Epigenetic Influences and Disease

By: Danielle Simmons, Ph.D. (Write Science Right) © 2008 Nature

Education
Citation: Simmons, D. (2008) Epigenetic influence and
disease. Nature Education 1(1)
The behavior of a person's genes doesn't just depend on the genes'
DNA sequence--it's also affected by so-called epigenetic factors.
Changes in these factors can play a critical role in disease.
The external environment's effects upon genes can influence disease,
and some of these effects can be inherited in humans. Studies
investigating how environmental factors impact the genetics of an
individual's offspring are difficult to design. However, in certain
parts of the world in which social systems are highly centralized,
environmental information that might have influenced families can
be obtained. For example, Swedish scientists recently conducted
investigations examining whether nutrition affected the death rate
associated with cardiovascular disease and diabetes and whether
these effects were passed from parents to their children and
grandchildren (Kaati et al., 2002). These researchers estimated how
much access individuals had to food by examining records of annual
harvests and food prices in Sweden across three generations of
families, starting as far back as the 1890s. These researchers found
that if a father did not have enough food available to him during
a critical period in his development just before puberty, his sons
were less likely to die from cardiovascular disease. Remarkably,
death related to diabetes increased for children if food was plentiful
during thiscritical period for the paternal grandfather, but it
decreased when excess food was available to the father. These
findings suggest that diet can cause changes to genes that are passed
down though generations by the males in a family, and that these
alterations can affect susceptibility to certain diseases. But what are
these changes, and how are they remembered? The answers to
questions such as these lie in the concept of epigenetics.
What Is Epigenetics? How Do Epigenetic Changes Affect Genes?

Figure 1: Interaction between RNA, histone modification and DNA

methylation in heritable gene silencing.
Epigenetics involves genetic control by factors other than an
individual's DNAsequence. Epigenetic changes can switch genes on
or off and determine which proteins are transcribed.
Epigenetics is involved in many normal cellular processes. Consider
the fact that our cells all have the same DNA, but our bodies contain
many different types of cells: neurons, liver cells, pancreatic cells,
inflammatory cells, and others. How can this be? In short, cells,
tissues, and organs differ because they have certain sets of genes that
are "turned on" or expressed, as well as other sets that are "turned
off" or inhibited. Epigenetic silencing is one way to turn genes off,
and it can contribute to differential expression. Silencing might also
explain, in part, why genetic twins are not phenotypically identical.
In addition, epigenetics is important for X-chromosome inactivation
in female mammals, which is necessary so that females do not have
twice the number of X-chromosome gene products as males
(Egger et al., 2004). Thus, the significance of turning genes off via
epigenetic changes is readily apparent.
Within cells, there are three systems that can interact with each other
to silence genes: DNA methylation, histone modifications, and
RNA-associatedsilencing (Figure 1; Egger et al., 2004).
DNA Methylation
DNA methylation is a chemical process that adds a methyl group
to DNA. It is highly specific and always happens in a region in
which a cytosinenucleotide is located next to
a guanine nucleotide that is linked by a phosphate; this is called a
CpG site (Egger et al., 2004; Jones & Baylin, 2002; Robertson,
2002). CpG sites are methylated by one of three enzymes
called DNA methyltransferases (DNMTs) (Egger et al., 2004;
Robertson, 2002). Inserting methyl groups changes the appearance
and structure of DNA, modifying a gene's interactions with the
machinery within a cell's nucleus that is needed for transcription.
DNA methylation is used in some genes to differentiate
which gene copy is inherited from the father and which gene copy is
inherited from the mother, a phenomenon known as imprinting.
Histone Modifications
Histones are proteins that are the primary components of chromatin,
which is the complex of DNA and proteins that makes up
chromosomes. Histonesact as a spool around which DNA can wind.
When histones are modified after they are translated
into protein (i.e., post-translation modification), they can influence
how chromatin is arranged, which, in turn, can determine whether
the associated chromosomal DNA will be transcribed.
If chromatin is not in a compact form, it is active, and the
associated DNA can be transcribed. Conversely, if chromatin is
condensed (creating a complex calledheterochromatin), then it is
inactive, and DNA transcription does not occur.
There are two main ways histones can be
modified: acetylation and methylation. These are chemical processes
that add either an acetyl or methyl group, respectively, to the amino
acid lysine that is located in the histone. Acetylation is usually
associated with active chromatin, while deacetylation is generally
associated with heterochromatin. On the other
hand, histone methylation can be a marker for both active and
inactive regions of chromatin. For example, methylation of a
particular lysine (K9) on a specific histone (H3) that marks
silent DNA is widely distributed throughout heterochromatin. This is
the type of epigenetic change that is responsible for the inactivated X
chromosome of females. In contrast, methylation of a different lysine
(K4) on the same histone (H3) is a marker for active genes (Egger et
al., 2004).
RNA-Associated Silencing
Genes can also be turned off by RNA when it is in the form of
antisense transcripts, noncoding RNAs,
or RNA interference. RNA might affect geneexpression by
causing heterochromatin to form, or by
triggering histone modifications and DNA methylation (Egger et al.,
2004).
Epigenetics and Disease: Some Examples
While epigenetic changes are required for normal development and
health, they can also be responsible for some disease states.
Disrupting any of the three systems that contribute to epigenetic
alterations can cause abnormal activation or silencing of genes. Such
disruptions have been associated withcancer, syndromes involving
chromosomal instabilities, and mental retardation (Table 1).

Epigenetics and Cancer

The first human disease to be linked to epigenetics was cancer, in
1983. Researchers found that diseased tissue from patients with
colorectal cancerhad less DNA methylation than normal tissue from
the same patients (Feinberg & Vogelstein, 1983). Because
methylated genes are typically turned off, loss of
DNA methylation can cause abnormally high gene activation by
altering the arrangement of chromatin. On the other hand, too
much methylationcan undo the work of protective tumor
suppressor genes.
As previously mentioned, DNA methylation occurs at CpG sites, and
a majority of CpG cytosines are methylated in mammals. However,
there are stretches of DNA near promoter regions that have higher
concentrations of CpG sites (known as CpG islands) that are free
of methylation in normal cells. These CpG islands become
excessively methylated in cancer cells, thereby causing genes that
should not be silenced to turn off. This abnormality is the trademark
epigenetic change that occurs in tumors and happens early in
the development of cancer (Egger et al., 2004; Robertson, 2002;
Jones & Baylin, 2002). Hypermethylation of CpG islands can cause
tumors by shutting off tumor-suppressor genes. In fact, these types
of changes may be more common in
human cancer than DNA sequence mutations (Figure 2).
Furthermore, although epigenetic changes do not alter the sequence
of DNA, they can cause mutations. About half of the genes that
cause familial or inherited forms of cancer are turned off
by methylation. Most of these genes normally suppress tumor
formation and help repair DNA, including O6-methylguanine-DNA
methyltransferase (MGMT), MLH1 cyclin-dependent
kinase inhibitor 2B (CDKN2B), and RASSF1A. For example,
hypermethylation of the promoter of MGMT causes the number of
G-to-A mutations to increase (Figure 2).
Hypermethylation can also lead to instability of microsatellites,
which are repeated sequences of DNA. Microsatellites are common
in normal individuals, and they usually consist of repeats of the
dinucleotide CA. Too much methylation of the promoter of
the DNA repair gene MLH1 can make a microsatelliteunstable and
lengthen or shorten it (Figure 2). Microsatellite instability has been
linked to many cancers, including colorectal, endometrial, ovarian,
and gastric cancers (Jones & Baylin, 2002).

Figure 2: Mechanism of action of nucleoside analogue inhibitors.

Deoxynucleoside analogues such as 5-aza-2-deoxycytidine (depicted
by Z) are converted into the triphosphate inside S-phase cells and are
incorporated in place of cytosine into DNA. Ribonucleosides such as
5-azacytidine or zebularine are reduced at the diphosphate level by
ribonucleotide reductase for incorporation (not shown). Once in
DNA, the fraudulent bases form covalent bonds with DNA
methyltransferases (DNMTs), resulting in the depletion of active
enzymes and the demethylation of DNA. Pink circles, methylated
CpG; cream circles, unmethylated CpG.
© 2002, Nature Publishing Group, Jones, P. A., et. al., The
fundamental role of epigenetic events in cancer, Nature Reviews
Genetics 3, 415-428
Epigenetics and Mental Retardation

Figure 3: The marker X chromosome.

