Molecular Diagnostics PDF
Preview Molecular Diagnostics
Molecular Diagnostics Third Edition Editor-in-Chief George P. Patrinos Associate Editors Wilhelm J. Ansorge Phillip B. Danielson AMSTERDAM lBOSTON lHEIDELBERGlLONDON lNEWYORK lOXFORD lPARIS SANDIEGO lSANFRANCISCO lSINGAPORE lSYDNEY lTOKYO AcademicPressisanimprintofElsevier AcademicPressisanimprint ofElsevier 125LondonWall,LondonEC2Y5AS,UnitedKingdom 525BStreet,Suite1800,SanDiego, CA92101-4495,UnitedStates 50HampshireStreet,5th Floor,Cambridge,MA02139,UnitedStates TheBoulevard, LangfordLane,Kidlington,OxfordOX51GB,UnitedKingdom Copyright ©2017,2010,2005Elsevier Ltd.Allrightsreserved. Nopartofthispublication maybereproducedor transmitted inanyformor byanymeans,electronicormechanical, including photocopying,recording,or anyinformationstorage andretrieval system,without permissioninwritingfrom thepublisher. 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Neitherthepublishernor theauthorsassume anyliability foranyinjury and/ordamagetopersonsor propertyarising fromthispublication. LibraryofCongress Cataloging-in-Publication Data Acatalogrecordforthisbookisavailablefrom theLibraryofCongress British LibraryCataloguing-in-Publication Data Acataloguerecordfor thisbookisavailablefrom theBritishLibrary ISBN:978-0-12-802971-8 ForinformationonallAcademic Presspublications visit ourwebsiteathttps://www.elsevier.com/ Publisher: MicaHaley AcquisitionEditor: TariBroderick EditorialProjectManager:TracyTufaga ProductionProjectManager:Kirsty HaltermanandKaren East Designer:GregHarris TypesetbyTNQBooksandJournals List of Contributors B.R. Ali United Arab Emirates University, Al-Ain, United L.R. Ferguson Nutrigenomics New Zealand, Auckland, Arab Emirates New Zealand; The University of Auckland, Auckland, New Zealand W.J. Ansorge Ecole Polytechnique Federal Lausanne, EPFL, Lausanne, Switzerland D.A. Forero Universidad Antonio Nariño, Bogotá, Colombia D.S. Atherton University of Alabama at Birmingham, Birmingham, AL, United States J. Fredenburgh Scigen Scientific Inc., Gardena, CA, United States M.P.G. Barnett AgResearch Limited, Palmerston North, New Zealand; Nutrigenomics New Zealand, Auckland, B.K. Gale University of Utah, Salt Lake City, UT, New Zealand United States W.C. Bell University of Alabama at Birmingham, J. Göransson Uppsala University, Uppsala, Sweden Birmingham, AL, United States W.E. Grizzle University of Alabama at Birmingham, S.R. Brand Dana-Farber Cancer Institute, Boston, MA, Birmingham, AL, United States United States I. Grundberg Uppsala University, Uppsala, Sweden G. Cane Uppsala University, Uppsala, Sweden I.G. Gut Barcelona Institute for Science and Technology, W. Chantratita Mahidol University, Bangkok, Thailand Barcelona, Spain T. Conze Uppsala University, Uppsala, Sweden D.HeidemanVUUniversityMedicalCenter,Amsterdam, The Netherlands D. Corach Universidad de Buenos Aires, Buenos Aires, Argentina I. Helbing Uppsala University, Uppsala, Sweden F. Coun KU Leuven, Leuven, Belgium S. Henriksson Uppsala University, Uppsala, Sweden P.B.DanielsonUniversityofDenver,Denver,CO,United I.Hernández-Neuta Stockholm University, Solna, Sweden States; Center for Forensic Science Research and F. Innocenti University of North Carolina at Chapel Hill, Education, Willow Grove, PA, United States Chapel Hill, NC, United States J.T. den Dunnen Leiden University Medical Center, M. Isaksson Uppsala University, Uppsala, Sweden Leiden, Netherlands M. Jarvius Uppsala University, Uppsala, Sweden E. Dequeker University of Leuven, Leuven, Belgium H. Jayamohan University of Utah, Salt Lake City, UT, M.De Rycke Universitair Ziekenhuis& Vrije Universiteit United States Brussel (VUB), Brussels, Belgium M. Kamali-Moghaddam Uppsala University, Uppsala, A. Deshpande Los Alamos National Laboratory, Los Sweden Alamos, NM, United States K. Kampourakis University of Geneva, Geneva, S. Drmanac Complete Genomics, Inc., Mountain Switzerland View, CA, United States; BGI-Shenzhen, Shenzhen, T. Katsila Universityof Patras School of Health