Post-Genomic Cardiology

653 Pages·2007·27.369 MB·English

by  José Marín-García, M.D., Michael J. Goldenthal, Ph.D., and Gordon W. Moe, M.D. (Auth.)

Preface
Heart disease is an endemic health problem of great mag (2) The mechanisms governing the early specification of
nitude in the world. In spite of considerable clinical and cardiac chambers in the developing heart tube have not yet
research effort during the last decade and the development of been precisely delineated but are thought to involve novel
new drugs and surgical modalities of therapy, the mortality cell-to-cell signaling among migrating cells, as well as the
and morbidity rates remain very high. Moreover, many fun triggering of chamber-specific gene expression programs,
damental questions regarding the basic underlying mecha mediated by specific transcription factors and growth factors
nisms and pathophysiology of most cardiovascular diseases such as bone morphogenetic protein (BMP). Future areas of
(CVDs), including congenital and acquired defects, remain study will focus on elucidating the role of signaling mol
unanswered. Breakthroughs in molecular genetic technol ecules (e.g., WNT) using conditional gene knock-outs (in
ogy have just begun to be applied in studies of cardiovascular a variety of genetic backgrounds) and accessing their inter
disease, allowing chromosomal mapping and the identifica action with critical transcription factors such as dHAND,
tion of many genes involved in both the primary etiology and NKX2.5, GATA4, and TBX. Similar approaches may also
also as significant risk factors in the development of these prove informative in probing the origins of the cardiac con
anomalies. Identification of genes responsible for rare familial duction system, and in deciphering the role of signaling
forms of cardiovascular disease has proved to be informative systems as participants in vascular formation in endothelial
in the study of nonsyndromic patients with cardiac pathol cells, focusing on the interaction of VEGF, angiopoietin,
ogy. Common cardiovascular anomalies (e.g., cardiomyopa TGF, and the Notch pathway.
thy, congenital heart disease, atherosclerosis, hypertension,
(3) Another critical area of research is the identification of
cardiac arrhythmias) seem to be united by association with
molecular regulators that control cardiomyocyte prolifera
distinct subsets of genes. These include genes responsible for
tion. Cardiomyocytes are mitotically active during embryo-
subcellular structures (e.g., sarcomere, cytoskeleton, chan
genesis and generally cease proliferation shortly after birth.
nels), metabolic regulatory enzymes (e.g., renin-angiotensin
Understanding the molecular basis of cardiomyocyte prolif
system, cholesterol metabolic pathway), or intracellular sig
eration could greatly impact on our clinical attempts to repair
naling pathways (e.g., calcineurin, CaMK, TNFa).
the damaged heart. Mechanism of cell growth regulation is
At present, the following areas of research appear par
being investigated by careful comparison of comprehensive
ticularly promising:
gene expression profiles of embryonic and post-natal myo
(1) With the completion of the Human Genome Project cytes, as well as by the generation of myocyte cell culture
the likely identification of novel genes involved in non lines with the capacity to respond to proliferative inducers.
syndromic cardiac disease, cardiac organogenesis, and vas
(4) Cellular transplantation is an alternative mechanism with
cular development will serve as an important foundation
which to augment myocyte number in diseased or ischemia
for our understanding of how specific gene defects gener
damaged hearts. New research efforts will be necessary to
ate their cardiovascular phenotypes. Bioinformatic methods
further define the optimal conditions necessary for cardio
can be employed to search existing databases with the rou
myocyte differentiation and proliferation and for the fully
tine use of reverse genetics techniques, allowing subsequent
functional integration of stem cells in the myocardium, as well
cloning of novel genes/cDNAs of interest, followed by the
as to investigate the ability of transplanted stem cells to repair
characterization of spatial-temporal patterns of specific
defects in the young and adult heart. It will be critical to learn
gene expression. Moreover, post-genomic analysis includ
whether cardiac failure secondary to myocardial ischemia or
ing both transcriptome and proteomic methodologies can be
dilated cardiomyopathy, myocardial infarct in adults, or chil
used to further delineate the functions of the gene products,
dren with Kawasaki disease and myocardial damage or with
defining their precise role in pathogenesis, elucidating their
ARVD, can be treated with stem-cell transplantation.
