A Comprehensive Strategy for Accurate Mutation Detection of the Highly Homologous PMS2
2015; Elsevier BV; Volume: 17; Issue: 5 Linguagem: Inglês
10.1016/j.jmoldx.2015.04.001
ISSN1943-7811
AutoresJianli Li, Hongzheng Dai, Yanming Feng, Jia Tang, Stella Chen, Tian Xia, Elizabeth Gorman, Eric Schmitt, Terah A.A. Hansen, Jing Wang, Sharon E. Plon, Victor Wei Zhang, Lee‐Jun C. Wong,
Tópico(s)Genomic variations and chromosomal abnormalities
ResumoGermline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3′ region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Germline mutations in the DNA mismatch repair gene PMS2 underlie the cancer susceptibility syndrome, Lynch syndrome. However, accurate molecular testing of PMS2 is complicated by a large number of highly homologous sequences. To establish a comprehensive approach for mutation detection of PMS2, we have designed a strategy combining targeted capture next-generation sequencing (NGS), multiplex ligation-dependent probe amplification, and long-range PCR followed by NGS to simultaneously detect point mutations and copy number changes of PMS2. Exonic deletions (E2 to E9, E5 to E9, E8, E10, E14, and E1 to E15), duplications (E11 to E12), and a nonsense mutation, p.S22*, were identified. Traditional multiplex ligation-dependent probe amplification and Sanger sequencing approaches cannot differentiate the origin of the exonic deletions in the 3′ region when PMS2 and PMS2CL share identical sequences as a result of gene conversion. Our approach allows unambiguous identification of mutations in the active gene with a straightforward long-range-PCR/NGS method. Breakpoint analysis of multiple samples revealed that recurrent exon 14 deletions are mediated by homologous Alu sequences. Our comprehensive approach provides a reliable tool for accurate molecular analysis of genes containing multiple copies of highly homologous sequences and should improve PMS2 molecular analysis for patients with Lynch syndrome. Heterozygous germline mutations in DNA mismatch repair genes MLH1, MSH2, MSH6, and PMS2 contribute to hereditary nonpolyposis colorectal cancer, or Lynch syndrome, which accounts for 2% to 4% of all colorectal cancers.1Barrow E. Hill J. Evans D.G. Cancer risk in Lynch Syndrome.Fam Cancer. 2013; 12: 229-240Crossref PubMed Scopus (141) Google Scholar Carrier individuals are at increased risk of developing cancers, including colorectal and endometrial cancers.2Lynch H. Chapelle A. Hereditary colorectal cancer.N Engl J Med. 2003; 348: 14Google Scholar In rare situations, biallelic mutations in PMS2 cause constitutional mismatch repair deficiency syndrome, which is characterized by childhood-onset malignancy, including brain cancers, hematological malignancies, colorectal cancers, and multiple intestinal polyposis.3Herkert J.C. Niessen R.C. Olderode-Berends M.J. Veenstra-Knol H.E. Vos Y.J. van der Klift H.M. Scheenstra R. Tops C.M. Karrenbeld A. Peters F.T. Hofstra R.M. Kleibeuker J.H. Sijmons R.H. Paediatric intestinal cancer and polyposis due to bi-allelic PMS2 mutations: case series, review and follow-up guidelines.Eur J Cancer. 2011; 47: 965-982Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 4Bougeard G. Olivier-Faivre L. Baert-Desurmont S. Tinat J. Martin C. Bouvignies E. Vasseur S. Huet F. Couillault G. Vabres P. Le Pessot F. Chapusot C. Malka D. Bressac-de Paillerets B. Tosi M. Frebourg T. Diversity of the clinical presentation of the MMR gene biallelic mutations.Fam Cancer. 2014; 13: 131-135Crossref PubMed Scopus (19) Google Scholar, 5Stark Z. Campbell L.J. Mitchell C. James P.A. Heath J.A. Boussioutas A. Lynch C. Trainer A.H. Clinical problem-solving. Spot diagnosis.N Engl J Med. 2014; 370: 2229-2236Crossref PubMed Scopus (7) Google Scholar PMS2 is the most common cause of this recessive condition. Accurate molecular testing of these genes will improve the diagnosis and help with appropriate genetic counseling and medical management. It was originally thought that most of Lynch syndrome–associated mutations were due to defective MLH1 and MSH2.6Peltomaki P. Lynch syndrome genes.Fam Cancer. 