Sciences, China Patras, Greece R. Drmanac Complete Genomics, Inc., Mountain View, B. Koos Uppsala University, Uppsala, Sweden CA, United States; BGI-Shenzhen, Shenzhen, China L. Koumakis Foundation for Research and Technology e T. Ebai Uppsala University, Uppsala, Sweden Hellas (FORTH), Heraklion, Greece J.S.FarrarVirginia CommonwealthUniversity School of P. Laissue Del Rosario University, Bogotá, Colombia Medicine, Richmond, VA, United States xv xvi List of Contributors U. Landegren Uppsala University, Uppsala, Sweden G. Potamias Foundation for Research and Technology e Hellas (FORTH), Heraklion, Greece C. Larsson Uppsala University, Uppsala, Sweden N.ReisdorphUniversityofColoradoDenver,Aurora,CO, K.M. Legg Center for Forensic Science Research and United States Education, Willow Grove, PA, United States V. Romanov University of Utah, Salt Lake City, UT, K.-J. Leuchowius Uppsala University, Uppsala, Sweden United States H.LiUniversityofUtah,SaltLakeCity,UT,UnitedStates R. Samuel University of Utah, Salt Lake City, UT, J.S. Liu Complete Genomics, Inc., Mountain View, CA, United States United States K.C. Sexton University of Alabama at Birmingham, A. Llerena Extremadura University Hospital and Medical Birmingham, AL, United States School, Badajoz, Spain A. Sgourou Hellenic Open University, Patras, Greece C.Lopez-CorreaGenomeQuebec,Montreal,QC,Canada D. Sie VU University Medical Center, Amsterdam, The C. Mathieu KU Leuven, Leuven, Belgium Netherlands H.E. McKiernan Center for Forensic Science Research E.SistermansVUUniversityMedicalCenter,Amsterdam, and Education, Willow Grove, PA, United States The Netherlands E. Mendrinou University of Patras School of Health O. Söderberg Uppsala University, Uppsala, Sweden Sciences, Patras, Greece J.SonUniversityofUtah,SaltLakeCity,UT,UnitedStates A. Mezger Stockholm University, Solna, Sweden A. Squassina University of Cagliari, Cagliari, Italy K. Mitropoulos The Golden Helix Foundation, London, C. Staessen Universitair Ziekenhuis & Vrije Universiteit United Kingdom Brussel (VUB), Brussels, Belgium C. Mizzi University of Malta, Msida, Malta J. Stenberg Uppsala University, Uppsala, Sweden L. Moens Uppsala University, Uppsala, Sweden P.E.M. Taschner University of Applied Sciences Leiden, Z. Mohamed University of Malaya, Kuala Lumpur, Leiden, Netherlands Malaysia H. Tönnies Robert Koch-Institute, Berlin, Germany J. Nelson University of Utah, Salt Lake City, UT, J. Tost CEAeInstitut de Génomique, Evry, France United States G. Tzimas Technological Educational Institute of Western M.NilssonStockholmUniversity,Solna,Sweden;Uppsala Greece, Patras, Greece University, Uppsala, Sweden V. Velissariou Bioiatriki Health Services, Athens, Greece L. Overbergh KU Leuven, Leuven, Belgium E.ViennasUniversityofPatrasSchoolofHealthSciences, A. Papachatzopoulou University of Patras School of Patras, Greece Health Sciences, Patras, Greece S. Vig KU Leuven, Leuven, Belgium K. Pardali Uppsala University, Uppsala, Sweden P.S.WhiteLosAlamosNationalLaboratory,LosAlamos, A.F. Patenaude Dana-Farber Cancer Institute, Boston, NM, United States MA, United States C.T.WittwerUniversityofUtahSchoolofMedicine,Salt G.P. Patrinos University of Patras School of Health Lake City, UT, United States Sciences, Patras, Greece; Erasmus University Medical Center, Rotterdam, The Netherlands; United Arab A.WonkamUniversityofCapeTown,CapeTown,South Emirates University, Al-Ain, United Arab Emirates Africa P.C. Patsalis The Cyprus Institute of Neurology and X. Xun Complete Genomics, Inc., Mountain View, CA, Genetics, Nicosia, Cyprus United States; BGI-Shenzhen, Shenzhen, China B.A. Peters Complete Genomics, Inc., Mountain View, B. Ylstra VU University Medical Center, Amsterdam, CA, United States; BGI-Shenzhen, Shenzhen, China The Netherlands Preface, Third Edition We are delighted to deliver to the scientific community the third edition of Molecular Diagnostics, perhaps the most successful textbook in the field of molecular genetic testing, almost 12years from its first appearance