Development and Characterization of Reference Materials for MTHFR, SERPINA1, RET, BRCA1, and BRCA2 Genetic Testing
2009; Elsevier BV; Volume: 11; Issue: 6 Linguagem: Inglês
10.2353/jmoldx.2009.090078
ISSN1943-7811
AutoresShannon Barker, Sherri J. Bale, Jessica K. Booker, Arlene Buller, Soma Das, Kenneth J. Friedman, Andrew K. Godwin, Wayne W. Grody, Edward W. Highsmith, Jeffery A. Kant, Elaine Lyon, Rong Mao, Kristin G. Monaghan, Deborah Payne, Victoria M. Pratt, Iris Schrijver, Antony E. Shrimpton, Elaine Spector, Milhan Telatar, Lorraine H. Toji, Karen E. Weck, Barbara A. Zehnbauer, Lisa V. Kalman,
Tópico(s)Genomics and Rare Diseases
ResumoWell-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website. Well-characterized reference materials (RMs) are integral in maintaining clinical laboratory quality assurance for genetic testing. These RMs can be used for quality control, monitoring of test performance, test validation, and proficiency testing of DNA-based genetic tests. To address the need for such materials, the Centers for Disease Control and Prevention established the Genetic Testing Reference Material Coordination Program (GeT-RM), which works with the genetics community to improve public availability of characterized RMs for genetic testing. To date, the GeT-RM program has coordinated the characterization of publicly available genomic DNA RMs for a number of disorders, including cystic fibrosis, Huntington disease, fragile X, and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population. Genotypic information about a number of other cell lines has been collected and is also available. The present study includes the development and commutability/genotype characterization of 10 DNA samples for clinically relevant mutations or sequence variants in the following genes: MTHFR; SERPINA1; RET; BRCA1; and BRCA2. DNA samples were analyzed by 19 clinical genetic laboratories using a variety of assays and technology platforms. Concordance was 100% for all samples, with no differences observed between laboratories using different methods. All DNA samples are available from Coriell Cell Repositories and characterization information can be found on the GeT-RM website. The use of genetic tests in medical practice has increased rapidly over the past few years. There are currently over 1400 genetic tests offered in clinical laboratories and hundreds of additional tests are available in research settings (National Institutes of Health, http://www.genetests.com, last accessed March 2, 2009). As for all medical testing, laboratories performing genetic tests must adhere to established quality assurance practices to ensure confidence in test integrity and accuracy.1Dequeker E Ramsden S Grody WW Stenzel T Barton DE Quality control in molecular genetic testing.Nat Rev. 2001; 2: 717-723Crossref Scopus (103) Google Scholar An integral part of quality assurance is the use of characterized and readily available reference materials (RMs). RMs, such as characterized DNA or cell lines that have a defined property, such as commutability (the equivalency of results between different measurement procedures) or genotype, can be used for quality control, monitoring of test performance, detection of errors, and proficiency testing of DNA-based genetic tests.1Dequeker E Ramsden S Grody WW Stenzel T Barton DE Quality control in molecular genetic testing.Nat Rev. 2001; 2: 717-723Crossref Scopus (103) Google Scholar2Emons H Fajgelj A van der Veen AMH Watters R New definitions on reference materials.Accred Qual Assur. 2006; 10: 576-578Crossref Google Scholar3Emons H Linsinger TPL Gawlik BM Reference materials: terminology and use. Can't one see the forest for the trees?.Trends Anal Chem. 2004; 23: 442-449Crossref Scopus (72) Google ScholarA hierarchy of RMs has previously been described based on the degree of characterization of each material.3Emons H Linsinger TPL Gawlik BM Reference materials: terminology and use. Can't one see the forest for the trees?.Trends Anal Chem. 2004; 23: 442-449Crossref Scopus (72) Google Scholar4Walker R Lumley I Pitfalls in terminology and use of reference materials.Trends Anal Chem. 1999; 18: 594-616Crossref Scopus (45) Google Scholar5Winn-Deen ES Standards and controls for genetic testing.Cancer Biomark. 