2005; 4: 227-232Crossref PubMed Scopus (193) Google Scholar PMS2, identified in 1994,7Nicholaides N. Papadopoilos N. Liu B. Wei Y.-F. Cater K. Ruben S. Mutations of two PMS homologues in hereditary nonpolyposis colon cancer.Nature. 1994; 371: 6PubMed Google Scholar was considered to play a minor role in Lynch syndrome.6Peltomaki P. Lynch syndrome genes.Fam Cancer. 2005; 4: 227-232Crossref PubMed Scopus (193) Google Scholar, 8van der Klift H. Wijnen J. Wagner A. Verkuilen P. Tops C. Otway R. Kohonen-Corish M. Vasen H. Oliani C. Barana D. Moller P. Delozier-Blanchet C. Hutter P. Foulkes W. Lynch H. Burn J. Moslein G. Fodde R. Molecular characterization of the spectrum of genomic deletions in the mismatch repair genes MSH2, MLH1, MSH6, and PMS2 responsible for hereditary nonpolyposis colorectal cancer (HNPCC).Genes Chromosomes Cancer. 2005; 44: 123-138Crossref PubMed Scopus (106) Google Scholar, 9Hendriks Y.M. Jagmohan-Changur S. van der Klift H.M. Morreau H. van Puijenbroek M. Tops C. van Os T. Wagner A. Ausems M.G. Gomez E. Breuning M.H. Brocker-Vriends A.H. Vasen H.F. Wijnen J.T. Heterozygous mutations in PMS2 cause hereditary nonpolyposis colorectal carcinoma (Lynch syndrome).Gastroenterology. 2006; 130: 312-322Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar This appears to be an underestimate because of the relatively lower penetrance for PMS2,10Clendenning M. Hampel H. LaJeunesse J. Lindblom A. Lockman J. Nilbert M. Senter L. Sotamaa K. de la Chapelle A. Long-range PCR facilitates the identification of PMS2-specific mutations.Hum Mutat. 2006; 27: 490-495Crossref PubMed Scopus (81) Google Scholar, 11Senter L. Clendenning M. Sotamaa K. Hampel H. Green J. Potter J.D. Lindblom A. Lagerstedt K. Thibodeau S.N. Lindor N.M. Young J. Winship I. Dowty J.G. White D.M. Hopper J.L. Baglietto L. Jenkins M.A. de la Chapelle A. The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.Gastroenterology. 2008; 135: 419-428Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar causing kindreds to be less likely to fit the Amsterdam criteria for Lynch syndrome.11Senter L. Clendenning M. Sotamaa K. Hampel H. Green J. Potter J.D. Lindblom A. Lagerstedt K. Thibodeau S.N. Lindor N.M. Young J. Winship I. Dowty J.G. White D.M. Hopper J.L. Baglietto L. Jenkins M.A. de la Chapelle A. The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.Gastroenterology. 2008; 135: 419-428Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar In addition, the presence of a large number of highly homologous sequences in other genomic regions and the frequent sequence exchange between the active and pseudogenes hindered the accurate molecular analysis of PMS2.12De Vos M. Hayward B.E. Picton S. Sheridan E. Bonthron D.T. Novel PMS2 pseudogenes can conceal recessive mutations causing a distinctive childhood cancer syndrome.Am J Hum Genet. 2004; 74: 954-964Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Nakagawa H. Lockman J. Negative for PMS2 protein, but paralogous genes obscure mutations are frequent in patients whose tumors stain mismatch repair gene PMS2: disease-causing germline mutation detection and interpretation.Cancer Res. 2004; 64: 7Crossref PubMed Scopus (58) Google Scholar, 14Hayward B.E. De Vos M. Valleley E.M. Charlton R.S. Taylor G.R. Sheridan E. Bonthron D.T. Extensive gene conversion at the PMS2 DNA mismatch repair locus.Hum Mutat. 2007; 28: 424-430Crossref PubMed Scopus (46) Google Scholar, 15Ganster C. Wernstedt A. Kehrer-Sawatzki H. Messiaen L. Schmidt K. Rahner N. Heinimann K. Fonatsch C. Zschocke J. Wimmer K. Functional PMS2 hybrid alleles containing a pseudogene-specific missense variant trace back to a single ancient intrachromosomal recombination event.Hum Mutat. 