in the scientific literature. In 2003, just 2years after the publication of the first draft of the Human Genome sequence, we proposed to edit a textbook exclusively devoted to the description of molecular techniques used to identify the underlying genetic hetero- geneityleadingtoinheriteddisorders.ThefirsteditionofMolecularDiagnosticswaspublishedinApril2005,followedby the second edition in October 2009. This textbook has been available to the scientific community for over 10years now, anditisclearlyconsideredtobethekeyreferenceinthefield,judgingfromthefollowing:(1)thelargenumberofcopies soldworldwide;(2)thevariouspostgraduateandspecialisttrainingcoursesonMolecularDiagnosticsthathavebeenused as syllabus and course material; (3) the adoption from universities as the textbook for related undergraduate courses and curricula, which also led to its translation in 2008; and (4) the various positive reviews obtained not only from external reviewers in scientific journals and elsewhere but also from fellow academics and students. This has prompted Elsevier/Academic Press to request the compilation of a third edition, justified not only from technological advances, particularly in high-throughput methods, but also from the intellectual revolution in biomedical sciences.Inthisthirdeditionwedecidedtokeeptheoriginalstructureoftheprevioustwoeditions,sincethiswasoneofits main innovative aspects, but we opted to expand the editorial team. We also decided to reshuffle the table of contents completely, providing a succinct outline and a historical perspective of the low-throughput methods that set a solid basis for the recent discoveries of the high-throughput methods together with a detailed overview of modern high-throughput methodologies, such as next-generation sequencing and microarray-based methods, along with examples of their appli- cations in a modern molecular genetic testing laboratory. The contents of this book are divided into three parts. The first part is dedicated to the battery of the most modern molecularbiologytechniquesandahistoricalperspectiveofthelow-throughputmethods.Inordertokeeppacewithrecent developments, the majority of the chapters from the previous editions have been either merged into a few chapters or omitted altogether, while being mentioned and fully referenced in the updated chapters. A large number of chapters pertaining to high-throughput molecular diagnostic approaches, for example, microarrays, next-generation sequencing, massspectrometry, next-generationsequencingcytogenomics,etc., havebeenincluded.The remainingchaptersfromthe previouseditionshavebeencomprehensivelyupdatedtoincludenotonlytechnologyinnovationsbutalsonoveldiagnostic applications. This resulted in the book being completely revamped with over half of its content being new compilations. The second part attempts to integrate previously analyzed technology with different aspects of molecular diagnostics, such as pharmacogenomics, molecular forensics and victim identification in mass disasters, and preimplantation genetic diagnosis,whilenewemergingdisciplines,suchasnutrigenomics,genomeinformatics,andgenomicdatabases,havebeen included. Finally, various everyday issues in a diagnostic laboratory, from genetic counseling to related ethical and psychological issues to safety and quality management, are discussed in the third and final part of the book. As with the previoustwoeditions,wefeelthattheinclusionofthelatterissuesinthisreferencebookhasgreatrelevancetooursociety. Aswiththeprevioustwoeditions,ourefforthasbeenassistedbymanyinternationallyrenownedexpertsintheirfields fromfivecontinentswhokindlyacceptedourinvitationtocompilethe29chaptersofthisbookandsharewithusandour readerstheirexpertise,experience,andresults.Inaddition,wemadeanefforttoformulatethebookcontentssuchthatthe notions described are explained in a simple language and terminology for the book to be useful not only to experienced physicians, healthcare specialists, and academics but also to undergraduate medical and