Development of Genomic Reference Materials for Cystic Fibrosis Genetic Testing
2009; Elsevier BV; Volume: 11; Issue: 3 Linguagem: Inglês
10.2353/jmoldx.2009.080149
ISSN1943-7811
AutoresVictoria M. Pratt, Michele Caggana, Christina Bridges, Arlene Buller, Lisa DiAntonio, W. Edward Highsmith, Leonard M. Holtegaard, Kasinathan Muralidharan, Elizabeth M. Rohlfs, Jack Tarleton, Lorraine H. Toji, Shannon Barker, Lisa V. Kalman,
Tópico(s)Genomics and Rare Diseases
ResumoThe number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812–1G>A (c.1680–1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories. The number of different laboratories that perform genetic testing for cystic fibrosis is increasing. However, there are a limited number of quality control and other reference materials available, none of which cover all of the alleles included in commercially available reagents or platforms. The alleles in many publicly available cell lines that could serve as reference materials have neither been confirmed nor characterized. The Centers for Disease Control and Prevention-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the genetic testing community as well as Coriell Cell Repositories, have characterized an extended panel of publicly available genomic DNA samples that could serve as reference materials for cystic fibrosis testing. Six cell lines [containing the following mutations: E60X (c.178G>T), 444delA (c.312delA), G178R (c.532G>C), 1812–1G>A (c.1680–1G>A), P574H (c.1721C>A), Y1092X (c.3277C>A), and M1101K (c.3302T>A)] were selected from those existing at Coriell, and seven [containing the following mutations: R75X (c.223C>T), R347H (c.1040G>A), 3876delA (c.3744delA), S549R (c.1646A>C), S549N (c.1647G>A), 3905insT (c.3773_3774insT), and I507V (c.1519A>G)] were created. The alleles in these materials were confirmed by testing in six different volunteer laboratories. These genomic DNA reference materials will be useful for quality assurance, proficiency testing, test development, and research and should help to assure the accuracy of cystic fibrosis genetic testing in the future. The reference materials described in this study are all currently available from Coriell Cell Repositories. Cystic fibrosis (CF) is an autosomal recessive disorder that affects approximately one of every 2500 live births (Caucasian). The CFTR gene was cloned in 1989 and to date over 1500 mutations linked to CF have been identified (The Hospital for Sick Children, http://www.genet.sickkids.on.ca/cftr, accessed May, 13, 2008). In 2001 the American College of Medical Genetics (ACMG) and the American College of Obstetricians and Gynecologists (ACOG) recommended CFTR mutation carrier screening be offered during pregnancy or in anticipation of a pregnancy to couples who were non-Jewish Caucasian or Ashkenazi Jewish.1Grody WW Cutting GR Klinger KW Richards CS Watson MS Desnick RJ Laboratory standards and guidelines for population-based cystic fibrosis carrier screening.Genet Med. 2001; 3: 149-154Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar A panel of 25 CFTR mutations with an allele frequency ≥0.1% in the general U.S. population was recommended for screening. The recommendation was updated by ACOG in 2005 to offer screening to all couples regardless of race or ethnicity.2Committee on Genetics, American College of Obstetricians and Gynecologists ACOG committee opinion. Number 325, December 2005. Update on carrier screening for cystic fibrosis.Obstet Gynecol. 2005; 106: 1465-1468Crossref PubMed Google Scholar In 2004, the mutation screening panel was revised to 23 mutations3Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.Genet Med. 2004; 6: 387-391Crossref PubMed Scopus (374) Google Scholar and the ACMG developed a recommended newborn screening panel that included CF.4American College of Medical Genetics' Newborn Screening Expert Group toward a uniform screening panel and system.Genet Med. 2006; 8: 1S-252SPubMed Google Scholar In response to these initiatives both CFTR testing volumes and the available commercial platforms for testing have undergone extensive growth. Suggestions for more standardization and the expansion beyond the recommended CF testing panels have generated much discussion in the genetic testing community.5Grody WW Cutting GR Watson MS The cystic fibrosis mutation "arms race": when less is more.Genet Med. 2007; 9: 739-744Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar Although inclusion of additional CF alleles does not significantly increase the detection frequency in European Caucasians, many commercial reagents and platforms exceed the recommended ACMG/ACOG 23 mutation panel. Two out of the four Food and Drug Administration-cleared CF assays exceed the recommended screening panel, suggesting that platform manufacturers perceive a competitive edge for screening additional CF mutations. Reference materials are needed for test development and validation, lot-testing of new reagent batches and for performance evaluation (proficiency testing/external quality assessment) programs. In addition, international, federal, and state regulations and professional guidelines require the use of reference or quality control materials alongside patient samples,6Amos J Feldman GL Grody WW Monaghan K Palomaki GE Prior TW Richards CS Watson MS Technical Standards and Guidelines for CFTR Mutation Testing. American College of Medical Genetics, 2006: 1-28Google Scholar7Association for Molecular Pathology Recommendations for in-house development and operation of molecular diagnostic tests.Am J Clin Pathol. 