino BC patients with relatives afflicted with BC.

52 Families with Relatives Afflicted with OC (Figure 12) High prior probability estimates are also observed for probands PtOl, PtO4, and Ptl' in the models utilized (Myriad.com and BRCAPRO). This corroborates with the highest occurrence of BRCA I mutations in HBOC families (Table 42). These also coincide with presence of putative polymorphisms at exons 13, 15, and/or 16 in these patients (Tables 19 and 20).

Low posterior probabilities are calculated for MI and P03 if these are Ust polymorphism but will be 1.000 if these are mutations. The higher value given by Myriad.com for PtO4 is due to the Page 105 of 126

presence of a relative with BC aside from her mother with OC. Moreover, the truncating mutation at exon 11 (1445insGT) observed in PtO4 would definitely affect the conformation of Brcal protein which could result in an inactive tumor suppressor giving a definite posterior probability of 1.000 (Table 20). Utilization of various applicable models increases predictive efficiency for mutation prevalence. Although, PtOl generally has the highest probability values in this group, the high probability value given by Myriad.com for PtO4 coincided with the presence of a deleterious mutation (1 445 insGT) in this patient.

All of the patients with first- or second-degree relatives afflicted with ovarian cancer either have mutation or putative polymorphism which could suggest high occurrence of BRCAJ mutation in Filipino HBOC families.

5.5.3 Families with Relatives Afflicted with Other Primary Cancers (Figure 13) Six out of the 7 probands have putative polymorphisms at exons 2 3, 15, 6, or 22, two of which (Pt22 and Pt3 3) are rare. Thus, probands PtO2, Pt 4, Pt 5, Pt22, PO 3, Pt34 have posterior probabilities of 1.000 if these are mutations (Table 20).

Since Pt28 showed no putative polymorphism. in all the exons studied, her posterior probability remains at 0002. P03 has two putative polymorphisms, one of which is also exhibited by her niece (Pt' )4) and was probably inherited through her mother. However, the rare putative polymorphism at exon 2 only existed in Pt33. Only PtI5 has a high prior probability through BRCAPRO whereas Myriad.com. values are above 0.100 for all the probands except Pt34 (Tables 19 and 20). Existence of other primary cancers in first- or second-degree relatives of Filipino BC patients may be an indicator of BRCA I mutation.

5.6 Effect of Screening Results on BRCAPRO Posterior Carrier and Unconditional Probabilities

Apparent decrease in the carrier prior probability value is observed once the individual has shown negativity or presence of polymorphism (instead of a mutation) in the screening tests performed (Tables 20 and 22). This is the sole reason why sequence analysis is inevitable in future studies to validate such prior probability models used. Presence of a mutation confirmed through sequencing would definitely give a carrier probability of 1.000 to the proband studied. However, relatives of the proband who was found to be BRCAI(+) carrier would still have carrier probabilities different from the proband. Page 106 of 126

Similarly, posterior unconditional probabilities for developing BC in unaffected individuals decrease if the screening results indicate BRCAI(-) and increase if the individual is found to be a BRCAP carrier (Table 21).

Not until all the coding exons of BRCA I (and BRCA2) have been screened, a BRCA P-) result will not be an assurance that the patient is not a carrier. However, due to the development of prior probability models, researchers can be more confident to assume "non-carrier" status of the individual if the estimated prior probability is almost zero.

5.7 520% Gradient vs. 14% Standard Polyacrylamide Gel for Separation of In Vitro Transcribed and Translated Protein Products

The standard (or fixed) 14% gel, or sometimes 12%, would be useful for separating fragments within the range of about kDa to 6kDa. This would mean, however, that more fragments will be generated from one large exon like exon 11 (3427bp) rendering the analysis to be more expensive due to the number of primers for amplification needed as well as the number of reactions that have to be performed. Furthermore, T7/Kozak sequence of about 39bp would be needed for each of the forward primers for PCR. However, with the 520% gradient gel, broader range of MWs can be separated by a single gel. Once the laboratory has a gradient gel caster, it would be beneficial to use the gradient gel over the standard gel as shown in the results of the study. Mutation controls with truncated protein products above 100 kDa, i.e., 3819de15, E125OX, and 3875del4 having 112 kDa, 114 kDa, and 115 kDa bands, respectively, were clearly separated with the 520% gradient gel when compared with the standard 14% gel Figures 20 and 2, Tables 17 and 18). Probable low molecular weight truncated protein products as low as IO kDa could still be resolved because of the high percentage of acrylamide 20%) in the lower portion of the gel. This was clearly manifested by the molecular weight markers used in this study. Lastly, the standard curve generated with a gradient gel is linear as opposed to the semi- logarithmic graph needed for standard percentage gels which could result to deviated estimates of the apparent molecular weight.

In summary, gradient gels would (a) have a higher resolution than standard gels when running protein solutions containing broad range MWs of its constituents and consequently detect truncated protein products that are almost of the same size with the wild-type protein, (b be more cost-effective since the number of primer pairs needed would be minimal, (c) have a higher chance of detecting even low molecular weight proteins that result from truncation, and (d be more appropriate in generating a more accurate standard curve. Page 107 of 126

5.8 Cost-Effectiveness and Significance of Utilizing Prior Probability Models

Because BRCA gene mutation testing is expensive, occasionally uninformative, and frequently associated with ethical and legal issues, careful patient selection is required before testing. However, in this study, sampling and PTT were already performed prior to acquisition of these prior probability models. With the favorable results of various studies done to validate specificity and accuracy of BRCAPRO (Berry et al., 2002; Euhus et al., 2002), it has been concluded in their respective geographic regions that BRCAPRO is an accurate counseling tool for determining the probability of carrying mutations of BRCAI and BRCA2.