life science students. The numerous self-explanatory illustrations and glossary clearly contribute to this end. Last, but not least, we provided the means to resolve the previously reported deficiencies in variant nomenclature by including a chapter on the official gene and genetic variation nomenclature at the very beginning of the textbook. xvii xviii Preface, Third Edition We are grateful to those colleagues who provided constructive comments and criticisms on the previous two editions andidentifieddeficienciesthathavebeen,hopefully,rectifiedinthisthirdedition.However,weexpectthatsomepointsin thisbookcanstillbefurtherimproved.Thereforewewouldagainwelcomecommentsandcriticismfromattentivereaders, whichwillcontributetoimprovingthecontentsofthisbookevenfurtherinitsfutureeditions.Wearealsogratefultothe editors, Drs. Tari Broderick, Jeffrey Rossetti, and Tracy Tufaga at Elsevier, who helped us in close collaboration to overcome encountered difficulties. We also express our gratitude to all of the contributors for delivering outstanding compilationsthatsummarizetheirexperienceandmanyyearsofhardworkintheirfieldsofresearch.Weareindebtedto GregHarris,whowasresponsibleforthedesignandthecoverofthisbook,andtotheproductionprojectmanagersKirsty HaltermanandKarenEast,whohaverefinedthefinalmanuscriptpriortogoingintoproduction.Weoweourthankstothe academic reviewers for their constructive criticisms on the chapters and their positive evaluation of our proposal for this third edition. Last,butnotleast,wewishtocordiallythankourfamilies,fromwhomwehavetakenaconsiderableamountoftimeto devote to this project, for their patience and continuous support over the years. Editor-in-Chief George P. Patrinos Department of Pharmacy, Faculty of Health Sciences University of Patras, Patras, Greece Associate Editors Wilhelm J. Ansorge École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland Phillip B. Danielson Department of Biological Sciences, University of Denver Denver, CO, United States of America December 2016 Chapter 1 Molecular Diagnostics: Past, Present, and Future G.P. Patrinos1, P.B. Danielson2,3 and W.J. Ansorge4 1UniversityofPatrasSchoolofHealthSciences,Patras,Greece;2UniversityofDenver,Denver,CO,UnitedStates;3CenterforForensicScience ResearchandEducation,WillowGrove,PA,UnitedStates;4EcolePolytechniqueFederalLausanne,EPFL,Lausanne,Switzerland 1.1 INTRODUCTION recurrentepisodesofacutepainduetovesselocclusion.In principle, their findings set the foundations of molecular Molecular (or nucleic acid-based) diagnosis of human diagnostics, although the big revolution occurred many disordersisreferredtoasthedetectionofgenomicvariants years later.Atthat time, when molecular biologywasonly that are pathogenic and/or benign in DNA and/or RNA hecticallyexpanding,theprovisionofmoleculardiagnostic samples in order to facilitate detection, diagnosis, sub- services was inconceivable and technically not feasible. classification, prognosis, and monitoring response to ther- The first seeds of molecular diagnostics were provided in apy. Molecular diagnostics combines laboratory medicine theearlydaysofrecombinantDNAtechnology,withmany with the knowledge and technology of molecular genetics scientists from various disciplines working in concert. and has been enormously revolutionized over the last de- cDNAcloningandsequencingwereatthattimeinvaluable cades,benefitingfromdiscoveriesinthefieldsofmolecular tools for providing basic knowledge on the primary biology and genomic technologies (Table 1.1). The iden- sequenceofvariousgenes.Thelatterprovidedanumberof tification and fine characterization of the genetic basis of DNA probes, allowing for analysis via southern blotting inherited diseases is vital for the accurate provision of of genomic regions, leading to the concept and application diagnosis. Gene discovery, via high-throughput methods, of restriction fragment length polymorphism (RFLP) to such as next-generation sequencing or genome-wide asso- track a variant allele from heterozygous parents