1999; 111: 449-463PubMed Google Scholar8Chen 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 (46) Google Scholar9Code of Federal Regulations. The clinical laboratory improvement amendments (CLIA) 2007, 42 CFR Part 493Google Scholar10MM01-A2 Molecular diagnostic methods for genetic diseases-approved guideline. The clinical and laboratory standards institute (CLSI). 2nd ed. CLSI, Wayne PA2006Google Scholar11Potter NT Spector EB Prior TW Technical standards and guidelines for Huntington disease.Genet Med. 2004; 6: 61-65Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar (European Molecular Genetics Quality Network, http://emqn.org/eqmn/bestpractice.html, 05/13/2008; Washington State Legislature, http://apps.leg.wa.gov/WAC/default.aspx?cite_246-338, 05/13/2008; New York State Clinical Laboratory Evaluation Program, http://www.wadsworth.org/labcert/clep/clep.html, 05/13/2008; College of American Pathologists, http://www.cap.org, 05/13/2008; American College of Medical Genetics, http://www.acmg.net/Pages/ACMG_Activities/stds-2002/g.htm, 06/02/2008). Despite the increase in the number of laboratories performing CF testing and the ever expanding cadre of CF mutation panels, characterized genomic DNA reference materials for mutations outside of the ACMG/ACOG 23 mutation panel are not available for laboratory quality assurance purposes. In the absence of these materials, laboratories, test developers and proficiency test providers must rely on residual patient specimens, which are often difficult to find and not consistently available (General recommendations for quality assurance programs for laboratory molecular genetic tests, http://wwwn.cdc.gov/dls/genetics/qapt.aspx, 12/11/2008).12Williams LO Cole EC Lubin IM Iglesias NI Jordan RL Elliott LE Quality assurance in human molecular genetics testing-Status and recommendations.Arch Pathol Lab Med. 2003; 127: 1353-1358PubMed Google Scholar The Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program (http://wwwn.cdc.gov/dls/genetics/rmmaterials/default.aspx, 05/13/2008), together with the clinical genetics laboratory community initiated a project to obtain and characterize additional publicly available cell lines and genomic DNA reference materials for CF genetic testing. These materials supplement the available characterized genomic DNA reference materials, which cover the recommended mutation screening panel. When we began this study, the National Institute of General Medical Sciences Human Genetic Cell Repository at the Coriell Cell Repositories already had cell lines covering the 23 alleles recommended by ACMG/ACOG, which were characterized previously by DNA sequence analysis (Coriell Cell Repositories, Camden, NJ). We selected additional alleles commonly included in commercial CF reagents that are not included in the 23 ACMG/ACOG alleles but are present in relatively high frequency in ethnic populations,3Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.Genet Med. 2004; 6: 387-391Crossref PubMed Scopus (374) Google Scholar alleles that were already available from Coriell but that had not been independently confirmed and alleles representing polymorphisms that could interfere with detection of F508del (c.1521_1523delCTT), which is one of the 23 alleles recommended by ACMG, and a mutation that could interfere with the detection of S549N (c.1647G>A), an allele common in some ethnic populations. Six cell lines were selected from those existing at Coriell and nine new cell lines were created specifically for this project. DNA samples were prepared from these cell lines by Coriell. The CF alleles in these materials were confirmed by testing in six volunteer laboratories using a variety of assay platforms, including DNA sequence analysis. We also documented the previously unpublished characterization of DNA from a number of other Coriell CF cell lines. These genomic DNA samples are publicly available from Coriell and can be used for quality assurance, assay development, and validation, as well as for proficiency testing. The availability of these reference materials will support accurate clinical CF testing. After patient testing, residual