However, BRCAPRO has its own limitations as specified in the review of related literature. It only includes relatives up to the second degree. In my experience as a researcher, family history can sometimes be unraveled by looking up to the third-degree relatives. Despite several limitations, BRCAPRO still proves to be a very important tool in genetic counseling especially in the developing countries. However, the degree of penetrance and prevalence in the country should be carefully and accurately established to obtain the optimum results from this model run within CancerGene. In the presence of Myriad.com mutation prevalence table (which proved to be very useful in this study) within this single software, clinicians can corroborate the results of these models and choose which values are acceptable based on the study population of their respective researches.

Prior probability models will be very useful in the country once the penetrance and prevalence values are well established and validated. Construction of accurate pedigrees would also be vital in analyzing family histories of the different high-risk individuals

Would Screening Exon II Be Sufficientfor BRCA I Genetic Testing in the Philippines? Genetic screening of BRCAI exon I I together with CancerGene is still needed in the country to establish and validate its capability as a probability model for applications in BRCA genetic testing. In the future, models will definitely emerge based on the various parameters that are slightly different in the country like the recent development reported by Matsuda et al 2002) indicating the BRCA2 prevalence is much higher than that of BRCAI in the country. However, this is not yet a well-established fact since unique putative polymorphisms or mutations have been detected in our study at exons 2 5, 13, 15, 16, 17, and 22 of BRC I using SSCP analysis. Five (5) of these putative polymorphisms are probable BRCAI mutations. It would definitely be more suitable to include screening of the BCAI exons -Nhere putative polymorphisms have been detected in the Filipino population. Page 108 of 126

5.9 Status of BRCA1 Genetic Testing in the Philippines

After these studies on BRC,41 mutation in Filipino BC patients have transpired, it is expected that various research attempts of the same objective would emerge from their findings. Other researches may start to arise from various parts of the country. However, with the recent capabilities of the different laboratories in the country-side, we hope that scientists, medical practitioners, and professionals in related institutions nationwide will collaborate towards the ultimate goal of establishing a national genetic testing center.

5.9. 1 The Irony of Sampling Collection Approximately only 17% of the hospitals contacted finally agreed to collaborate with the program geared towards free genetic testing for individuals who have been diagnosed of breast cancer at an early age 40years at disease diagnosis). Although there are so many breast cancer cases in the country 47.7 in 100,000 as of 1997; 44 in 100,000 for the mean age-standardized incidence from 1980-1995), it has been noted that without active interaction of the hospitals throughout the country, the chances of catching a BC case that would fit the specified criteria for early-onset and familial breast cancer would be minimal (Ngelangel et al., 1997; Parkin et al., 2001). Furthermore, once an index case qualifies in the criteria given, the ability to explain and finally convince the prospective collaborating high-risk individual to join the free genetic test would be of great impact to the final outcome of the study. This is because even if the genetic test would be very expensive for the patient when undergone abroad, some of the patients opt to decline from joining this free genetic test merely due to lack of knowledge and awareness regarding the possible benefits that these tests can provide for future approaches towards cancer cure. t is the utmost desire of the researchers to help build a nationwide resource or database for breast cancer genetics. However, collaborative approaches to establish tese cancer registries and genetic testing centers for a nationwide database should be well structured and adequately funded from the very beginning to definitely achieve these goals.

5.9.2 Low Socio-Economic Profile Demands Cheaper Genetic Testing Based on the profile of the BC cases in this study, more than half (55.8%) have a net monthly income below P30,000 44.1% have incomes below P20,000) even if the about 82.4% have at least an undergraduate degree. Any test which would eventually be commercially available in the future would have to consider the income bracket of the market where the test will be conducted. Page 109 of 126

5 93 Information ObtainedRegarding Pathologic Manifestationsand Socio-Economic, Dietary, and Other Factors:Still at a Very Early Stage Pathologic data on the patients is relatively limited. About half of the patients have known histology type, majority of which had IDC. Approximately 40% of the patients had their ER and PR assayed, 40% of which are ER- and PR-. Though pathologic manifestations in other studies indicated abundance of ER- and PR- in BCAI(-") patients, additional samples with more comprehensive data is needed to assess the molecular pathology of BRCAl in Filipino BC patients. Various distributions obtained from the questionnaire that was filled up by the BC patients, with regards to different factors is still at a very early stage for deten-nining any correlation with the risk of developing BC. With the recent developments of mutation screening in the country, in the event that more samples have been collected and processed, gene- environment interactions for these cancer cases could then be studied. Page II 0 of 126

VI. CONCLUSIONS AND RECOMMENDATIONS

6.1 Low Incidence of Germline Mutation in BCAI Exon 11 among Suspected Familial Filipino Breast Cancer Cases: The Only Mutation Detected is the Second Documented Filipino BC Case with BRCA1 Mutation at Exon 11.