Metaphase chromosomes showing the peculiar constriction at the
end of the long arm of chromosome X that is characteristic in fragile
X (FX) individuals. The black arrowhead marks the marker X
chromosome in the upper right hand quadrant.
Fragile X syndrome is the most frequently inherited mental
disability, particularly in males. Both sexes can be affected by this
condition, but because males only have one X chromosome, one
fragile X will impact them more severely. Indeed, fragile
X syndrome occurs in approximately 1 in 4,000 males and 1 in 8,000
females. People with this syndrome have severe intellectual
disabilities, delayed verbal development, and "autistic-like" behavior
(Penagarikano et al., 2007).
Fragile X syndrome gets its name from the way the part of the X
chromosomethat contains the gene abnormality looks under a
microscope; it usually appears as if it is hanging by a thread and
easily breakable (Figure 3). Thesyndrome is caused by an
abnormality in the FMR1 (fragile X mental retardation 1) gene.
People who do not have fragile X syndrome have 6 to 50 repeats of
the trinucleotide CGG in their FMR1 gene. However, individuals
with over 200 repeats have a full mutation, and they usually show
symptoms of the syndrome. Too many CGGs cause the CpG islands
at the promoterregion of the FMR1 gene to become methylated;
normally, they are not. Thismethylation turns the gene off, stopping
the FMR1 gene from producing an important protein called fragile X
mental retardation protein. Loss of this specific protein causes fragile
X syndrome. Although a lot of attention has been given to the CGG
expansion mutation as the cause of fragile X, the epigenetic change
associated with FMR1 methylation is the real syndromeculprit.
Fragile X syndrome is not the only disorder associated with mental
retardation that involves epigenetic changes. Other such conditions
include Rubenstein-Taybi, Coffin-Lowry, Prader-
Willi, Angelman, Beckwith-Wiedemann, ATR-X, and Rett
syndromes (Table 1).
Combating Diseases with Epigenetic Therapy
Because so many diseases, such as cancer, involve epigenetic
changes, it seems reasonable to try to counteract these modifications
with epigenetic treatments. These changes seem an ideal target
because they are by nature reversible, unlike DNA sequence
mutations. The most popular of these treatments aim to alter
either DNA methylation or histone acetylation.
Inhibitors of DNA methylation can reactivate genes that have been
silenced. Two examples of these types of drugs are 5-azacytidine and
5-aza-2′-deoxycytidine (Egger et al., 2004). These medications work
by acting like the nucleotide cytosine and incorporating themselves
into DNA while it is replicating. After they are incorporated
into DNA, the drugs block DNMT enzymes from acting, which
inhibits DNA methylation.
Drugs aimed at histone modifications are called histone deacetylase
(HDAC) inhibitors. HDACs are enzymes that remove the acetyl
groups from DNA, which condenses chromatin and
stops transcription. Blocking this process with HDAC inhibitors
turns on gene expression. The most common HDAC inhibitors
include phenylbutyric acid, SAHA, depsipeptide, and valproic acid
(Egger et al., 2004).
Caution in using epigenetic therapy is necessary because epigenetic
processes and changes are so widespread. To be successful,
epigenetic treatments must be selective to irregular cells; otherwise,
activating gene transcription in normal cells could make them
cancerous, so the treatments could cause the very disorders they are
trying to counteract. Despite this possible drawback, researchers are
finding ways to specifically target abnormal cells with minimal
damage to normal cells, and epigenetic therapy is beginning to look
increasingly promising..

Epistasis: Gene Interaction and the Phenotypic Expression of

Complex Diseases like Alzheimer's
By: Ingrid Lobo, Ph.D. (Write Science Right) © 2008 Nature
Education
Citation: Lobo, I. (2008) Epistasis: Gene interaction and the
phenotypic expression of complex diseases like
Alzheimer's. Nature Education 1(1
Did you know that genes can mask and alter the effects of other
genes? Could this process, called epistasis, be a key to understanding
complex conditions like Alzheimer’s disease and diabetes?
When we think about factors that cause disease, we often think
about specific mutations in individual genes or the environmental
factors that contribute to adisease's phenotype. It is also important
to consider epistasis, which involves the interaction between two
or more genes (Figure 1; Carlborg & Haley, 2004). In fact,
understanding epistatic interactions may be the key to understanding
complex diseases, such as Alzheimer's disease, diabetes,
cardiovascular disease, and cancer.

How Common Is Epistasis in Disease Susceptibility?

Epistatic gene-gene interactions are perhaps more common than we
think. Indeed, some scientists believe that epistasisis ubiquitous in
biology and has been ignored for too long in studies of complex
traits (Moore, 2003; Carlborg & Haley, 2004). Research has shown
that genes don't function alone; rather, they constantly interact with
one another. These biological interactions are critical
for gene regulation, signal transduction, biochemical networks,
and numerous other physiological and developmental pathways
(Moore, 2003; Greenspan, 2001). As depicted in the schematic in
Figure 2, some genes (depicted as grey hexagons) have positive
interactions with one another (blue lines), while other gene pairs
have negative interactions (red lines). Together these gene-gene
interactions result in an output phenotype. Certain genes are known
to modify the phenotype of other genes, which results in
differences in disease penetrance and expressivity.
Epistatic interactions can complicate a scientist's search for
the gene responsible for a complex disease. For instance, the results
of most studies focusing on an initially promising
candidate gene have not been able to fully explain
complexdisease phenotypes in patients with the same disease once
more individuals were studied (Moore, 2003). This implies that
multiple genes may be involved, and that multiple genes may
interact to increase or decrease disease susceptibility. If the effect of
the disease-bearing gene is masked or altered by the effects of a
second gene, then identifying the first genecan be complicated. In
addition, if more than one epistatic interaction occurs to cause
a disease, then identifying the genes involved and defining their
relationships becomes even more difficult. There are, however, a
number of ways to studyepistasis in populations by adapting
methods used to detect quantitative trait loci (Carlborg & Haley,
2004).
Epistasis in Alzheimer’s Disease
To better understand how epistasis affects disease development,
it helps to consider an example of a complex disease.
Alzheimer's disease, for instance, is a progressive
neurodegenerative disorder that causes memory loss and dementia.
In the early 1990s, a number of scientists found that a gene called
apolipoprotein E4 was associated with a higher risk of developing
Alzheimer's disease (Corder et al., 1993; Saunders et al., 1993;
Strittmatter et al., 1993). However, the researchers also noted that
while having one or two copies of apolipoprotein E4 increase one's
risk of Alzheimer's, not all carriers of apolipoprotein E4 develop
the disease. This suggested that other genes and/or gene-gene
interactions were involved in the development of Alzheimer's.
Onofre Combarros and his colleagues thus set out to study the role
of epistasis in the onset of Alzheimer's disease(Combarros et al.,
2008). Because the research team realized that studying candidate
genes individually had met with little success, the team instead opted
to measure interactions between genes. In fact, Combarros et
al. evaluated the likelihood of over 100 published suggestions of
epistatic association in sporadic Alzheimer's disease. Some of these
alleged epistatic effects had been hypothesized to occur between
pairs of genes, but they had never been statistically tested. Thus, in
order to evaluate whether epistasis occurred, the researchers
measured both the size and the statistical significance of interactions
between pairs of implicated genes.
Eventually, Combarros et al. confirmed 27 different significant
epistatic interactions using this method, which were grouped into
five categories: cholesterol metabolism, beta-amyloid production,
inflammation, oxidative stress, and other networks. Some
interactions were synergistic, while others were antagonistic. The
synergistic interactions indicate that the pair of involved genes
together increase the risk of Alzheimer's disease. Meanwhile, the
antagonistic relationships indicate a protective relationship between
two genes. The strongest interactions involved the pairing of
apolipoprotein E4 with three different genes: alpha(1)-
antichymotrypsin, β-secretase, and butyrylcholinesterase K
(Combarros et al., 2008). Thus, it is clear that epistatic interactions
are involved in complex diseases like Alzheimer's disease, and that
these genes are not acting alone, but in pathways that affect one
another.
Many of the other predictions of epistasis between genes could also
prove to be significant if a larger population of Alzheimer's patients
was studied. Indeed, now that there is a foundation for understanding
epistatic interactions between pairs of genes in sporadic
Alzheimer's disease, future studies can focus on epistatic
interactions between combinations of three or more genes and
between additional pairs of genes.
Evidence for Epistasis in Other Diseases
Diabetes is another complex disease that is influenced by both
epistatic and environmental factors. Only in rare cases does
the disease appear to be monogenic, and generally, multiple genes
seem to be involved (Florez et al., 2003). While it is known that
diabetics have insufficient levels of insulin and high blood sugar
levels, the specific factors underlying disease susceptibility are still
being researched. For instance, interactions have been detected
between loci on chromosomes 2 and 15, as well as between loci on
chromosomes 1 and 10, in patients with type II diabetes (Cox et al.,
1999; Wiltshire et al., 2006). While we do not know the identities of
these genes now, it is hoped that they can eventually be mapped and
identified.
Evidence also exists that epistasis is involved with other complex
diseases, including cardiovascular disease, hypertension, autism,
cleft lip and/or palate, and schizophrenia and other neurological
disorders, as well as sporadic breast cancer, bladder cancer, and
other types of cancer (Combarros et al., 2008; Vieira, 2008).
Understanding the causes and genetic basis behind these diseases
proved elusive when using single-gene studies. However, now that
there is a greater focus on epistatic interactions, there may be more
progress toward understanding the manifestation of these complex
human diseases.
Making Sense of the Complex
It is now becoming possible to identify gene relationships,
networks, and epistatic interactions on a systems level. Today, high-
throughput experimental tools are available to measure molecular
and biochemical data. For example, DNA microarrays allow
scientists to gather hundreds of thousands of data points from cells,
with transcription level used as the measured phenotype. Then,
computational and bioinformatics methods can be used to sift and
sort though the massive amounts of biological data to search for
epistatic interactions. Once we identify and understand epistatic
relationships using techniques such as these, we can apply this
knowledge to better diagnose and treat complex diseases.
The Use of Animal Models in Studying Genetic Disease:
Transgenesis and Induced Mutation
By: Danielle Simmons, Ph.D. (Write Science
Right) © 2008 Nature Education
Citation: Simmons, D. (2008) The use of animal models in
studying genetic disease: Transgenesis and induced
mutation. Nature Education 1(1)
You are more like a mouse than you might think! Today, scientists
are creating models of human genetic disease using mice, flies,
worms, and other animals. But what do these models reveal about
us?
Except in the case of highly controlled and regulated clinical trials,
geneticists and scientists do not use humans for their experimental
investigations because of the obvious risk to life. Instead, they use
various animal, fungal, bacterial, and
plant species as model organisms for their studies. Some
suchspecies are described in Table 1.
Table 1: Models Used to Study Genetic Principles and Human
Diseases
Model Common
Research Applications
Organism Name