interaction with other proteins in the subcellular pathways,
and potentially enabling their application as clinical markers With the beginning and eventually rapid progress in cell
of specific CVDs. engineering, we expect to see the end of many of these
VIII Preface
cardiac abnormalities that weaken human Hfe and bankrupt to cardiologists and researchers in diverse fields, with specific
the health care system. Moreover, insight into the cardio attention paid to pediatric, aging, and gender-based cardiovas
vascular consequences of abnormal gene function and cular medicine, eyeing new therapeutic modalities that may
expression should ultimately impact the development of tar improve currentiy available therapies and interventions in the
geted therapeutic strategies and disease management and management of human cardiac diseases. Furthermore, we are
may replace less effective treatment modalities directed witnessing the transition from the cardiology of the past to the
solely at rectifying structural cardiac defects and temporal study of systems biology, the constructive cycle of computa
improvement of function. tional model building, and experimental verification capable
As the role of genetic screening in cardiology is strength to provide the input for exciting new discoveries and hope.
ened and as research on the multiple signaling pathways
involved in cardiac organogenesis and pathology progresses, ''Tomorrow is here and...there, keep looking up at it.''
the time seems appropriate for a book that comprehensively
integrates known facts, current developments, and future
knowledge. In addition to providing a recount of past discov Jose Marin-Garcia
eries, this book deals with areas that are of emerging interest Highland Park, 2006
CHAPTER
Introduction to Post-Genomic Cardiology
OVERVIEW GENE STRUCTURE, TRANSCRIPTION,
AND TRANSLATION
At the beginning of the 2 P^ century, the appHcation of molec
ular cellular biology and genetics to clinical cardiology has The gene is the fundamental unit of inherited information and
become increasingly compelling. The great strides that have can be defined as a segment of DNA involved in producing
been made in our understanding of the gene and its expres a polypeptide chain (protein) by virtue of its synthesis of
sion are now being applied to our improved understanding of RNA by transcription. Only a small number of the DNA
normal cardiovascular development, physiology, and aging, sequences carried on the human chromosomes are organized
as well as to abnormalities that occur during physiological as genes, whereas other sequences act as regulatory ele
insults and cardiac disease. ments; however, no function has yet been found for most of
The information derived from high-resolution stud the chromosomal DNA.
ies of the human genome together with the development The DNA of the chromosomes consists primarily of two
of animal models of cardiac disease, including trans long strands that wind around each other in the form of a
genic studies, and from increased bioengineering of the double helix. Each strand is composed of a chain of nucleo
cell (e.g., stem cells) has begun to be used in earnest in tides (each containing a phosphate group, a base, and a sugar
the diagnosis and treatment of cardiovascular disease. called deoxyribose); these chains are millions of nucleotides
The complete delineation of the human genome and its long. The sugar and phosphate groups of each nucleotide and
approximately 75,000 gene products clearly represents a the covalent phosphodiester bond that links them are said to
first and important step in unraveling the complexity of the form the backbone of each strand and lie on the outside of
cardiomyocyte and of cardiovascular disease. Moreover, the helix; they carry a negative charge from the phosphate
the next stage of analysis, including the understanding of groups. The hydrophobic bases are arranged on the inside of
the gene and its interactions within its cellular environ the helix structure.