2010; 31: 552-560PubMed Google Scholar, 16van der Klift H.M. Tops C.M. Bik E.C. Boogaard M.W. Borgstein A.M. Hansson K.B. Ausems M.G. Gomez Garcia E. Green A. Hes F.J. Izatt L. van Hest L.P. Alonso A.M. Vriends A.H. Wagner A. van Zelst-Stams W.A. Vasen H.F. Morreau H. Devilee P. Wijnen J.T. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.Hum Mutat. 2010; 31: 578-587PubMed Google Scholar The high frequency of negative staining of PMS2 immunohistochemistry in colorectal cancer tissues without identifiable PMS2 mutations suggests that PMS2 mutations have been grossly underestimated as a result of these technical difficulties.11Senter L. Clendenning M. Sotamaa K. Hampel H. Green J. Potter J.D. Lindblom A. Lagerstedt K. Thibodeau S.N. Lindor N.M. Young J. Winship I. Dowty J.G. White D.M. Hopper J.L. Baglietto L. Jenkins M.A. de la Chapelle A. The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.Gastroenterology. 2008; 135: 419-428Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 13Nakagawa H. Lockman J. Negative for PMS2 protein, but paralogous genes obscure mutations are frequent in patients whose tumors stain mismatch repair gene PMS2: disease-causing germline mutation detection and interpretation.Cancer Res. 2004; 64: 7Crossref PubMed Scopus (58) Google Scholar, 17Baudhuin L.M. Burgart L.J. Leontovich O. Thibodeau S.N. Use of microsatellite instability and immunohistochemistry testing for the identification of individuals at risk for Lynch syndrome.Fam Cancer. 2005; 4: 255-265Crossref PubMed Scopus (90) Google Scholar, 18Truninger K. Menigatti M. Luz J. Russell A. Haider R. Gebbers J.-O. Bannwart F. Yurtsever H. Neuweiler J. Riehle H.-M. Cattaruzza M.S. Heinimann K. Schär P. Jiricny J. Marra G. Immunohistochemical analysis reveals high frequency of PMS2 defects in colorectal cancer.Gastroenterology. 2005; 128: 1160-1171Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar Recent advancements in technologies and reagents have significantly improved the molecular diagnostic yield of PMS2 in cancer patients with tumor specimens that demonstrate negative PMS2 immunohistochemistry and normal MLH1 staining.19Bowles K, Leclair B, Rajamani S: Representation of PMS2 mutations in patients seeking genetic testing for Lynch syndrome [abstract]. Presented at the 2012 ACMG Annual Clinical Genetics Meeting, 2012 March 29, Charlotte, NCGoogle Scholar, 20Schofield L. Grieu F. Goldblatt J. Amanuel B. Iacopetta B. A state-wide population-based program for detection of lynch syndrome based upon immunohistochemical and molecular testing of colorectal tumours.Fam Cancer. 2012; 11: 1-6Crossref PubMed Scopus (17) Google Scholar, 21Vaughn C.P. Baker C.L. Samowitz W.S. Swensen J.J. The frequency of previously undetectable deletions involving 3' exons of the PMS2 gene.Genes Chromosomes Cancer. 2013; 52: 107-112Crossref PubMed Scopus (22) Google Scholar However, a comprehensive approach to unequivocally identify PMS2 mutations remains to be developed. Molecular testing of PMS2 is especially challenging because of the presence of the PMS2CL pseudogene, which has more than 98% sequence identity with the 3′ region of PMS2, including exons 9 and 11 to 15 (Supplemental Table S1).12De Vos M. Hayward B.E. Picton S. Sheridan E. Bonthron D.T. Novel PMS2 pseudogenes can conceal recessive mutations causing a distinctive childhood cancer syndrome.Am J Hum Genet. 2004; 74: 954-964Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 13Nakagawa H. Lockman J. Negative for PMS2 protein, but paralogous genes obscure mutations are frequent in patients whose tumors stain mismatch repair gene PMS2: disease-causing germline mutation detection and interpretation.Cancer Res. 2004; 64: 7Crossref PubMed Scopus (58) Google Scholar PMS2CL, a 100-kb inverted duplication of PMS2, is located about 700 kb centromeric of PMS2. Several studies suggest that extensive sequence exchange as a result of gene conversion is still ongoing between PMS2 and PMS2CL.14Hayward B.E. De Vos M. Valleley E.M. Charlton R.S. Taylor G.R. Sheridan E. Bonthron D.T. Extensive gene conversion at the PMS2 DNA mismatch repair locus.Hum Mutat. 