Molecular Medicine. Genomics to Personalized Healthcare

332 Pages·2012·9.58 MB·English

by  R. Trent (Auth.)

MOLECULAR MEDICINE
Genomics to Personalized Healthcare
MOLECULAR
MEDICINE
Genomics to Personalized
Healthcare
FOURTH EDITION
Ronald J Trent
PhD, BSc(Med), MBBS (Sydney), DPhil (Oxon), FRACP, FRCPA, FFSc, FTSE
Professor of Medical Molecular Genetics, Sydney Medical School, University of Sydney and Director,
Department of Molecular & Clinical Genetics, Royal Prince Alfred Hospital, NSW 2050, Australia
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Acknowledgments and Dedications
I would like to thank members of the Mary Preap and Julia Haynes from Elsevier
Molecular Genetics Laboratory at RPA Hospital. have been very supportive.
Their skills and dedication made molecu- I dedicate the 4th Edition to my family – Pit,
lar medicine a lot more interesting. Prof. John Charlotte and Timothy. They have constantly
Buchanan in Auckland understood early on provided support and understanding when I
that Molecular Medicine was important for needed to do “home work” for this book. Also
patient care and steered me towards the educa- my Executive Assistant Carol Yeung, who has
tional aspects. My mother Ninette and my sister drawn the illustrations for all four editions and
Lynette have always been there when needed. still remains enthusiastic.
vii
Preface
There have been six major developments professionals are suitably engaged. The first edi-
since the third edition of Molecular Medicine: tion was subtitled: An introductory text for stu-
dents. This was left out in subsequent editions
1. Growth of omics particularly genomics;
on the assumption that the clinical applications
2. The start of whole genome sequencing for
of DNA-based medicine were being taught in
patient care;
the universities. However, new developments
3. Broader acceptance of personalized medicine in
in omics are occurring rapidly, and there is some
selecting the right drug or its dose based on
concern that their educational aspects are not
molecular typing of patient DNA;
being addressed in many of the modern cur-
4. A shift to somatic cell genetics particularly
ricula. Governments and major research funders
solid cancers;
are attempting to fast track the translational
5. Expansion in the Direct-to-Consumer DNA
aspects of molecular medicine but this will not
testing market, and
be enough without linking their initiatives to the
6. Recognition of a roadblock to the effective
education of tomorrow’s health practitioners.
translation of molecular medicine research
This edition no longer has a Glossary or
including the need for better bioinformatics to
Methodology because this material can be found
understand the significance of DNA variants
on the Internet. Nevertheless, Methodology
and the many changes in DNA, RNA or even
remains important, since patients and fami-
chromosomes now detectable through omics
lies are interested and will go to the Internet,
strategies.
so the health professional may be asked techni-
The title to this edition has subtly changed cal questions. In the era of open yet personal-
to include reference to personalized medi- ized medicine, there is no reason why the health
cine, which, as explained in Chapter 1, is not professional and the patient or family can-
new with some taking it as another example of not sit down and work through the technical
inappropriate hype. Nevertheless, it attracts issues using the computer as a component of the
attention and so is useful if it helps to push the consultation.
translational components of molecular medi- Ronald J Trent
cine and ensures the next generation of health Sydney, December 2011
ix
C H A P T E R
1
Genes to Personalized Medicine
O U T L I N E
Introduction 1 10 Years On 28
Genome Anatomy 2 Genome Variation 31
DNA 2 1 000 Genome Project 31
Protein-Coding Genes 9 Encyclopedia of DNA Elements
Junk DNA 11 (ENCODE) Project 32
RNA 14
Personalized Medicine 32
ncRNA 15
Education and Resources 33
Chromosomes 18
Roadmap 34
Human Genome Project 22
References 36
Goals 24
The 10 Year Project 25
INTRODUCTION l Molecular genetics – the discipline within
genetics that deals with the structure and
There are many definitions of molecular function of DNA and RNA.
medicine. In this book the term predominantly
The common thread in these names is
describes the effect that knowledge of DNA
the way in which an understanding of DNA
(and increasingly RNA) is having on medical
and the ability to manipulate it in vitro or
practice. Some other terms which overlap with
in vivo – and increasingly now to interrogate it
molecular medicine include:
in silico – has greatly expanded the options that
l Molecular biology – the application of DNA or are available in clinical practice, public health,
RNA knowledge in research or industry. research and industry.
l Genetic engineering or recombinant DNA Single gene Mendelian disorders are relatively
(rDNA) technology – the manipulation of an uncommon and are traditionally considered
organism’s DNA using DNA or RNA-based under genetics. Examples include cystic fibro-
techniques. sis, hemophilia, Huntington disease and genetic
Molecular Medicine. DOI: http://dx.doi.org/1 0.1016/B978-0-12-381451-7.00001-3 1 © 2012 Elsevier Inc. All rights reserved.