ment (termed a post-genomic approach), is now under Each strand has a polarity, a 5' and a 3' end; the two
way. In this book, we will attempt to describe where we strands are aligned in an antiparallel fashion so that they
have been, where we are, and provide a vision of where have opposite polarities (Fig. 1). There are only four types
we are going on this journey. of nucleotide—each with its own distinct base: adenine, gua
The basic concepts and principles of molecular biol nine, cytosine, and thymine. The sequence of the covalently
ogy and genetics focusing on the gene, its environment joined nucleotides in each DNA strand constitutes the DNA
within the cell, and the regulation of its expression will be primary structure. In double-stranded DNA, the bases in one
presented in this chapter. It will include a brief review and DNA strand are arranged in such a way as to interact with the
recapitulation to provide the reader with enough back bases in the other strand by the formation of hydrogen bonds
ground and terminology to readily understand specific between complementary bases. These essentially form the
techniques and their applications in clinical cardiology. rungs on a ladder structure. Adenine (A) always pairs with
The information in this introduction includes background thymine (T), forming two hydrogen bonds, whereas guanine
material available in a variety of different textbooks dedi (G) base pairs with cytosine (C), forming a slightly stron
cated to molecular and cell biology, several of which are ger three-hydrogen bond. The double-stranded structure of
cited in the reference section.^""^ An effort has been made DNA, which is stabilized by weak hydrogen bonds and by
to present the material in an accessible way to readers who hydrophobic base-stacking, constitutes the DNA secondary
may not have had recent coursework in these subjects, structure; this allows the structure to be easily opened during
reinforcing in this way the basic concepts of molecular either DNA replication or transcription so that polymeriz
and cell biology and highlighting the terminology that is ing enzymes can have access to read the sequence of each
used throughout the text. strand's DNA as a template for making more DNA or RNA.
Copyright © 2007 by Academic Press.
Post-Genomic Cardiology All rights of reproduction in any form reserved.
SECTION I • Post-Genomic Cardiology
Thymine — — Adenine
(T) - " (A)
Cytosine Guanine
(C) - - (G)
Guanine ~ ~ Cytosine
(G) - - (C)
Thymine — Adenine
Sugar-phosphate
(T) (A)
backbone
FIGURE 1 Double-strand structure
Adenine — — Thymine
of DNA. Depicted is the complementary
(A) (T)
base pairing of two strands of DNA
located at the center of the double helix
with the sugar-phosphate backbone of
the two chains oriented on the outside of
Cytosine Guanine the helix. Also shown is the antiparallel
(C) (G) nature of the strands with respect to
their 5' to 3' orientation. The structure
Hydrogen-bonding
is largely held together by the hydrogen
Complementary base pairing bonding between guanine (G) and
cytosine (C), which involves three bonds
and the double hydrogen bonds between
B 5TCGTAC 5'GTACGA adenine (A) and thymine (T) as shown.
In DNA replication, the sequence of each parental strand which largely confer the structural and functional characteris
determines the complementary nucleotide sequence on the tics specific to each cell type, are genetically controlled. The
nascent strand and, therefore, defines its new partner. The flow of information from DNA to protein can be reversed from
invariant base-pairing rule ensures that an exact complement mRNA to DNA only in certain viruses (e.g., retroviruses) that
will be present on the daughter strand during replication and contain specialized enzymes called reverse transcriptase and
forms the basis for both RNA transcription and DNA repair. in retrotransposons but never from protein to mRNA. Another
RNA produced from the DNA is also a linear polynucleotide unique exception to the normal pathway of information is found
molecule containing four kinds of bases, three the same as in prions (proteins replicating themselves).
DNA (A, C, and G), whereas the fourth is uracil (U) instead The first step in gene expression is transcription, a process
of thymine; it is generally single stranded and has a different that, like DNA replication, involves DNA as a template and
sugar (ribose) in its backbone. takes place accordingly in the nucleus. Transcription is medi
The central dogma of molecular biology deals with the ated by RNA polymerase, which in conjunction with spe
detailed residue-by-residue transfer of sequential informa cific transcription factors binds with high affinity to specific
tion, and this information cannot be transferred from protein sequence elements (promoter regions) on the DNA usually
to either protein or nucleic acid. The flow goes from DNA to located in front (5') or upstream of genes (Fig. 3A). After
RNA to protein. In Fig. 2, we show the flow of genetic infor binding, the RNA polymerase enzyme initiates, elongates,
mation going from DNA to mRNA (transcription)', the mRNA and terminates the synthesis of a discrete mRNA chain com
copy is sent out of the nucleus to specialized structures in the plementary to a portion of one of the two strands (i.e., the
cytoplasm (i.e., ribosomes), where the mRNA is used to form antisense strand) (Fig. 3B). The RNA made is essentially an
proteins from amino acids joined together by peptide bonds exact copy of the untranscribed "sense" strand (except for the
(translation). In this way, the types of protein a cell makes, substitution of a U for every T). The growing RNA chain is
CHAPTER 1 • Introduction
DNA replication
Nuclear localized, requires a DNA template,
oligonucleotide primer, deoxyribonucleotides,
DNA polymerase and additional factors
DNA
Nuclear localized, in addition to DNA
Transcription template requires an enzyme RNA polymerase,
A ribonucleotides, transcription factors
Reverse transcription (only viral?)