SaccharomycesYeast Used for biological studies

cerevisiae of cell processes
(e.g., mitosis) anddiseases (e.g., cancer
 Used by Gregor Mendel to describe
Pisum sativum Pea plant
patterns of inheritance

Employed in a wide variety of studies

ranging from early genemapping
Drosophila
Fruit fly via linkage and recombination studies, to
melanogaster
large scalemutant screens to identify genes
related to specific biological functions

Valuable for studying the development

Caenorhabditis
Roundworm
simple nervous systems and
elegans (nematode)
the aging process

Used for mapping and identifying genes

Danio rerio Zebra fish
involved in organdevelopment

Commonly used to study genetic principles

Mus musculusHouse mouse
and human disease

Commonly used to study genetic principles

Rattus norvegicus
Brown rat
and human disease
When animal models are employed in the study of human disease,
they are frequently selected because of their similarity to humans in
terms of genetics, anatomy, and physiology. Also, animal models are
often preferable for experimental disease research because of their
unlimited supply and ease of manipulation. For example, to obtain
scientifically valid research, the conditions associated with an
experiment must be closely controlled. This often means
manipulating only one variable while keeping others constant, and
then observing the consequences of that change. In addition, to test
hypotheses about how a disease develops, an adequate number of
subjects must be used to statistically test the results of the
experiment. Therefore, scientists cannot conduct research on just one
animal or human, and it is easier for scientists to use sufficiently
large numbers of animals (rather than people) to attain significant
results.
Rodents are the most common type of mammal employed in
experimental studies, and extensive research has been conducted
using rats, mice, gerbils, guinea pigs, and hamsters. Among these
rodents, the majority of genetic studies, especially those
involving disease, have employed mice, not only because their
genomes are so similar to that of humans, but also because of their
availability, ease of handling, high reproductive rates, and relatively
lowcost of use. Other common experimental organisms include fruit
flies, zebra fish, and baker's yeast.
Methods of Inducing Human Disease in Other Organisms
Despite their genomic similarities to humans,
most model organisms typically do not contract the same genetic
diseases as people, so scientists must alter their genomes to induce
human disease states. In attempting to engineer a genetic
mouse model for a human disorder, for example, it is important to
know what kind of mutation causes the disease (for example, is
the disease null, hypomorphic, or dominant negative?), so that the
same kind of mutationcan be introduced into the corresponding
mouse gene.
Scientists approach this task in two main ways: one that is directed
and disease driven, and the other that is nondirected
and mutation driven (Hardouin & Nagy, 2000). The nondirected,
mutation-driven method uses radiation and chemicals to cause
mutations. One common technique associated with this method is the
large-scale mutation screen. On the other hand, the directed,
disease-driven approach can employ any one of a number of
techniques, depending on the exact type of mutation involved in
the disease under study. Common directed techniques include
transgenesis, single-gene knock-outs and knock-ins,
conditional gene modifications, and chromosomal
rearrangements.
Large-Scale Mutation Screens
As the name might imply, indirect approaches attempt to randomly
make mutations in animal models' genomes. Then, the animals are
screened in an attempt to determine which ones show phenotypes
that are similar to human diseases. Thus, instead of being driven by
the disease mutation, these methods are based on screening the
phenotypes.
Two of the most effective ways to generate mutations are by
exposing organisms to X-rays or to the chemical N-ethyl-N-
nitrosourea (ENU). X-rays often cause
large deletion and translocation mutations that involve multiple
genes (Bedell et al., 1997a), whereas ENU treatment is linked to
mutations within single genes, such as point mutations (Hardouin &
Nagy, 2000). ENU can produce mutations with many different types
of effects, such as loss and gain offunction (Rosenthal & Brown,
2007), and it is frequently employed in screens in model organisms
such as zebra fish. These types of models are particularly useful for
identifying new genes and pathways that contribute to disease.
Transgenesis
As opposed to the use of X-rays and ENU, transgenesis is a directed
approach. Transgenic animals are generated by adding foreign
genetic information to the nucleus of embryonic cells, thereby
inhibiting gene expression. This can be achieved by either injecting
the foreign DNA directly into the embryo or by using a retroviral
vector to insert the transgene into an organism's DNA. The first
mouse gene transfers were performed in 1980 (Hardouin & Nagy,
2000); however, at that time, the methods for transgenesis were not
optimal. For instance, the foreign DNA was incorporated into only a
small percentage of embryos and was inconsistently passed to the
next generation. Also, small transgenes were inserted into random
sites in the genome, and depending on their location, they weren't
always expressed. More recently, scientists have developed a way to
increase the size of the DNA fragments used in transgenesis by
cloning them in yeast or bacterial artificial chromosomes (YACs or
BACs, respectively). These larger transgenes are more likely to
contain regulatory sequences necessary for normal gene expression
and are usually more comparable to the endogenous gene (Bedell et
al., 1997a). As a result, the use of transgenic mice has dramatically
increased in the past two decades, and this type of animal model has
contributed greatly to our knowledge ofdisease development.
Single-Gene Knock-Outs and Knock-Ins
Both knock-out and knock-in models are ways to target
a mutation to a specific gene locus. These methods are particularly
useful if a single gene is shown to be the primary cause of
a disease. Knock-out mice carry a gene that has been inactivated,
which creates less expression and loss of function; knock-in mice
are produced by inserting a transgene into an exact location where
it is overexpressed. Over the years, more than 3,000 genes have been
knocked out of mice, and most of these genes have been related to
disease (Hardouin & Nagy, 2000).
Both knock-out and knock-in animals are created in the same way: a
specific mutation is inserted into the endogenous gene, and then it
is conveyed to the next generation through breeding. The use of
embryonic stem (ES) cells is required for this technology. This is
because ES cells can contribute to all celllineages when injected into
blastocysts, and they can be genetically modified and selected for the
desired gene changes. Homologous recombinationcreates the
mutations; this is a process that physically rearranges two strands
of DNA for the exchange of genetic material. Many types of
mutations can be introduced into a model gene in this way,
including null or point mutations and complex chromosomal
rearrangements such as large deletions, translocations, or
inversions (Bedell et al., 1997a). Many knock-out and knock-in mice
have similar, if not identical, phenotypes to human patients and are
therefore good models for human disease.
Conditional Gene Modifications
One drawback to using transgenic, knock-in, and knock-out mice to
study human diseases is that many disorders occur late in life, and
when genes are altered to model such diseases, the mutations can
profoundly affect development and cause early death. These effects
would preclude using animal models to study adult diseases.
Thankfully, new technology has made it possible to generate
mutations in specific tissues and at different stages ofdevelopment,
including adulthood. To do this, mice with two different types of
genetic alterations are needed: one that contains a conditional vector,
which is like an "on switch" for the mutation, and one that contains
specific sites (called loxP) inserted on either side of a whole gene,
or part of a gene, that encodes a certain component of a protein that
will be deleted (Bedell et al., 1997a). A conditional vector for
the gene is made by inserting recognition sequences for the bacterial
Cre recombinase (loxP sites) using homologous recombination in
ES cells. The vector contains a drug-resistant marker genethat
allows only the targeted ES cells to survive when exposed to
the drug. Thus, the mutant ES cells can be selected and injected
into the host mouseembryo, which is implanted into a
foster mother. The resulting offspring are chimeras and have
multiple populations of genetically distinct cells.
Chimericoffspring are then crossed, and the resulting generation
of offspring has the recombinase effector gene. The mice
containing the Cre recombinase under the control of tissue-specific
or inducible regulatory elements are crossed to the mice with the
desired loxP sites. When Cre is expressed, recombinationoccurs at