to a high- ciation studies, provides invaluable insights into the risk pregnancy. In 1976, Kan and coworkers carried out, mechanisms of disease, and genomic markers allow phy- forthefirsttime,prenataldiagnosisofa-thalassemia,using sicianstonotonlyassessdiseasepredispositionbutalsoto hybridizationonDNAisolatedfromfetalfibroblasts.Also, design and implement accurate diagnostic methods. The Kan and Dozy (1978), implemented RFLP analysis to latter is of great importance, as the plethora and variety of pinpoint sickle cell alleles of African descent. This break- moleculardefectsdemandstheuseofmultipleratherthana through provided the means of establishing similar diag- singlevariantdetectionplatform.Moleculardiagnosticshas nostic approaches for the characterization of other genetic gradually become a clinical reality with its roots deep into diseases,suchasphenylketonurea(Wooetal.,1983),cystic the basic science of gene expression and gene function. fibrosis (Farrall et al., 1986), and so on. Atthattime,however,asignificanttechnicalbottleneck had to be overcome. The identification of the pathogenic 1.2 HISTORY OF MOLECULAR variant was possible only through the construction of a DIAGNOSTICS: INVENTING THE WHEEL genomicDNAlibraryfromtheaffectedindividual,inorder In 1949, Pauling and his coworkers introduced the term to first clone the variant allele and then determine its moleculardiseaseinthemedicalvocabulary,basedontheir nucleotide sequence. Again, many human globin gene discovery that a single amino acid change at the b-globin mutations were among the first to be identified through chain leads to sickle cell anemia, characterized mainly by such approaches (Busslinger et al., 1981; Treisman et al., 1 MolecularDiagnostics.http://dx.doi.org/10.1016/B978-0-12-802971-8.00001-8 Copyright©2017ElsevierLtd.Allrightsreserved. 2 MolecularDiagnostics TABLE1.1 Timelineofthe PrincipalDiscoveriesintheFieldofMolecularBiology,Which Influencedthe DevelopmentofMolecularDiagnostics Date Discovery 1949 Characterizationofsicklecellanemiaasamoleculardisease 1953 DiscoveryoftheDNAdoublehelix 1958 IsolationofDNApolymerases 1960 Firsthybridizationtechniques 1969 Insituhybridization 1970 Discoveryofrestrictionenzymesandreversetranscriptase 1975 Southernblotting 1977 DNAsequencing 1983 Firstsynthesisofoligonucleotides 1985 Restrictionfragmentlengthpolymorphismanalysis 1985 Inventionofthepolymerasechainreaction 1986 Developmentoffluorescentinsituhybridization 1988 DiscoveryofthethermostableDNApolymerasedoptimizationofthepolymerasechainreaction 1992 Conceptionofthereal-timepolymerasechainreaction 1993 Discoveryofstructure-specificendonucleasesforcleavageassays 1996 FirstapplicationofDNAmicroarrays 2001 Firstdraftversionsofthehumangenomesequence 2001 Applicationofproteinprofilinginhumandiseases 2002 LaunchoftheHapMapproject 2005 Introductionofhigh-throughputnext-generationsequencingtechnology 2008 Launchofthe1000GenomesProject 2013 IntroductionoftheCRISPRsystemforgeneediting 2014 Announcementofthesequencingofthehumangenomefor$1000 2015 LaunchofthePrecisionMedicineInitiativebyUSPresidentBarackObama 1983). In 1982, Orkin and his coworkers showed that a developed, setting the basis for variant screening and number of sequence variations were linked to specific scanning methods. The first methods involved mismatch pathogenic HBB gene variants. These groups of RFLPs, detection in DNA/DNA or RNA/DNA heteroduplexes termed haplotypes (both intergenic and intragenic), have (Myers et al., 1985a,b) or differentiation of mismatched provided a first-screening approach in order to detect a DNA heteroduplexes using gel electrophoresis, according disease-causing variant. Although this approach enabled to their melting profile (Myers et al., 1987). Using this researchers to predict which HBB allele was pathogenic, laborious and time-consuming approach, a number of significantly facilitating mutation screening, no one was in variant sequence alleles have been identified, which made the position to determine the exact nature of the disease- possible the design of short synthetic oligonucleotides that causing mutation, as many different HBB gene variants were used as allele-specific probes onto genomic Southern werelinkedtoaspecifichaplotypeindifferentpopulations blots. This experimental design was quickly implemented (furtherinformationisavailableathttp://globin.bx.psu.edu/ for the detection of b-thalassemia mutations (Orkin et al., hbvar; Patrinos et al., 2004; Giardine et al., 2014). 