whole blood containing CF mutations was sent to the Coriell Cell Repositories, under an existing institutional review board research protocol at the collection site, for Epstein-Barr virus transformation of B-lymphocytes as previously described.13Beck JC Beiswanger CM John EM Satraiano West D Successful transformation of cryopreserved lymphocytes: a resource for epidemiological studies.Cancer Epidemiol Biomarkers Prev. 2001; 10: 551-554PubMed Google Scholar,14Bernacki 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 samples were placed into culture and expanded to yield approximately 2 × 108 total viable cells. The culture medium was antibiotic-free to increase the likelihood that contamination would be readily detected. The cell suspension was dispersed in 40 1-ml vials so that each contained 5 × 106 viable cells. Cultures were cryopreserved in heat-sealed borosilicate glass ampoules and stored in liquid nitrogen (liquid phase). Successful cultures were free from bacterial, fungal, and mycoplasmal contamination and were viable after cryopreservation in liquid nitrogen, as evidenced by a doubling of the cell number within four days of recovery. Approximately 2 mg of DNA was prepared from each of the selected cell lines by Coriell Cell Repositories using Gentra/Qiagen Autopure per manufacturer's instructions (Valencia, CA) or previously described methods.15Miller 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 (18101) Google Scholar A total of six clinical genetic laboratories that offer CF testing volunteered to participate in the study. Laboratories were solicited based on their current CF assay methods, so that each of the DNA samples was tested by all of the commonly used platforms. The assay method used, alleles included in this study, and whether or not they can be detected by the assay are shown in Table 1. The assays used in this study detect the 23 alleles recommended by ACMG/ACOG as well as additional CF alleles. All of the laboratories are located in the United States and have CLIA certification or are accredited by the College of American Pathologists (CAP).Table 1Specificity of Detection MethodsStudy allelesMethods*Methods used: Luminex Tag-It CFTR 40 + 4 Mutation Detection Kit (FDA-cleared; IVD); Asuragen Signature 2.0 CF Expand [laboratory developed test (LDT)]; Laboratory developed assay; Third Wave InPlex (LDT); Abbott/Celera OLA V3.0 (LDT); sequencing (LDT).cDNA sequence change†HGVS nomenclature http://www.hgvs.org/mutnomen/ (accessed December 9, 2008). All cDNA sequence change nomenclature is based on the GenBank cDNA reference sequence NM_000492.3.Common nameLuminex Tag-ItAsuragen SignatureLaboratory developed test (Luminex)Third Wave InPlexAbbott/Celera OLASequencingc.3302T>AM1101Kyes‡Yes indicates allele is detected by the method, no indicates allele is not detected by method.noyesnonoyesc.3277C>AY1092Xyesnoyesyesnoyesc.1721C>AP574Hnonoyesnonoyesc.532G>CG178Ryesnoyesnonoyesc.312delA444delAyesnoyesnonoyesc.1680-1G>A (AJ574983.1:g.92G>A)1812-1G>Ayesyesyesnonoyesc.1040G>AR347Hyesyesyesyesyesyesc.3744delA3876delAyesyesyesyesyesyesc.1647G>AS549Nyesyesyesyesyesyesc.3773_3774insT3905insTyesnoyesyesyesyesc.1646A>CS549Ryesnoyesyesyesyesc.1516A>GI506Vyesnoyesyesyesyesc.1519A>GI507Vyesnoyesnoyesyesc.223C>TR75Xnonoyesnonoyesc.178G>TE60Xnonoyesyesnoyes* Methods used: Luminex Tag-It CFTR 40 + 4 Mutation Detection Kit (FDA-cleared; IVD); Asuragen Signature 2.0 CF Expand [laboratory developed test (LDT)]; Laboratory developed assay; Third Wave InPlex (LDT); Abbott/Celera OLA V3.0 (LDT); sequencing (LDT).† HGVS nomenclature http://www.hgvs.org/mutnomen/ (accessed December 9, 2008). All cDNA sequence change nomenclature is based on the GenBank cDNA reference sequence NM_000492.3.‡ Yes indicates allele is detected by the method, no indicates allele is not detected by method. Open table in a new tab The assays and platforms used in this study are as follows: Luminex Tag-It CFTR 40 + 4 Platform The Tag-It CFTR 40 + 4 Mutation Detection Kit (Luminex Molecular Diagnostics, Austin TX) simultaneously screens for 40 mutations and four variants. Briefly, genomic DNA was amplified and alleles were discriminated using allele-specific primer extension and hybridization to a universal microsphere array. Genotypes were detected on a Luminex 100 IS System and called using the proprietary TM Data Analysis Software as previously described.16Strom CM Janeszco R Quan F Wang S Buller A McGinness M Sun W Technical validation of a Tm biosciences luminex-based multiplex assay for detecting the American College of Medical Genetics-recommended cystic fibrosis mutation panel.J Mol Diagn. 