2005; 1: 217-220PubMed Google Scholar6Chen B O'Connell CD Boone DJ Amos JA Beck JC Chan MM Farkas DH Lebo RV Richards CS Roa BB Silverman LM Barton DE Bejjani BA Belloni DR Bernacki SH Caggana M Charache P Dequeker E Ferreira-Gonzalez A Friedman KJ Greene CL Grody WW Highsmith Jr, WE Hinkel CS Kalman LV Lubin IM Lyon E Payne DA Pratt VM Rohlfs E Rundell CA Schneider E Willey AM Williams LO Willey JC Winn-Deen ES Wolff DJ Developing a sustainable process to provide quality control materials for genetic testing.Genet Med. 2005; 7: 534-549Crossref PubMed Scopus (43) Google Scholar The top hierarchy includes Standard Reference Materials (SRMs), which are produced by the National Institute of Standards and Technology (NIST); Certified Reference Materials (CRMs), produced by several organizations including the Institute of Reference Materials and Measurements; and other materials available from independent organizations, such as the World Health Organization. These RMs possess properties that are certified by a procedure that establishes metrological traceability and degree of uncertainty.3Emons H Linsinger TPL Gawlik BM Reference materials: terminology and use. Can't one see the forest for the trees?.Trends Anal Chem. 2004; 23: 442-449Crossref Scopus (72) Google Scholar4Walker R Lumley I Pitfalls in terminology and use of reference materials.Trends Anal Chem. 1999; 18: 594-616Crossref Scopus (45) Google Scholar5Winn-Deen ES Standards and controls for genetic testing.Cancer Biomark. 2005; 1: 217-220PubMed Google Scholar6Chen B O'Connell CD Boone DJ Amos JA Beck JC Chan MM Farkas DH Lebo RV Richards CS Roa BB Silverman LM Barton DE Bejjani BA Belloni DR Bernacki SH Caggana M Charache P Dequeker E Ferreira-Gonzalez A Friedman KJ Greene CL Grody WW Highsmith Jr, WE Hinkel CS Kalman LV Lubin IM Lyon E Payne DA Pratt VM Rohlfs E Rundell CA Schneider E Willey AM Williams LO Willey JC Winn-Deen ES Wolff DJ Developing a sustainable process to provide quality control materials for genetic testing.Genet Med. 2005; 7: 534-549Crossref PubMed Scopus (43) Google Scholar These materials have also been extensively characterized to ensure their homogeneity and stability. On the next level of the hierarchy are those RMs whose properties are sufficiently homogeneous and established for use in quality control applications.4Walker R Lumley I Pitfalls in terminology and use of reference materials.Trends Anal Chem. 1999; 18: 594-616Crossref Scopus (45) Google Scholar These materials include some commercially available RMs, which have either been cleared by the US Food and Drug Administration (FDA) or CE-marked (a mandatory conformity mark for products placed within the European Union market), and are therefore cleared for in vitro diagnostic use. For the purposes of this article, we will call SRMs, CRMs, FDA-approved, and CE-marked RMs "higher order" RMs. The use of "higher order" in this article does not represent a technical definition but only a categorization. Some manufacturers and governmental organizations have developed higher order RMs for a small number of diseases (Table 1), but many more are needed. In the absence of widely available higher order RMs, laboratories must use other types of materials as controls such as residual patient specimens, which are not reliably available or renewable. Other materials exist such as genomic DNA or cell lines that are available from sources such as repositories. Ideally these renewable materials are characterized by DNA sequence analysis or evaluated in multiple independent laboratories.5Winn-Deen ES Standards and controls for genetic testing.Cancer Biomark. 2005; 1: 217-220PubMed Google Scholar Though not higher order RMs, these genomic DNA or cell line materials play a vital role in quality control by providing a publicly available, renewable, and inexpensive source of RMs for various laboratory quality assurance needs.Table 1Publicly Available Higher Order RMsOrganizationCRM/SRMFDA-cleared*Information provided by manufacturer.CE marked*Information provided by manufacturer.