2007; 28: 424-430Crossref PubMed Scopus (46) Google Scholar, 22Auclair J. Leroux D. Desseigne F. Lasset C. Saurin J.C. Joly M.O. Pinson S. Xu X.L. Montmain G. Ruano E. Navarro C. Puisieux A. Wang Q. Novel biallelic mutations in MSH6 and PMS2 genes: gene conversion as a likely cause of PMS2 gene inactivation.Hum Mutat. 2007; 28: 1084-1090Crossref PubMed Scopus (55) Google Scholar Such gene conversion is one of the mechanisms introducing pathogenic alleles into the active gene.14Hayward B.E. De Vos M. Valleley E.M. Charlton R.S. Taylor G.R. Sheridan E. Bonthron D.T. Extensive gene conversion at the PMS2 DNA mismatch repair locus.Hum Mutat. 2007; 28: 424-430Crossref PubMed Scopus (46) Google Scholar, 15Ganster C. Wernstedt A. Kehrer-Sawatzki H. Messiaen L. Schmidt K. Rahner N. Heinimann K. Fonatsch C. Zschocke J. Wimmer K. Functional PMS2 hybrid alleles containing a pseudogene-specific missense variant trace back to a single ancient intrachromosomal recombination event.Hum Mutat. 2010; 31: 552-560PubMed Google Scholar, 16van der Klift H.M. Tops C.M. Bik E.C. Boogaard M.W. Borgstein A.M. Hansson K.B. Ausems M.G. Gomez Garcia E. Green A. Hes F.J. Izatt L. van Hest L.P. Alonso A.M. Vriends A.H. Wagner A. van Zelst-Stams W.A. Vasen H.F. Morreau H. Devilee P. Wijnen J.T. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.Hum Mutat. 2010; 31: 578-587PubMed Google Scholar Multiplex ligation-dependent probe amplification (MLPA) assay using probes targeting paralogous sequence variants (PSVs) of PMS2 and PMS2CL has been designed to evaluate the copy number variation (CNV) of these two genes (MRC Holland, Amsterdam, the Netherlands). By combining gene-specific long-range PCR (LR-PCR) of the active gene with Sanger sequencing analysis, it is possible to differentiate CNVs at the 3′ end of PMS2 from these of PMS2CL.11Senter L. Clendenning M. Sotamaa K. Hampel H. Green J. Potter J.D. Lindblom A. Lagerstedt K. Thibodeau S.N. Lindor N.M. Young J. Winship I. Dowty J.G. White D.M. Hopper J.L. Baglietto L. Jenkins M.A. de la Chapelle A. The clinical phenotype of Lynch syndrome due to germ-line PMS2 mutations.Gastroenterology. 2008; 135: 419-428Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar, 16van der Klift H.M. Tops C.M. Bik E.C. Boogaard M.W. Borgstein A.M. Hansson K.B. Ausems M.G. Gomez Garcia E. Green A. Hes F.J. Izatt L. van Hest L.P. Alonso A.M. Vriends A.H. Wagner A. van Zelst-Stams W.A. Vasen H.F. Morreau H. Devilee P. Wijnen J.T. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.Hum Mutat. 2010; 31: 578-587PubMed Google Scholar, 23Schofield L. Watson N. Grieu F. Li W.Q. Zeps N. Harvey J. Stewart C. Abdo M. Goldblatt J. Iacopetta B. Population-based detection of Lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test.Int J Cancer. 2009; 124: 1097-1102Crossref PubMed Scopus (63) Google Scholar, 24Vaughn C.P. Hart K.J. Samowitz W.S. Swensen J.J. Avoidance of pseudogene interference in the detection of 3' deletions in PMS2.Hum Mutat. 2011; 32: 1063-1071Crossref PubMed Scopus (32) Google Scholar However, CNVs cannot be unambiguously differentiated when PMS2 and PMS2CL share identical PSVs because of sequence exchange.24Vaughn C.P. Hart K.J. Samowitz W.S. Swensen J.J. Avoidance of pseudogene interference in the detection of 3' deletions in PMS2.Hum Mutat. 2011; 32: 1063-1071Crossref PubMed Scopus (32) Google Scholar Indeed, a previous study16van der Klift H.M. Tops C.M. Bik E.C. Boogaard M.W. Borgstein A.M. Hansson K.B. Ausems M.G. Gomez Garcia E. Green A. Hes F.J. Izatt L. van Hest L.P. Alonso A.M. Vriends A.H. Wagner A. van Zelst-Stams W.A. Vasen H.F. Morreau H. Devilee P. Wijnen J.T. Quantification of sequence exchange events between PMS2 and PMS2CL provides a basis for improved mutation scanning of Lynch syndrome patients.Hum Mutat. 