ColoSeq Provides Comprehensive Lynch and Polyposis Syndrome Mutational Analysis Using Massively Parallel Sequencing
2012; Elsevier BV; Volume: 14; Issue: 4 Linguagem: Inglês
10.1016/j.jmoldx.2012.03.002
ISSN1943-7811
AutoresColin C. Pritchard, Christina Smith, Stephen J. Salipante, Ming K. Lee, Anne Thornton, Alex S. Nord, Cassandra Gulden, Sonia S. Kupfer, Elizabeth M. Swisher, Robin L. Bennett, Akiva P. Novetsky, Gail P. Jarvik, Olufunmilayo I. Olopade, Paul J. Goodfellow, Mary‐Claire King, Jonathan F. Tait, Tom Walsh,
Tópico(s)Colorectal Cancer Screening and Detection
ResumoLynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of mutation detection between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits. Lynch syndrome (hereditary nonpolyposis colon cancer) and adenomatous polyposis syndromes frequently have overlapping clinical features. Current approaches for molecular genetic testing are often stepwise, taking a best-candidate gene approach with testing of additional genes if initial results are negative. We report a comprehensive assay called ColoSeq that detects all classes of mutations in Lynch and polyposis syndrome genes using targeted capture and massively parallel next-generation sequencing on the Illumina HiSeq2000 instrument. In blinded specimens and colon cancer cell lines with defined mutations, ColoSeq correctly identified 28/28 (100%) pathogenic mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH, including single nucleotide variants (SNVs), small insertions and deletions, and large copy number variants. There was 100% reproducibility of mutation detection between independent runs. The assay correctly identified 222 of 224 heterozygous SNVs (99.4%) in HapMap samples, demonstrating high sensitivity of calling all variants across each captured gene. Average coverage was greater than 320 reads per base pair when the maximum of 96 index samples with barcodes were pooled. In a specificity study of 19 control patients without cancer from different ethnic backgrounds, we did not find any pathogenic mutations but detected two variants of uncertain significance. ColoSeq offers a powerful, cost-effective means of genetic testing for Lynch and polyposis syndromes that eliminates the need for stepwise testing and multiple follow-up clinical visits. Defects in mismatch repair (MMR) are responsible for hereditary nonpolyposis colorectal cancer, also known as Lynch syndrome. Inherited loss of function mutations in MLH1, MSH2, MSH6, PMS2, and EPCAM result in a 25% to 75% lifetime risk of colon cancer and up to a 60% lifetime risk of endometrial cancer in women.1Jenkins M.A. Hayashi S. O'Shea A.M. Burgart L.J. Smyrk T.C. Shimizu D. Waring P.M. Ruszkiewicz A.R. Pollett A.F. Redston M. Barker M.A. Baron J.A. Casey G.R. Dowty J.G. Giles G.G. Limburg P. Newcomb P. Young J.P. Walsh M.D. Thibodeau S.N. Lindor N.M. Lemarchand L. Gallinger S. Haile R.W. Potter J.D. Hopper J.L. Jass J.R. Pathology features in Bethesda guidelines predict colorectal cancer microsatellite instability: a population-based study.Gastroenterology. 2007; 133: 48-56Google Scholar The population prevalence of Lynch syndrome is estimated to be as high as 1 in 440,2Kohlmann W. Gruber S.B. Lynch Syndrome In GeneReviews [Internet]. Copyright University of Washington, Seattle1993–2012http://www.ncbi.nlm.nih.gov/books/NBK1211Google Scholar making it the most common inherited cancer predisposition syndrome. Germline mutations in APC and MUTYH result in adenomatous polyposis syndromes that are also associated with a very high lifetime risk of colorectal cancer. Mutations in APC cause familial adenomatous polyposis, Gardner's syndrome, Turcot's syndrome, and attenuated familial adenomatous polyposis, whereas MUTYH mutations are the cause of autosomal recessive MUTYH-associated polyposis syndrome.3Jasperson K.W. Tuohy T.M. Neklason D.W. Burt R.W. Hereditary and familial colon cancer.Gastroenterology. 