†It is important to note that while higher order RMs are available for a small number of diseases, CE-marked RMs are currently unavailable.NIBSC‡For more information, please refer to the National Institute for Biological Standards and Controls (NIBSC) website (http://www.nibsc.ac.uk, last accessed December 19, 2008).World Health Organization Reference Reagent Factor V Leiden, Human gDNAWorld Health Organization Reference Reagent Prothrombin Mutation G20210A, Human gDNAIRMM§For more information, please refer to the Institute for Reference Materials and Measurements (IRMM) website (http://irmm.jrc.ec.europa.eu/html/homepage.htm, last accessed December 19, 2008).Plasmid DNA carrying human prothrombin gene (G2021A mutation) CRMPlasmid DNA carrying human prothrombin gene (Wildtype) CRMPlasmid DNA carrying human prothrombin gene (heterozygous for G2021A mutation)NIST¶For more information, please refer to the NIST website (http://ts.nist.gov/measurementservices/referencematerials/index.cfm, last accessed December 19, 2008).SRM Mitochondrial DNA Sequencing (Human HL-60 DNA)SRM Mitochondrial DNA Sequencing (Human DNA)SRM Fragile X Human DNA Triplet Repeat StandardSRM Heteroplasmic Mitochondrial DNA Mutation Detection StandardParagonDx∥ParagonDx (Morrisville, NC).CYP2D6 *4a/*2AXNCYP2D6 *2 mol/L/*17CYP2D6 *29/*2AXNCYP2D6 *6B/*41CYP2D6 *1/*5CYP2D6 *3A/*4AMaine Molecular Quality Controls**Maine Molecular Quality Controls (Scarborough, ME).INTROL Cystic Fibrosis Panel I* Information provided by manufacturer.† It is important to note that while higher order RMs are available for a small number of diseases, CE-marked RMs are currently unavailable.‡ For more information, please refer to the National Institute for Biological Standards and Controls (NIBSC) website (http://www.nibsc.ac.uk, last accessed December 19, 2008).§ For more information, please refer to the Institute for Reference Materials and Measurements (IRMM) website (http://irmm.jrc.ec.europa.eu/html/homepage.htm, last accessed December 19, 2008).¶ For more information, please refer to the NIST website (http://ts.nist.gov/measurementservices/referencematerials/index.cfm, last accessed December 19, 2008).∥ ParagonDx (Morrisville, NC).** Maine Molecular Quality Controls (Scarborough, ME). Open table in a new tab To address the need for improved publicly available characterized RMs for genetic testing (on all levels of the RM hierarchy), the Centers for Disease Control and Prevention (CDC), the National Institutes of Health, and NIST held a series of meetings to discuss possible solutions.6Chen B O'Connell CD Boone DJ Amos JA Beck JC Chan MM Farkas DH Lebo RV Richards CS Roa BB Silverman LM Barton DE Bejjani BA Belloni DR Bernacki SH Caggana M Charache P Dequeker E Ferreira-Gonzalez A Friedman KJ Greene CL Grody WW Highsmith Jr, WE Hinkel CS Kalman LV Lubin IM Lyon E Payne DA Pratt VM Rohlfs E Rundell CA Schneider E Willey AM Williams LO Willey JC Winn-Deen ES Wolff DJ Developing a sustainable process to provide quality control materials for genetic testing.Genet Med. 2005; 7: 534-549Crossref PubMed Scopus (43) Google Scholar Based on resulting recommendations, the CDC established the Genetic Testing Reference Material (GeT-RM) Coordination Program, which coordinates a process to improve the availability of appropriate RMs for the genetic testing community (GeT-RM Program, http://www.cdc.gov/dls/genetics/rmmaterials/default.aspx, last accessed March 2, 2009). Though sponsored by the CDC, much of the work performed by the GeT-RM, including RM priority decisions, specimen collection, material development, and molecular genetic characterization, occurs through voluntary collaborations with various clinical genetic laboratories. To date, the GeT-RM program has coordinated the development and/or commutability/genotype characterization of RMs for cystic fibrosis,7Pratt VM Caggana M Bridges C Buller AM DiAntonio L Highsmith WE Holtegaard LM Muralidharan K Rohlfs EM Tarleton J Toji L Barker SD Kalman LV Development of genomic reference materials for cystic fibrosis genetic testing.J Mol Diagn. 