2006; 8: 371-375Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar The Asuragen Signature 2.0 CF Expand reagents (Asuragen Inc., Austin, TX) simultaneously screens for 44 mutations and four variants. Genomic DNA was amplified in a single multiplex PCR reaction using fluorescently labeled primers. The PCR products were denatured and hybridized to allele-specific oligonucleotides coupled to Luminex x-Map Beads (Luminex, Austin, TX). Genotypes were detected on a Luminex 100 IS System and called using the proprietary Signature Script software.17Hadd AG Laosinchai-Wolf W Novak CR Badgett MR Isgur LA Goldrick M WalkerPeach CR Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms.J Mol Diagn. 2004; 6: 348-355Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar This proprietary method for the detection of 97 CF mutations includes the amplification of genomic DNA followed by allelic discrimination using allele-specific primer extension and hybridization to a universal microsphere panel. Genotypes were detected on the Luminex 100 xMAP system and assigned using TM Data Analysis Software. Alleles on multiplexed microspheres were resolved by bidirectional sequencing. The CFTR InPlex (Third Wave Technologies, Madison, WI) assay detects 38 clinically relevant mutations and six variants. Briefly, genomic DNA was amplified using an abbreviated PCR and the products (15 μL) were added to the CFTR InPlex cards containing fluorescent resonance energy transfer cassettes to detect the mutations after isothermal signal amplification. Results were read on a plate reader and genotypes were called by the Invader® Data Analysis Worksheet software.18Johnson MA Yoshitomi MJ Richards CS A comparative study of five technologically diverse CFTR testing platforms.J Mol Diagn. 2007; 9: 401-407Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar The Abbott/Celera oligonucleotide ligation assay reagents (Alameda, CA) detect 31 clinically relevant mutations and six variants. Briefly, genomic DNA was amplified by PCR and the products were subsequently used in the oligonucleotide ligation assay reaction. The oligonucleotide ligation assay products were detected on the Applied Biosystems, Inc. (ABI) 3100 Genetic Analyzer and interpreted using the ABI CF Genotyper software (Foster City, CA).19Brinson EC Adriana T Bloch W Brown CL Chang CC Chen J Eggerding FA Grossman PD Iovannisci DA Madonik AM Sherman DG Tam RW Winn-Deen ES Woo SL Fung S Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.Genet Test. 1997; 1: 61-68PubMed Google Scholar DNA sequence analysis was performed in two different laboratories. One laboratory used Big Dye terminator chemistry with detection on an ABI3100 and/or ABI 3730 Genetic Analyzer. Primers were designed to amplify the exons containing the mutations under standard amplification conditions. Sequencing was performed in the forward and reverse directions and compared with a normal in-house control. The second laboratory performed DNA sequence analysis as previously described.20Strom CM Huang D Chen C Buller A Peng M Quan F Redman J Sun W Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test.Genet Med. 2003; 5: 9-14Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar Each of the six testing laboratories received one 50-μg aliquot of DNA from each of the 15 CF cell lines tested. The expected CF allele(s) in each of the samples were not revealed to the laboratories except to those who were performing DNA sequence analysis (laboratory staff was told which exons to sequence, but blinded to the specific mutation). The laboratories assayed each DNA sample using their standard assay methods (Table 1). One laboratory performed testing of the DNA from the cell lines using two different CF assays as it was validating a new platform at the time of the study. These results were sent to the study coordinators (LVK and VMP), who examined the data for quality and discrepancies. In addition, several DNA variants present in the publicly available cell lines, which were not specifically targeted in this study, were confirmed by DNA sequence analysis performed previously at the Wadsworth Center, New York State Dept. of Health. These cell lines and the alleles that they contain are indicated in Table 2.Table 2Characterized Cell LinescDNA sequence change name*All cDNA sequence change HCVS nomenclature is based on the GenBank cDNA reference sequence NM_000492.3.Common nameCoriell #Allele 1Allele 2Allele 1Allele 2Methods†Methods used: A, sequencing; B, reverse hybridization; C, oligonucleotide ligation assay (Abbott/Celera); D, mass spectrometry; E, allele specific amplification assay with gel electrophoresis; F, Invader Assay (Third Wave Technologies); G, restriction fragment length polymorphism; H, restriction fragment length polymorphism + sequencing; I, heteroduplex analysis; J, restriction fragment length polymorhpism + heteroduplex analysis + sequencing; K, eSensor chip (Motorola Life Sciences) + sequencing; L, 97 Mutation allele-specific assay (proprietary LDT); M, heteroduplex analysis + sequencing; N, restriction fragment length polymorphism + heteroduplex analysis + oligonucleotide ligation assay. (# labs)Population frequency of mutated allele(s)‡CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population.Refs.