Nucleic Acids as Molecular Diagnostics E-Book

• Herausgeber: Wiley-VCH
• Kategorie: Wissenschaft und neue Technologien
• Sprache: Englisch

Seitenzahl: 841

Veröffentlichungsjahr: 2014

Beschreibung

By integrating technology, supporting infrastructure and efficient application, the all-in-one guide presents molecular diagnostics as an essential component of modern, personalized clinical practice. It considers all important aspects, from the hardware and software needed, to recent improvements in blood- and non-blood-based biomarker tests. Chapters on ethical challenges and a look at current trends and the latest innovations are also included.
Bridging the gap between industry and academia, this is a highly useful resource for practitioners as well as for developers of modern, DNA- and RNA-based molecular diagnostics.

Leseprobe

CONTENTS

Cover

Related Titles

Title Page

Copyright

List of Contributors

Preface

Chapter 1: Next-Generation Sequencing for Clinical Diagnostics of Cardiomyopathies

1.1 Introduction

1.2 Cardiomyopathies and Why Genetic Testing is Needed

1.3 NGS

1.4 NGS for Cardiomyopathies

1.5 Sample Preparation

1.6 Bioinformatics Analysis Pipeline

1.7 Interpretation of Results and Translation into Clinical Practice

References

Chapter 2: MicroRNAs as Novel Biomarkers in Cardiovascular Medicine

2.1 Introduction

2.2 miRNAs are Associated with Cardiovascular Risk Factors

2.3 miRNAs in Coronary Artery Disease

2.4 miRNAs in Cardiac Ischemia and Necrosis

2.5 miRNAs as Biomarkers of Heart Failure

2.6 Future Challenges

Acknowledgments

References

Chapter 3: MicroRNAs in Primary Brain Tumors: Functional Impact and Potential Use for Diagnostic Purposes

3.1 Background

3.2 Gliomas

3.3 Meningiomas

3.4 Pituitary Adenomas

3.5 Medulloblastomas

3.6 Other Brain Tumors

3.7 Summary and Outlook

References

Chapter 4: Genetic and Epigenetic Alterations in Sporadic Colorectal Cancer: Clinical Implications

4.1 Introduction

4.2 Chromosomal Instability

4.3 Microsatellite Instability

4.4 Driver Somatic Mutations in CRC

4.5 Epigenetic Instability in CRC

4.6 Hypomethylation

4.7 CpG Island Methylator Phenotype

4.8 Concluding Remarks

References

Chapter 5: Nucleic Acid-Based Markers in Urologic Malignancies

5.1 Introduction

5.2 Bladder Cancer

5.3 Prostate Cancer

5.4 Renal Cell Carcinoma

5.5 Summary

References

Chapter 6: From the Genetic Make-Up to the Molecular Signature of Non-Coding RNA in Breast Cancer

6.1 Introduction

6.2 Molecular Breast Cancer Detection

6.3 Molecular Breast Cancer Subtypes and Prognostic/Predictive Molecular Biomarkers

References

Chapter 7: Nucleic Acid-Based Diagnostics in Gynecological Malignancies

7.1 Introduction

7.2 Cervix, Vulva, and Vaginal Carcinoma

7.3 Endometrial Carcinoma (Carcinoma Corpus Uteri)

7.4 Ovarian Carcinoma

7.5 Breast Cancer

7.6 Conclusion

References

Chapter 8: Nucleic Acids as Molecular Diagnostics in Hematopoietic Malignancies – Implications in Diagnosis, Prognosis, and Therapeutic Management

8.1 Introduction

8.2 Methodological Approaches

8.3 Cytogenetic Analysis to Molecular Diagnostics

8.4 Minimal Residual Disease

8.5 Chronic Myeloid Leukemia

8.6 Acute Myeloid Leukemia

8.7 Acute Lymphocytic Leukemia

8.8 Chronic Lymphocytic Leukemia

8.9 Outlook and Perspectives

References

Chapter 9: Techniques of Nucleic Acid-Based Diagnosis in the Management of Bacterial and Viral Infectious Diseases

9.1 Importance of Nucleic Acid-Based Molecular Assays in Clinical Microbiology

9.2 Nucleic Acid Amplification Techniques

9.3 Post-Amplification Analyses

9.4 General Overview and Concluding Remarks

Acknowledgments

References

Chapter 10: MicroRNAs in Human Microbial Infections and Disease Outcomes

10.1 Introduction

10.2 General Aspects of miRNAs in Infectious Diseases

10.3 miRNAs as Biomarkers and Therapeutic Agents in Tuberculosis and Hepatitis C Infections