RNA
RNA processing at 5' and 3' ends, poly A addition,
capping, removal of intervening sequences (introns),
splicing to make mature messenger RNA; all occur in
the nucleus prior to export to cytoplasm
Translation Cytoplasmically localized requires mRNA
template, amino acids, tRNAs, ribosomes,
numerous factors, amino-acyl synthetases
Protein FIGURE 2 Flow of genetic information.
Gene
General transcription factors
regulatory
proteins RNA polymerase Regulatory
proteins
CO pSi^
I Promoter Structural gene
Regulatory TATA
sequence
Start codon Stop codon
Transcription initiation site Transcription termination site
>
RNA transcript
B
AGCTAAG T Chain growth
TCGATTC/^ A ^ Nascent RNA chain
CUCGAU UC
^ GAGCTAAG
FIGURE 3 Transcription involves both regulatory proteins and DNA sequence. Elements in the formation of new
RNA. (A) Involvement of RNA polymerase and transcription factors in binding both TATA and the promoter region
upstream of the gene to be transcribed. Other regulatory elements, which bind regulatory proteins and modulate the
levels of transcription, are located both further upstream, as well as downstream of the gene coding region. Also
shown are the relative positions of the transcription initiation and termination sites and the start and stop codons.
(B) The separation of the DNA strands required for transcription to occur and the relative orientation of the growing
nascent RNA strand (5' to 3') relative to its DNA template strand.
SECTION I • Post-Genomic Cardiology
polymerized in the 5' to 3' direction from the antisense strand scripts are rapidly excised, and the exon RNAs are spliced
template (3' to 50- Discrete regulatory signals are present on together to form the mature transcript, making the coding
the DNA molecule called cis-acting elements, which are rec region continuous for translation. Although apparently it
ognized by proteins (e.g., transcription factors) termed trans would seem wasteful to transcribe large stretches of DNA
acting factors that can either enhance or inhibit the initiation into RNA only to excise major fragments, what is gained is
or termination of the RNA transcript. Genes contain mul versatility in structure and in regulation that arises from the
tiple cis-acting elements responsive to a variety of intracel variable splicing patterns that can produce different mRNAs,
lular and extracellular physiological and pathophysiological depending on the cell type or developmental stage, allow
stimuli. The interaction between trans-2iC\mg factors and cis- ing the production of multiple distinct proteins (isoforms)
elements constitutes the primary basis for the regulation of from the same gene. Before the RNA is exported from the
transcription that can govern gene activity levels during cell nucleus for translation, other processing occurs at both the
differentiation, development, and in response to physiologi 5' end ("capping" with 7-methyl guanosine) and at the 3'
cal and pathophysiological stimuli and is discussed through end, a trimming accompanied by the addition of several ade
out much of this book. After the transcription of a gene is nine bases generating a poly A tail. These modifications to
complete, the RNA is separated from the DNA template. the mRNA increase translation efficiency and contribute to
Most nuclear genes also contain intervening sequences, message stability and may play a role in its transport from
which are noncoding (introns), varying in number and size, nucleus to cytoplasm. Modulation of transcript stability and
often larger than the coding region (exons); these sequences or turnover can be an effective mechanism influencing the
are not found in the mature mRNA to be translated into control of gene expression.