2010; 138: 2044-2058Google Scholar Even with clinical guidelines such as the Revised Bethesda and Amsterdam II criteria,4American Gastroenterological Association medical position statement: hereditary colorectal cancer and genetic testing.Gastroenterology. 2001; 121: 195-197Google Scholar it can be challenging to distinguish Lynch syndrome from adenomatous polyposis syndromes on the basis of clinical features, particularly with regard to attenuated familial adenomatous polyposis and Lynch syndrome, which present at a similar median age of cancer onset (∼45 to 60 years), and with similar numbers of colonic polyps.5Cao Y. Pieretti M. Marshall J. Khattar N.H. Chen B. Kam-Morgan L. Lynch H. Challenge in the differentiation between attenuated familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer: case report with review of the literature.Am J Gastroenterol. 2002; 97: 1822-1827Google Scholar, 6Jasperson K.W. Blazer K.R. Lowstuter K. Weitzel J.N. Working through a diagnostic challenge: colonic polyposis Amsterdam criteria, and a mismatch repair mutation.Fam Cancer. 2008; 7: 281-285Google Scholar Clinicians are therefore frequently faced with the dilemma of which gene or genes to test first when ordering expensive genetic testing using standard Sanger sequencing.A complex battery of tumor-based screening tests have been developed for Lynch syndrome, in part because of the high cost of conventional germline genetic testing. These tests include functional assessment of defective MMR genes either by demonstration of genomic microsatellite instability (MSI) or loss of MLH1, MSH2, MSH6, or PMS2 protein expression by immunohistochemistry (IHC), BRAF V600E mutational analysis, and MLH1 promoter hypermethylation analysis (screening algorithms are reviewed in Pritchard and Grady7Pritchard C.C. Grady W.M. Colorectal cancer molecular biology moves into clinical practice.Gut. 2011; 60: 116-129Google Scholar). Loss of a specific MMR protein revealed by IHC can be helpful in suggesting which gene most likely harbors a germline mutation. MMR proteins are often lost as pairs (MLH1/PMS2 or MSH2/MSH6) because they function as heterodimers. However, mutations in PMS2 and MSH6 are more likely to result in isolated loss of the corresponding protein by IHC because they are minor partners in the heterodimer.8Hall G. Clarkson A. Shi A. Langford E. Leung H. Eckstein R.P. Gill A.J. Immunohistochemistry for PMS2 and MSH6 alone can replace a four antibody panel for mismatch repair deficiency screening in colorectal adenocarcinoma.Pathology. 2010; 42: 409-413Google Scholar No tumor-based screening tests are currently available for polyposis syndromes.Massively parallel next-generation sequencing technology has dramatically increased throughput and reduced the cost per nucleotide sequenced compared with traditional Sanger methods, enabling cost-effective sequencing of multiple genes simultaneously in the clinical laboratory setting.9Hu H. Wrogemann K. Kalscheuer V. Tzschach A. Richard H. Haas S.A. Menzel C. Bienek M. Froyen G. Raynaud M. Van Bokhoven H. Chelly J. Ropers H. Chen W. Mutation screening in 86 known X-linked mental retardation genes by droplet-based multiplex PCR and massive parallel sequencing.Hugo J. 2009; 3: 41-49Google Scholar, 10Meder B. Haas J. Keller A. Heid C. Just S. Borries A. Boisguerin V. Scharfenberger-Schmeer M. Stahler P. Beier M. Weichenhan D. Strom T.M. Pfeufer A. Korn B. Katus H.A. Rottbauer W. Targeted next-generation sequencing for the molecular genetic diagnostics of cardiomyopathies.Circ Cardiovasc Genet. 2011; 4: 110-122Google Scholar, 11Voelkerding K.V. Dames S. Durtschi J.D. Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology.J Mol Diagn. 2010; 12: 539-551Google Scholar, 12Walsh T. Lee M.K. Casadei S. Thornton A.M. Stray S.M. Pennil C. Nord A.S. Mandell J.B. Swisher E.M. King M.C. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.Proc Natl Acad Sci U S A. 2010; 107: 12629-12633Google Scholar, 13Morgan J.E. Carr I.M. Sheridan E. Chu C.E. Hayward B. Camm N. Lindsay H.A. Mattocks C.J. Markham A.F. Bonthron D.T. Taylor G.R. Genetic diagnosis of familial breast cancer using clonal sequencing.Human Mutat. 2010; 31: 484-491Google Scholar Target enrichment is generally required to achieve adequate read depth for accurate identification of the spectrum of mutations, including large genomic rearrangements, small insertions and deletions (indels), and SNVs.14Mamanova L. Coffey A.J. Scott C.E. Kozarewa I. Turner E.H. Kumar A. Howard E. Shendure J. Turner D.J. Target-enrichment strategies for next-generation sequencing.Nat Methods. 2010; 7: 111-118Google Scholar We recently reported a proof-of-principle study demonstrating the accuracy and feasibility of solution-based targeted capture and next-generation sequencing for 21 genes that are associated with breast and ovarian cancer risk.12Walsh T. Lee M.K. Casadei S. Thornton A.M. Stray S.M. Pennil C. Nord A.S. Mandell J.B. Swisher E.M. King M.C. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.Proc Natl Acad Sci U S A. 2010; 107: 12629-12633Google Scholar Here we report the validation results of ColoSeq, a clinical diagnostic assay for hereditary colon cancer that detects single nucleotide, indel, and deletion/duplication mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH. The ColoSeq assay can be performed for approximately the same cost as performing sequencing and deletion/duplication analysis of a single gene by traditional methods, and with equivalent or better sensitivity and accuracy, eliminating the need for stepwise molecular genetic testing in patients with suspected Lynch or polyposis syndromes.Materials and MethodsDNA SamplesWe tested a total of 82 unique DNA samples, including 23 peripheral blood DNA samples from patients with known mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, or MUTYH; 31 peripheral blood DNA samples from patients with a clinical history suggestive of Lynch or polyposis syndrome; 19 peripheral blood DNA samples from patients without a known family history of cancer; 6 publicly available DNA samples from the HapMap project;15Frazer K.A. Ballinger D.G. Cox D.R. Hinds D.A. Stuve L.L. Gibbs R.A. et al.A second generation human haplotype map of over 3.1 million SNPs.Nature. 2007; 449: 851-861Google Scholar and 3 DNA samples from colon cancer cell lines known to harbor mutations in MMR genes and/or APC. Tumor cell lines (LoVo, HCT116, LS174T) were obtained from William M. Grady (University of Washington). HapMap samples (NA18507, NA18558, NA07019, NA07348, NA10857, NA10851) were obtained from Coriell Cell Repositories (Camden, New Jersey). Clinical specimens were obtained in accordance with the Declaration of Helsinki and the study was approved by the Human Subjects Division of the University of Washington (protocol 34173) and the University of Chicago Institutional Review Board.Library Construction, Gene Capture, and Massively Parallel SequencingThree micrograms of DNA was sonicated to a peak of 200 bp on a Covaris S2 instrument (Covaris, Woburn, MA) in 1× low TE (10 mmol/L Tris/0.1 mmol/L EDTA) for 6 minutes using frequency sweeping mode with duty cycle, 10%; intensity, 5; cycles per burst, 200 at a temperature 4° to 7°C. After sonication, DNA was purified with AMPure XP beads (Beckman Coulter, Brea CA) and subjected to three enzymatic steps: end repair, A-tailing, and ligation to Illumina paired-end adapters as described in the SureSelectXT Target Enrichment for Illumina multiplexed sequencing that is available for free download. All liquid handling steps were performed in 96-well plates on a Bravo liquid-handling instrument (Agilent Technologies, Santa