Molecular Diagnostics

George P. Patrinos, George P. Patrinos, Wilhelm Ansorge

• Publisher: Academic Press
• Year: 2009
• eBook ISBN:
  9780080923185
• Edition: 2
• Topic: Biological Sciences
• Subtopic: Human Anatomy & Physiology
• Index: Biological Sciences

The 2e of Molecular Diagnostics, the only book dealing with diagnosis on a molecular level, discusses current molecular biological techniques used to identify the underlying molecular defects in inherited disease. The book delves further into the principle and brief description of the technique, followed by examples from the authors' own expertise. Contributors to the 2e are well-known experts in their field, and derive from a variety of disciplines, to ensure breadth and depth of coverage. Molecular Diagnostics, 2e , is a needed resource for graduate students, researchers, physicians and practicing scientists in molecular genetics and professionals from similar backgrounds working in diagnostic laboratories in academia or industry, as well as academic institutions and hospital libraries. - Deals exclusively with the currently used molecular biology techniques to identify the underlying molecular defect of inherited diseases - Includes pharmacogenetics and pharmacogenomics relating to new cancer therapies - Provies a comprehensive guide through emerging concepts and demonstrates how the available mutation screening technology can be implemented in diagnostic laboratories and provide better healthcare

Chapter 1. Molecular Diagnostics

Past, Present, and Future

George P. Patrinos¹² and Wilhelm J. Ansorge³

¹Department of Pharmacy, School of Health Sciences, University of Patras, Patras, Greece;

²Erasmus University Medical Center, Faculty of Medicine and Health Sciences, Department of Bioinformatics, Rotterdam, The Netherlands;

³Ecole Polytechnique Federal Lausanne, EPFL, Lausanne, Switzerland

1.1. Introduction

Molecular or nucleic acid-based diagnosis of human disorders is referred to as the detection of the various pathogenic mutations 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 the discoveries in the field of molecular biology (see Table 1.1). The identification and fine characterization of the genetic basis of the disease in question is vital for accurate provision of diagnosis. Gene discovery provides invaluable insights into the mechanisms of disease, and gene-based markers allow physicians not only to assess disease predisposition but also to design and implement improved 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 mutation detection platform. Molecular diagnostics is currently a clinical reality with its roots deep into the basic study of gene expression and function.

Table 1.1 The 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 PCR
1986	Development of fluorescent in situ hybridization (FISH)
1988	Discovery of the thermostable DNA polymerase – Optimization of PCR
1992	Conception of real-time PCR
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
2005	Introduction of the high-throughput next-generation sequencing technology

1.2. History of Molecular Diagnostics: Inventing the Wheel

In 1949, Pauling and his coworkers introduced the term molecular disease into the medical vocabulary, based on their discovery that a single amino acid change at the β-globin chain leads to sickle cell anemia, characterized mainly by recurrent episodes of acute pain due to vessel occlusion. In principle, their findings have 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 the basic knowledge on the primary sequence of various genes. The latter provided a number of DNA probes, allowing the analysis via Southern blotting of genomic regions, leading to the concept and application of restriction fragment length polymorphism (RFLP) to track a mutant 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, in 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 disease causing mutation was possible only through the construction of a genomic DNA library from the affected individual, in order first to clone the mutated 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 β-globin gene mutations. These groups of RFLPs, termed haplotypes (both intergenic and intragenic), have provided a first-screening approach in order to detect a disease-causing mutation. Although this approach enabled researchers to predict which β-globin gene would contain a mutation, significantly facilitating mutation screening, no one was in the position to determine the exact nature of the disease-causing mutation, as many different β-globin gene mutations were linked to a specific haplotype in different populations (further information is available at http://globin.bx.psu.edu/hbvar; Hardison et al., 2002; Patrinos et al., 2004; Giardine et al., 2007).

