Molecular Diagnostics

George P. Patrinos, Wilhelm Ansorge, Phillip B. Danielson

• Publisher: Academic Press
• Year: 2016
• eBook ISBN: 9780128029886
• Edition: 3
• Topic: Medicine
• Subtopic: Immunology

Molecular Diagnostics, Third Edition, focuses on the technologies and applications that professionals need to work in, develop, and manage a clinical diagnostic laboratory. Each chapter contains an expert introduction to each subject that is next to technical details and many applications for molecular genetic testing that can be found in comprehensive reference lists at the end of each chapter.Contents are divided into three parts, technologies, application of those technologies, and related issues. The first part is dedicated to the battery of the most widely used molecular pathology techniques. New chapters have been added, including the various new technologies involved in next-generation sequencing (mutation detection, gene expression, etc.), mass spectrometry, and protein-specific methodologies.All revised chapters have been completely updated, to include not only technology innovations, but also novel diagnostic applications. As with previous editions, each of the chapters in this section includes a brief description of the technique followed by examples from the area of expertise from the selected contributor.The second part of the book attempts to integrate previously analyzed technologies into the different aspects of molecular diagnostics, such as identification of genetically modified organisms, stem cells, pharmacogenomics, modern forensic science, molecular microbiology, and genetic diagnosis. Part three focuses on various everyday issues in a diagnostic laboratory, from genetic counseling and related ethical and psychological issues, to safety and quality management.- Presents a comprehensive account of all new technologies and applications used in clinical diagnostic laboratories- Explores a wide range of molecular-based tests that are available to assess DNA variation and changes in gene expression- Offers clear translational presentations by the top molecular pathologists, clinical chemists, and molecular geneticists in the field

Chapter 1

Molecular Diagnostics

Past, Present, and Future

G.P. Patrinos¹, P.B. Danielson^2,3, and W.J. Ansorge^{4 1}University of Patras School of Health Sciences, Patras, Greece ²University of Denver, Denver, CO, United States ³Center for Forensic Science Research and Education, Willow Grove, PA, United States ⁴Ecole Polytechnique Federal Lausanne, EPFL, Lausanne, Switzerland

Abstract

Molecular diagnostics is referred to as the detection of genomic variants, aiming to facilitate detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. Molecular diagnostics is the outcome of the fruitful interplay among laboratory medicine, genomics knowledge, and technology in the field of molecular genetics, especially with significant discoveries in the field of molecular genomic technologies. All these factors contribute to the identification and fine characterization of the genetic basis of inherited diseases which, in turn, is vital for the accurate provision of diagnosis. High-throughput methods, such as next-generation sequencing or genome-wide association studies, provide invaluable insights into the mechanisms of disease, and genomic biomarkers allow physicians to not only assess disease predisposition but also to design and implement accurate diagnostic methods and to individualize therapeutic treatment modalities. In this chapter, we provide an overview of the field of molecular diagnostics and of the molecular diagnostic methods that are described in the chapters that are included in this textbook.

Keywords

Genomic variants; Inherited disorders; Molecular diagnostics; Next-generation sequencing; Personalized medicine

1.1. Introduction

Molecular (or nucleic acid-based) diagnosis of human disorders is referred to as the detection of genomic variants that are pathogenic and/or benign in DNA and/or RNA samples in order to facilitate detection, diagnosis, subclassification, prognosis, and monitoring response to therapy. Molecular diagnostics combines laboratory medicine with the knowledge and technology of molecular genetics and has been enormously revolutionized over the last decades, benefiting from discoveries in the fields of molecular biology and genomic technologies (Table 1.1). The identification and fine characterization of the genetic basis of inherited diseases is vital for the accurate provision of diagnosis. Gene discovery, via high-throughput methods, such as next-generation sequencing or genome-wide association studies, provides invaluable insights into the mechanisms of disease, and genomic markers allow physicians to not only assess disease predisposition but also to design and implement accurate diagnostic methods. The latter is of great importance, as the plethora and variety of molecular defects demands the use of multiple rather than a single variant detection platform. Molecular diagnostics has gradually become a clinical reality with its roots deep into the basic science of gene expression and gene function.