The one and only BRCAJ exon 11 mutation among 34 early-onset and familial breast cancer patients was detected through PTT, validated by DHPLC, and confirmed by cycle sequencing. The mutation, designated as 1445insGT, when corroborated with the Bayesian risk modeling results, indicates validated low incidence of BRCAJ mutation in the Philippines (Matsuda et al., 2002; Euhus et al., 2002). This would, however, be the second documented mutation in BRCAI exon I I in a Filipino BC patient. Mutations in BRCAI exon 1 1 are generally covered within the OC region (Huusko, 1999). This is evident in the pedigree of PtO4 as manifested by her mother who had ovarian cancer. The fact that only two mutations have been detected at BRCAI exon 1 1 in Filipino BC cases is in itself unique since majority of mutations detected in BC cases worldwide is in exon I .

6.2 Eight (8) Unique Putative Polymorphisms Detected at BRCAI: Prevalence May Indicate Probable Mutation or Biological Insignificance

SSCP analysis revealed unique putative polymorphisms, 5 of which are probable mutations at exons 2 5, 17, and 22 due to low prevalence and distinct aberrant migrating patterns in a non- denaturing gel. High prevalence of putative polymorphisms at exons 13, 15, and 16 may indicate biological insignificance. This is further exhibited at exon 15 wherein Hardy-Weinberg distribution is manifested by the presence of 2 different homozygous alleles and a heterozygous allele which is a combination of the 2 homozygous alleles despite the small sample size and the biases caused b the highly stringent inclusion criteria. Pooled SSCP analysis proved to be an efficient and sensitive technique to perform genetic screening cost-effectively. However, it pooled samples cannot be used for exon 1 1 due to the high polymorphic rate.

6.3 Probable Mutations at Exons 2 5, 17, and 22 May Suggest Initial Genotype-Phenotype Correlations in Filipino Breast Cancer Patients

P 3 (Br5 1) who has a probable mutation at exon 2 has a sister with breast cancer and maternal aunt with colon cancer. Third- to fourth-degree relatives have ovarian cancer, prostate cancer, colon cancer, and pancreatic cancer. Pt')6 (Br32) who has a probable mutation at exon 5 has a Page III of 126

maternal aunt with BC and another maternal aunt with cancer. Ptl I (Br 42) and Ptl2 (Br38), who probably have the same mutation at exon 17, have no relatives diagnosed of B(O)C. Pt22 (Br4l) and Pt26 (Br47) are not particularly early-onset cases but both have relatives afflicted with BC. Further data is needed to verify that these probable mutations could specify certain genotype-phenotype correlations in Filipino breast cancer patients.

6.4 Gail, Claus, and BRCAPRO Models can be Utilized for Estimating BC Risks of Unaffected Individuals

The Gail, Claus, and BRCAPRO models can be utilized in determining lifetime risk or unconditional probabilities of unaffected individuals. BRCAPRO can also be utilized to estimate posterior unconditional probability after incorporation of screening results. Further studies need to be performed to validate their applicability in the Filipino population.

6.5 Patterns in Age at Disease Onset Observed in Families with Cancer-Afflicted Relatives

Observable patterns in age at disease onset of probands and their relatives were elucidated. Further studies have to be performed to confirm these observations.

6.6 Most of the BRCAPRO and Myriad.com Prior Probability Estimates Coincide with the Presence of Mutation and/or Putative Polymorphisms in Suspected Familial Filipino Breast Cancer Patients

Coordination of the prior probability values obtained through BRCAPRO and Myriad.com for the BC patients with first- or second-degree relatives diagnosed of BC, OC, or other primary cancers coincided with the mutation and/or putative polymorphism in most of the families studied. These two prior probability models may therefore be utilized as good indicators for existence of BRCAI mutation in a Filipino BC patient. Sequence analysis is therefore needed to validate the results in the Filipino population.

6.7 Screeninc, Results Apparently Affect Posterior Probabilities

Positive results in the screening tests confirmed by sequence analysis which indicate "carrier" status of patient will increase the carrier probability to 1.000. Negative results definitely decrease the prior probability value. Unconditional posterior probabilities of unaffected individuals are also definitely affected by the screening results. Page 112 of 126

6.8 Cost-Effectiveness of using Gradient Gel Electrophoresis Coupled with Only One Primer Pair for Protein Truncation Test

Utilization of the gradient 520%) polyacrylamide gel electrophoresis will ensure higher resolution of truncated proteins generated in a particular gene test being developed. This setup, coupled with the design of only one primer pair for only one exon would definitely decrease the amount spent for these techniques. However, it should be noted that mutations/polymorphisms in the other exons will always be present.

6.9 CA-1 Genetic Testing in the Philippines Needs Nationwide Support from Various Sectors

After studies on BRCAI mutation in Filipino BC patients have transpired, we hope that the scientists, medical practitioners, and professionals in related institutions nationwide will collaborate towards the ultimate goal of establishing a national genetic testing center. Such nationwide resource or database center for BC genetics could be built to increase chances of catching BC patients who fit the inclusion criterion and ultimately effectively tackle the irony of sample collection in the country. Low socioeconomic profile of the BC cases in this study indicates the demand for cheaper gen6tic testing. This could only be achieved by a nationwide cooperation of leaders from various sectors with the scientists and medical professionals.