proteins. However, introns are initially transcribed in their In the translation process, each consecutive three nucle
entirety in a short-lived primary nuclear transcript along otides or triplet on the mRNA forms a codon read by the
with the contiguous sequences that are found in the mature ribosome to specify a specific amino acid, which is then
message (exons) (Fig. 4). Before the transcript is exported inserted into a growing polypeptide chain. This process uses
from the nucleus, the intron portions of the primary tran an adaptor transfer RNA (tRNA) molecule that carries both a
i-globin gene
Intron 1 Intron 2
Exon 1 Exon 2 Exon 3
Transcription, cap formation
and poly A addition
FIGURE 4 Processing of the RNA
transcript. The structure of most eucaryotic
genes involves the presence of both exons
and introns (intervening sequences) whose
number and size vary greatly in different
genes. The example shown here is with
the P-globin gene. After transcription
Cap AAAAAAAA
in the nucleus to form a large precursor
Splicing to remove Primary transcript primary transcript, the RNA is capped
introns and join exons
at its 5' terminus and polyadenylated at a
site near its 3' terminus. A series of RNA
splicing events follows to excise the intron
sequences and rejoin the exons to form the
mature transcript; these processing events
are required before the export of the RNA
from the nucleus to the cellular location
Cap AAAAAAAA
where it will eventually be translated on
Mature transcript (mRNA) ribosomes.
CHAPTER 1 • Introduction
Specific amino acid (e.g., tRNA^^^ carries glycine etc.) and tone proteins. Under specific conditions, the histones wrapped
an anticodon region that can recognize specific codons on around the nuclear DNA can be arranged in a regularly repeat
the mRNA. There are at least 20 types of tRNA molecules; ing unit called a nucleosome. Modifications to the charged
the covalent linkage of each adjoining peptide releases the residues of the histone proteins (e.g., by phosphorylation,
amino acid from the tRNA, allowing the "free" uncharged acetylation, or methylation of the lysine residues) can strik
tRNA to bind more amino acid for further translation. The ingly alter the assembly or condensation of chromatin. T\iis
codon AUG usually is the initiating codon, which encodes remodeling of chromatin can modulate the global expression
methionine. The first amino acid has a free NH2 group and of genes, largely by impeding the progression of their tran
defines the amino or N-terminus, whereas the growing end scription into RNA. Recent studies have also identified the
of the peptide chain has a free COOH group defining the presence of small RNA species in chromatin that play a criti
carboxyl or C-terminus. cal role in RNA processing and transport from the nucleus. In
The nearly universal genetic code, which is found through contrast, prokaryotes (e.g., bacteria) have neither a separate
out the entire plant and animal kingdoms as well as in bacte nuclear compartment nor chromatin and frequently have non-
ria, specifies which amino acid corresponds to which of the chromosomal DNA molecules cdXl^diplasmids.
64 possible 3-nucleotide codons. The code shows degeneracy
(i.e., 61 of the possible 64 triplet codons represent 20 amino
acids), with almost every amino acid represented by several CELL CYCLE
codons. Three codons (UAA, UGA, and UAG) do not repre
sent amino acids and play a role in terminating protein syn The genes on each chromosome are said to be linked and
thesis and are thereby termed stop codons. The initiation site are generally inherited as a unit; recombination can occur
on the mRNA determines the reading frame of the codons as a fundamental function of crossing-over during meiosis,
(uninterrupted coding region) and, therefore, determines the with increased recombination found between more distally
amino-acid sequence of the synthesized proteins. Most mature located genes. The cell cycle constitutes the period between
mRNA contains a single reading frame to generate a specific the release of a cell as one of the progeny of a division and its
protein. The deletion or insertion of a single base can cause a own subsequent division by mitosis into two daughter cells.
shift in the reading frame (frameshift); mutations with such It is composed of two major phases, an interphase during
an insertion/deletion can lead to the formation of a protein which there is little visible change, although the cell is active
with altered sequence (beyond the modified site) and size (the synthetically and bioenergetically, and the mitotic stage (M)
termination sites will likely be different) and can lead to dys during which cell division actually occurs (Fig. 5A).