§References for characterization: O, Coriell ACMG23 Panel (MUTCF); P, Reference #23, Q, DNA sequencing Michele Caggana, NYSDOH.GM07441¶Cell line included in Coriell MUTCF Panel.c.2988 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ575003.1:g.305G>T)c.489 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ574942.1:g.240G>T)3120+G>A∥Allele included in the ACMG 23 Panel.621 + 1G>T∥Allele included in the ACMG 23 Panel.A(4), C(1), E(1), F(1), G(2), H(1)0.86%/1.30%**See reference # 3.O,PGM13591¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.350G>A∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.R117H∥Allele included in the ACMG 23 Panel.A(5), C(1), E(1), F(1), G(2), H(1)66.31%/0.54%**See reference # 3.O,P,QGM18799¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.2052delA∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.2184delA∥Allele included in the ACMG 23 Panel.A(1)66.31%/0.15%**See reference # 3.O,QGM18800¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1766 + 1G>A∥Allele included in the ACMG 23 Panel. (AJ574983.1:g.179G>A)F508del∥Allele included in the ACMG 23 Panel.1898 + 1G>A∥Allele included in the ACMG 23 Panel.A(1)66.31%/0.13%**See reference # 3.O,QGM11277¶Cell line included in Coriell MUTCF Panel.c.1519_1521delATC∥Allele included in the ACMG 23 Panel.delI507∥Allele included in the ACMG 23 Panel.A(1)0.90%**See reference # 3.O,QGM01531¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.A(1)66.31%/66.31%**See reference # 3.O,QGM11496¶Cell line included in Coriell MUTCF Panel.c.1624G>T∥Allele included in the ACMG 23 Panel.c.1624G>T∥Allele included in the ACMG 23 Panel.G542X∥Allele included in the ACMG 23 Panel.G542X∥Allele included in the ACMG 23 Panel.A(1)2.64%/2.64%**See reference # 3.O,QGM07552¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1657C>T∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.R553X∥Allele included in the ACMG 23 Panel.A(1)66.31%/1.21%**See reference # 3.O,QGM08338¶Cell line included in Coriell MUTCF Panel.c.1652G>A∥Allele included in the ACMG 23 Panel.G551D∥Allele included in the ACMG 23 Panel.A(1)1.93%**See reference # 3.O,QGM11859¶Cell line included in Coriell MUTCF Panel.c.2657 + 5G>A∥Allele included in the ACMG 23 Panel. (AJ574995.1:g.216G>A)c.2657 + 5G>A∥Allele included in the ACMG 23 Panel. (AJ574995.1:g.216G>A)2789 + 5G>A∥Allele included in the ACMG 23 Panel.2789 + 5G>A∥Allele included in the ACMG 23 Panel.A(1)0.38%/0.38%**See reference # 3.O,QGM11723¶Cell line included in Coriell MUTCF Panel.c.3846G>A∥Allele included in the ACMG 23 Panel.W1282X∥Allele included in the ACMG 23 Panel.A(1)2.20%**See reference # 3.O,QGM11860¶Cell line included in Coriell MUTCF Panel.c.3718-2477C>T∥Allele included in the ACMG 23 Panel. (AY848832.1:g.40725C>T)c.3718-2477C>T∥Allele included in the ACMG 23 Panel. (AY848832.1:g.40725C>T)3849 + 10kbC>T∥Allele included in the ACMG 23 Panel.3849 + 10kbC>T∥Allele included in the ACMG 23 Panel.A(1)0.85%/0.85%**See reference # 3.O,QGM11280¶Cell line included in Coriell MUTCF Panel.c.579 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ574943.1:g.261G>T)c.489 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ574942.1:g.240G>T)711 + 1G>T∥Allele included in the ACMG 23 Panel.621 + 1G>T∥Allele included in the ACMG 23 Panel.A(1)0.35%/1.30%**See reference # 3.O,QGM11282¶Cell line included in Coriell MUTCF Panel.c.254G>A∥Allele included in the ACMG 23 Panel.c.489 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ574942.1:g.240G>T)G85E∥Allele included in the ACMG 23 Panel.621 + 1G>T∥Allele included in the ACMG 23 Panel.A(1)0.26%/1.30%**See reference # 3.O,QGM12585¶Cell line included in Coriell MUTCF Panel.c.3484C>T∥Allele included in the ACMG 23 Panel.R1162X∥Allele included in the ACMG 23 Panel.A(1)0.3%**See reference # 3.O,QGM12444¶Cell line included in Coriell MUTCF Panel.c.1585-1G>A∥Allele included in the ACMG 23 Panel. (AJ574980.1:g.116G>A)1717-1G>A∥Allele included in the ACMG 23 Panel.A(1)0.44%**See reference # 3.O,QGM11472¶Cell line included in Coriell MUTCF Panel.c.4046G>Ac.3909C>G∥Allele included in the ACMG 23 Panel.G1349DN1303K∥Allele included in the ACMG 23 Panel.A(1)no data/1.27%**See reference # 3.O,QGM12785¶Cell line included in Coriell MUTCF Panel.c.1652G>A∥Allele included in the ACMG 23 Panel.c.4040G>C∥Allele included in the ACMG 23 Panel.G551D∥Allele included in the ACMG 23 Panel.R347P∥Allele included in the ACMG 23 Panel.A(1)1.93/0.36%**See reference # 3.O,QGM12960¶Cell line included in Coriell MUTCF Panel.c.1000C>T∥Allele included in the ACMG 23 Panel.R334W∥Allele included in the ACMG 23 Panel.A(1)0.37%**See reference # 3.O,QGM11275¶Cell line included in Coriell MUTCF