6.10 Utilization of Prior Probability Models in the Philippines: A Pioneering Endeavor for Cost-Effective Pre-Genetic Test Screening

This is the first time that prior probability models are utilized with BRCA I mutation screening in the Philippines. These models prove to be efficient in minimizing genetic testing costs. However, more pedigrees and other pertinent details of other BC patients who fit the inclusion criteria should be obtained in the future to come up with the best estimate of the carrier probability of an individual. Page 113 of 126

VII SUMMARY OF FINDINGS

Nine mutations/putative polymorphisms have been detected in BRCA I exons 2 5, 11, 13, 15, 16, 17, and 22 among 34 early-onset and familial Filipino breast cancer cases, eight of which are putative polymorphisms observed through SSCP analysis. The only mutation detected at exon 1 1, using PTT and validated by DHPLC, is a GT insertion at nucleotide 1445 resulting in a stop codon at amino acid 452 (1445insGT) which constitutes a novel mutation. This is the second documented mutation at exon I I in a Filipino breast cancer patient since 1998.

Unusually low incidence of BRCAI mutation at exon I I has been corroborated by this study. However, the presence of putative polymorphisms in exons 2 5, 13, 15, 16, 17, and 22 may be due to the use of higher stringency in patient prospecting.

Prevalence of unique putative polymorphisms may indicate probable mutation or biological insignificance. Low prevalence of unique putative polymorphisms at exons 2 5, 17, and 22 may indicate that these polymorphisms are probable mutations. On the other hand, high prevalence of unique putative polymorphisms at exons 13, 15, and 16 may suggest that these putative polymorphisms are true polymorphisms and are therefore biologically insignificant. Probable mutations may suggest initial genotype-phenotype correlations in Filipino breast cancer patients.

The Gail, Claus, and BRCAPRO models can be utilized to estimate BC risks of unaffected individuals. However, validation should still be performed in the Filipino population.

Most of the BRCAPRO and Myriad.com prior probability estimates coincide with the presence of mutation and/or putative polymorphisms. This pioneering endeavor of utilizing these prior probability models, as pre-genetic test screening procedures, have huge potential in minimizing expenses of relatives of breast cancer patients who want to undergo genetic testing, as well as, in hampering unnecessary tests performed in genetic screening laboratories. Screening results definitely affect BRCAPRO posterior carrier and unconditional probabilities.

The utilization of both gradient gel electrophoresis coupled with only one primer pair for PTT as well as pooled SSCP analysis is definitely cost-effective.

The success of BRCA] genetic testing depends on the collaboration among scientists, medical practitioners, and professionals in related institutions nationwide. Page 114 of 126

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Szabo, C, Masiello, A, Ryan, J E, The BIC Consortium, Brody, L C 2000 The Breast Cancer Information Core: Database Design, Structure, and Scope. Hunian Mutation 16 123-131 Tavtigian, S V, Simard, J , Rommens, J , Couch, F, Shattuck-Eidens, D, Neuhausen, S, Merajver, S , Thoflacius, S, Offit, K, Stoppa-Lyonnet, D Belanger, C, Bell, R, Berry, , Bogden, R, Chen, Q, Davis, T, Dumont, M, Frye, C, Hattier, T,, Jammulapati, S, Janecki, T, han, P, Kehrer, R, Leblanc, J F, Gldgar, D E, et at 1996 The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds. Nat Genet 23) 333- 337 Theodor, L, Bar-Sade, R, Krughkova, A, Ben-Baruch, G, Rise], S, Shirt-Sverdlov, R, Hirsh Yechezkel, G, Modan, B, Papa, MZ, Rechavi, G, Friedman, E 1998 An identical novel mutation in BRCA1 and a common haplotype in familial ovarian cancer in non-Ashkenazi ews. Br Cancer 77(11) 880-1883 Thorlacius, S , Struewing, J P, Hartge, P, lafsdottir, G H, Sigvaldason, H , Tryggvadottir, L, Wacholder, S , Tulimus, H , Eyfjord, J E i999 Population-based sudy of risk of breast cancer in carriers orBRCA2 mutation. Lancet 352(9137) 1337-1339 Verhoog, L C, van den Cuweland A MW, Berns, , van Veghel-Plandsoen, MM van Staveren I L, Wagner, A, Bartels, C C M, Tilanus-Linthorst, M MA Devilee, P, Seynaeve, C, Halley, D J J, Niertneijer, M F, Klign, J G M, Meijers-HeUboer, H 2001 Large regional differences in the frequency of distinct BRCAI/BRCA2 mutations in 517 Dutch breast and/or ovarian cancer families. Eur J Cancer 37 2082-2090 Vidal-Puig A and Moller, D E 1994 Comparative sensitivity of alternative single-strand conformation polymorpbism (SSCP) methods. BioTechniques 17 490-492,494,496