Clara, CA). The adapter-ligated library was amplified by polymerase chain reaction (PCR) for five cycles with Illumina primers 1.0 and 2.0 and quantified by a DNA1000 chip on a Bioanalyzer 2100 instrument (Agilent Technologies). Individual paired-end libraries (500 ng) were hybridized to a custom design of complementary RNA (cRNA) biotinylated oligonucleotides targeting 31 genes in 30 genomic regions (see Supplemental Table S1 at http://jmd.amjpathol.org). The 120-mer oligonucleotide baits were designed in Agilent's eArray web portal with the following parameters: centered tiling, 3x bait overlap and a maximum overlap of 20 bp into repetitive regions. The custom design targets a total of 1.1 Mb of DNA including 209 kb in MLH1, MSH2, MSH6, PMS2, EPCAM, APC, and MUTYH (Table 1). The BED file of probe sequences is available on request. After 24 hours of hybridization at 65°C, the library-bait hybrids were purified by incubation with streptavidin-bound T1 Dynabeads (LifeTechnologies, Carlsbad CA) and washed with increasing stringency to remove nonspecific binding. After capture, each library was amplified by PCR directly on the Dynabeads for 13 cycles with primers containing a unique 6-bp index (Table 2). After PCR amplification, the libraries were quantified by a high-sensitivity chip on a Bioanalyzer 2100 instrument (Agilent Technologies). Equimolar concentrations of 96 libraries were pooled to a final concentration of 10 pM, denatured with 3N NaOH, and cluster amplified with a cBot instrument on a single lane of an Illumina v3 flow cell. Sequencing was performed with 2 × 101-bp paired-end reads and a 7-bp index read using SBS v3 chemistry on a HiSeq2000 (Illumina, Inc, San Diego, CA).Table 1Genes Validated for ColoSeqGeneChromosomeKb targeted⁎Repetitive DNA elements were not targeted.Genomic region targeted (Hg 19)StartEndMLH13353702997937097337MSH2 + EPCAM2484759526347715360MSH62194800522148039092PMS271760078706053737APC577112038202112186936MUTYH1144578991445811142A total of 30 genomic regions representing 31 genes related to hereditary cancer risk were targeted19Walsh T. Casadei S. Lee M.K. Pennil C.C. Nord A.S. Thornton A.M. Roeb W. Agnew K.J. Stray S.M. Wickramanayake A. Norquist B. Pennington K.P. Garcia R.L. King M.C. Swisher E.M. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.Proc Natl Acad Sci U S A. 2011; 108: 18032-18037Google Scholar, but here we focus on validation of these 7 genes. Repetitive DNA elements were not targeted. Open table in a new tab Table 2Postindex BarcodesATCACGAACAAAAATAGGACTCTCCGATGTCACGATCCACGCCGGAATTTAGGCGATATAGCTCCAGTGGCCTGACCATATAATTCGGCATGCTGGACAGTGAACCCCACAAACACTGATGCCAATCACTCACCCATGCTAGCTCAGATCGATGCTGGCACAGTTTCGACTTGATCATTCTCTACCTGGCGCGATCAGAACTTGACATCTAGAAGATAGCTTCAGGCGCCCCCTCTATACGGCTACGCAAGGGGCCTGCGTACGCTTGTAATAATTATCCTAATGAGCAAACATAAGACTACCCAGAGATAGCAAAAGCATGGCCCGCAACTCAGAGAAACCGCACTTGTAGAGGAGTGGTAATCGTCCCGATGAATGTTCGAAAAAGCAAAGCGAACCGGCAGCATCCAACTACATTTTCCTTAGCTGCTGGAATAAGCCGCGGTCCGCGGTAGCTACAGCTCGAAGTGCCATTTCTCCAAATGCAAGGACACGATAAGCGCTCACCGGCCAACACGAGAACCGTCCGACGGAGCCTTAGTGAAAATTCCTAGGCCGATACGGATCTATAGGTTT Open table in a new tab Mutation AnalysisSequence alignment and variant calling were performed against the reference human genome (UCSC hg19). Sequencing reads were aligned using MAQ,16Li H. Ruan J. Durbin R. Mapping short DNA sequencing reads and calling variants using mapping quality scores.Genome Res. 2008; 18: 1851-1858Google Scholar and SNVs and insertions and deletions were detected as previously described.12Walsh T. Lee M.K. Casadei S. Thornton A.M. Stray S.M. Pennil C. Nord A.S. Mandell J.B. Swisher E.M. King M.C. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.Proc Natl Acad Sci U S A. 2010; 107: 12629-12633Google Scholar Each variant was annotated with respect to gene location and predicted function in Human Genome Variation Society nomenclature. Deletions and duplications of exons were detected by depth of coverage analysis.17Nord A.S. Lee M. King M.C. Walsh T. Accurate and exact CNV identification from targeted high-throughput sequence data.BMC Genomics. 