At the same time, in order to provide a shortcut to DNA sequencing, a number of exploratory methods for pinpointing mutations in patients' DNA were developed. The first methods involved mismatch detection in DNA/DNA or RNA/DNA heteroduplexes (Myers et al., 1985a and Myers et al., 1985b) 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 mutations or polymorphic sequence variations 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 the intense efforts from different laboratories worldwide, 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 PCR Revolution: Getting More Out of Less

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 (see Fig. 1.1), which allows the identification of a known mutation within a single day, rather than months. Also, PCR has markedly decreased or even diminished the need for 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 screening for known mutations, prenatal diagnosis of inherited diseases, or in recent years, identification of unknown mutations, in close collaboration with research laboratories. Therefore, being moved to their proper environment, the clinical labora...

Table of contents

• Cover Image
• Table of Contents
• Copyright
• Contributors
• Preface – First Edition
• Preface – Second Edition
• Foreword – First Edition
• Chapter 1. Molecular Diagnostics
• Chapter 2. Allele-Specific Mutation Detection
• Chapter 3. Enzymatic and Chemical Cleavage Methods to Identify Genetic Variation
• Chapter 4. Mutation Detection by Single Strand Conformation Polymorphism and Heteroduplex Analysis
• Chapter 5. Capillary Electrophoresis
• Chapter 6. Temperature and Denaturing Gradient Gel Electrophoresis
• Chapter 7. Real-Time Polymerase Chain Reaction
• Chapter 8. Pyrosequencing
• Chapter 9. Application of Padlock and Selector Probes in Molecular Medicine
• Chapter 10. Molecular Cytogenetics in Molecular Diagnostics
• Chapter 11. Analysis of Human Splicing Defects Using Hybrid Minigenes
• Chapter 12. Detection of Genomic Duplications and Deletions
• Chapter 13. Multiplex Ligation-Dependent Probe Amplification (MLPA) and Methylation-Specific (MS)-MLPA
• Chapter 14. Molecular Techniques for DNA Methylation Studies
• Chapter 15. High-Resolution Melting Curve Analysis for Molecular Diagnostics
• Chapter 16. DNA Microarrays and Genetic Testing
• Chapter 17. Arrayed Primer Extension Microarrays for Molecular Diagnostics
• Chapter 18. Application of Proteomics to Disease Diagnostics
• Chapter 19. RNA-Based Variant Detection
• Chapter 20. Protein Diagnostics by Proximity Ligation
• Chapter 21. Mass Spectrometry and its Applications to Functional Proteomics
• Chapter 22. Pharmacogenetics and Pharmacogenomics
• Chapter 23. Nutrigenomics
• Chapter 24. Novel Next-Generation DNA Sequencing Techniques for Ultra High-Throughput Applications in Bio-Medicine
• Chapter 25. Locus-Specific and National/Ethnic Mutation Databases
• Chapter 26. Molecular Diagnostic Applications in Forensic Science
• Chapter 27. Mass Disaster Victim Identification Assisted by DNA Typing
• Chapter 28. Detection of Highly Pathogenic Viral Agents
• Chapter 29. Identification of Genetically Modified Organisms
• Chapter 30. Molecular Diagnostics and Comparative Genomics in Clinical Microbiology
• Chapter 31. Genetic Monitoring of Laboratory Rodents
• Chapter 32. Safety Analysis in Retroviral Gene Therapy
• Chapter 33. Preimplantation Genetic Diagnosis
• Chapter 34. Automated DNA Hybridization and Detection
• Chapter 35. The Use of Microelectronic-Based Techniques in Molecular Diagnostic Assays
• Chapter 36. Human Gene Patents and Genetic Testing
• Chapter 37. Genetic Counseling and Ethics in Molecular Diagnostics
• Chapter 38. Genetic Testing and Psychology
• Chapter 39. General Considerations Concerning Safety in Biomedical Research Laboratories
• Chapter 40. Quality Management in the Laboratory
• Glossary
• Index