1983; Pirastu et al., 1983). Atthesametime,inordertoprovideashortcuttoDNA Despite intense efforts from different laboratories sequencing, a number of exploratory methods for pin- worldwide,thediagnosisofinheriteddiseasesontheDNA pointing pathogenic variants in patients’ DNA were level was still underdeveloped and therefore still not ready MolecularDiagnostics:Past,Present,andFuture Chapter | 1 3 to be implemented in clinical laboratories for routine activity of resolvase enzymes T4 endonuclease VII analysisofpatientsduetothecomplexities,costs,andtime and T7 endonuclease I to digest heteroduplex DNA requirementsofthetechnologyavailable.Itwasonlyaftera formed by annealing wild type and mutant DNA few years that molecular diagnosis entered its golden era (Mashal et al., 1995). Digestion fragments indicate withthediscoveryofthemostpowerfulmolecular biology the presence and the position of any variants. A vari- tool since cloning and sequencing, the polymerase chain ationofthe themeinvolvesthe use of chemical agents reaction (PCR). for the same purpose (Saleeba et al., 1992). Another enzymatic approach for variant detection is the oligo- nucleotide ligation assay (Landegren et al., 1988; 1.3 THE POST-POLYMERASE CHAIN Chapter 3); ligation was also one of the main princi- REACTION REVOLUTION ples of one of the most widely used next-generation The discovery of PCR (Saiki et al., 1985; Mullis and sequencing approaches (see also Chapter 8). Faloona, 1987) and its quick optimization, using a ther- 2. Electrophoretic-based techniques. This category is mostable Taq DNA polymerase from Thermus aquaticus characterized by a plethora of different approaches (Saiki et al., 1988), has greatly facilitated and in principle designedforthescreeningofknownorunknownmuta- revolutionized molecular diagnostics. The most powerful tions, based on the different electrophoretic mobility of feature of PCR is the large amount of copies of the target the mutant alleles, under denaturing or nondenaturing sequencegeneratedbyitsexponentialamplification,which conditions. Single-strand conformation polymorphism allows the identification of a known mutation within a (SSCP) and heteroduplex analyses (HDA; Orita et al., single day, rather than months. Also, PCR has markedly 1989; see Chapter 3) were among the first methods decreased or even diminished the use of radioactivity for designed to detect molecular defects in genomic loci. routine molecular diagnosis. This has allowed molecular In combination with capillary electrophoresis, SSCP diagnosticstoentertheclinicallaboratoryfortheprovision and HDA analysis now provide an excellent, simple, of genetic services, such as carrier or population genetic andrapidvariantdetectionplatform withlowoperation screening, prenatal diagnosis of inherited diseases, or, in costs and, most interestingly, the potential of easily be- recent years, the identification of unknown variants, in ing automated, thus allowing for high-throughput anal- closecollaborationwithresearchlaboratories.Thereforeby ysis of patients’ DNA. Similarly, denaturing gradient being moved to their proper environment, the clinical lab- gel electrophoresis (DGGE) and temperature gradient oratory, molecular diagnostics could provide the services gel electrophoresis can be used equally well for variant for which they have been initially conceived. alleledetection(seeChapter3).Inthiscase,electropho- The discovery of PCR also has provided the founda- retic mobility differences between a wild type and tions for the design and