functional phenotypes and hereditary disorders. During M, when the chromosomes are segregated before
The process of translation can also be subject to regu division, the chromatin becomes highly condensed and vis
lation. Several key initiating and elongating factors play ible as chromosomes because of this condensation (they take
pivotal roles in the regulation of protein synthesis. Proteins up stain more easily). The chromatin structure enables the
translated on the ribosomes can undergo a variety of cotrans- efficient packaging and distribution of the nuclear genetic
lational and post-translational modifications, which can material, which is segregated for distribution within the cell
affect their ultimate placement within the cell, as well as to the new daughter cells at mitosis. During the metaphase
their function. For example, specific signal sequences (usu stage of mitosis, a karyotype, essentially a snapshot of the
ally 15-60 amino acids long) at either the N-terminus or C- entire chromosome complement of the cell, can be made,
terminus can target a protein for import into the nucleus or because the entire complement of chromosomes are highly
the mitochondria. These signal sequences may or may not visible as an ordered array lined up. During interphase, the
be cleaved from the protein on entry to the appropriate cell chromatin is reorganized into areas that are either more con
compartment. Proteins may also undergo modifications such densed (and stainable), called heterochromatin associated
as glycosylation, proteolytic cleavage, and phosphoryla with less active genes, and less condensed regions termed
tion, which can affect their functional activity, stability, or euchromatin in which the DNA is more actively expressed
translocation between compartments in the cell. (Fig. 5B).
Within the cell cycle interphase, nuclear DNA replication
occurs during a discrete period, S phase. In nuclear DNA
THE NUCLEAR ENVIRONMENT replication and regulation, all sequences are replicated in a
tightly regulated process. Both strands are completely cop
Within the nucleus of the animal cells the chromosomes ied—albeit in different regions (or replication units)—on the
reside, each a collection of genes linearly arrayed on a long same chromosome and replicate at distinct times. Highly con
DNA molecule surrounded by a protein structure or scaffold densed chromatin replicates late in S phase, whereas genes in
ing structure called chromatin. The chromatin is composed active chromatin replicate early. Replication requires a DNA
primarily of proteins including five basic (positively charged) polymerase acting in concert with various enzymes, which
histones (HI, H2A, H2B, H3, and H4) and numerous non-his- are present to unwind the strands for initiation and elongation
SECTION I • Post-Genomic Cardiology
Hours
B
Condensed chromatin
(heterochromatin)
FIGURE 5 Eucaryotic cell cycle. (A) The relative timing of interphase (composed of S, Gp and G2 phases and the mitotic
(M) events of division. Many quiescent or non-dividing cells such as adult cardiomyocytes are withdrawn from the cell cycle
and remain at GQ. (B) Less condensed chromatin with more actively transcribing genes.
to occur and ligate the newly replicated fragments together the linear chromosome during cell division, an enzyme called
(e.g., helicases and DNA ligases, respectively). The accu telomerase recognizes the G-rich strand and elongates it using
rate duplication of DNA is profoundly important, and the an RNA template that is a component of the enzyme itself.
polymerizing enzymes such as DNA polymerase also pos Both the length of telomere repeats and the integrity of the
sess proofreading functions. Moreover, the nucleus contains telomere-binding proteins is also important for telomere pro
a dedicated group of enzymes to repair DNA damage as it tection, preventing chromosome ends from being detected as
occurs either during replication or post-replication. As we damaged DNA. In cultured cells lacking telomerase, the short
will discuss later, DNA damage is frequently associated with ening of telomeres results in both senescence-associated gene
cellular oxidative injury and aging and can be induced by a expression and inhibition of cell replication. Age-dependent
variety of physical and chemical agents or mutagens such as telomere shortening in most somatic cells, including vascular
ultraviolet hght, gamma irradiation, and a host of chemicals. endothehal cells, smooth muscle cells, and cardiomyocytes, is
The cell cycle is a central feature of proliferative or divid associated with impaired cellular function and viability of the
ing cells. A number of critical proteins (e.g., cyclins) have aged organism. In later chapters, we shall review the evidence
been identified that regulate the progression of events at sev that telomere dysfunction is implicated in the aging heart and
eral discrete stages (or checkpoints) of the cycle. Terminally is also a contributory factor in the cardiovascular dysfunction
differentiated cells (e.g., cardiomyocytes), which are said to associated with heart failure and hypertension.