the loxP sites, which delete the intervening sequences, and the
resulting mutation is induced in specific regions and times. These
conditionalmutant models are becoming increasingly popular, and
international initiatives have been created to accommodate their
demand (Rosenthal & Brown, 2007).
Chromosomal Rearrangement
The aforementioned advances in ES cells and Cre/loxP conditional
mutations have helped pave the way for the creation of models for
complex human diseases involving chromosomal rearrangements.
Mouse models of these disorders can be created using indirect
approaches, such as radiation, but their usefulness is restricted
because pathological endpoints are unpredictable and undefined (Yu
& Bradley, 2001). Using the Cre/loxP recombination system
overcomes these setbacks by allowing site-specific mutations
necessary to produce accurate models of defects caused by
human chromosomal rearrangements. These mutations can
include chromosome deletions, duplications, inversions, and
translocations, as well as nested chromosomedeletions.
Mouse Models Exist for Many Human Genetic Diseases, but Are
They Effective?
Human genetic diseases affect a wide range of tissues throughout the
body and are caused by numerous types of mutations. Creating
mouse models for all these disorders is understandably a daunting
task for scientists. Nevertheless, over 1,000 mutant strains exist, and
most of these mutants are models for inherited genetic diseases
(Hardouin & Nagy, 2000). Models for human disease have been
made by mutating the same gene in mice that is responsible for the
human condition for about 100 genes (Bedell et al., 1997b), and in
most cases, these models replicate many of the corresponding
human diseasephenotypes. These diseases include several types
of cancer, heart disease, hypertension, metabolic and hormonal
disorders, diabetes, obesity, osteoporosis, glaucoma, skin
pigmentation diseases, blindness, deafness, neurodegenerative
disorders (such as Huntington's or Alzheimer's disease), psychiatric
disturbances (including anxiety and depression), and birth
defects (such as cleft palate and anencephaly) (Rosenthal & Brown,
2007).
Animal models have greatly improved our understanding of the
cause and progression of human genetic diseases and have proven to
be a useful tool for discovering targets for therapeutic drugs.
Nonetheless, despite promising results with certain preclinical
treatments in animal models, the same treatments do not always
translate to human clinical trials. As a result, many diseases are still
incurable. Most available animal models are made in mice, and they
recreate some aspects of the particular disease. However, few, if
any, replicate all the symptoms. This statement is particularly true
for neurodegenerative diseases, most of which involve cognitive
deficits. One reason that mouse models might not completely mimic
human disorders is that mice simply might not be capable of
expressing some cognitive human disease symptoms that are
apparent to the observer. For example, Huntington's disease patients
show dyskinesia (involuntary movements), whereas mice do not.
Perhaps using nonhuman primates might alleviate some of these
discrepancies because their physiology is closer to that of humans. In
fact, some researchers have pursued this possibility despite the
technical difficulties and additional costs to perform transgenesis in
primates (Wolfgang & Golos, 2002). For example, a
transgenic model of Huntington's disease was recently developed
using rhesus macaques that replicated some of the characteristic
pathologies of the disorder as it occurs in humans (Yang et al.,
2008). Indeed, because of the tremendous genetic resources that are
currently available, use of nonhuman primate models might become
more accessible and might lead us into a new era
of disease research and drug discovery.
Rare Genetic Disorders: Learning About Genetic Disease through
Gene Mapping, SNPs, and Microarray Data
By: Heidi Chial, Ph.D. (Write Science Right) © 2008 Nature
Education
Citation: Chial, H. (2008) Rare genetic disorders: Learning about
genetic disease through gene mapping, SNPs, and microarray
data. Nature Education 1(1)
Most diseases are caused by mutations in more than one gene. So
what clues can monogenic, or single-gene disorders provide?
Researchers have made dramatic inroads into the study
of polygenic and other complex human diseases, due in large part
to knowledge of the human genome sequence, the generation of
widespread markers of genetic variation, and the development of
new technologies that allow investigators to
associate disease phenotypes with genetic loci.
Although polygenic diseases are more common than single-gene
disorders, studies of monogenic diseases provide an invaluable
opportunity to learn about underlying molecular mechanisms,
thereby contributing a great deal to our understanding of all forms of
genetic disease.
Mendel Revisited: Monogenic Diseases
The human genome contains an estimated total of 20,000-25,000
genes that serve as blueprints for building all of our proteins
(International Human Genome Sequencing Consortium, 2004). In
single-gene diseases, a mutation in just one of these genes is
responsible for disease. Single-gene diseases run in families and
can be dominant or recessive, and autosomal or sex-
linked. Pedigree analyses of large families with many affected
members are very useful for determining the inheritance pattern of
single-gene diseases. Table 1 includes some examples of single-gene
diseases.
OMIM, Online Mendelian Inheritance in Man, is a regularly
updated, online database established in 1997 by Dr. Victor A.
McKusick that is focused on inherited genetic diseases in humans.
As of June 15, 2008, OMIM reported 387 human genes of known
sequence with a known phenotype, and 2,310 human phenotypes
with a known molecular basis. However, OMIM also reported 1,621
confirmed Mendelian phenotypes for which the molecular basis is
not known. Furthermore, OMIM reported 2,084 phenotypes for
which a Mendelian basis is suspected but has not been fully
established, or that may exhibit overlap with other characterized
phenotypes. As you can see, more questions than answers remain
regarding the identity of single genes and their role in
human disease.
Trends in Gene Discovery
After the human genome was sequenced, researchers began to shift
their focus from monogenic diseases to polygenic diseases, which
involve many genes. There are several reasons for this movement
toward polygenic diseases. For one, many of the 1,621 monogenic
disorders without known genes are very rare. As a result, researchers
face difficulties in identifying families with the disease and in
obtaining sufficient numbers of DNA samples for comparison to
unaffected family members. Also, funding
agencies, biotechnology companies, and pharmaceutical companies
are often less likely to invest financial resources in research efforts
focused on rare diseases.
However, studies of monogenic diseases contribute a great deal to
knowledge of polygenic forms of human disease (Antonarakis &
Beckmann, 2006). To this end, monogenic diseases are most worthy
of our attention.
Back to the Future: Using New Technologies to Find Old Genes
Before the human genome was sequenced, researchers relied on
labor-intensive, slow-going techniques for mapping and isolating
disease-associated genes. For instance, although efforts to isolate
the gene associated with Huntington's disease began in the late
1970s, the gene was not identified until 1993.
With the sequence of the human genome available, researchers have
been able to generate maps of each chromosome, showing the
precise location of every gene and determining areas of
the genome that can differ from one person to the next (termed
"polymorphic"). Of special interest to researchers are
single nucleotide polymorphisms (SNPs), which are
single base pair polymorphic regions. SNPs occur throughout the
human genome at an average rate of one SNP per every
1,000 base pairs. The International HapMap Project has mapped
SNPs along the length of every human chromosome and has made
this information freely available to scientists worldwide.
These polymorphic SNP markers can be used to map disease-
associated genes.
Researchers have developed methods for the simultaneous analysis
of up to one million different SNPs throughout the entire
human genome using genomic DNA isolated from a
blood sample (or any other biological source of DNA) and a
single DNA chip, which is a small silicon glass wafer onto which
single-stranded DNA fragments can be adhered in a grid-like
pattern. A SNP chip contains short, single-stranded DNA molecules
called oligonucleotides that correspond to known SNP variants.
The DNA isolated from the blood sample is broken into fragments,
labeled with a fluorescent dye, and converted into single-
stranded DNA. The single-stranded, fluorescently labeled
genomic DNA fragments are then incubated with the SNP chip, and
only those DNAfragments that are a perfect match will bind to
their complementary SNP oligonucleotide on the SNP chip grid. A
laser is then used to scan each grid position to determine which SNP
variants are represented.