Panel.c.3437delC∥Allele included in the ACMG 23 Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.3659delC∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.A(1)0.28%/66.31%**See reference # 3.O,QGM11281¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.489 + 1G>T∥Allele included in the ACMG 23 Panel. (AJ574942.1:g.240G>T)F508del∥Allele included in the ACMG 23 Panel.621 + 1G>T∥Allele included in the ACMG 23 Panel.A(1)66.31%1.30%**See reference # 3.O,QGM11283¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1364C>A∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.A455E∥Allele included in the ACMG 23 Panel.A(1)66.31%/0.26%**See reference # 3.O,QGM11284¶Cell line included in Coriell MUTCF Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1679G>C∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.R560T∥Allele included in the ACMG 23 Panel.A(1)66.31%/0.30%**See reference # 3.O,QGM07461c.1657C>T∥Allele included in the ACMG 23 Panel.R553X∥Allele included in the ACMG 23 Panel.A(1)1.21%**See reference # 3.QGM08345c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.F508del∥Allele included in the ACMG 23 Panel.A(1)66.31%/66.31%**See reference # 3.QGM11278c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1477C>TF508del∥Allele included in the ACMG 23 Panel.Q493XA(1)66.31%/0.17%**See reference # 3.QGM11287c.1521_1523delCTT∥Allele included in the ACMG 23 Panel.c.1721C>AF508del∥Allele included in the ACMG 23 Panel.P574HA(1)66.31%¶Cell line included in Coriell MUTCF Panel./very lowSee reference # 21.QGM12961c.1558G>TV520FA(1)0.09%**See reference # 3.QGM13033c.1523T>GF508CA(1)no dataQGM13423c.254G>A∥Allele included in the ACMG 23 Panel.c.3454G>CG85E∥Allele included in the ACMG 23 Panel.D1152HA(1)0.26%/0.03%**See reference # 3.QCD00003c.948delT1078delTB(1), C(1), L(1)0.03%**See reference # 3.PCD00004c.1766 + 1G>A∥Allele included in the ACMG 23 Panel. (AJ574983.1:g.179G>A)1898 + 1G>A∥Allele included in the ACMG 23 Panel.B(1), C(1), L(1)0.13%**See reference # 3.PCD00005c.1766 + 1G>A∥Allele included in the ACMG 23 Panel. (AJ574983.1:g.179G>A)1898 + 1G>A∥Allele included in the ACMG 23 Panel.B(1), C(1), L(1)0.13%**See reference # 3.PCD00006c.1766 + 1G>A∥Allele included in the ACMG 23 Panel. (AJ574983.1:g.179G>A)1898 + 1G>A∥Allele included in the ACMG 23 Panel.B(1), C(1), L(1)0.13%**See reference # 3.PCD00007c.1766 + 1G>A∥Allele included in the ACMG 23 Panel. (AJ574983.1:g.179G>A)1898 + 1G>A∥Allele included in the ACMG 23 Panel.B(1), C(1), L(1)0.13%**See reference # 3.PCD00008c.2052delA+2184delA∥Allele included in the ACMG 23 Panel.B(1), C(1), L(1)0.15%**See reference # 3.PCD00009c.262delTT394delTTB(1), C(1), L(1)0.09%See reference # 21.PCD00010c.443T>CI148TB(1), C(1), L(1)0.08%**See reference # 3.PCD00012c.443T>CI148TB(1), C(1), L(1)0.08%**See reference # 3.PCD00013c.3703A>CS1235RA(6), D(1), J(1), K(2)1.6%See reference # 22.P* All cDNA sequence change HCVS nomenclature is based on the GenBank cDNA reference sequence NM_000492.3.† Methods used: A, sequencing; B, reverse hybridization; C, oligonucleotide ligation assay (Abbott/Celera); D, mass spectrometry; E, allele specific amplification assay with gel electrophoresis; F, Invader Assay (Third Wave Technologies); G, restriction fragment length polymorphism; H, restriction fragment length polymorphism + sequencing; I, heteroduplex analysis; J, restriction fragment length polymorhpism + heteroduplex analysis + sequencing; K, eSensor chip (Motorola Life Sciences) + sequencing; L, 97 Mutation allele-specific assay (proprietary LDT); M, heteroduplex analysis + sequencing; N, restriction fragment length polymorphism + heteroduplex analysis + oligonucleotide ligation assay.‡ CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population.§ References for characterization: O, Coriell ACMG23 Panel (MUTCF); P, Reference #23Bernacki SH Beck JC Stankovic AK Williams LO Amos J Snow-Bailey K Farkas DH Friez MJ Hantash FM Matteson KJ Monaghan KG Muralidharan K Pratt VM Prior TW Richie KL Levin BC Rohlfs EM Schaefer FV Shrimpton AE Spector EB Stolle CA Strom CM Thibodeau SN Cole EC Goodman BK Stenzel TT Genetically characterized positive control cell lines derived from residual clinical blood samples.Clin Chem. 2005; 51: 2013-2024Crossref PubMed Scopus (9) Google Scholar, Q, DNA sequencing Michele Caggana, NYSDOH.¶ Cell line included in Coriell MUTCF Panel.∥ Allele included in the ACMG 23 Panel.** See reference # 3Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.Genet Med. 2004; 6: 387-391Crossref PubMed Scopus (374) Google Scholar.†† See reference # 21Heim RA Sugarman EA Allitto BA Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel.Genet Med. 2001; 3: 168-176Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar.‡‡ See reference # 22Monaghan KG Feldman GL Barbarotto GM Manji S Desai TK Snow K Frequency and clinical significance of the S1235R mutation in the cystic fibrosis transmembrane conductance regulator gene: results from a collaborative study.Am J Med Genet. 