be post-mitotic generally, do not enter mitosis, remain in a In addition to the nuclear DNA organized within the chro
perpetual interphase, and are, therefore, said to be withdrawn mosomes (23) present in haploid human cells (e.g., germUne
from the cell cycle. As we will see, this topic with respect to cells such as sperm and tgg) and 46 in human diploid cells
cardiomyocyte replication remains a highly contentious, but (e.g., somatic cells), DNA is also present in mitochondria
important, question to which we will return. {mtDNA). The mtDNA (Fig. 6) encodes a small set of pro
teins (13 subunits) involved in the mitochondrial respiratory
chain and oxidative phosphorylation, as well as components
TELOMERES AND MTDNA of mitochondrial-specific ribosomes (i.e., 2 rRNAs and 22
tRNA molecules) for the translation of the mtDNA-encoded
At the end of the chromosomes, specialized cap structures proteins. Interestingly, its genetic code is slightly different
called telomeres are present. They contain tandem repeats than the nuclear code. Most proteins (estimated at more than
of a short G-rich sequence (GGGTTA in humans) bound to 1000 kinds of protein) that define mitochondrial structure and
an array of specialized proteins, which constitute this hetero- function are encoded by nuclear DNA, translated on cytosolic
chromatin-associated cap structure. To replicate the ends of ribosomes, and imported into the mitochondrial organelle.
CHAPTER 1 • Introduction
D-loop larger areas of sequence are modified either by insertion,
deletion, or transposition of sequences. Point mutations can
be nucleotide substitutions, single insertions, or deletions. A
substitution can cause a change in the codon, resulting in a dif
ferent amino acid replacement in the protein, or it may cause
no change; an insertion or deletion can cause a change in the
reading frame, resulting in changed amino acids downstream
ND5
of the mutation site. Changes in protein caused by substituted
ND1
amino acid residues are referred to as missense mutations; the
severity of the change in regard to phenotype depends on the
kind of amino acid replaced (e.g., a charged amino acid for a
neutral one). A mutation can also change a codon from one
ND2
ND4 specifying an amino-acid residue to one that is a stop codon;
this causes a premature termination of the polypeptide chain
and is termed a nonsense mutation. The effect on the mutated
protein depends on its location within the chain; if it is located
near the N-terminus, the protein will be drastically shorter with
attendant effects on its function and, depending on the role of
con ATPasee
ATPaseS the protein within the cell, the cellular phenotype. Nonsense
mutations residing near the C-terminus of the protein may
FIGURE 6 Human mitochondrial double-stranded circular DNA. have limited effects on function and phenotype.
This circular DNA encodes 13 protein components of four of the
Some traits or phenotypes are determined by a single
five enzyme complexes involved in electron transport and OXPHOS,
two ribosomal RNAs (12S and 16S), and 22 tRNAs, as shown. The nuclear gene {monogenic). A specific defect in single genes
noncoding D-loop region is also shown. can cause a number of cardiovascular disorders, including
familial forms of hypertension, hypertrophic and dilated car
diomyopathy, long QT syndrome, structural anomalies of
PHENOTYPE/GENOTYPE
the heart and large vessels, and atherosclerosis because of
defects in lipoprotein metabolism.
Visible or observable properties that define the appearance of
Many traits are caused by more complex genotypes involv
a trait or organism constitute what is termed the phenotype,
ing several genes (polygenic). Cardiovascular diseases such
whereas the genetic factors, which are largely responsible
as hypertrophic cardiomyopathy (HCM) and hypertension
for creating the phenotype, are called the genotype. A gene
have been shown to have complex genotypes encompass
may exist in alternate forms that determine the expression
ing several genetic loci. In addition, the expression of spe
of particular phenotypes termed alleles. Diploid organisms,
cific phenotypes can be variable because of other influences
which carry two identical alleles for a specific gene, are said
including environment. Moreover, a single mutant allele can
to be homozygous with respect to that gene, whereas the
often have pleiotropic consequences affecting more than just
possession of two different alleles is termed heterozygous. In
the protein that it encodes. In addition to mutations in pro
some cases, one allele is said to be dominant when it deter
tein-encoding genes (i.e., structural gene mutations), muta
mines the phenotype irrespective of the presence of the other
tions can also be found in nonprotein genes such as those
allele (recessive). Alleles are said to be codominant when
involved in coding for the small RNAs (e.g., tRNAs) or
they contribute equally to the phenotype. Penetrance refers
larger RNA (e.g., ribosomal RNAs) that can effect protein
to the proportion of individuals with a specific genotype that
synthesis or in the noncoding regulatory areas of structural
expresses the related phenotype. A highly penetrant geno
genes such as regulatory mutations, which can impact the
type is more amenable to mapping by linkage analysis.
levels of specific gene expression.