10.4 miRNA-Targeting Therapeutics

10.5 Concluding Remarks

Acknowledgments

References

Chapter 11: Towards the Identification of Condition-Specific Microbial Populations from Human Metagenomic Data

11.1 Introduction

11.2 Nucleic Acid-Based Methods in Diagnostic Microbiology

11.3 Need for Comprehensive Microbiome Characterization in Medical Diagnostics

11.4 Challenges for Metagenomics-Based Diagnostics: Read Lengths, Sequencing Library Sizes, and Microbial Community Composition

11.5 Deconvolution of Population-Level Genomic Complements from Metagenomic Data

11.6 Need for Comparative Metagenomic Data Analysis Tools

11.7 Future Perspectives in Microbiome-Enabled Diagnostics

Acknowledgments

References

Chapter 12: Genome, Exome, and Gene Panel Sequencing in a Clinical Setting

12.1 Introduction

12.2 Genetic Diagnostics from a Laboratory Perspective – From Sanger to NGS

12.3 NGS Diagnostics in a Clinical Setting – Comparison Between Genome, Exome, and Panel Diagnostics

12.4 Conclusion and Outlook

References

Chapter 13: Analysis of Nucleic Acids in Single Cells

13.1 Introduction

13.2 Isolating Single Cells

13.3 Looking at the DNA of a Single Cancer Cell

13.4 Molecular DNA Analysis in Single Cells

13.5 Approaches to Analyze RNA of a Single Cell

13.6 Expression Analysis in Single Cells and its Biological Relevance in Cancer

13.7 Thoughts on Bioinformatics Approaches

13.8 Future Impact of Single-Cell Analysis in Clinical Diagnosis

References

Chapter 14: Detecting Dysregulated Processes and Pathways

14.1 Introduction

14.2 Measuring and Normalizing Expression Profiles

14.3 Biological Networks

14.4 Measuring the Degree of Deregulation of Individual Genes

14.5 Over-Representation Analysis and Gene Set Enrichment Analysis

14.6 Detecting Deregulated Networks and Pathways

14.7 miRNA Expression Data

14.8 Differential Network Analysis

14.9 Conclusion

References

Chapter 15: Companion Diagnostics and Beyond – An Essential Element in the Puzzle of Transforming Healthcare

15.1 Introduction

15.2 The Healthcare Environment

15.3 What is Companion Diagnostics?

15.4 What are the Drivers for Companion Diagnostics?

15.5 Companion Diagnostics Market

15.6 Partnerships and Business Models for Companion Diagnostics

15.7 Regulatory Environment for Companion Diagnostics Tests [6]

15.8 Outlook – Beyond Companion Diagnostics Towards Holistic Solutions

References

Chapter 16: Ethical, Legal, and Psychosocial Aspects of Molecular Genetic Diagnosis

16.1 General Peculiarities of Genetic Diagnoses

16.2 Informed Consent and Genetic Counseling

16.3 Medical Secrecy and Data Protection

16.4 Predictive Diagnosis

16.5 Prenatal Diagnosis

16.6 Multiparameter Testing

References

Index

End User License Agreement

List of Tables

Table 2.1

Table 2.2

Table 2.3

Table 2.4

Table 5.1

Table 6.1

Table 6.2

Table 7.1

Table 10.1

Table 10.2

Table 10.3

Table 12.1

Table 13.1

Table 15.1

Table 15.2

Table 15.3

Table 15.4

Table 15.5

List of Illustrations

Figure 3.1

Figure 4.1

Figure 6.1

Figure 6.2

Figure 9.1

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5

Figure 12.1

Figure 12.2

Figure 12.3

Figure 13.1

Figure 14.1

Figure 14.2

Figure 14.3

Figure 14.4

Figure 15.1

Figure 15.2

Figure 15.3

Guide

Cover

Table of Contents

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Nucleic Acids as Molecular Diagnostics

Edited by Andreas Keller

Eckart Meese

All books published by Wiley-VCH are carefully produced. Nevertheless, authors, editors, and publisher do not warrant the information contained in these books, including this book, to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

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© 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Boschstr. 12, 69469 Weinheim, Germany