2000; 95: 361-365Crossref PubMed Scopus (14) Google Scholar. Open table in a new tab Twenty-three cell lines (Table 2, cell lines with †) containing the 23 mutations recommended for screening by ACMG/ACOG (Table 2, alleles with *) have been characterized previously and are available as a reference material panel from Coriell (Coriell Cell Repositories, Camden, NJ). For this study, we selected DNA samples containing 15 alleles that are not part of the 23 recommended by ACMG/ACOG to characterize based on their inclusion in a number of commercially available CF reagents. Seven of the selected alleles [E60X (c.178G>T), R347H (c.1040G>A), S549N (c.1647G>A), 1812-1G>A (c.1680-1G>A), Y1092X (c.3277C>A), 3876delA (c.3744delA), and 3905insT (c.3773_3774insT)] have also been suggested for inclusion in carrier screening panels based on their frequency in minority populations.3Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.Genet Med. 2004; 6: 387-391Crossref PubMed Scopus (374) Google Scholar Other alleles selected for study were already available from Coriell [R75X (c.223C>T), 444delA (c.312delA), G178R (c.532G>C), P574H (c.1721C>A), M1101K (c.3302T>A)], or are mutations that would interfere with the detection of F508del (c.1521_1523delCTT) [I506V (c.1516A>G), I507V (c.1519A>G)] or S549N [S549R (c.1646A>C)]. The fifteen experimental cell lines containing the 16 additional alleles were chosen for study based on the submitter's description of the CF mutation present. Six pre-existing Epstein-Barr virus-transformed lymphoblast cell lines were selected from the National Institute of General Medical Sciences Repository at the Coriell Cell Repositories (GM07857, GM11285, GM11287, GM11288 GM07732, and GM11370; Table 3). Nine new CF cell lines described in Table 3 (GM20737, GM20741, GM20745, GM20836, GM20925, GM20928, GM21551, GM20924, and GM20929) were generated for this study.Table 3Results of Multi-Laboratory Characterization StudiescDNA sequence change name*All cDNA sequence change HGVS nomenclature is based on the GenBank cDNA reference sequence NM_00492.3.Common nameCoriell #Allele 1 (# labs†Number of laboratories that successfully detected the mutation.)Allele 2 (# labs†Number of laboratories that successfully detected the mutation.)Allele 1 (# labs†Number of laboratories that successfully detected the mutation.)Allele 2 (# labs*All cDNA sequence change HGVS nomenclature is based on the GenBank cDNA reference sequence NM_00492.3.)Population frequency of allele(s)‡CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population.GM07857c.3302T>A (3)c.3302T>A (3)M1101K (3)M1101K (3)0.20%/0.20%§See Reference #21.GM11285c.1521_1523delCTT (6)c.3277C>A (4)F508del (6)Y1092X (4)66.31%‡CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population./0.12%¶See Reference #3.GM11287c.1521_1523delCTT (6)c.1721C>A (2)F508del (6)P574H (2)66.31%‡CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population./very low§See Reference #21.GM11288c.1521_1523delCTT (6)c.532G>C (3)F508del (6)G178R (3)66.31%‡CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population./0.2%§See Reference #21.GM11370c.312delA (3)c.1680-1G>A (AJ574983.1:g.92G>A (4)444delA (3)1812-1G>A (4)0.05§See Reference #21./0.02%¶See Reference #3.GM20737c.1040G>A (6)Wt (6)R347H (6)wt (6)0.06%¶See Reference #3.GM20741c.3744delA (6)Wt (6)3876delA (6)wt (6)0.03%¶See Reference #3.GM20745c.1647G>A (6)Wt (6)S549N (6)wt (6)0.14%¶See Reference #3.GM20836c.3773_3774insT (5)Wt (6)3905insT (5)wt (6)0.12%¶See Reference #3.GM20925c.1646A>C (5)Wt (6)S549R (5)wt (6)0.12%§See Reference #21.GM20928c.1521_1523delCTT (6)wt∥Cell line was tested for I506V mutation; however, the mutation was not detected and all laboratories reported as not detected (wt) instead. (6)F508del (6)wt∥Cell line was tested for I506V mutation; however, the mutation was not detected and all laboratories reported as not detected (wt) instead. (6)66.31%¶See Reference #3.GM21551c.1521_1523delCTT (5)c.1519A>G (4)F508del (5)I507V (4)66.31%¶See Reference #3./no dataGM20924c.223C>T (2)Wt (5)R75X (2)wt (5)no dataGM20929c.1521_1523delCTT (5)wt∥Cell line was tested for I506V mutation; however, the mutation was not detected and all laboratories reported as not detected (wt) instead. (5)F508del (5)wt∥Cell line was tested for I506V mutation; however, the mutation was not detected and all laboratories reported as not detected (wt) instead. (5)66.31%¶See Reference #3.GM07732c.1521_1523delCTT (3)c.178G>T (3)F508del (3)E60X (3)66.31%/0.12%¶See Reference #3.* All cDNA sequence change HGVS nomenclature is based on the GenBank cDNA reference sequence NM_00492.3.† Number of laboratories that successfully detected the mutation.‡ CTFR mutation frequency among clinically diagnosed cystic fibrosis individuals in a pan-ethnic U.S. population.