Wagner, , Stoppa-Lyonnet, D, Fleischmann, E, Muhr, d, Pages, S , Sandberg, T, Caux, V, Moeslinger, R, Langbauer, G, Borg, A Oefher, P 999 Denaturing high-performance liquid chromatography detects reliably BRC41 and CA2 mutations. Genomics 62:369-376 Wagner, T M U, Moslinger, R A, Muhr, D_ Langbauer, G, Hirtenlehner, K, Concin, H, Doeller, W, Haid, A, Lang, A H, Mayer, P, Ropp, E, Kubista, E, Amirimani, B, Helbich, T, Becherer, A, Schemer, 0, Bretteneder, H, Borg, A Devilee, P, Oeftier, P, Zielinski, C 1998 RCAI- related breast cancer in Austrian breast and ovarian cancer families: specific BRCAJ mutations and pathological characteristics. nt Cancer 77:354-360 Warburg, and Christian, W 1942. Isolation and crystallization of enolase. Biochenj 310:384-421 Wooster, R, Bignell, G, Lancaster, J, Swift, S , Seal, S, Mangion, J, Collins, N, Gregory, S, Gumbs, C, Micklem, G 1995 Identification of the breast cancer susceptibility gene BRCA2. Nature 378(6559) 789-792 Published erratum appears inNature 39(6567) 749 Worsham, M Nathanson, D, Pals, G, Christopherson, P, Strunk, M, Wolman, S R 1998 A new BRCA1 mutation in a Filipino woman with a family history of breast and ovarian cancer. Dragn Mol Pathol 73) 164-167 Xu, C F, Chambers, J A, Nicolai, H, Brown, M A, Hujenat, Y, Mohammed, , Hodgson, S, Kelsell, D P, Spurr, N K, Bishop, D T, and Solomon, E 1997 Mutations and alternative splicing of the BRCAJ gene in UK breast/ovarian cancer families. Genes Chromosomes Cancer 18(2) 102- 110 Yazici, H, Bitisik, 0, Akisik, E, Cabioglu, N , Saip, P, Muslumanoglu, M, Glendon, G, Bengisu, E, Ozbilen, S, Dincer, M, Turkmen, S, Andruhs, I L, Dalay, N, Ozcelik, H 2000 BRCA1 and BRCA2 mutations in Turkish breast/ovarian families and young breast cancer patients. Br J Cancer 83(6) 737-742 IX. Appendices

A. Bayes' Theorem Utilized in BRCAPRO

B. Informed Consent

C. BRCAI Mutation Screening Program Questionnaire

D. Concentration and Purity of Isolated Genomic DNA Page 120 of 126

Appendix A Bayes' Theorem Utilized in BRCAPRO

BRCAPRO is a computer program tat implements a statistical model for calculating an individual's probability of carrying a deleterious mutation of BRC,41, BRC,42, neither, or both (also called as "carrier probability") based on the individual's cancer status and the history of B(O)C among the first- and second-degree relatives (Parmigiam, Berry, Aguilar, 1998, Berry el a, 1997) The model uses the autosomal dominant Mendelian characteristics of the genes and incorporates prevalence and penetrance based on published results (Iversen el at, 2000 It also employs Bayesian updating which exploits the fundamental tool for finding the probability of a enotype based on empiric information (Berry, 1996, Murphy & Mutalik, 1969)

Population-based inputs of BRCAPRO are mutation prevalence and disease penetrance Prevalence of mutations of Ashkenazi Jewish (AJ) descent is estimated to be 22% for BRC,41 and 36% for BRC,42 (Oddoux et a, 1996, Roa et a, 1996) These figures are estimated to be 0 12% and 044% for non-AJ individuals (Ford et a, 1998). Penetrance is the probability of diagnosis of cancer up to and including each age Penetrance for B(O)C is estimated by combining estimates for A and non-AJ individuals Penetrances at age 70 for both A and non-A.1 carriers of mutations of RRCAI and BRC,42 are 691 and 671 for BC and 296 and 191 for OC Derences are greater at some other ages versen, Parmigiam, & Berry, 2000, Ford et a, 998, Struewing el a, 1997) Genetic testing may be imperfect and BRCAPRO predicts genotype rather than genetic testing result Some individuals who carry a mutation and whose BRCAPRO probabilities are close to 100% will test negative However, Euhus et al 2002) observed that sensitivity for identifying BRCA gene mutation carriers is similar for experienced risk counselors and the Cmputer model BRCAPRO Since BRCAPRO consistently demonstrated sperior specificity, overall discrimination between BRCA gene mutation carriers and BRCA ene mutation non-carriers was slightly better for BRCAPRO

The fundamental tool for finding the probability of a genotype based on empiric information is Bayes' theorem (Berry, 996, Murphy Mutalik, 1969) Let M stand for "individual carries a mutation," N for its complement "individual does not carry a mutation," and H for "individual's family history" Bayes' theorem relates PMH), the probability of M conditional on family history H (called posterior probability), with its unconditional or prior probability, PAl) Family history enters through its "likelihood" under M and under N PM* and P(IAN) By Bayes' theorem,

P(M I H = P(H I M)P(M) (6) P(H) which is also called the theorem of inverse probabilities since it relates roles of P(MH) and P(IAM) According to the law of total probability,

P(H = P(H I M)P(M) (7) P(H I N)P(N)

Some agebra reveals that P(IAM and P(I-AN) enter Bayes' theorem only through their ratio, called lkelihood ratio (LR)

LR = P(H M) (8) P(H N)

The version of Bayes' theorem used is

P(M I H) LR (9) LR + P(N) / P(M) where P(N)IP(M is the prior odds against being a carrier Calculating the numerator of LR, P(MM, means assuming that the individual of interest carries a mutation On the other hand, calculating the denominator-, PAN), means assuming that she is not a carrier Both of these assume Mendelian genetics (Euhus et at, 2002, Parmigiani, Berry, Aar, 1998, Berry et a, 1997) In finding PAAH), the information in a family history enters trough the LR Te LR compares the probability of the individual's actual family history assuming that she carries a mutation with the probability of her family history assuming that she does not carry a mutation Large values of LRs indicate that the first probability is large wen compared to the second, LR=l is the break-even point at which family history is non-informative as regards M, i e, LR=I means that the posterior probability of carrying a mutation does not change from the prior probability PMH)= PM (Berry et a/, 1997)