2011; 12: 184Google Scholar All previously unidentified frameshift, nonsense, and splice site mutations predicted to be deleterious to protein function were confirmed by PCR amplification and Sanger sequencing. Exonic deletions and duplications were confirmed by multiplex ligation-dependent probe amplification or gap-PCR and direct sequencing.18Walsh T. Casadei S. Coats K.H. Swisher E. Stray S.M. Higgins J. Roach K.C. Mandell J. Lee M.K. Ciernikova S. Foretova L. Soucek P. King M.C. Spectrum of mutations in BRCA1, BRCA2, CHEK2, and TP53 in families at high risk of breast cancer.JAMA. 2006; 295: 1379-1388Google ScholarResultsColoSeq AssayThe objective of our study was to evaluate the performance of targeted DNA capture and massively parallel sequencing for the detection of inherited mutations in colon cancer in the clinical laboratory setting. We designed oligonucleotides to target all exons, introns, and approximately 10 kb of 5′ and 3′ flanking genomic regions of the seven genes that are most commonly responsible for inherited risk of colon cancer (Table 1). Our capture panel also includes 24 other genes that rarely harbor mutations causing colon cancer, endometrial cancer, and other solid tumors,19Walsh T. Casadei S. Lee M.K. Pennil C.C. Nord A.S. Thornton A.M. Roeb W. Agnew K.J. Stray S.M. Wickramanayake A. Norquist B. Pennington K.P. Garcia R.L. King M.C. Swisher E.M. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.Proc Natl Acad Sci U S A. 2011; 108: 18032-18037Google Scholar for a total of 31 captured genes and 1.1 Mb of captured DNA after removal of repetitive sequences. Here we focus on validation results of the seven genes listed in Table 1 that constitute the ColoSeq assay for Lynch and polyposis syndromes.Workflow of the ColoSeq assay is summarized in Figure 1. We begin by fragmenting genomic DNA extracted from peripheral blood and preparing libraries with an average insert size of approximately 200 bp. ColoSeq genes are captured via solution hybridization with complementary oligonucleotide RNA (cRNA) baits (Agilent SureSelect). Each captured sample library is amplified by PCR to incorporate a unique index barcode, and up to 96 barcoded samples are pooled and run on a single lane of an Illumina HiSeq2000 instrument.It is unusual to pool patient samples in the clinical setting because of the risks of sample misidentification and analytic interference. To control for specimen mix-up and for potential barcode-specific sequencing biases, each clinical sample is indexed with two distinct barcodes and run in duplicate. Barcodes were selected such that a single base change could not lead to one barcode being misidentified as another (Table 2). Less than 0.005% of sequencing reads were assigned to the incorrect patient because of barcode misidentification.Paired-end 2 × 101-bp reads are generated at a median of 320-fold coverage per nucleotide across the entire targeted region (range of median coverage between samples 145-fold to 556-fold; Figure 2A). The median coverage specifically across the seven ColoSeq genes is 475-fold (Figure 2B). Raw sequence of 0.3 gigabases (Gb) (range 0.15 to 0.56 Gb) of high-quality (>Q30) sequence per sample is generated when the maximum 96 samples are pooled within a lane. On average, 62% of sequence reads are on target, mapping specifically to the captured regions. We align sequences to the reference genome (build Hg 19) and select variants for further analysis that meet the following criteria: i) variant is present in sequence reads from both strands (mean ± SD, 433 ± 97 variants/sample), ii) population frequency of the variant is less than 5% (44 ± 31 