2009; 11: 186-193Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar Huntington disease,8Kalman L Johnson MA Beck J Berry-Kravis E Buller A Casey B Feldman GL Handsfield J Jakupciak JP Maragh S Matteson K Muralidharan K Richie KL Rohlfs EM Schaefer F Sellers T Spector E Richards CS Development of genomic reference materials for Huntington disease genetic testing.Gen Med. 2007; 9: 719-723Abstract Full Text Full Text PDF Scopus (18) Google Scholar fragile X syndrome,9Amos Wilson A Pratt VM Phansalkar A Muralidharan K Highsmith WE Beck JC Bridgeman S Courtney EM Epp L Ferreira-Gonzalez A Hjelm NL Holtegaard LM Jama MA Jakupciak JP Johnson MA Labrousse P Lyon E Prior TW Richards CS Richie KL Roa BB Rohlfs EM Sellers T Sherman SL Siegrist KA Silverman LM Wiszniewska J Kalman LV Fragile Xperts Working Group of AMP Consensus characterization of 16 FMR1 reference materials: a consortium study.J Mol Diagn. 2008; 10: 2-12Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and several genetic conditions with relatively high prevalence in the Ashkenazi Jewish population10Kalman L, Amos Wilson J, Buller A, Dixon J, Edelmann L, Geller L, Highsmith WE, Holtegaard L, Kornreich R, Rohlfs EM, Payeur TL, Sellers T, Toji L, Muralidharan K: Development of genomic DNA reference materials for genetic testing of disorders common in people of Ashkenazi Jewish descent. J Mol Diagn, in pressGoogle Scholar; and has also provided information for several pharmacogenetic markers, including members of the CYP450 gene family, VKORC1, and UGT1A1.In this study, the GeT-RM coordinated RM characterization studies of 10 genomic DNA samples containing sequence changes associated with hyperhomocysteinemia (MTHFR), and clinically relevant gene mutations in α-1 antitrypsin deficiency (SERPINA1), multiple endocrine neoplasia type 2A (RET), and hereditary breast and ovarian cancer (BRCA1 and BRCA2). The genomic DNA samples used in this study were characterized by using a variety of assays in a total of 18 College of American Pathologists-accredited and Clinical Laboratory Improvement Amendment-certified laboratories performing clinical genetic testing. These samples are publicly available from the Coriell Cell Repositories (Camden, NJ) and can be used for various quality assurance purposes and for research.Materials and MethodsMutation SelectionThese specific loci were chosen for this study because cell lines containing these clinically relevant mutations were recently made available at the Coriell Cell Repositories. Because of their clinical relevance, the GeT-RM aimed to conduct characterization studies on them and make these data publicly available in a timely fashion, which is why they are grouped together in a single study.The MTHFR gene encodes a key enzyme in homocysteine metabolism. Certain sequence variants in this gene (677C>T and 1298A>C) have been associated with hyperhomocysteinemia, which is characterized by abnormally high levels of homocysteine in the blood.11Ilhan N Kucuksu M Kaman D Ilhan N Ozbay Y The 677 C/T MTHFR polymorphism is associated with essential hypertension, coronary artery disease, and higher homocysteine levels.Arch Med Res. 2008; 39: 125-130Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar12Frederiksen J Juul K Grande P Jensen GB Schroeder TV Tybjaerg-Hansen A Nordestgaard BG Methylenetetrahydrofolate reductase polymorphism (C677T), hyperhomocysteinemia, and risk of ischemic cardiovascular disease and venous thromboembolism: prospective and case-control studies from the Copenhagen City Heart Study.Blood. 2004; 104: 3046-3051Crossref PubMed Scopus (107) Google Scholar13Blom HJ Genetic determinants of hyperhomocysteinemia: the roles of cystathionine beta-synthase and 5,10-methylenetetrahydrofolate reductase.Eur J Pediatr. 