By using SNP chips, researchers can obtain a SNP profile of an
individual that spans the entire genome. SNP profiles can be
compared between affected and unaffected family members (or
unaffected, unrelated individuals) to determine which SNPs
segregate with a disease (or are associated with thedisease).
Because the SNP sequences have already been mapped to specific
chromosomal locations, researchers can also immediately map the
disease-associated gene to a specific region of a given
human chromosome.
The Bioinformatics Era: Genomics and Proteomics
Bioinformatics is the genome-inspired field of biology that
analyzes genomic information to
predict gene and protein function. Bioinformaticists can easily
examine a region of a chromosome and determine which segments
correspond to protein-encoding genes. Furthermore, they can
compare the sequence of a gene of unknown function to the rest of
the genome and find similar genes with known functions. Based on
similarity between genes, researchers can often predict how gene-
encoded proteins may function within a cell.
Large-scale studies of genes and proteins are referred to
as genomics and proteomics, respectively. Researchers can now
use gene chips to simultaneously examine the expression (mRNA)
levels of all human protein-encoding genes from a
given cell population. Gene chips are similar in concept to SNP
chips, but their grids contain single-stranded DNA fragments that
correspond to protein-encoding genes. In order to
study gene expression, researchers first isolate mRNA from a tissue
of interest, then convert it into single-
stranded complementary DNA (cDNA) and label it with a
fluorescent dye. The single-stranded, fluorescently labeled cDNA is
then incubated with the gene chip, allowing hybridization between
the cDNA molecules and theircomplementary sequences on
the gene chip grid. A laser is used to scan the chip and determine the
fluorescent signal associated with every mRNArepresented on
the gene chip grid system to yield a gene expression profile for a
given individual. By comparing gene expression profiles from
normal and diseased individuals, scientists can also determine
changes in gene expression associated with human disease.
More recently, researchers have developed chip-based methods for
the simultaneous examination of thousands of proteins within a
given cell population. In these approaches, the protein chip grid
contains adhered antibodies that recognize and bind to specific
human proteins. The protein chip is incubated with a fluorescently
labeled protein sample from a given individual, and a laser is used
to scan the chip to determine the levels of each protein represented
by the antibodies on the grid. In this way, researchers can determine
the proteomic profile associated with a given form of
human disease, and they can see which proteins show altered
expression.
Databases
As you can imagine, genomic and proteomic approaches, which
simultaneously examine thousands of genes and proteins, generate a
tremendous amount of data. Furthermore, scientists are publishing
new data at a very fast pace. In order to make meaningful
connections among worldwide scientific discoveries, a number of
databases have been established. Examples of useful databases
include OMIM and Entrez Gene, which provide a number of useful
links to other databases.
From Man to Mouse: Using Genetic Model Organisms to
Understand Single-Gene Diseases in Humans
Due to the remarkable level of homology between genomes across
the evolutionary tree, scientists can learn a lot about the underlying
molecular mechanisms associated with single-gene diseases in
humans by studying organisms that are much simpler: mice, frogs,
worms, flies, and even yeast. Many of the genes found in humans
are also present in these other types of organisms. Moreover, several
of the same basic cellular processes are shared among humans and
these organisms, including metabolism, cell division, growth
regulation, and more. Although this discussion focuses on mouse
models, many seminal discoveries relevant to our understanding of
human disease have come from studies of the same type
of yeast used to make bread and beer.
Similar to that of humans, the entire sequence of the
mouse genome is known. Many human genes are also found in
mice, and using mice as a modelorganism for genetic studies has
contributed to our understanding of human disease. Today,
researchers can generate mice with a mutation or deletion of a
disease-associated gene. They can carry out detailed phenotypic
analyses of the mutant mice and learn how the
corresponding gene may function in humans. For example,
researchers have developed a mouse model of Huntington's disease,
in which the mutant mice carry the expanded CAG repeat within the
Huntington's disease-associated gene. Although genetically
engineered mice are not perfect models of human disease, they can
offer valuable insights into the function of disease-associated genes.
Studies of single-gene diseases in humans have led to many
completely unexpected findings. One such example is the discovery
of trinucleotide repeat expansions and their association with several
forms of neurodegenerativedisease, including
Huntington's disease (HTT gene), myotonic dystrophy
(DMPK gene), fragile X syndrome (FMR1 gene), Friedreich's
ataxia (FRDAgene), and spinocerebellar ataxias
(SCA1, SCA2, SCA3, and ATXN1 genes).
Research regarding single-gene human diseases has also uncovered
"modifier" genes that can alter the severity of phenotypes associated
with mutations in the primary disease-associated gene. For instance,
for many years, cystic fibrosis was considered a single-gene disease
associated with mutations in the cystic fibrosis-
associated gene, CFTR. However, the initial discovery of
the CFTR gene was followed by the identification of several
additional genes that contribute to cystic fibrosis; several modifier
genes have also been identified that can modulate the phenotypes
associated with mutations in CFTR (Guggino & Stanton, 2006).
Cystic fibrosis-associated phenotypes due to mutations in
the CFTR gene are in turn modulated by mutations in the following
genes: gastrointestinal phenotypes (MUC1), pulmonary phenotypes
(TNF, TGFB1, and MBL2), bowel obstruction at birth/meconium
ileus (CFM1), and microbial infections (NOS1).
In addition, studies of monogenic disease transmission in identical
twins have uncovered various nongenetic mechanisms associated
with disease. For example, identical twins with the
same mutation in the gene associated with Duchenne muscular
dystrophy, called DMD, can exhibit strikingly
different disease phenotypes due to different patterns of X
chromosome inactivation (Abbadi et al., 1994).
Finally, monogenic syndromes can sometimes serve as models for
complex diseases. Consider the example of Van der
Woude syndrome, which is characterized by lower lip pits,
orofacial clefts, and even occasional hypodontia. This disorder is
caused by dominant mutations in the IRF6 (interferon regulatory
factor 6) gene (Kondo et al., 2002). Scientists have proposed
that IRF6 variation may also contribute to isolated cleft lip with or
without cleft palate (Zucchero et al., 2004), a complex birth
defect suggested to be caused by as many as three to 14 genes
(Schliekelman & Slatkin, 2002). Indeed, studies with a
large sample data set have shown that IRF6 variation contributes to
an expressive proportion of isolated cleft lip with or without cleft
palate cases (Zucchero et al., 2004). In related studies, researchers
isolated tooth agenesis, another complex phenotype commonly
found in the generalpopulation and that is present in a subset of
cases of Van der Woude syndrome, and they showed
that IRF6 variation contributes to this condition as well (Vieira et
al., 2007). Such results are of interest because they indicate that the
same gene can cause a disease as rare as Van der
Woude syndrome (with a frequency of 1:100,000 births; Figure 1)
and also contribute to much more common defects, such as isolated
cleft lip with or without cleft palate (frequencyof 1:700 births) and
isolated tooth agenesis (frequency of 1:100 births), that have more
complex genetic etiologies.
From Simple Beginnings to Complex Endings
Armed with knowledge of the human genome sequence and an
arsenal of new molecular tools for gene discovery,
today's gene hunters are prepared to greatly expand our knowledge
of disease-associated genes. Most certainly, our collective
knowledge of single-gene diseases, with the help of databases and
reference systems, has the potential to advance our understanding of
all types of human disease in ways far greater than imagined at the
time of each individual discovery.
Cytogenetic Methods and Disease: Flow Cytometry, CGH, and FISH
By: Heidi Chial, Ph.D. (Write Science Right) © 2008 Nature
Education
Citation: Chial, H. (2008) Cytogenetic methods and disease: Flow
cytometry, CGH, and FISH. Nature Education 1(1)
Some diseases involve regions of chromosomes that have been
flipped or damaged. Find out what techniques scientists are using to
dissect these chromosomes at the molecular level.
Cytogenetic approaches to studying chromosomes and their