2010; 31: 578-587PubMed Google Scholar estimated that 22% of cases have identical PSVs because of sequence transfer events between PMS2 and PMS2CL. Here, we describe a novel approach that combines targeted capture/next-generation sequencing (NGS), MLPA, and LR-PCR/NGS to unambiguously identify sequence variants and copy number changes of PMS2. Eight positive samples with known PMS2 mutations and six negative samples were used as control specimens for validation. The validated method incorporating MLPA, capture NGS, and LR-PCR/NGS was then applied to 32 clinical samples for molecular diagnosis. These samples were sent to us for genetic testing of hereditary cancer panels. Total genomic DNA was extracted from blood using a commercially available DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer's instructions. This study was performed in accordance with protocols approved by the institutional review board at the Baylor College of Medicine. A comprehensive workflow incorporating MLPA, capture NGS, and LR-PCR/NGS was designed for sequence and CNV analyses. Three methods were performed in parallel (Figure 1). MLPA was used to detect CNVs in PMS2 and PMS2CL. Capture sequence detects PSVs and CNVs from both PMS2 and PMS2CL. To distinguish mutations in the active gene from changes in the pseudogene, the active gene was specifically enriched by LR-PCR, followed by NGS analysis using previously described protocols.25Zhang W. Cui H. Wong L.J. Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing.Clin Chem. 2012; 58: 1322-1331Crossref PubMed Scopus (109) Google Scholar, 26Cui H. Li F. Chen D. Wang G. Truong C.K. Enns G.M. Graham B. Milone M. Landsverk M.L. Wang J. Zhang W. Wong L.J. Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.Genet Med. 2013; 15: 388-394Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 27Wong L.J. Next generation molecular diagnosis of mitochondrial disorders.Mitochondrion. 2013; 13: 379-387Crossref PubMed Scopus (59) Google Scholar Consistent results of four PSVs at the 3′ region of PMS2 must be observed by all three different methods to avoid false positives (Supplemental Table S2). Copy number analysis for PMS2 was performed using MCR Holland SALSA MLPA Kit P008-C1 according to the manufacturer's recommendations.28Schouten J. McElgunn C. Waaijer R. Zwijnenburg D. Diepvens F. Pals G. Relative quantification of 40 nucleic acid sequences by multiplex ligation-dependent probe amplification.Nucleic Acids Res. 2002; 30: e57Crossref PubMed Scopus (2092) Google Scholar The P008-C1 commercial kit includes probes specific for PMS2 exons 1 to 11, exons 11 to 15, and PSVs in exons 11 to 15 targeting both PMS2 and PMS2CL. We selected a reference DNA (NA12878 from the Coriell Institute, Camden, NJ) that has five pairs of different PSVs in exons 11 to 15. The MLPA data are analyzed using Coffalyzer software version 140429.1057 (MRC Holland). A probe library (Roche NimbleGen SeqCap; Roche NimbleGen, Madison, WI) targeting all coding exons and 50 bp of flanking intronic regions of 197 hereditary cancer genes, including PMS2, was custom designed followed by NGS analysis according to published procedures.25Zhang W. Cui H. Wong L.J. Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing.Clin Chem. 2012; 58: 1322-1331Crossref PubMed Scopus (109) Google Scholar, 26Cui H. Li F. Chen D. Wang G. Truong C.K. Enns G.M. Graham B. Milone M. Landsverk M.L. Wang J. Zhang W. Wong L.J. Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.Genet Med. 2013; 15: 388-394Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 29Wang J. Schmitt E.S. Landsverk M.L. Zhang V.W. Li F.Y. Graham B.H. Craigen W.J. Wong L.J. An integrated approach for classifying mitochondrial DNA variants: one clinical diagnostic laboratory's experience.Genet Med. 2012; 14: 620-626Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 30Wang J. Cui H. Lee N.C. Hwu W.L. Chien Y.H. Craigen W.J. Wong L.J. Zhang V.W. Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin.Genet Med. 2013; 15: 106-114Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Feng Y. Chen D. Wang G.L. Zhang V.W. Wong L.J. Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing.Genet Med. 2015; 17: 99-107Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Wang J. Zhang V.W. Feng Y. Tian X. Li F.Y. Truong C. Wang G. Chiang P.W. Lewis R.A. Wong L.J. Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa.Invest Ophthalmol Vis Sci. 2014; 55: 6213-6223Crossref PubMed Scopus (56) Google Scholar The sample preparation was performed according to the manufacturer's recommendation in the SeqCap EZ Choice Library User's Guide (Roche NimbleGen). Equal molar ratios of 15 indexed samples were pooled to loaded to one lane of the flow cell for sequencing on a HiSeq2000 sequencer (Illumina, San Diego, CA) with 100-cycle single-end reads. For NGS data analysis, the raw data in base call files (.bcl format) were conveyed to qseq files before demultiplexing using CASAVA software version 1.7 (Illumina). Demultiplexed sequence reads were aligned to the PMS2 reference sequence NM_000535.5 using the NextGENe software version 2.3 (SoftGenetics, State College, PA). The mean coverage per base was extracted from the aligned data. The normalized average coverage depth per individual exon was used to detect exonic CNVs.31Feng Y. Chen D. Wang G.L. Zhang V.W. Wong L.J. Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing.Genet Med. 2015; 17: 99-107Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar The script for the detection of CNVs is deposited online at https://sourceforge.net/projects/cnvanalysis (last accessed April 29, 2015). The identification of breakpoints is similar to previously published procedures.26Cui H. Li F. Chen D. Wang G. Truong C.K. Enns G.M. Graham B. Milone M. Landsverk M.L. Wang J. Zhang W. Wong L.J. Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders.Genet Med. 2013; 15: 388-394Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Variant annotation and copy number analyses using capture NGS data were performed.25Zhang W. Cui H. Wong L.J. Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing.Clin Chem. 2012; 58: 1322-1331Crossref PubMed Scopus (109) Google Scholar, 30Wang J. Cui H. Lee N.C. Hwu W.L. Chien Y.H. Craigen W.J. Wong L.J. Zhang V.W. Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin.Genet Med. 2013; 15: 106-114Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Feng Y. Chen D. Wang G.L. Zhang V.W. Wong L.J. Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing.Genet Med. 2015; 17: 99-107Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Wang J. Zhang V.W. Feng Y. Tian X. Li F.Y. Truong C. Wang G. Chiang P.W. Lewis R.A. Wong L.J. Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa.Invest Ophthalmol Vis Sci. 2014; 55: 6213-6223Crossref PubMed Scopus (56) Google Scholar Short and long interspersed repeat sequences were identified using online software (RepeatMasker Open-4.0 2013-2015, http://repeatmasker.org, last accessed April 29, 2015). The alignment of Alu sequences was completed using web tool ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2, last accessed April 29, 2015). Previously published primers were used to amplify both PMS2 and PMS2CL genes.10Clendenning M. Hampel H. LaJeunesse J. Lindblom A. Lockman J. Nilbert M. Senter L. Sotamaa K. de la Chapelle A. Long-range PCR facilitates the identification of PMS2-specific mutations.Hum Mutat. 2006; 27: 490-495Crossref PubMed Scopus (81) Google Scholar, 33Vaughn C.P. Robles J. Swensen J.J. Miller C.E. Lyon E. Mao R. Bayrak-Toydemir P. Samowitz W.S. Clinical analysis of PMS2: mutation detection and avoidance of pseudogenes.Hum Mutat. 