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List of Contributors

Saskia Biskup

CeGaT GmbH Paul-Ehrlich-Strasse 23

72076 Tübingen

Germany

Nikolaus Blin

Wroclaw Medical University

Department of Genetics

Marcinkowskiego 1

50-368 Wroclaw

Poland

Johannes Dietl

University of Würzburg Medical School

Department of Obstetrics and Gynecology

Josef-Schneider-Strasse 4

97080 Würzburg

Germany

Juana Díez

Universitat Pompeu Fabra

Department of Experimental and Health Sciences

Molecular Virology Laboratory

Doctor Aiguader 88

08003 Barcelona

Spain

Claudia Durand

CeGaT GmbH

Paul-Ehrlich-Strasse 23

72076 Tübingen

Germany

Peter J. Goebell

Universitätsklinikum Erlangen

Urologische Klinik

Krankenhausstrasse 12

91054 Erlangen

Germany

Jan Haas

University of Heidelberg

Department of Internal

Medicine III

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

and

University of Heidelberg

DZHK (German Centre for Cardiovascular Research)

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

Sebastian F.M. Häusler

University of Würzburg Medical School

Department for Obstetrics and Gynecology

Josef-Schneider-Strasse 4

97080 Würzburg

Germany

Wolfram Henn

Universität des Saarlandes

Institut für Humangenetik

Campus Homburg, Gebäude 68

66421 Homburg

Germany

Pawel Karpinski

Wroclaw Medical University

Department of Genetics

Marcinkowskiego 1

50-368 Wroclaw

Poland

Hugo A. Katus

University of Heidelberg

Department of Internal

Medicine III

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

and

University of Heidelberg

DZHK (German Centre for Cardiovascular Research)

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

Stefan Kirsch

Fraunhofer Institut für Toxikologie und Experimentelle Medizin

Personalisierte Tumortherapie

Josef-Engert-Straße 9

93053 Regensburg

Germany

Jan Kirsten

Merck Serono/Merck KGaA

Fertility Technologies

Frankfurter Straße 250

64293 Darmstadt

Germany

and

Merck Serono Diviison of Merck KGaA

Head of Fertility Technologies – Global Business Franchise Fertility

Frankfurter Str. 250

64293 Darmstadt

Germany

Christoph A. Klein

Fraunhofer Institut für Toxikologie und Experimentelle Medizin

Personalisierte Tumortherapie

Josef-Engert-Straße 9

93053 Regensburg

Germany

and

University Regensburg

Chair of Experimental Medicine and Therapy Research

Franz-Josef-Strauß-Allee 11

93053 Regensburg

Germany

Cédric C. Laczny

University of Luxembourg

Luxembourg Centre for Systems Biomedicine

7 Avenue des Hauts-Fourneaux

4362 Esch-sur-Alzette

Luxembourg

Irene Latorre

Saarland University

Human Genetics Department

Kirrberger Strasse, 66424

Homburg

Germany

and

Universitat Pompeu Fabra

Infection Biology Laboratory

Department of Experimental and Health Sciences

Doctor Aiguader 88, 08003

Barcelona

Spain

and

CIBER Enfermedades Respiratorias (CIBERES)

Barcelona

Spain

Hans-Peter Lenhof

Universität des Saarlandes

Zentrum für Bioinformatik

Gebäude E2.1

66041 Saarbrücken

Germany

Benjamin Meder

University of Heidelberg Department of Internal

Medicine III

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

and

University of Heidelberg

DZHK (German Centre for Cardiovascular Research)

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

Andreas Meyerhans

Universitat Pompeu Fabra

Infection Biology Laboratory

Department of Experimental and

Health Sciences

Doctor Aiguader 88, 08003

Barcelona

Spain

and

Institució Catalana de Recerca i Estudis Avançats (ICREA)

Barcelona

Spain

Christian P. Pallasch

University of Cologne

Department I of Internal Medicine and Center of Integrated Oncology (CIO)

Kerpener Strasse 62

50937 Cologne

Germany

Bernhard Polzer

Fraunhofer Institut für Toxikologie und Experimentelle Medizin

Personalisierte Tumortherapie Josef-Engert-Straße 9

93053 Regensburg

Germany

Patrick Roth

University Hospital Zurich Department of Neurology and Brain Tumor Center

Frauenklinikstrasse 26

8091 Zurich

Switzerland

Verónica Saludes

Universitat Pompeu Fabra

Molecular Virology Laboratory

Department of Experimental and Health Sciences

Doctor Aiguader 88, 08003

Barcelona

Spain

and

CIBER Epidemiología y Salud Pública (CIBERESP)

Barcelona

Spain

Maria M. Sasiadek

Wroclaw Medical University Department of Genetics Marcinkowskiego 1

50-368 Wroclaw

Poland

Michael G. Schrauder

Universitätsklinikum Erlangen

Frauenklinik

Universitätsstrasse 21–23

91054 Erlangen

Germany

Janine Schwamb

University of Cologne

Department I of Internal Medicine and Center of Integrated Oncology (CIO)