1.2. History of Molecular Diagnostics: Inventing the Wheel

In 1949, Pauling and his coworkers introduced the term molecular disease in the medical vocabulary, based on their discovery that a single amino acid change at the b-globin chain leads to sickle cell anemia, characterized mainly by recurrent episodes of acute pain due to vessel occlusion. In principle, their findings set the foundations of molecular diagnostics, although the big revolution occurred many years later. At that time, when molecular biology was only hectically expanding, the provision of molecular diagnostic services was inconceivable and technically not feasible. The first seeds of molecular diagnostics were provided in the early days of recombinant DNA technology, with many scientists from various disciplines working in concert. cDNA cloning and sequencing were at that time invaluable tools for providing basic knowledge on the primary sequence of various genes. The latter provided a number of DNA probes, allowing for analysis via southern blotting of genomic regions, leading to the concept and application of restriction fragment length polymorphism (RFLP) to track a variant allele from heterozygous parents to a high-risk pregnancy. In 1976, Kan and coworkers carried out, for the first time, prenatal diagnosis of α-thalassemia, using hybridization on DNA isolated from fetal fibroblasts. Also, Kan and Dozy (1978), implemented RFLP analysis to pinpoint sickle cell alleles of African descent. This breakthrough provided the means of establishing similar diagnostic approaches for the characterization of other genetic diseases, such as phenylketonurea (Woo et al., 1983), cystic fibrosis (Farrall et al., 1986), and so on.

At that time, however, a significant technical bottleneck had to be overcome. The identification of the pathogenic variant was possible only through the construction of a genomic DNA library from the affected individual, in order to first clone the variant allele and then determine its nucleotide sequence. Again, many human globin gene mutations were among the first to be identified through such approaches (Busslinger et al., 1981; Treisman et al., 1983). In 1982, Orkin and his coworkers showed that a number of sequence variations were linked to specific pathogenic HBB gene variants. These groups of RFLPs, termed haplotypes (both intergenic and intragenic), have provided a first-screening approach in order to detect a disease-causing variant. Although this approach enabled researchers to predict which HBB allele was pathogenic, significantly facilitating mutation screening, no one was in the position to determine the exact nature of the disease-causing mutation, as many different HBB gene variants were linked to a specific haplotype in different populations (further information is available at http://globin.bx.psu.edu/hbvar; Patrinos et al., 2004; Giardine et al., 2014).

Table 1.1

Timeline of the Principal Discoveries in the Field of Molecular Biology, Which Influenced the Development of Molecular Diagnostics

Date	Discovery
1949	Characterization of sickle cell anemia as a molecular disease
1953	Discovery of the DNA double helix
1958	Isolation of DNA polymerases
1960	First hybridization techniques
1969	In situ hybridization
1970	Discovery of restriction enzymes and reverse transcriptase
1975	Southern blotting
1977	DNA sequencing
1983	First synthesis of oligonucleotides
1985	Restriction fragment length polymorphism analysis
1985	Invention of the polymerase chain reaction
1986	Development of fluorescent in situ hybridization
1988	Discovery of the thermostable DNA polymerase—optimization of the polymerase chain reaction
1992	Conception of the real-time polymerase chain reaction
1993	Discovery of structure-specific endonucleases for cleavage assays
1996	First application of DNA microarrays
2001	First draft versions of the human genome sequence
2001	Application of protein profiling in human diseases
2002	Launch of the HapMap project
2005	Introduction of high-throughput next-generation sequencing technology
2008	Launch of the 1000 Genomes Project
2013	Introduction of the CRISPR system for gene editing
2014	Announcement of the sequencing of the human genome for $1000
2015	Launch of the Precision Medicine Initiative by US President Barack Obama

At the same time, in order to provide a shortcut to DNA sequencing, a number of exploratory methods for pinpointing pathogenic variants in patients' DNA were developed, setting the basis for variant screening and scanning methods. The first methods involved mismatch detection in DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985a,b) or differentiation of mismatched DNA heteroduplexes using gel electrophoresis, according to their melting profile (Myers et al., 1987). Using this laborious and time-consuming approach, a number of variant sequence alleles have been identified, which made possible the design of short synthetic oligonucleotides that were used as allele-specific probes onto genomic Southern blots. This experimental design was quickly implemented for the detection of β-thalassemia mutations (Orkin et al., 1983; Pirastu et al., 1983).