development of many variant variantallelecan be “visualized” in a gradientof dena- detection schemes, based on amplified DNA. In general, turing agents, such as urea and formamide, or of PCRiseitherusedforthegenerationofDNAfragmentsto increasing temperature. A less common variant detec- be analyzed or is part of the detection method. The first tiontechniqueistwo-dimensionalgenescanning,based attempt was the use of restriction enzymes (Saiki et al., ontwo-dimensionalelectrophoreticseparationofampli- 1985) or oligonucleotide probes, immobilized onto mem- fied DNA fragments, according to their size and base branes or in solution (Saiki et al., 1986), in order to detect pair sequence. The latter involves DGGE, following the existing genetic variation, in particular the sickle cell the size separation step. disease-causing mutation. In the following years, an even 3. Solid phase-based techniques. This set of techniques larger number of variant detection approaches have been consists of the basis for most of the present-day muta- developed and implemented (see also Chapter 3). These tion detection technologies, since they have the extra techniques can be divided roughly into three categories, advantage of being easily automated and hence are depending on the basis for discriminating the allelic highly recommended for high-throughput mutation variants: detection orscreening.Afast,accurate,andconvenient methodforthedetectionofknownmutations isreverse 1. Enzymatic-based methods. RFLP analysis was histori- dot-blot, initially developed by Saiki et al. (1989) and cally the first widely used approach, exploiting the al- implemented for the detection of HBB gene variants terations in restriction enzyme sites, leading to the leading to b-thalassemia. The essence of this method gain or loss of restriction events (Saiki et al., 1985). is the utilization of oligonucleotides, bound to a mem- Subsequently, a number of enzymatic approaches for brane, as hybridization targets for amplified DNA. variant allele detection have been conceived, based Some ofthistechnique’sadvantagesarethatonemem- onthedependenceofa secondary structure on thepri- brane strip can be used to detect many different known mary DNA sequence. These methods exploit the mutationsinasingleindividual(aonestrip-one patient 4 MolecularDiagnostics typeofassay),thepotentialofautomation,andtheease disciplines to intensify their research efforts to improve by of interpretation of the results, using a classical avidin- orders of magnitude the existing methods for genomic biotin system. However, this technique cannot be used variantdetection,tomakeavailabledatasetswithgenomic for the detection of unknown mutations. Continuous variationandanalyzethesesetsusingspecializedsoftware, development has given rise to allele-specific hybridiza- to standardize and commercialize genetic tests for routine tion of amplified DNA [PCR-ASO (Allele Specific diagnosis, and to improve the existing technology in order Oligonucleotide), Chapter 3] on filters, recently to provide state-of-the-art automated devices for high- extended to DNA oligonucleotide microarrays (see throughput genetic analysis. Chapter 18) for high-throughput mutation analysis The biggest challenge, following the publication of the (Gemignani et al., 2002; Cremonesi et al., 2007). In humangenomedraftsequence,wastoimprovetheexisting particular, oligonucleotides of known sequence are variant detection technologies to achieve robust, cost- immobilized onto appropriate surfaces, and hybridiza- effective, rapid, and high-throughput analysis of genomic tion of the targets to the microarray is detected, mostly variation. Also, the increased pace of novel variant detec- using fluorescent dyes. tionandgenediscoverydictatedtheharmonizationofgene nomenclature,aneffortthatwasspearheadedbytheHuman Thechoiceofthevariantdetectionmethodisdependent Genome Variation Society (http://www.hgvs.org; see also upon a number of variables, including the variation spec- Chapter2).Since 