Several potential limitations of BRCAPRO include () the utilization of published penetrance/incidence and prevalence estimates, which may be inaccurate, 2 te fact tat it considers only te first- and second-degree rlatives, 3) the integration of breast and ovarian cancer only, and 4) the consideration of only two known BC Ssceptibility genes, BRCAl and BRCA2 Probable presence of other ssceptibility enes with disease penetrance comparable to those of BRC,41 and BRC,42 will result to instances where individuals with large BRCAPRO carrier probability will harbor mutations of te other genes but will test negative for BRC,41 and BRCA2 De to these potential limitations alongside with the wide applicability of te odels various studies ave been performed to validate its performance Consistency of results is observed in these studies making thern conclude that BRCAPRO provides an accurate assessment of the probability of arrying a deleterious mtation of BRCAI and BRC,42 (Berry et at, 2002, Iversen, Parmigiani, & Berry, 1999, Hiller et a, 1997, Hilsenbeck el a, 1997)

Source ofEcIns 69 Berry et a 1997 J Nail Cancer Inst 89(3) 227-238 Page 121 of 126

Appendix B Informed Consent PHILIPPINE EREDITARY BREAST CANCER REGISTER Cancer Research Laboratory, Philippine Nuclear Research Institute Commonwealth Avenue, Diliman I 01 Quezon City CONSENT FOPW agree to participate in the Hereditary Breast Cancer Register. The purposes and benefits of the register have been outlined to me, and I understand that participation will include:

a allowing the Register to build up a family tree related to the disease of Hereditary Breast Cancer.

b. understanding that such a family tree will allow the Register and my doctor to determine whether or not other family members are at-risk for breast cancer, and consequently, would benefit from regular surveillance checks.

c. permitting the Register, if I feel it is suitable, either through their doctor, or through me, to contact other members of my family for relevant information.

d. permitting the results of my breast examinations and other health information, including tissue samples, to be obtained from my doctor, hospitals and other relevant institutions, to update my records on the Register

e obtaining required information from me by asking general questions about my health, and that of other family members

f. requesting the Register by the doctor to send out reminder notices of upcoming checks to myself and my doctor if I am put on a regular check-up program by my doctor

g providing blood sample which will be stored for genetic (DNA) testing and that these tests rely on being able to obtain samples from as many family members as possible I understand that my blood sample is important since I have breast cancer

I have been made aware that when and if enough samples have been obtained from my family, genetic testing will be offered to my relatives if they are thought to be at-risk for breast cancer If I accept the genetic testing, my family and I may be required to undergo a preliminary group counseling session with Hereditary Breast Cancer Register staff. Once the results are available, I shall be required to attend another individual session, at which my doctor may also be present if he/she or I so wish. I understand that the results of this genetic testing may be inconclusive. I also understand that if I decline genetic testing that this will in no way effect my medical treatment nor the services provided by the Register

h realization that participating in the Register will mean regular (though not necessarily frequent) contact with the Registrar or other Register staff to update details

I optional participation in some research studies investigating Hereditary Breast Cancer in the future

I understanding that all data pertaining to me (personal details. medical records, specimens provided, etc will be treated in a strictly confidential manner.

Consent Gven Consigned Witnessed

Signature of Collaborating Patient Name and Signature of Medical Doctor Name and Signature ofWaness

Date _ / Date Date min dd yyyy mm dd yyy mm dd yyyy

Guardian's si-nature over printed ame (if<18 yo)

PHILIPPINE HEREDITARY BREAST CANCER REGISTER STAFF USE ONLY

Received by Date Received - / Date Noted Reference Number -01 Page 122 of 126

Appendix C BRCAI Mutation Screening Program Questionnaire PHILIPPINE HEREDITARY BREAST CANCER REGISTER Cancer Research Laboratory, Philippine Nuclear Research Institute Commonwealth Avenue, Diliman I 01 Quezon City

El IFFIlp WN ------HRCA1 Mutation Screening Program Questionnaire_

Project 7-iffe BRC41 Germline Mut3tion Among Filipino Hereditary Breast Cancer Patients Using Radioactive Protein Truncation Test PTr) Unique Identifier Proect1-tvator Alejandro Q. Nato, Jr. Date Collaborating Proband's General Information Name

Birthdate Sex Height __:k_ inches (_ m) Weight - Ibs (- kg)

Current Address

Permanent Address

Tel #1Cf #/pager

Email Address

Office Address

Tel Fax

Email Address

Contact Person

Address

Tel #/Cf #/pager

Email Address

Civil Status LI Single U Separated 0 Others 0 Married LI Widow/Widower Religion El Roman Catholic 0 Apostolic 0 Jehovah's Witness 0 Protestant 0 Muslim 0 Seventh Day Adventist LI Trinitarian 13 ElShaddai 0 Others LI Pentecostal 0 Iglesia ni Cristo CI None Educational Attainment

C3No schooling C3Attended elementary school 0 Graduated from elementary school El Attended high school El Graduated from high school El Atte nded vocational school El Graduated from vocational school 0 Attended university or college 0 Graduated with degreeldiploma Field C] Attended graduate school LI With Masters' or Doctoral Degree Field Occupation/Nature of Work 0 Agriculture (farming/fishing) 0 Bar/massage parlor 0 Nuclear related work El Industrial manufacturing El Textile manufactunng/industry Q Domestic (helper) • Health (hospitals/laboratones) El Administration/office El Others • Services and sales

Position Net Monthly Family Income

El

Family Present Medical History

How many siblings do you have? _ brother(s) and _ sister(s)

Do you have family member(s) or blood relathre(s) with cancer? • Yes Type of Cancer Family Member/Relative Occupation (-) here 40.011cable Age of onset Breast Cancer Ovarian Cancer Other Cancers Father/Mother BrotherlSister Son/Daughter Mother ; s1de Greatgrandpa/Greatqrandma Grandfather/Grandmother Uncle/Aunt Male Cousin/Female Cousin Nephew/Niece Father; side Greatgrandpa/Greatqrandma GrandfatherIGrandmother Uncle/Aunt Male Cousin/Female Cousin Nephew/Niece 4 - Others No

Do you have a doctor that you see for general medical care?