Despite intense efforts from different laboratories worldwide, the diagnosis of inherited diseases on the DNA level was still underdeveloped and therefore still not ready to be implemented in clinical laboratories for routine analysis of patients due to the complexities, costs, and time requirements of the technology available. It was only after a few years that molecular diagnosis entered its golden era with the discovery of the most powerful molecular biology tool since cloning and sequencing, the polymerase chain reaction (PCR).

1.3. The Post-Polymerase Chain Reaction Revolution

The discovery of PCR (Saiki et al., 1985; Mullis and Faloona, 1987) and its quick optimization, using a thermostable Taq DNA polymerase from Thermus aquaticus (Saiki et al., 1988), has greatly facilitated and in principle revolutionized molecular diagnostics. The most powerful feature of PCR is the large amount of copies of the target sequence generated by its exponential amplification, which allows the identification of a known mutation within a single day, rather than months. Also, PCR has markedly decreased or even diminished the use of radioactivity for routine molecular diagnosis. This has allowed molecular diagnostics to enter the clinical laboratory for the provision of genetic services, such as carrier or population genetic screening, prenatal diagnosis of inherited diseases, or, in recent years, the identification of unknown variants, in close collaboration with research laboratories. Therefore by being moved to their proper environment, the clinical laboratory, molecular diagnostics could provide the services for which they have been initially conceived.

The discovery of PCR also has provided the foundations for the design and development of many variant detection schemes, based on amplified DNA. In general, PCR is either used for the generation of DNA fragments to be analyzed or is part o...

Table of contents

• Cover image
• Title page
• Table of Contents
• Copyright
• List of Contributors
• Preface, Third Edition
• Chapter 1. Molecular Diagnostics: Past, Present, and Future
• Chapter 2. Describing DNA Variants (Mutation Nomenclature)
• Chapter 3. Low- and Medium-Throughput Variant Detection Methods: A Historical Perspective
• Chapter 4. Quantitative Polymerase Chain Reaction
• Chapter 5. Integrated Polymerase Chain Reaction Technologies (Sample-to-Answer Technologies)
• Chapter 6. High-Resolution Melting Curve Analysis for Molecular Diagnostics
• Chapter 7. Molecular Techniques for DNA Methylation Studies
• Chapter 8. Perspectives for Future DNA Sequencing Techniques and Applications
• Chapter 9. Advanced Personal Genome Sequencing as the Ultimate Diagnostic Test
• Chapter 10. Application of Padlock and Selector Probes in Molecular Medicine
• Chapter 11. Advances in Microfluidics and Lab-on-a-Chip Technologies
• Chapter 12. Protein Diagnostics by Proximity Ligation: Combining Multiple Recognition and DNA Amplification for Improved Protein Analyses
• Chapter 13. Application of Proteomics to Medical Diagnostics
• Chapter 14. Molecular Cytogenetics in Molecular Diagnostics
• Chapter 15. Cytogenomics of Solid Tumors by Next-Generation Sequencing: A Clinical Perspective
• Chapter 16. Pharmacogenomics in Clinical Care and Drug Discovery
• Chapter 17. Nutrigenomics: Integrating Genomic Approaches Into Nutrition Research
• Chapter 18. DNA Microarrays and Genetic Testing
• Chapter 19. Bioinformatics Tools for Data Analysis
• Chapter 20. Genomic Databases: Emerging Tools for Molecular Diagnostics
• Chapter 21. Molecular Diagnostic Applications in Forensic Science
• Chapter 22. New Perspectives in Mass Disaster Victim Identification Assisted by DNA Typing and Forensic Genomics
• Chapter 23. Preimplantation Genetic Diagnosis
• Chapter 24. Noninvasive Cell-Free DNA Prenatal Testing for Fetal Aneuploidy in Maternal Blood
• Chapter 25. Genetic Testing and Psychology
• Chapter 26. Genomic Medicine in Developing Countries and Resource-Limited Environments
• Chapter 27. Public Understanding of Genetic Testing and Obstacles to Genetics Literacy
• Chapter 28. Safety and the Biorepository
• Chapter 29. Quality Assurance in Genetic Laboratories
• Index