relationship to human disease have improved greatly over the past
several decades. Modern cytogenetic approaches enable researchers
to do the following, among other things:
Precisely label the chromosomal location of any gene using
different colored dots
Examine cells from any type of tissue, even tumor cells
Identify cells that have lost or gained a specific chromosome,
undergone a translocation event involving a specific set of
chromosomes, or lost or gained a copy of a given gene or genes
Determine whether specific regions of chromosomes have been lost
or gained without ever looking at the chromosomes under a
microscope
Clearly, the field of cytogenetics has developed into a vital tool for
studying and diagnosing human disease.
The Emergence of a New Field
The field of human cytogenetics was initiated in 1956, when the
number of chromosomes in a diploid human cell was accurately
determined to be 46 (Tjio & Levan, 1956). Since then, our
knowledge of human cytogenetics and our ability to utilize
cytogenetic data to understand and diagnose human disease has
increased by leaps and bounds (Speicher & Carter, 2005; Trask,
2002).
As the field of human cytogenetics emerged, researchers developed
methods to visualize chromosome structure and organization.
Scientists quickly realized that not all chromosomes are created
equal: specifically, they differ in their length and in the position of
their centromere. Researchers also embarked on numerous studies
to determine the relationship between human disease and
chromosomes.
Early cytogenetic studies showed that an extra or missing copy of
certain human chromosomes could lead to disease. For example, in
1959, an extra copy of chromosome 21 was shown to be associated
with Down syndrome (also called trisomy 21) (Lejeune et al.,
1959). In the same year, several abnormalities
in sex chromosome number were linked to disease:
Turner's syndrome was shown to be associated with the presence of
a single X chromosome and no Y chromosome (45,X) (Ford et
al., 1959), whereas Klinefelter's syndrome was determined to be
associated with the presence of two copies of the X
chromosome and one copy of the Y chromosome (47,XXY)
(Jacobs & Strong, 1959). Both Turner's syndrome and
Klinefelter's syndromeaffect sexual differentiation in affected
invidivuals.
The Philadelphia Chromosome
Soon after discovering the link between chromosome number
and disease, researchers turned their attention to the role
of chromosome structure. Thus, in 1960, a collaborative study
between Peter Nowell, a new faculty member at the University of
Pennsylvania, and David Hungerford, a graduate student at the
Institute for Cancer Research in Philadelphia, uncovered a link
between chronic myelogenous leukemia (CML) and abnormal
chromosome structure. Specifically, the researchers discovered the
presence of a small chromosome, which they named the
"Philadelphia chromosome," that was unique to CML cells (Nowell
& Hungerford, 1960). Later, in 1968, Janet Rowley used
new chromosome staining techniques to show that the
Philadelphia chromosomearose as a result of a translocation event
involving chromosomes 9 and 22 (Rowley, 1973). Then, in 1985, the
breakpoint of the translocation was mapped and shown to result in
the fusion of parts of the BCR gene from chromosome 22 and
the ABL1 gene from chromosome 9, resulting in a gene fusion
product called BCR-ABL (Heisterkamp et al., 1985). The ABL1 half
of the encoded fusion protein exhibits high tyrosine kinase activity,
which is largely responsible for CML-associated phenotypes. Using
this information, scientists developed a drug called imatinib
mesylate (also called STI571 and Gleevec) to inhibit ABL1 kinase
activity (Druker, 2002). Imatinib treatment of CML has been
heralded as one of the biggest success stories
in cancer treatment over the past decade.
Cri du Chat Syndrome and Retinoblastoma
During the period in which Nowell and Hungerford discovered the
Philadelphia chromosome, other scientists were examining the link
between chromosomal deletions and disease. An important
breakthrough occurred in 1963, when cri du chat syndrome,
a congenital disorder that is associated with severe mental
retardation and a cat-like cry in affected infants, was found to result
from a loss of part of the short arm of chromosome 5 (Lejeune et
al., 1963). That same year, early studies of cells from patients with
retinoblastoma, a childhood form of retinal cancer, also
showed deletion of a specific chromosomal region (Lele et al.,
1963). This region was later determined to harbor the RB1 gene, the
first official tumor suppressor gene ever identified. Studies
of RB1-associated retinoblastoma led to the establishment of the
two-hit hypothesis, which is a cornerstone
of cancer biology (Knudson, 1971).
Using Cytogenetic Approaches to Map Genes
In addition to providing associations between chromosomal
abnormalities and disease, cytogenetic approaches have also
allowed researchers to map genes to particular chromosomes. For
example, in 1968, Roger Donahue used new methods to
study metaphase chromosomes in his own blood cells, and he noted
that one of his copies of chromosome 1 had a region near
the centromere that was loosely structured and uncoiled. Using
his extended familypedigree and conducting biochemical tests to
determine blood group markers, Donahue employed cytogenetic
techniques to map the Duffy blood
grouplocus to chromosome 1 (Donahue et al., 1968).
Shortly after the Duffy blood group locus was mapped, Maximo
Drets and Margery Shaw established methods to
stain metaphase chromosomes using a dye called Giemsa, which
produces a signature banding pattern, called G-bands, for each
of the 24 different human chromosomes (Drets & Shaw, 1971).G-
banding patterns can be used to detect chromosomal translocations,
deletions, and insertions, and they have made key advances
in gene discovery possible. For instance, as previously mentioned,
Rowley used G-banding patterns to determine that
a translocation event involving chromosomes 9 and 22 was
responsible for CML (Rowley, 1973). G-banding methods continue
to be widely used today, though such approaches have certain
drawbacks. For instance, G-
banding requires metaphase chromosomes, which are easily
obtained from blood samples but are more difficult to retrieve from
solid tissue samples. Furthermore, metaphase chromosomes are
highly condensed, which can lead to lower resolution in mapping.
Human-Mouse Somatic Cell Hybrids
Although cytogenetic approaches evolved over time such that
chromosomes could be easily distinguished from each other,
researchers also needed ways to study individual chromosomes in
more detail. In an effort to meet this need, researchers used the
Sendai virus to induce fusion between a human cell and a
mouse cell, resulting in a human-mouse somatic cell hybrid that
contained the complete mouse genome, as well as sparse numbers
of human chromosomes (Ephrussi & Weiss, 1965; Harris &
Watkins, 1965). An extensive series of human-
mouse hybrid cell lines that carried known combinations of human
chromosomes was thus developed, and this series greatly facilitated
the mapping of human genes to specific chromosomes prior to the
advent of the Human Genome Project.
Using Flow Cytometry to Sort Chromosomes
.
Yet another advance in cytogenetic techniques involved the process
known as flow cytometry, which was originally used to study
distinct cell populations within a mixture of different cell types.
With this technique, a fluorescent dye is used to specifically label
the cell population of interest. Individual cells can then be
examined one at a time as they are pulled through the flow cytometer
and subjected to laser-diffracted light to determine cell size and
shape. Fluorescently labeled cells can also be sorted into separate
tubes, based on their size and the intensity of their fluorescence
signal, using diffraction plates in a process called fluorescence-
activated cell sorting (FACS).
Although flow cytometry and FACS were initially used to isolate
populations of intact cells, researchers adapted these techniques to
isolate individual human chromosomes as shown in Figure 1. Such
techniques involve using mitotic cellsuspensions and disrupting
the cell membranes to release the condensed chromosomes that are
labeled using two different types of fluorescent dyes (Carrano et al.,
1979). The first dye, called Hoechst 33269, binds to A-T base pairs,
and the second dye, called chromomycin A, binds to G-C base pairs.
FACS is used to isolate individual fluorescently labeled
chromosomes in the solution, and with the exception of four
chromosomes (9, 10, 11, and 12), all of the human chromosomes can
be resolved based on their laser light-scattering properties. In
addition to isolating pools of individual chromosomes, this approach
can be used to determine changes in chromosome size and number.
Larger chromosomes contain a higher number of A-T or G-
C base pairs, leading to higher levels of Hoechst and chromomycin
staining, respectively. As illustrated in Figure 1, chromosome 21,
which is the smallest, shows the lowest Hoechst and chromomycin
staining intensity. Chromosomes 1 and 2, which are the largest, show