2000; 159: S208-S212Crossref PubMed Google Scholar14Zee RYL Mora S Cheng S Erlich HA Lindpaintner K Rifai N Buring JE Ridker PM Homocysteine, 5,10-Methylenetetrahydrofolate reductase 677C>T polymorphism, nutrient intake, and incident cardiovascular disease in 24,968 initially healthy women.Clin Chem. 2007; 53: 845-851Crossref PubMed Scopus (62) Google Scholar15Brown NM Pratt VM Buller A Pike-Buchanan L Redman JB Sun W Chen R Crossley B McGinniss MJ Quan F Strom CM Detection of 677CT/1298AC "double variant" chromosomes: implications for interpretation of MTHFR genotyping results.Genet Med. 2005; 7: 278-282Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar For this study, one DNA sample containing a heterozygous 677C>T change and a homozygous 1298A>C variant was characterized.Specific mutations in SERPINA1 lead to various levels of α-1 antitrypsin deficiency (AATD), which is characterized by pulmonary disease in adults and liver disease in children and adults (National Institutes of Health, http://www.genetests.com, last accessed March 2, 2009).16Ghebranious N Mallum J A single multiplexed allele-specific polymerase chain reaction for simultaneous detection of α1-antitrypsin S and Z mutations.Genet Testing. 2005; 9: 185-189Crossref PubMed Scopus (4) Google Scholar The normal allele, M, produces normal levels of the AAT protein. Two mutations, S and Z (representing the Glu264Val and Glu342Lys mutations, respectively), produce low or very low levels of circulating AAT protein, respectively, and patients carrying two of these alleles (ZZ or SZ) are at risk for developing disease related to AATD. Individuals who are MZ, MS, or SS produce enough AAT to function normally.16Ghebranious N Mallum J A single multiplexed allele-specific polymerase chain reaction for simultaneous detection of α1-antitrypsin S and Z mutations.Genet Testing. 2005; 9: 185-189Crossref PubMed Scopus (4) Google Scholar17Fregonese L Stolk J Hereditary alpha-1-antitrypsin deficiency and its clinical consequences.Orphanet J Rare Dis. 2008; 3: 16Crossref PubMed Scopus (163) Google Scholar18Cox DW Levinson H Emphysema of early onset associated with a complete deficiency of alpha-1 antitrypsin (null homozygotes).Am Rev Respir Dis. 1988; 137: 317-375Google Scholar19Turina GM Barker AF Brantly ML Clinical features of individuals with PiSZ phenotype of alpha-1-antitrypsin deficiency.Am J Respir Crit Care Med. 1996; 154: 1718-1725Crossref PubMed Scopus (152) Google Scholar Four genomic DNA samples with the MS, MZ, SZ, and ZZ genotypes were included in this study, covering both at-risk and nonrisk testing.Mutations in RET can lead to multiple endocrine neoplasia type 2A (MEN2A), MEN 2B and familial medullary thyroid carcinoma (National Institutes of Health, http://www.genetests.com, last accessed March 2, 2009).20Marini F Falchetti A Del Monte F Carbonell Sala S Tognarini I Luzi E Brandi ML Multiple endocrine neoplasia type 2.Orphanet J Rare Dis. 2006; 1: 45Crossref PubMed Scopus (132) Google Scholar21Mulligan LM Kwok JB Healey CS Elsdon MJ Eng C Gardner E Love DR Mole SE Moore JK Papi L Ponder MA Telenius H Tunnacliffe A Ponder BAJ Germ-line mutations of the RET proto-oncogene in multiple endocrine neoplasia type 2A.Nature. 1993; 363: 458-460Crossref PubMed Scopus (1717) Google Scholar22Donis-Keller H Dou S Chi D Carlson KM Toshima K Lairmore TC Howe JR Moley JF Goodfellow P Wells SA Mutations in the RET proto-oncogene are associated with MEN 2A and FMTC.Hum Mol Genet. 1993; 2: 851-856Crossref PubMed Scopus (1158) Google Scholar Mutations in cysteine residues at codons 609, 611, 618, 620, and 634 account for 95% of families with MEN2A and 85% of families with familial medullary thyroid carcinoma.23Edery P Eng C Munnich A Lyonett S RET in human development and oncogenesis.Bioassays. 1997; 19: 389-395Crossref PubMed Scopus (67) Google Scholar24Eng C Clayton D Schuffenecker I Lenoir G Cote G Gagel RF van Amstel HK Lips CJ Nishisho I Takai SI Marsh DJ Robinson BG Frank-Raue K Raue F Xue F Noll WW Romei C Pacini F Fink M Niederle B Zedenius J Nordenskjold M Komminoth P Hendy GN Mulligan LM The relationship between specific RET proto-oncogene mutations and disease phenotype in multiple endocrine neoplasia type 2: international RET mutation consortium analysis.JAMA. 1996; 276: 1575-1579Crossref PubMed Google Scholar25Mulligan LM Eng C Healey CS Clayton D Kwok JB Gardner E Ponder MA Frilling A Jackson CE Lehnert H Neumann HPH Thibodeau SN Ponder BAJ Specific mutations of the RET proto-oncogene are related to disease phenotype in MEN 2A and FMTC.Nat Genet. 