El Yes How long have you been going to this doctor7 0 s year El >1 to s2 years 0 >2 to --5 years El >5 years No

Do you have any of the following signs and symptoms? 0 Yes LI Pains in breast tssue 0 Alternabng diarrheaand constipation 0 Vomiting C3Blood tinged stools 13 Pallor/Anemia El Fever/Body malaise El Weight loss Q Abdominal distention Q Others 0 Anonexia 1 No Have you undergone any of the following modes of detection/screening? C1Yes • Palpation BSE El Thermcgraphy El Graphic Stress telethermometry • Mammography LJ Ultrasound El Tumor Markers No When were you first diagnosed with breast cancer? How were you diagnosed with breast cancer? Q Mammography 0 Needle Biopsy Q Surgical Biopsy 0 Others

Did you undergo any of te following tests after diagnosis? LI Yes 0 Unnalysis 0 Blood test 0 Estrogen Receptor Assay 0 Body Scans 0 Others El No Did you undergo surgery for your cancer?

• Yes How many mos _I weeks _days - from diagnosis was surgery done7 How was it done? El Elective basis El Emergency basis No Are you currently taking any medications/vitamins?

0 Yes Medicine/Vitamin Dose Frequency

No

Past Medical History When did you first experience menstrual activity? _ years old

How long is your usual menstrual cycle? _ days Describe the frequency of your menstrual cycle. (regular - pedictable Within 5days] 13 Always regular • Usualty regular When did your periods become regular? years old When did your eriods become irregular? years old • Never regular What were the symptoms associated with premenstrual cycle that you have experienced? LI Cramps C3very seldom El sometimes Q often 0 Irritability 0 very seldom LI sometimes 0 often El Bloating Q very seldom 0 sometimes 0 often 0 Headache 0 very seldom CI sometimes Q often ID None Page 124 of 126

Did you ever have sexual contact?

0 Yes. How old were you when you first had sexual Contact? _ years old No

Did your ever get pregnant?

Q Yes What were the details your preqnancieV Pregnancy Number Year of Brth Lenoth of Labor (hr) Mscarnage I Theapeubc Abortion StA Rom Uve Brn Months of Breast Feeding

2 3 4

6 7 Cl No

Have you experienced personally induced/ 'quack doctor' abortion?

C! Yes When? C No

Have you reached menopause?

0 Yes When? 1 No

Please indicate if you have any of the following medical illnesses:

• Allergy LI Heart problem C3 Pulmonary tuberculosis (PTB) • Asthma 0 Hepabbs B 13 Schizophrenia • Benign breast disease LI Hypertension (HPN)/sboke IJ Sexually transmitted disease (STD) esp. HPV CI Diabetes Q Jaundice El Others C3Goiter 0 Osteoporosis

Have you ever been hospitalized before?

0 Yes For what reason(s)7 - When) El No

Have you undergone any other form of surgery before?

LI Yes What surgical procedure(s)7 When) L No

Have you ever been diagnosed with any other form of cancer (aside from breast cancer)?

13 Yes Type of cancer No

Have you experienced previous exposure to radiation (occupation, x-rays, thyroid scan, etc ?

U Yes Specify El No Social History Have you ever smoked cigarettes?

C3Yes At what age did you start smoking regularly? How any sticks of cigarette do you smoke per day? Do you still smoke? C3Yes 0 No When did you stop smoking? No

Does someone that you have lived with for more than a year ever smoked cigarettes?

0 Yes. At what age did he/she start smoking regularly7 - How many sticks of agarette does he/she smoke per day7 Does he/she still smoke? (Answer this only dthe person still fives wth you) 0 Yes E3 No When did he/she stop smoking7 C) No

Do you drink alcoholic beverages?

• Yes What type of alcoholic beverage do you drink? How much do you consume per week? El Wine 0 53 glasses 0 >3 to = glasses Z > 0 to s2O glasses D >20 glasses El Beer El 53 glasses 0 >3 to 5 10 glasses : > 0 to !20 glasses El > 20 glasses Cl Gin El 53 glasses LI > 3 to !510 glasses 0 >10 to i20 glasses -1 >20 glasses No

Have you ever drunk coffee regularly?

El Yes At what age did you start drinking coffee regularly') _ years old Do you still drink coffee? Yes LI No When did you stop dnnking coffee7 On the average, over this period, how many cups of coffee did you nk a day? - Cups Caffeinated - Cups Decaffeiriated -Total Cups of Coffee Cl No Page 125 of 126

How often do you eat the following foods (Please answer only ji'applicable.)