the highest Hoechst and chromomycin staining. In general, the A-T
and G-C contents of a given chromosome are quite similar, and that
is why a diagonal line of chromosomes, each of increasing size,
connects the smallest (21) to the largest (1 and 2) in Figure 1.
Fluorescence In Situ Hybridization (FISH)
.
In further chromosomal studies, researchers used restriction enzymes
to cut pooled chromosome populations into
smallerDNA fragments. The resulting DNA fragments were ligated
into DNA plasmid vectors, which allowed them to be propagated
in bacteria or yeast host cells. This led to the generation of
chromosome-specific collections of DNAfragments, called libraries,
which served as a platform for the Human Genome Project.
Furthermore, the ability to isolate collections of DNA fragments
that span individual chromosomes led to the development of
chromosome-specific staining methods.
Researchers also wanted to study regions of individual chromosomes
within the nucleus of intact cells. Thus, they used a cytogenetic
method called fluorescence in situ hybridization (FISH) to
map DNA sequences to specific regions of human
chromosomes. FISH involves the use of fluorescently labeled DNA
probes that are capable of hybridizing
to complementary chromosomal regions. This technique allows
researchers to view the chromosomal location of a
particular gene or DNA sequence through a microscope; the net
result is a fluorescent dot at the chromosomal location where the
labeled probe binds. The first single-copy human gene to be
mapped using FISH was thyroglobulin in 1985(Landegent et al.,
1985). FISH allows a higher level of resolution than standard G-
banding approaches.
Figure 2a demonstrates an example of a standard FISH experiment,
in which the red fluorescent DNA probe, corresponding to a 150
kilobase pair region of chromosome 1, is used to label
a metaphase chromosome spread. In this case, two red fluorescent
dots can be observed, corresponding to the maternal and paternal
copies of chromosome 1. Probes to different genes
or DNA sequences can even be used simultaneously, as long as each
has a different color associated with it. Figure 2b shows
a FISH experiment in which a set of DNA probes that bind along
the length of a human chromosomewere each labeled with a
different color; the net result is a rainbow-colored chromosome.
FISH has been very useful in the characterization and diagnosis
of disease. For instance, FISH was used to show that acute
myelogenous leukemia (AML) is associated with
a chromosome 16 inversion event near the centromere that leads
to the fusion of two chromosome 16
genes: CBFB and MYH11 (Liu et al., 1993). FISH analyses have
also contributed greatly to our understanding of
Angelman syndrome and Prader-Willi syndrome (Knoll et al.,
1989). Researchers found that Angelman syndrome and Prader-
Willi syndrome were both associated with the
same deletion in chromosome 15 (from region q11 to q13).
However, they found that Angelman syndrome patients inherited
the deleted copy of chromosome 15 from their mother,
whereas Prader-Willi syndrome patients inherited the deleted
copy of chromosome 15 from their father; this is due
to chromosomal imprinting.
FISH can be used to identify genes with increased copy number or
to detect gene loss, as shown by more or fewer than two fluorescent
"dots" in a somaticcell, respectively. Furthermore, FISH can be
carried out using nondividing cells, which allows investigators to
examine nonmitotic cells. This is important, because DNA packing
is approximately 10,000 times less compact in nonmitotic
(interphase) cells, allowing researchers to achieve a higher level of
resolution. For example, the neurological disorder Charcot-Marie-
Tooth type 1A is associated with a duplication of one
million base pairs that can be resolved by interphase FISH, but not
by metaphase FISH (Lupski et al., 1991).
An extremely high-resolution form of FISH, called fiber-FISH, is
carried out using isolated chromosomes that are free from nuclear
architecture and exist as long, stretched-out DNA fibers (Parra &
Windle, 1993; Wiegant et al., 1992). By using DNA fibers as a
template for FISH, researchers can resolve generearrangements and
duplications with incredible precision.
Spectral Karyotyping (SKY) and Multiplex-FISH (M-FISH)
.
The ability to isolate individual human chromosomes using flow
cytometry, combined with knowledge of the
human genomesequence, has allowed cytogeneticists to develop 24-
color probe sets that are used to label each
human chromosome with a distinct color (Figure 3). Chromosome-
specific probes are made by labeling DNA fragments covering the
length of each individual chromosome with a distinctly colored
fluorescent dye, as shown in Figure 3a. The labeled DNA probes are
then pooled and used in hybridization experiments
with metaphase chromosome spreads. The
labeled DNA probe sets bind to
their complementary chromosomes, allowing each
individual chromosome to be labeled with a specific fluorescent
color along its entire length. In a somatic cell, the maternal and
paternal copy of each chromosome will be labeled with the same
colors, as shown in Figure 3b. This powerful approach, which
permits the simultaneous tracking of all human chromosomes, has
been called spectral karyotyping (SKY) or multiplex-FISH (M-
FISH) (Schrock et al., 1996; Speicher et al., 1996). As you can see
in the bladder cancer cell depicted in Figure 3c, SKY analysis can
be used to detect interchromosomal rearrangements (indicated by
arrows) and aneuploidy (abnormal chromosome number).
These techniques are probably the most significant development in
molecular cytogenetics in the past decade. Because
each chromosome has its own color, chromosomal translocations
are easily detected when a chromosome shows a region with a
different color; the second color reports the identity the
other chromosome involved in the translocation. SKY/M-FISH
techniques have allowed researchers to detect small chromosomal
rearrangements in individuals with seemingly normal karyotypes,
and also to determine more precisely the cytogenetic aberrations in
individuals with complex aberrant karyotypes.
Comparative Genomic Hybridization
Next on the horizon for cytogeneticists was the ability to perform
genome-wide scans to identify chromosome regions associated
with loss or gain of genetic information. In order to address this
need, researchers developed a technique
called comparative genome hybridization (CGH) (Figure 4).
This approach involves the isolation and fragmentation of
genomic DNA from a control subject and an experimental subject.
The fragmented control DNA sample is labeled with green
fluorescence, and the fragmented experimental DNA sample is
labeled with red fluorescence. The two DNAsamples are pooled and
used together as DNA probes in hybridization experiments with
normal chromosomes. The green and red probes then compete to
bind to the chromosomes.
For unaltered chromosomal regions, the green and red probes should
bind equally, resulting in an orange/yellow color. If a chromosomal
region was deleted in the experimental group, that region will appear
more green under the microscope. If a chromosomal region was
amplified in the experimental group, the corresponding
chromosomal region will appear more red under the microscope.
Researchers can then scan along the length of the chromosomes to
identify genomic alterations. Using this approach, researchers have
discovered that the gene encoding the catalytic subunit of
phosphatidylinositol 3-kinase (PIK3CA) was amplified in
ovarian cancer (Shayesteh et al., 1999), thus identifying PIK3CA as
an oncogene associated with ovarian cancer.
Standard CGH methods are very labor intensive and require the use
of metaphase chromosomes, which leads to limited resolution.
However, a more recent microarray-based CGH method does not
require the use of metaphase chromosomes (Pinkel et al., 1998).
This method uses arrays containing thousands of base pair
fragments of the human genome adhered to a microchip. Each
individual DNA fragment, which is located in a specific position on
the chip, corresponds to a known DNA sequence that has been
mapped to a specific chromosomal region. The same color-coded
probes (green for the control group, and red for the experimental
group) are used in hybridization experiments with the
CGH microarray platform, which can be scanned using an
automated approach. As described for standard CGH experiments,
unaltered chromosome regions would show equal binding of the
green and red probes and a resulting orange/yellow color, whereas
amplified and deleted chromosomal regions in the experimental
group would appear red and green, respectively. By using the arrays,
researchers can very precisely determine the chromosomal regions
and genes that are amplified or missing. The information derived
from a single array-CGH experiment is equal to that derived from
thousands of FISH experiments.
The powerful combination of cytogenetics and the
human genome sequence has permitted new views of human
genetic disease. The future of molecularcytogenetics is bright, and
this field will most certainly continue to uncover new and
unexpected insights.