1994; 6: 70-74Crossref PubMed Scopus (589) Google Scholar Two genomic DNA samples were included in this study, one heterozygous for the Cys620Phe mutation and one heterozygous for the Cys618Ser mutation.Women who inherit certain BRCA1 or BRCA2 mutations have an increased risk of developing breast and ovarian cancer compared with the general population.26Ford D Easton DF Stratton M Narod S Goldgar D Devilee P Bishop DT Weber B Lenoir G Chang-Claude J Sobol H Teare MD Struewing J Arason A Scherneck S Peto J Rebbeck TR Tonin P Neuhausen S Barkardottir R Eyfjord J Lynch H Ponder BA Gayther SA Birch JM Lindblom A Stoppa-Lyonnet D Bignon Y Borg A Hamann U Haites N Scott RJ Maugard CM Vasen H Seitz S Cannon-Albright LA Schofield A Zelada-Hedman M Breast Cancer Linkage Consortium Genetic heterogeneity and penetrance analysis of the BRCA1 and BRCA2 genes in breast cancer families.Am J Hum Genet. 1998; 62: 676-689Abstract Full Text Full Text PDF PubMed Scopus (2472) Google Scholar Mutations in these two genes are also associated with an increased risk for prostate, pancreatic, and colon cancer (National Institutes of Health, http://www.genetests.com, last accessed March 2, 2009).27Brose MS Rebbeck TR Calzone KA Stopfer JE Nathanson KL Weber BL Cancer risk estimates for BRCA1 mutation carriers identified in a risk evaluation program.J Nat Cancer Inst. 2002; 94: 1365-1372Crossref PubMed Google Scholar28Robles-Diaz L Goldfrank DJ Kauff ND Robson M Offit K Hereditary ovarian cancer in Ashkenazi Jews.Fam Cancer. 2004; 3: 259-264Crossref PubMed Scopus (35) Google Scholar29Antoniou A Pharoah PD Narod S Risch HA Eyfjord JE Hopper JL Loman N Olsson H Johannsson O Borg A Pasini B Radice P Manoukian S Eccles DM Tang N Olah E Anton-Culver H Warner E Lubinski J Gronwald J Gorski B Tulinius H Thorlacius S Eerola H Nevanlinna H Syrjakoski K Kallioniemi OP Thompson D Evans C Peto J Lalloo F Evans DG Easton DF Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case series unselected for family history: a combined analysis of 22 studies.Am J Hum Genet. 2003; 72: 1117-1130Abstract Full Text Full Text PDF PubMed Scopus (2755) Google Scholar Three Ashkenazi Jewish founder BRCA mutations, 185delAG and 5382insC (in BRCA1), and 6174delT (in BRCA2), which account for 90% of breast and ovarian cancer in Ashkenazi Jewish patients, were included in this study.30Roa BB Boyd AA Volcik K Richards CS Ashkenazi Jewish population frequencies for common mutations in BRCA1 and BRCA2.Nat Genet. 1996; 14: 185-187Crossref PubMed Scopus (623) Google Scholar31Struewing JP Hartage P Wacholder S Baker SM Berlin M McAdams M Timmerman MM Brody LC Tucker MA The risk of cancer associated with specific mutations of BRCA1 and BRCA2 among Ashkenazi Jews.N Engl J Med. 1997; 336: 1401-1408Crossref PubMed Scopus (1961) Google Scholar32Neuhausen S Gilewski T Norton L Tran T McGuire P Swensen J Hampel H Borgen P Brown K Skolnick M Shattuck-Eidens D Jhanwar S Goldgar D Offit K Recurrent BRCA2 6174delT mutation in Ashkenazi Jewish women affected by breast cancer.Nat Genet. 1996; 13: 126-128Crossref PubMed Scopus (257) Google ScholarCell Line Creation and DNA PreparationSeven cell lines used in this study were previously available from the Coriell Cell Repositories and were selected based on the description of genotype in the catalogue. Two of these lines were reported to contain mutations in SERPINA1 (GM03578 and GM03579), two contained mutations in RET (GM16658 and GM16660), two contained mutations in BRCA1 (GM13715 and GM14090), and one contained a BRCA2 mutation (GM14170). The only genotype information available for these cell lines was information obtained from the original submitter of the materials to the repository. The cell lines were selected for study based on this reported genotype. The reported genotype of each line was confirmed in each case. Three additional cell lines, one carrying a sequence change in MTHFR (GM20730) and two carrying SERPINA1 mutations (GM20835 and GM20918), were created