Food Daily 2X-3YI Occsnily Seldom Never Food Daily 2X-21Y 0ccsnIIy Seldom Never week week Rice 13 CI C3 0 Fruits 13 El 0 El E3 Bread Q_ Q a 0 13 Vegetable Q a El 13 0 Cereal Cl 0 0 El El Sweets 13 13 0 0 0 Red Meat Q Li U Q cl Softdrinks Q 0 Cl 0 El Poultry El 0 El El cl Preserved food' C! Q 0 0 0 Fish 0 I 13 1 13 1 El 0 1 Dried Fish' I a I Q 1 C3 1 CI 0

soyaEggs El0 U0 0C3 0a C30 BariZue"[la 5 1 QC] C13C 0 0 El Milk 0 1 Q E3 0 El Dried Seeds' I U U Li Li U toono, longanisa,bacon, ham, hot-spcy sud, hot-spcy dls 'o.11edfoods ancho,nes,dned sgud Watermelon, sua5h, peanuts, whae beans, 10amoy,champoy 'Otherfermented foods and condiments

Food Preference

How was the food usually prepared? (Check all that apply)

Q Deep ried E3 Sauteed CI Boiled 0 Broiled

What type of cooking oil do you usually use?

0 Soya 0 Corn 0 Olive El Animal

Do you reuse it?

C3Yes How many tirnes7 ND Opinions About Genetic Testing

In the future, if genetic tests for the following would be available, will you be willing to have yourself tested?

LI Yes • Breast-Ovarian Cancer Prediction El Homosexuality 0 Schizophrenia • Colorectal Cancer Prediction LI Intelligence 13 Stroke 13 Diabetes 0 Longevity C3Violence • Heart Disease LI Lung Cancer Susceptibility 0 Other hife-threatening diseases • HIV-resistance 13 Paternity Testing E No

If you can choose the genetic traits for your baby, would you choose to:

0 Determine sex Q Influence height/ weight 0 Test for a fatal disease El Ensure greater Intelligence 0 Rule out genes for violence 0 Not applicable 13 Have knowledge on the gender Cl Test for a disfiguring disease

If you are a carrier of a gene for a life-threatening incurable disease, would you have your unborn child tested? 0 Yes C No

If your baby carries a life-threatening disease gene, would you consider abortion? 0 Yes E No

Whom do you think should be prohibited from obtaining these genetic Information from your records?

• Spouse 0Court C3 Hospitals (for medical records) • Future spouse 0Employers QOther scientists local Cl Adult children QHealth Insurance C3foreign 13 Parents Cl Life Insurance 0None 0 Other relatives 0Pastor/Counselor

Do you believe people will benefit from cancer genetic tests? U Yes LI No

Should genetic tests be performed when there is no available treatment? U Yes No

If you are a carrier of a genetic disease, will you use the information for family planning? 0 Yes 1 No

Will you feet bothered of your children's future once you find out that you carry a genetic mutation for cancer? El Yes C) No

Please rate the contributions of the following factors toward getting a cancer:

Factor Strong Moderate Weak Does not contribute Genetics 0 0 El Cl Food El U El Water 0 C3 0 Pollution CI 0 0 Emotion E3 0 Economics Q 0 El Spiritual 11 Q 0 Q 1,ee I C3 Cl cl Lfegl, 0 a I i 0 Radiation 0 Q C33 0 I hereby certify that all the abovementioned data are true and correct to the best of my knowledge.

Signature of Collaborating Patient Page 126 of 126

Appendix D Concentration and Purity of Isolated Genomic DNA.

Stock Genomic DNA Working Genornic DNA Solution Patient ID [Stock] Purity [Working Solution) Purity (Pg/ml) (stock solution) (pg/ml) (working solution) Ptol 345 69 1 92 50 77 179 PtO2 1726 99 1 89 87 13 195 PtO3 1210 20 1 87 61 62 189 PtO4 1176 13 1 81 51 40 190 PtO5 983 75 1 95 49 49 193 PtO6 86 02 1 93 86 02 193 PtO7 377 80 1 83 82 70 184 PtO8 106 46 1 81 82 51 198 PtO9 66 72 1 95 66 72 195 Pilo 67 36 1 93 67 36 193 PO 67 81 1 85 67 81 185 P02 218 69 1 86 41 01 194 P03 237 57 1 81 52 42 190 Pt 14 87 31 1 97 87 3 197 P05 89 42 1 98 89 42 1.98 P06 81 42 1 91 52 51 1.89 Ptl7 72 03 1 98 72 03 19 8 P08 58 42 1.95 58.42 195 Pt19 141 22 1 94 44 23 195 Pt2O 71 51 1 96 71.51 196 Pt2l 87 97 1 97 87 97 197 Pt22 96 13 1 92 96 13 192 Pt23 62 1 9 62 81 19 Pt24 56 10 1 90 56 10 190 P125 103 28 1 91 47 62 2 0 Pt26 268 50 1 94 50 44 192 Pt27 156 04 1 99 29 79 18 8 Pt28 125 38 1 83 67 30 19 Pt29 127 58 1 82 46 84 193 POO 150 33 1 92 54 63 197 Pt31 332 79 1 95 42 87 194 P02 223 85 1 96 99 33 19 Pt33 388 64 1 93 56 87 196 Pt34 84 43 1 97 84 43 197 Pt35 1710 01 1 96 48 94 2 0 Pt36 74 93 1 79 74 93 79 P07 67 73 i 80 67 73 1 80 Buffer. IX TE (10mM Tris-HO, pH 7 , .ImM EDTA, pH 8.0)