2010; 31: 588-593PubMed Google Scholar LR-PCR primer pairs were used to amplify three fragments, E1 to E5, E6 to E10, and E11 to E15, specific for the active PMS2 gene and the pseudo gene PMS2CL. The LR-PCR was performed using TaKaRa LA Taq DNA polymerase Hot-Start Version (Takara Bio, Otsu, Japan) with 100 ng of total genomic DNA in a final volume of 50 μL. LR-PCR products of PMS2 were fragmented and indexed for the subsequent analysis by high-throughput sequencing on the HiSeq2000 sequencer (Illumina) as described above.25Zhang W. Cui H. Wong L.J. Comprehensive one-step molecular analyses of mitochondrial genome by massively parallel sequencing.Clin Chem. 2012; 58: 1322-1331Crossref PubMed Scopus (109) Google Scholar, 30Wang J. Cui H. Lee N.C. Hwu W.L. Chien Y.H. Craigen W.J. Wong L.J. Zhang V.W. Clinical application of massively parallel sequencing in the molecular diagnosis of glycogen storage diseases of genetically heterogeneous origin.Genet Med. 2013; 15: 106-114Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 31Feng Y. Chen D. Wang G.L. Zhang V.W. Wong L.J. Improved molecular diagnosis by the detection of exonic deletions with target gene capture and deep sequencing.Genet Med. 2015; 17: 99-107Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 32Wang J. Zhang V.W. Feng Y. Tian X. Li F.Y. Truong C. Wang G. Chiang P.W. Lewis R.A. Wong L.J. Dependable and efficient clinical utility of target capture-based deep sequencing in molecular diagnosis of retinitis pigmentosa.Invest Ophthalmol Vis Sci. 2014; 55: 6213-6223Crossref PubMed Scopus (56) Google Scholar All point mutations and breakpoints identified by NGS were confirmed by PCR-based Sanger sequencing analysis using BigDye terminator chemistry (Life Technologies, Carlsbad, CA) and Mutation Surveyor version 4 (SoftGenetics).34Yu H. van Karnebeek C. Sinclair G. Hill A. Cui H. Zhang V.W. Wong L.J. Detection of a novel intragenic rearrangement in the creatine transporter gene by next generation sequencing.Mol Genet Metab. 2013; 110: 465-471Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar The primers used for nested PCR to identify rearrangement of exons 11 and 12 are 5′-CAGAAAAATAATCTTGTAAAATGTCTG-3′ (E12F) and 5′-CCGGTATCTTCCTGGTTTGAA-3′ (E11R). The primers used for sequencing are 5′-ACTGGAATGTTTTACCCTGACT-3′ (forward) and 5′-TGTGACGAAGAGAAAAGGCC-3′ (reverse). Using the workflow described above, we were able to detect sequence variants and CNVs, as well as distinguish the mutations in PMS2 from its pseudogene PMS2CL. Table 1 lists the mutations detected in the positive validation samples and two clinical samples. The detection of point mutations by capture NGS is relatively straightforward. For example, a novel variant c.466A>G (p.T156A) was detected in patient C1 (Table 1) with colon and breast cancer diagnosed at age 52 and a positive family history of cancers. This variant is absent in the Exome Sequencing Project and 1000 Genomes Project. The computer-based algorithms SIFT (http://sift.jcvi.org, last accessed April 29, 2015) and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2, last accessed April 29, 2015) predict p.T156A to be deleterious. Because a pathogenic variant, c.466A>C (p.T156P), at the same amino acid position has been reported in a patient with colorectal cancer and a positive family history,35Brea-Fernandez A.J. Cameselle-Teijeiro J.M. Alenda C. Fernandez-Rozadilla C. Cubiella J. Clofent J. Rene J.M. Anido U. Mila M. Balaguer F. Castells A. Castellvi-Bel S. Jover R. Carracedo A. Ruiz-Ponte C. High incidence of large deletions in the PMS2 gene in Spanish Lynch syndrome families.Clin Genet. 2014; 85: 583-588Crossref PubMed Scopus (6) Google Scholar the p.T156A variant is interpreted as likely pathogenic according to newly revised American College of Medical Genetics and Genomics standards and guidelines.36Richards S. Aziz N. Bale S. Bick D. Das S. Gastier-Foster J. Grody W.W. Hegde M. Lyon E. Spector E. Voelkerding K. Rehm H.L. Stand
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