specifically for this project from residual patient specimens tested in a clinical laboratory as follows: after mutation identification, residual whole blood was sent to the Coriell Institute, under an existing institutional review board protocol, for B-lymphocyte transformation by Epstein-Barr virus as previously described.33Beck JC Beiswanger CM John EM Satraiano WD Successful transformation of cryopreserved lymphocytes: a resource for epidemiological studies.Cancer Epidemiol Biomarkers Prev. 2001; 10: 551-554PubMed Google Scholar,34Bernacki SH Stankovic AK Williams LO Beck JC Herndon JE Snow-Bailey K Prior TW Matteson KJ Wasserman LM Cole EC Stenzel TT Establishment of stably EBV-transformed cell lines from residual clinical blood samples for use in performance evaluation and quality assurance in molecular genetic testing.J Mol Diagn. 2003; 5: 227-230Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar All 10 cell lines were cultured and expanded in antibiotic-free media. Cell suspensions were aliquoted in 1-ml vials containing approximately 5 × 106 viable cells per vial. Cultures were cryopreserved in heat-sealed borosilicate glass ampoules and stored in liquid nitrogen. Successful cultures were bacterial, fungal, and mycoplasma-free and capable of doubling in cell number within 4 days of recovery. Coriell prepared approximately 2 mg of DNA from each cell line in this study by using Gentra/Qiagen Autopure (Valencia, CA) per manufacturer's instructions or as previously described.35Miller SA Dykes DD Polesky HF A simple salting out procedure for extracting DNA from human nucleated cells.Nucleic Acids Res. 1988; 16: 1215Crossref PubMed Scopus (17699) Google ScholarLaboratory ParticipationClinical laboratories were solicited for participation based on their capability to detect the mutations included in this study and on their current assay methods. This ensured that each of the DNA samples was tested in multiple laboratories using a variety of the commonly used assays and technology platforms. A total of 18 clinical genetic laboratories agreed to participate in this study. Table 2 shows the gene(s) for which individual laboratories performed testing and the assay(s) or platform(s) they used. All laboratories are located within the United States and are accredited by the College of American Pathologists and Clinical Laboratory Improvement Amendment certified.Table 2Investigators and Assays UsedLaboratoryMTHFRSERPINA1 (AATD)RET (MEN2)BRCA1/BRCA21PCR/LuminexPCR/Luminex2PCR with hybridization probes and melting curve analysisDNA sequence analysis3DNA sequence analysis4PCR with hybridization probes and melting curve analysis5DNA sequence analysis6DNA sequence analysis7PCR/RE/electrophoresisDNA sequence analysis8DNA sequence analysis9DNA sequence analysisAllele-specific oligonucleotide hybridization10Third Wave Technologies Invader ASR11Autogenomics INFINITI Assay12PCR/RE/ABI310013Third Wave Technologies Invader ASR14Heteroduplex mobility assay15PCR with hybridization probes and melting curve analysisPCR with hybridization probes and melting curve analysisDNA sequence analysis16PCR/RE/electrophoresisDNA sequence analysis17Mutliplex allele-specific PCR/electrophoresis18PCR/RE/electrophoresisDNA sequence analysisRE, restriction enzyme analysis; ASR, analyte specific reagent; ABI3100, Genetic Analyzer 3100 (Applied Biosystems, Foster City, CA). Open table in a new tab ProtocolEach of the 18 laboratories received a 50-μg aliquot of extracted DNA from each cell line they were asked to test. With the exception of those laboratories performing DNA sequence analysis, the expected mutation(s) for each DNA sample were not disclosed. Laboratories assayed each DNA sample by using their routine clinical assays. Results were sent directly to study coordinators (L.V.K. and S.D.B.), who reviewed the data for quality and discrepancies.Assays/Platforms UsedMTHFRA total of eight laboratories tested for nucleotide sequence variants in MTHFR. One laboratory used a commercial kit (INFINITI Assay, Autogenomics, Carlsbad, CA) and followed manu
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