The inheritance pattern can be highly informative as to the
MUTATIONS chromosomal location of the gene causing dysfunction (Fig. 7).
For instance, an X-linked inheritance pattem (indicating the pres
Differences in alleles are primarily due to mutation, a change ence of the gene of interest on the X chromosome) can be readily
in the sequence of the gene. Mutations can be deleteri distinguished from an autosomal inheritance pattem, in which
ous, advantageous, or neutral with respect to the organism. the gene is present on any of the non-sex chromosomes. Either of
Variations in nucleotide sequence that are frequently present these inheritance pattems follows the Mendelian rules of trans
in the population (>1%) are designated pofymorp/z/c alleles, mission. In contrast, a maternal pattem of inheritance in which
which can either be harmful or neutral. Mutations can be the patemal contribution is almost nil is also readily distinguish
either point mutations involving a single nucleotide change able and is a possible indicator of an mtDNA location, because
or multiple nucleotide changes or rearrangements in which mammalian mitochondria are derived mostly from the mother.
10 SECTION I • Post-Genomic Cardiology
30 kb allele on X 8 kb allele on X
chromosome with chromosome with
normal dystrophin mutant dystrophin
gene gene
30 8
30
8 kb allele on X
chromosome with
normal dystrophin
gene
30
30 8 8 30 8 8 8
FIGURE 7 Inheritance pattern of an X-linked disease (Duchenne muscular disease or DMD), with a
molecular mutation in the X chromosome indicated by a restriction fragment length polymorphism (RFLP).
Southern blotting was used to detect a polymorphism of 30 kb and 8 kb fragments; in this family, the mutation
causing DMD was located in 8 kb fragment.
The genetic background in which deleterious mutations may have an impact on the choice of diagnostic and treatment
occur can significantly modulate their phenotypic expres options (e.g., pharmacogenomics). Furthermore, the intensive
sion. For instance, a large number of recent studies have effort that is underway to identify SNPs in the human genome
reported the presence of modifier genes in the genetic is also noteworthy, because these SNPs can be used as specific
background that influence the phenotypic expression and gene-mapping markers. It is hoped that this genetic informa
severity of pathogenic HCM genes. The identification of tion will prove to be clinically useful in the development of
modifier genes, which will markedly improve the elucida highly individualized cardiovascular medicine.
tion of genetic risk factors, has been advanced by large-scale
genome-wide approaches to detect polymorphic variants References
correlated with disease severity.
A variety of molecular techniques are currently available 1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and
Walter, R (2002). "Molecular Biology of the Cell." 4th ed.
for the detection of single nucleotide polymorphisms (SNPs).
Garland Publishing, New York.
SNP association studies have identified several candidate
2. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore,
modifier genes for various cardiac disorders, and a number of
D., and Darnell, J. E. (1999). "Molecular Cell Biology." 4th ed.
specific DNA polymorphisms have been reported in associa
W. H. Freeman & Co., New York.
tion with myocardial infarction, coronary artery disease, and
3. Watson, J. D., Baker, T. A., Bell, S. B., Gann, A., Levine, M.,
HCM. With the increased cataloging of SNPs, either alone or
and Losick, R. (2004). "Molecular Biology of the Gene." 5th
within a larger chromosomal region (haplotypes) in available ed. Benjamin Cummings, San Francisco.
shared databases, these modifier loci can be evaluated for 4. Lewin B. (2004). "Genes VIII." Pearson Prentice Hall, Upper
their effects in predisposing to specific cardiac defects and Saddle River, NJ.