variants/sample remain), and iii) variant represents at least 5% of the sequence reads at a particular site (26 ± 21 variants/sample remain). We chose a threshold of 5% variant reads to ensure that variants in highly segmentally duplicated regions of genes would be detected. Frequencies of variants among individuals without colon cancer are based on an internal database of more than 1000 unique patients from different ethnic backgrounds who have had targeted capture and massively parallel sequencing performed for these genes.12Walsh T. Lee M.K. Casadei S. Thornton A.M. Stray S.M. Pennil C. Nord A.S. Mandell J.B. Swisher E.M. King M.C. Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.Proc Natl Acad Sci U S A. 2010; 107: 12629-12633Google Scholar, 19Walsh T. Casadei S. Lee M.K. Pennil C.C. Nord A.S. Thornton A.M. Roeb W. Agnew K.J. Stray S.M. Wickramanayake A. Norquist B. Pennington K.P. Garcia R.L. King M.C. Swisher E.M. Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.Proc Natl Acad Sci U S A. 2011; 108: 18032-18037Google Scholar By using an internal frequency database, we can quickly filter out both common benign variants and potential recurrent artifactual variant calls, leaving an average of 20 noncoding and only 6 rare coding or splice junction variants per patient across the seven genes. These filtered variants are then compared with the exome variant server (NHLBI Exome Sequencing Project [ESP], Seattle, WA), dbSNP, and public mutation databases such as InSight20Kohonen-Corish M.R. Macrae F. Genuardi M. Aretz S. Bapat B. Bernstein I.T. Burn J. Cotton R.G. den Dunnen J.T. Frebourg T. Greenblatt M.S. Hofstra R. Holinski-Feder E. Lappalainen I. Lindblom A. Maglott D. Moller P. Morreau H. Moslein G. Sijmons R. Spurdle A.B. Tavtigian S. Tops C.M. Weber T.K. de Wind N. Woods M.O. Deciphering the colon cancer genes–report of the InSiGHT-Human Variome Project Workshop.Human Mutat. 2011; 32 (UNESCO, Paris 2010): 491-494Google Scholar (http://www.insight-group.org/mutations) and results reviewed by a laboratory director. Novel variants of uncertain significance (VUS) are analyzed by PolyPhen2,21Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat Methods. 2010; 7: 248-249Google Scholar SIFT,22Kumar P. Henikoff S. Ng P.C. Predicting the effects of coding non-synonymous variants on protein function using the SIFT algorithm.Nature Protoc. 2009; 4: 1073-1081Google Scholar and MutationTaster23Schwarz J.M. Rodelsperger C. Schuelke M. Seelow D. MutationTaster evaluates disease-causing potential of sequence alterations.Nat Methods. 2010; 7: 575-576Google Scholar to predict potential deleterious effects and are assessed for evolutionary conservation. Pathogenic mutations and VUS are confirmed by Sanger sequencing or multiplex ligation-dependent probe amplification (for deletions/duplications). Missense variants that are not well characterized are reported as VUS when they meet the following criteria: i) population frequency 1000-fold reflect capture of highly homologous genomic regions.View Large Image Figure ViewerDownload Hi-res image Download (PPT)SensitivityTo assess the sensitivity of the ColoSeq assay to detect pathogenic mutations, we blindly tested peripheral blood DNA samples from 23 cancer patients and 3 colon cancer cell lines (LS174T, HCT116, LoVo) with previously defined germline mutations in MLH1, MSH2, MSH6, PMS2, EPCAM, MUTYH, or APC. Some cell lines harbored more than one mutation. ColoSeq correctly identified all mutations (23/23 patient mutations, 5/5 cell line mutations, 100% sensitivity) including nonsense, missense, frameshift, in-frame deletions, splice site, and large deletions and duplications (Table 3, cohorts: known and cell line).24Rowan A.J. Lamlum H. Ilyas M. Wheeler J. Straub J. Papadopoulou A. Bicknell D. Bodmer W.F. Tomlinson I.P. 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