2005,genomictechnologyhasimproved trum of a given inherited disorder, the available infra- rapidly, and new high-throughput variant detection tech- structure, the number of tests performed in the diagnostic niques have become available, whereas old methodologies laboratory, and issues of intellectual properties (see also have evolved to fit into the increasing demand for auto- Section 1.5.1). Most of the clinical diagnostic laboratories mated and high-throughput screening or were gradually have not invested in expensive high-technology in- abandoned.Thechromatographicdetectionofpolymorphic frastructures, since the test volumes (the number of tests) changes of pathogenic variants using denaturing high- expected to be performed have not been large enough to performance liquid chromatography (DHPLC; for review, justify the capital investment. Therefore simple “home- seeXiaoandOefner,2001)isoneofthenewtechnologies brew”screening tests such as SSCP and HDA were and that emerged. DHPLC reveals the presence of a genetic still are the methods of choice for many clinical labora- variation by the differential retention of homo- and het- tories,astheyallowforrapidandsimultaneousdetectionof eroduplex DNA on reversed phase chromatography under different sequence variations at a detection rate of close to partial denaturation. Single-base substitutions, deletions, 100%. Although DNA amplification has significantly andinsertionscanbedetectedsuccessfullybyultravioletor facilitated the expansion of molecular diagnostics, it fluorescence monitoring within 2 to 3min in unpurified nonetheless has a number of limitations, such as amplifi- PCR products as large as 1.5-kilo bases. These features, cation of cytidine-guanine repeat-rich regions, the error- prone features of Taq polymerase (at a range of 10(cid:1)4 to together with its low cost, make DHPLC one of the most 10(cid:1)5 per nucleotide), and so on. Finally, it is noteworthy powerful tools for mutational analysis. Also, pyrose- quencing,anongel-basedgenotypingtechnology,provides that despite the wealth of variant detection methodologies, a very reliable method and an attractive alternative to DNA sequencing, particularly in the era of whole genome DHPLC. Pyrosequencing detects de novo incorporation of sequencing and the breakthroughs in next-generation nucleotides based on the specific template. The incorpora- sequencing technology, is considered the golden standard tion process releases a pyrophosphate, which is converted and the definitive experimental procedure for variant call- to ATP and followed by luciferase stimulation. The light ing. However, the costs for the initial investment and the produced, detected by a charge couple device camera, is difficulties for standardization and interpretation of “translated” to a pyrogram, from which the nucleotide ambiguousresultshaverestricteditsuseespeciallytobasic sequence can be deducted (Ronaghi et al., 1998). This research laboratories. approach constituted the basis for the development of the first next-generation sequencing approaches by 454 Life 1.4 MOLECULAR DIAGNOSTICS IN THE Technologies (see also Chapter 8). POST-GENOMIC ERA One of the major advances was the invention of the In February 2001, with the announcement of the first draft real-time PCR and the numerous variations of this theme sequence of the human genome (International Human (Holland et al., 1991; see Chapter 4). The method allows Genome Sequencing Consortium, 2001; Venter et al., for the direct detection of the PCR product during the 2001) and subsequently with the genomic sequence of exponential (mid-log) phase of the reaction, therefore other organisms, molecular biology has entered into a new combining amplification and detection in one single step. erawithunprecedentedopportunitiesandchallenges.These The increased speed of real-time PCR is due largely to tremendous developments put pressure on a variety of reduced cycles, the removal of post-PCR detection
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