2 1. GENEs TO PERsONALIzED MEDICINE
forms of cancer. Complex genetic disorders are com- Today, medical research and clinical prac-
mon and comprise important public health chal- tice underpinned by molecular medicine
lenges both in the developed and the developing continue to provide novel insights into our
world. Included here are diabetes, heart disease understanding of disease pathogenesis. From
and dementia. The emerging health issues related these concepts, new therapies to prevent or
to aging and obesity also have a complex genetic treat important and common human disorders
component underlying their pathogenesis. are starting to emerge. The consequences of the
The understanding of complex genetic dis- Human Genome Project (described later in this
orders requires a new level of sophistication chapter) are many. One of the significant but
now possible through omics which describes less publicized outcomes has been the increas-
an approach that characterizes all or many mol- ing trend to form large multi-centered interna-
ecules within a cell, tissue or organism. The catalyst tional research collaborations that can ask very
for omics has been the Human Genome Project ambitious research questions.
which has rewritten the way research is con-
ducted, and has enabled impressive technologica l
developments. While genomics (all or many
GENOME ANATOMY
genes) will be the predominant theme of this
book, it is important to acknowledge that other
Most of what was considered the core com-
omics particularly transcriptomics (all or many
ponent of the human genome actually occu-
RNA transcripts), metabolomics (all or many
pies a relatively small portion of it. Only about
metabolites), proteomics (all or many proteins),
1–2% contains protein-coding genes. The func-
epigenomics (the complete epigenetic profile)
tion of the remaining 98% is now starting to be
and phenomics (the composite of the phenotypes)
explored. This includes:
contribute to molecular medicine. Thus genomic
medicine overlaps with molecular medicine but
1. Intronic sequences;
has a narrower brief.
2. Copy number variations;
To store and analyze the large data sets gen-
3. Non-coding (nc) RNA genes;
erated by omics requires sophisticated compu-
4. Regulatory elements, and
ter power and software. This is bioinformatics
5. Repetitive DNA.
(also called informatics, or computational biol-
ogy). Related to bioinformatics is the concept of
For convenience the term gene will gener-
systems biology which attempts to join the dots
ally describe segments of DNA that code for
between the seemingly unrelated data that are
proteins (these are also called structural genes),
emerging (Chapter 4).
although this does not distinguish other genes
The emergence of molecular medicine may
particularly the ncRNA genes described later.
broadly be considered over three time periods:
(1) The discovery of DNA structure in 1953 fol-
lowed by developments in recombinant DNA
DNA
(rDNA) technologies; (2) The Human Genome
Project 1990–2000, and (3) The launch of omics Many discoveries led to the uncovering of
(Figure 1.1). Another way to track the mile- the double-stranded structure of DNA, pro-
stones in molecular medicine is to consider posed by J Watson and F Crick in 1953, and
the Nobel Prizes awarded for work in this area more followed to build the foundations for
(Table 1.1). Key developments in molecular molecular medicine (Table 1.2). DNA comprises
medicine are summarized in Table 1.2. two polynucleotide strands twisted around each
MOLECULAR MEDICINE
1. GENEs TO PERsONALIzED MEDICINE 3
Double-stranded DNA can be DNA can be Automated DNA
DNA sequenced amplified with PCR sequencing becomes
available
DNA Discovery 1953 1975 1985 1987
Beginning of "Book of Life" DNA diagnostics - Critical development for
molecular medicine (human genome) can unlimited potential Human Genome Project
be read base by base
Human Genome Project Controversy - DNA sequence for NIH policy
starts, and first NIH patents first model organism that human genome
successful gene therapy anonymous (H.influenzae) sequences are
DNA sequences published freely available
Human Genome
1990 1991 1995 1996
Project
Modern molecular Commercialization Success with model Two models:
medicine era increasingly prominent organisms fuels public (free) &
enthusiasm for completing commercial (user pay)
human genome
First draft of Annotated final version First diploid Alternative fuels &
human genome sequence of human genome human genome artificial bacterium
publicly announced sequence now available published
2000 2003 2007 2010
Omics
Complete sequences Beginning of Beginning of Synthetic biology
for fruit fly and a genomics era next generation on the march
plant are published DNA sequencing
FIGURE 1.1 Three major developments in the evolution of molecular medicine. Various time periods are depicted
with discoveries above and their implications below.
other in the form of a double helix (Figure 1.2). These are called DNA variants, or point muta-
In biological terms, the double-stranded DNA tions when they lead to genetic disease.
structure is essential for replication to ensure Deletions or insertions affecting the codons
that each dividing cell receives an identical copy can produce a smaller truncated protein or a
of the DNA. frameshift abnormality (Chapter 3).