Kerpener Strasse 62

50937 Cologne

Germany

Daniel Stöckel

Universität des Saarlandes

Zentrum für Bioinformatik

Gebäude E2.1

66041 Saarbrücken

Germany

Reiner Strick

Universitätsklinikum Erlangen

Frauenklinik

Universitätsstrasse 21–23

91054 Erlangen

Germany

Helge Taubert

Universitätsklinikum Erlangen Urologische Klinik

Krankenhausstrasse 12

91054 Erlangen

Germany

Britta Vogel

University of Heidelberg Department of Internal

Medicine III

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

and

University of Heidelberg

DZHK (German Centre for Cardiovascular Research)

Im Neuenheimer Feld 410

69120 Heidelberg

Germany

Sven Wach

Universitätsklinikum Erlangen

Urologische Klinik

Krankenhausstrasse 12

91054 Erlangen

Germany

Michael Weller

University Hospital Zurich Department of Neurology and Brain Tumor Center

Frauenklinikstrasse 26

8091 Zurich

Switzerland

Paul Wilmes

University of Luxembourg

Luxembourg Centre for Systems Biomedicine

7 Avenue des Hauts-Fourneaux

4362 Esch-sur-Alzette

Luxembourg

Jörg Wischhusen

University of Würzburg Medical School

Department for Obstetrics and Gynecology

Section for Experimental Tumor Immunology

Josef-Schneider-Strasse 4

97080 Würzburg

German

Bernd Wullich

Universitätsklinikum Erlangen

Urologische Klinik

Krankenhausstrasse 12

91054 Erlangen

Germany

Preface

With the selected contributions presented in this volume we set out to shed light on the role of nucleic acids as molecular diagnostic tools from different perspectives. We invited clinicians, biologists, and bioinformaticians to present their views on this intriguing topic. Their contributions offer a broad coverage of methods, biological targets, and clinical applications.

As for the different biological targets, the diagnostic roles of nucleic acids are addressed on a systemic level (e.g., body fluids), on an organ level (e.g., different cancer tissues), and, most challenging, on the single-cell level. On the molecular level, the different targets include DNA as well as coding and non-coding RNA (ncRNA). From the clinical perspective, the chapters address different human diseases, including the most lethal diseases (i.e., cardiovascular and cancer diseases). The diagnostics of infectious diseases as one of the leading healthcare challenges is addressed with specific emphasis on nucleic acids for the detection of viral and bacterial pathogens. The methods addressed include array-based and next-generation sequencing (NGS)-based techniques. All of the aforementioned topics (i.e., methods, biological targets, and clinical applications) may be used legitimately for an overall book structure; however, we chose the clinic/biology topics for structuring since the clinical application is the crucial endpoint of any nucleic acid-based diagnostic.

The first group of chapters (Chapters 1–8) address cardiovascular and cancer diseases, and the specific challenges for nucleic acid-based diagnoses for these diseases. Chapter 1 by Haas et al. describes the application of NGS for the genetic diagnostics of cardiomyopathies. The roles of microRNAs (miRNAs) as biomarkers for cardiomyopathies are described in Chapter 2 by Vogel et al., who specifically address their diagnostic potential in coronary artery disease, cardiac ischemia and necrosis, and heart failure. In Chapter 3, Roth and Weller address the diagnostic potential of miRNAs in various brain tumors, including the generally benign meningiomas. As an example for one of the most common and lethal cancer diseases, in Chapter 4, Karpinski et al. focus on sporadic colon cancer and its specific genetic and epigenetic alterations, including chromosomal and microsatellite instability. Wullich et al., in Chapter 5, address biomarkers for the three most prominent urologic malignancies: bladder cancer, prostate cancer, and renal cell carcinoma. In Chapter 6 on molecular markers in breast cancer, Schrauder and Strick specifically address long intergenic ncRNAs, which are increasingly recognized as important ncRNAs in addition to miRNAs. Other tumors also treated by gynecological oncologists are addressed by Häusler et al. in Chapter 7, who summarize the emerging role of DNA-, RNA-, and miRNA-based diagnostics in gynecological oncology. While the aforementioned contributions concern solid tumors, Chapter 8 by Schwamb and Pallasch addresses nucleic acid-based approaches in the diagnosis of hematopoetic malignancies.

The second group of contributions (Chapters 9–11) deals with infectious diseases. Latorre et al. give a summary of nucleic acid-based diagnostic methods in the management of bacterial and viral infectious diseases in Chapter 9. Chapter 10 by Saludes et al. addresses questions of nucleic acid-based diagnostics in infectious diseases, specifically the diagnostic potential of miRNAs in Mycobacterium tuberculosis and chronic hepatitis C virus infections. Laczny and Wilmes take a broader microbiology approach in Chapter 11 in that they address compositional and functional changes in endogenous microbial communities. They use metagenomic data for a microbiome-based diagnostics and modified therapeutic intervention.