§ See Reference #21Heim RA Sugarman EA Allitto BA Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel.Genet Med. 2001; 3: 168-176Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar.¶ See Reference #3Watson MS Cutting GR Desnick RJ Driscoll DA Klinger K Mennuti M Palomaki GE Popovich BW Pratt VM Rohlfs EM Strom CM Richards CS Witt DR Grody WW Cystic fibrosis population carrier screening: 2004 revision of American College of Medical Genetics mutation panel.Genet Med. 2004; 6: 387-391Crossref PubMed Scopus (374) Google Scholar.∥ Cell line was tested for I506V mutation; however, the mutation was not detected and all laboratories reported as not detected (wt) instead. Open table in a new tab Since the purpose of this project was to develop characterized reference materials that will be useful for many possible applications, we wanted to ensure that the characterization of these materials was as comprehensive and included as many assay platforms as possible. We compared the alleles detected in all commercially available CF reagents/platforms and selected those with the ability to detect the alleles included in our study. Some of the commercially available reagents/platforms (eg, from Osmetech and from Nanogen) were not used in this study because they detect primarily the ACMG/ACOG 23 panel, and not the additional alleles included in this study. We also included DNA sequence analysis and a laboratory developed assay based on the Luminex platform (LDT, Table 1) because of the ability to detect additional CF mutations. Clinical genetics laboratories performing CF testing using the various reagents/platforms were recruited to participate in this study. The assays used by the participating laboratories and the alleles detected by each are indicated in Table 1. The results of this study are summarized in Table 3. The expected genotype of each DNA sample was confirmed by all assay platforms designed to detect the alleles. The genotype of each DNA sample, and the number of laboratories/assays that detected each allele, and the published population frequency of each allele, are shown in Table 3. DNA from GM20928 and GM20929 was expected to carry the I506V (c.1516A>G) allele based on information from the submitter. However, this variant was not present when DNA was tested on all four of the assay platforms capable of detecting it. In addition, DNA sequence analysis did not detect the I506V (c.1516A>G) allele in either cell line. No false-positive or other discordant results were reported among the laboratories. A panel of DNA from 23 cell lines representing the 23 CF alleles recommended by ACMG/ACOG is available from Coriell (Coriell Cell Repositories, Camden, NJ). The mutations in some these DNA samples had been confirmed by analysis in numerous laboratories as part of the project that created them.23Bernacki SH Beck JC Stankovic AK Williams LO Amos J Snow-Bailey K Farkas DH Friez MJ Hantash FM Matteson KJ Monaghan KG Muralidharan K Pratt VM Prior TW Richie KL Levin BC Rohlfs EM Schaefer FV Shrimpton AE Spector EB Stolle CA Strom CM Thibodeau SN Cole EC Goodman BK Stenzel TT Genetically characterized positive control cell lines derived from residual clinical blood samples.Clin Chem. 2005; 51: 2013-2024Crossref PubMed Scopus (9) Google Scholar Alleles in the remaining ACMG/ACOG 23 samples and DNA samples from some additional CF cell lines (not included in the ACMG/ACOG 23) have been characterized by DNA sequence analysis (reported in this work). These results are summarized in Table 2. CF carrier screening is widespread with at least 66 laboratories in the United States currently offering some form of CF testing (National Institutes of Health, www.genetests.com, 05/13/2008). A higher estimate of CF laboratories can be made by examining the number of participants in the CAP CF surveys [150 laboratories in 2007 based on CAP Participant Summaries, MGL 2007 (reviewed by VMP)]. Note the number of laboratories performing CF testing in the CAP surveys is different from the number of laboratories listed in GeneTests. GeneTests is a voluntary registry and the CAP participating laboratories may include laboratories outside of the United States and laboratories that do not offer clinical CF testing. In addition, CF testing is offered in many hospital laboratories and, in the public health setting, to 3 to 4 million newborns per year (National Newborn Screening and Genetics Resource Center, http://genes-r-us.uthscsa.edu/nbsdisorders.htm, 08/14/2008). Due to a lack of available materials for use in quality assurance, proficiency testing, assay validation, and research, we undertook this study to expand the number of characterized mutations in cell lines that can be used as reference materials for CF genetic testing.
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