The genetic code in DNA is represented by The genetic code needs to be read from the
nucleotide triplets called codons (Table 1.3). sense strand. Hence, transcription to give the
Each individual amino acid is represented by a appropriate mRNA sequence is taken from
different triplet combination. Thus, the codons the antisense strand so that the single-stranded
for a polypeptide such as: glycine-serine-valine- mRNA will have the sense sequence (anti-
alanine-alanine-tryptophan will read: GGT TCT sense RNA is discussed later in this chapter).
GTT GCT GCT TGG. The positions indicating More information on DNA structure, includ-
where a polypeptide starts and where it ends ing its various A, B and Z forms can be found
are also defined by triplet codons. For example, in reference [1]. The unit for measurement of
3
ATG is found at the start, and the end or stop DNA is the base pair (bp). Thus 10 bp  1 Kb
6
codons are TAA or TAG or TGA. Single base (kilobase); 10 bp  1 Mb (megabase); and
9
changes in the DNA sequence occur regularly. 10 bp  1 Gb (gigabase).
MOLECULAR MEDICINE
4 1. GENEs TO PERsONALIzED MEDICINE
a
TABLE 1.1 Molecular medicine and Nobel Prize winners (1953–2011).
Year Recipients Subject
a
1957 A R Todd Work on nucleotides and nucleotide co-enzymes
1958 G W Beadle, E L Tatum and J Lederberg Regulation and genes, and genetic recombination in bacteria
1959 S Ochoa, A Kornberg In vitro synthesis of nucleic acids
1962 J D Watson, F H Crick, M H Wilkins Structure of DNA
1965 F Jacob, A L Woff, J Monod Genetic control enzyme and virus synthesis
1968 R W Holley, H B Khorana, Interpretation of the genetic code
M W Nirenberg
1975 D Baltimore, H M Temin, R Dulbecco Reverse transcriptase and oncogenic viruses
1978 W Arber, D Nathans, H O Smith Restriction endonucleases
a
1980 P Berg and W Gilbert, F Sanger Creation of first recombinant DNA molecule and
DNA sequencing
1989 J M Bishop, H E Varmus Oncogenes
a
1989 S Altman, T R Cech RNA ribozymes
1993 R J Roberts, P A Sharp Gene splicing
a
1993 K Mullis and M Smith Polymerase chain reaction (PCR) and site directed
mutagenesis
1995 E B Lewis, C Nusslein-Volhard, Genetic mechanisms in early embryonic development
E F Wieschaus
2001 L H Hartwell, T Hunt, P M Nurse Key regulators of the cell cycle
2002 S Brenner, J E Sulston, H R Horvitz Genetic regulation of organ development and programmed
cell death
2004 R Axel, L B Buck Discoveries of odorant receptors and the organization of the
olfactory system
2006 A Z Fire, C C Mello Discovery of RNA interference
a
2006 R D Kornberg Studies on the molecular basis of eukaryotic transcription
2007 M R Capecchi, M J Evans, O Smithies Targeted gene insertion into ES cells to produce transgenic
mice
2009 E H Blackburn, C W Greider, J W Szostak Telomeres and telomerases in chromosome protection
a
Nobel Prize in Chemistry; all others Nobel Prize in Physiology or Medicine. The list starts at 1953 when the structure of DNA was described.
DNA Replication
strand and a second complementary new strand.
DNA replication involves the separation of The first step in replication is to unwind the
the double-stranded DNA and then the duplica- double-stranded DNA using DNA helicase. DNA
tion of each strand. The final product is two DNA polymerase then synthesizes the new strand in a
copies, each of which has one original parental 5 to 3 direction. However, since the two DNA
MOLECULAR MEDICINE
1. GENEs TO PERsONALIzED MEDICINE 5
TABLE 1.2 The evolution of molecular medicine.
Discoveries and achievements
1869: A Swiss physician named F Miescher isolated an acidic material from cell nuclei which he called nuclein. From this
came nucleic acid.
1940s–1950s: O Avery and colleagues showed that genetic information in the Pneumococcus was found within its DNA.