Chapters 12–14 focus on technical approaches, including bioinformatics tools. In Chapter 12, Durand and Biskup deal with challenges of sequencing in a clinical setting. Chapter 13 by Kirsch et al. is dedicated to one of the ultimate challenges in nucleic acid-based diagnostics – the analysis of single cells. Single-cell analysis allows us to both address challenges associated with a heterogeneous cell population as found in tumor tissues and to utilize circulating tumor cells for diagnostic purposes. Chapter 14 on bioinformatics approaches by Stöckel and Lenhof is dedicated to problems that hamper the routine clinical application of biomarkers. Topics covered in this bioinformatics chapter include dealing with the noise of high-dimensional data produced by the applied biotechnological high-throughput, with batch effects by various experimental environments, and with frequent switchovers of the experimental platforms.

Finally, two chapters (Chapters 15 and 16) are dedicated to general healthcare and ethical questions. Kirsten addresses economical aspects, specifically the key drivers for the companion diagnostics that have become increasingly important beside the primary diagnostic, in Chapter 15. Henn emphasizes the importance of ethical and legal issues for molecular genetic diagnosis in Chapter 16. In his chapter, Henn addresses key aspects such as medical secrecy, data protection, problems associated with informed consent, and predictive/prenatal diagnosis.

Homburg/Saar

July 2014

Andreas Keller

Eckart Meese

1Next-Generation Sequencing for Clinical Diagnostics of Cardiomyopathies

Jan Haas, Hugo A. Katus, and Benjamin Meder

1.1 Introduction

The vast progress next-generation sequencing (NGS) has undergone during the past few years [1,2] has opened doors for a more advanced genetic diagnostic for many inherited diseases, such as Miller syndrome or Charcot–Marie–Tooth neuropathy [3,4]. Here, we want to describe the paradigm change in genetic diagnostics using the example of cardiomyopathies.

1.2 Cardiomyopathies and Why Genetic Testing is Needed

Cardiomyopathies are a heterogeneous group of cardiac diseases that can either be acquired through, for example, inflammation (myocarditis), be stress-induced (tako-tsubo), or be due to a genetic cause [5,6]. Examples of genetic forms are hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy, and left-ventricular non-compaction cardiomyopathy. Together with the channellopathies, such as long-QT syndrome and Brugada syndrome, they account for the most common heart diseases and belong to the most prevalent causes of premature death in western civilizations [7,8]. A point mutation in exon 13 of the β-myosin heavy chain gene was the first detected mutation diagnosed to be relevant for HCM in 1990 [9]. Driven by this finding, genetic research has progressed tremendously over the past two decades. Mutations in genes coding for a diverse set of proteins (e.g., sarcomeric, cytoskeletal, desmosomal, channel and channel-associated, membrane, and nuclear proteins, but also mitochondrial proteins or proteins relevant for mRNA splicing) have now been found to be implicated in disease onset and progression [10]. With currently more than 90 known disease genes with more than 1000 exons and multiple malign mutations per gene, the disease's heterogeneity is high and poses a challenge for classical Sanger-based sequencing. Although Sanger sequencing is able to detect mutations by testing only the most heavily affected genes, such as the β-myosin heavy chain gene (MYH7), where it is possible to find mutations in up to 30–50% of HCM patients, the mutation frequency in most genes is very low [10–12]. Therefore, new methods were needed to further improve genetic diagnostics in cardiomyopathy patients.

1.3 NGS

In contrast to Sanger sequencing, which is only capable of sequencing a few megabases, NGS is able to sequence hundreds of gigabases per run [13–15]. Currently, the most widely used NGS systems are the “sequencing by synthesis”-based sequencer HiSeq 2000 (Illumina), the “ligation and two-base coding”-based system SOLIDv4 (Life technologies), and the 454 GS FLX (Roche), which relies on “pyrosequencing” technology [7,16]. A detailed comparison of currently used systems including performance benchmarks, such as read lengths and output amounts, has been published recently by Liu [1,2,17]. In addition to the mature NGS systems, so-called benchtop sequencers have emerged. Those instruments, such as the MiSeq (Illumina), the Ion Torrent (PGM), or the GS Junior (Roche), benefit from a significantly shorter run time (hours compared to days) and a lower price, taking into account a reduced amount of sequenced bases [17,18].