E Chargaff demonstrated equal numbers of the nucleotide bases adenine and thymine as well as guanine and cytosine in
DNA. This and the X-ray crystallographic work by R Franklin and M Wilkins, enabled J Watson and F Crick to propose
the double-stranded structure of DNA in 1953. Complementary strands that made up the DNA helix were then shown to
separate during replication. DNA polymerase was discovered by A Kornberg in 1956. It enabled small segments of double-
stranded DNA to be synthesized. Fifty years later his son R Kornberg was awarded the Nobel Prize in Chemistry for his
work on the molecular basis of eukaryotic transcription.
1960s–1970s: Discoveries included: (1) Showing mRNA to be the link between the nucleus and the site of protein synthesis
in the cytoplasm. (2) Identification of autonomously replicating, extra-chromosomal DNA elements called plasmids. These
were shown to carry genes including those coding for antibiotic resistance in bacteria. (3) The genetic code for each amino
acid was shown to be a nucleotide triplet (Table 1.3). In 1961 M Lyon proposed that one of the two X chromosomes in
female mammals was normally inactivated. The process of X-inactivation enabled males and females to have equivalent
DNA content despite differing numbers of X chromosomes. Restriction endonuclease enzymes were isolated from bacteria
by H Smith, D Nathans, W Arber and colleagues. They digested DNA at specific sites determined by the underlying
nucleotide base sequences allowing DNA fragments of known sizes to be produced. In 1966 V McKusick published
Mendelian Inheritance in Man, a catalog of genetic disorders in humans. This became a forerunner to the many databases
or banks that would subsequently be created to store DNA, information or tissues.
1970s–1980s: The dogma that DNA → RNA → protein moved in only one direction was revised when H Temin and
D Baltimore showed that reverse transcriptase, an enzyme found in the RNA retroviruses, allowed RNA to be copied back
into DNA, i.e. RNA → DNA. This enzyme would later provide the researcher with a means to produce DNA copies (known
as complementary or cDNA) from RNA templates. Reverse transcriptase also explained how some viruses could integrate
genetic information into the host’s genome. DNA ligase was discovered and allowed DNA fragments to be joined. The first
recombinant DNA molecules comprising segments that had been stitched together were produced by P Berg and colleagues. S
Cohen and colleagues showed that DNA could be inserted into plasmids and then reintroduced back into bacteria. Replication
of the bacteria containing the foreign DNA enabled unlimited amounts of a single fragment to be produced, i.e. DNA could be
cloned. DNA sequencing methodologies were described by F Sanger and W Gilbert. Protein-coding genes were shown to be
discontinuous with coding regions (exons) split by non-coding regions (introns). From this splicing was described by R Roberts
and P Sharp to explain how introns were removed in the process of transcription. The importance of genes from evolutionary
conservation was demonstrated by E Lewis, C Nusslein-Volhard and E Wieschaus with their work on development in
Drosophila. Variations in the length of DNA segments between individuals (called DNA polymorphisms) were described.
Subsequently D Botstein showed that DNA polymorphisms allowed maps of the human genome to be developed. Using
mitochondrial DNA polymorphisms and DNA sequence information R Cann and colleagues proposed that homo sapiens evolved
from a common female ancestor in Africa. First use of DNA polymorphisms for forensic purposes reported by A Jeffreys.
1990s: Human Genome Project (HGP) starts with the publicly-funded initiative led by F Collins. The private sector later
becomes involved through J C Venter. New technologies for gene mapping and DNA cloning developed. YACs (yeast
artificial chromosomes) were the early choice but a better vector, BACs (bacterial artificial chromosomes) was developed.
Bioinformatics starts to play a critical role in both the storage of data and its analysis because of the increasingly complex
mapping and sequencing data that emerge. First model organism is sequenced. RNAi discovered by A Fire and C Mello.
2000s: The HGP is completed and the genomics era starts. This soon moves to the omics era as new analytic platforms
emerge. An increasing number of genomes are sequenced and by 2005 the NG (next generation) DNA sequencing platforms
and protocols emerge with the target being a whole human genome sequence costing $1 000. Metagenomics (sequencing
uncultured organisms from various environmental samples) using the omics/shot gun approach starts to gain traction.
2007–2011: Analytic platforms continue to evolve rapidly with the suggestion that 3rd generation sequencing now emerging
will reduce the cost of a whole genome sequence to around $100! Synthetic biology hits the headlines with the first synthetic
microbe Mycoplasma mycoides JCVI-syn1.0 published. Major national and international consortia are formed to study cancer
at the somatic cell DNA level. There is growing interest in the link between drug selection in cancer through DNA testing
(companion diagnostics) as well as the importance of pharmacogenetics in ensuring the right drug for the right person at
the right dose. The concept of Personalised Medicine takes hold.
MOLECULAR MEDICINE