Cytogenetic Methods in Diagnosing Genetic Disorders

By: Heidi Chial, Ph.D. (Write Science Right) © 2008 Nature
Education
Citation: Chial, H. (2008) Cytogenetic methods in diagnosing
genetic disorders. Nature Education 1(1)
Since genes are packed into chromosomes, abnormal chromosomes
can actually cause genetic diseases. What methods have scientists
invented to study these abnormalities?
To be able to map the location of disease-associated genes within
a genome, scientists first needed to determine the landscape of
human chromosomes. How many chromosomes are present in a
human cell? Are all human chromosomes the same size? How can
we tell them apart? We now know the answers to these questions,
but the first of these discoveries wasn't made until 1956, when Tjio
and Levan provided the first accurate count of the number of
chromosomes in a human cell: 46. This knowledge led
researchers in new directions that facilitated gene mapping and
discovery. In fact, one of the earliest methods used to study human
chromosomes - Giemsa staining, which yields signature banding
patterns for each of the 24 types of human chromosomes -is still in
use today. By combining Giemsa staining with
somatic cell hybrids, which contain a mixture of human and mouse
chromosomes, scientists were eventually able to map disease-
associated genes to individual chromosomes.
Human Chromosome Anatomy
Human chromosomes come in 24 varieties: 22 different autosomes
and two different sex chromosomes (X and Y). Moreover, somatic
cells contain 23 pairs of chromosomes, including 22 autosomal pairs
and one pair of sex chromosomes. Before various staining
techniques were developed, researchers were only able to distinguish
these different types of chromosomes from each other based on their
length and the position of their centromere, which is the "waistline"
of the chromosome where the two sister chromatids are attached
following DNA replication. Researchers were also aided in this
endeavor by the fact that human chromosomes are not symmetrical,
and the two arms that extend from the centromere are often
different lengths. The short arm of a human chromosome is
referred to as the p arm, while the long arm is called the q arm. The
ends of chromosomes are called telomeres.
G-Bands Distinguish Individual Human Chromosomes
.
In 1971, researchers Maximo Drets and Margery Shaw developed a
method for staining human chromosomes using Giemsa dye that led
to distinct banding patterns (called G-bands) for each of the 24 types
of human chromosomes (Drets & Shaw, 1971). In their experiments,
Drets and Shaw took cultures of human lymphocytes and fibroblasts
that were growing and undergoing mitosis and exposed them to
a drug called colchicine, which disrupts microtubules and causes
cells to arrest in metaphase with duplicated chromosomes
consisting of two sister chromatids. The cells were then exposed to
hypotonic conditions to permeabilize them. After that, the cells
were fixed in a mixture of methanol and acetic acid, which allowed
them to be flame dried and adhered to a glass microscope slide.
Next, the chromosomes from the cells were treated with sodium
hydroxide and increasing concentrations of sodium citrate, after
which Giemsa staining yielded distinct and reproducible G-
banding patterns.
Drets and Shaw then photographed and generated G-banding maps
for each human chromosome. They found that each pair of
chromosomes within a cell showed a similar G-banding pattern
(Figure 1). Furthermore, they found that the G-banding patterns
were the same for a given chromosome when they compared
different cells.
Drets and Shaw also carried out a "blind" experiment in which they
analyzed the G-banding patterns in 15 cells from three males (Drets
& Shaw, 1971). Here, the duo photographed the chromosomes in
each of the 15 cells, karyotyped them by cutting out individual
photographs of each chromosome, and labeled the back of the
photos to designate which cell each chromosome came from. Next,
they put the chromosomes into groups according to their length
and centromere position without looking at the banding patterns.
One group of three chromosomes, called group D, was examined in
more detail. Drets and Shaw wanted to determine whether the
number of types of G-banding patterns was the same as the number
of group D chromosomes, and whether each cell contained two
chromosomes with each G-banding pattern.

Figure 2: Analysis of banding patterns of D-group chromosomes

among 15 male cells.
Drets and Shaw then examined the G-banding patterns of three
pairs of group D chromosomes from each of the 15 cells in order to
identify similar patterns in each of the chromosomes. Immediately,
they noted three distinct types of G-banding patterns, which they
termed D13, D14, and D15. The duo then determined the G-
banding patterns within each of the 15 cells, and found that
each cell contained two copies apiece of the D13, D14, and D15
chromosomes. As shown in Figure 2, each of the 15 cells examined
(numbered 1-15) typically showed two chromosomes with each of
the three group D G-banding patterns (D13, D14, and D15).
Sometimes, however, not all of the group D chromosomes could be
identified, or they were not present in the photograph, a fact that
highlights the real-world difficulties associated with studying
chromosomes.
Collectively, the results of these experiments demonstrate that
each autosomeand sex chromosome can be clearly distinguished
when its length, centromereposition, and G-banding pattern are
taken into account. Furthermore, researchers found that banding
techniques made it possible to determine which chromosomes were
involved in various types of structural rearrangements. Indeed, the
ability to determine the nature of chromosomal translocations and
deletions contributed immensely to the discovery and mapping of
human disease-associated genes. By determining which part of
a chromosome was deleted or translocated, scientists could
determine the approximate chromosomal location of a disease-
associated gene. For example, the RB1tumor suppressor gene was
mapped to the X chromosome based on the ability to visualize a
deleted chromosomal region in cells from individuals with
retinoblastoma and a loss of chromosome band 13q14 (Francke &
Kung, 1976; Knudson et al., 1976).
Human-Mouse Somatic Cell Hybrids
Also in 1971, researchers Jacques Jami and Simone Grandchamp
carried out a series of experiments in which they used the
Sendai virus to induce the fusion of mouse and human somatic cells
to generate a human-mouse somatic cell hybrid (Jami &
Grandchamp, 1971). They found that the resulting hybridcells
rapidly lost many of the human chromosomes but retained the mouse
chromosomes. Indeed, after 20 cell divisions, the hybrid cells
retained only between 1 and 15 of the original human chromosomes.
Occasionally, the hybrid cells contained two complete sets of mouse
chromosomes; in this case, the human chromosomes were retained at
higher levels, as shown by a persistence of 10 or more human
chromosomes after 150 cell divisions. In the years that followed,
various researchers were able to generate a series of human-mouse
somatic cell hybrid lines that stably maintained a small, defined
number of human chromosomes.
At first glance, these efforts to create human-mouse
somatic cell hybrids may seem more like science fiction than true
science. However, being able to generate cells that contain small
numbers of human chromosomes and then use G-band staining
patterns to determine precisely which human chromosomes are
present greatly contributed to our ability to map and clone human
genes. For instance, human-mouse somatic cell hybrids played a key
role in the mapping and eventual cloning of the Huntington's
disease-associated gene, HTT. Indeed, researchers found that if
they could identify a DNAprobe linked to a human disease, they
could use this probe in Southern blot experiments with
chromosomal DNA from a series of well-defined human-mouse
somatic cell hybrids. They could then determine which
human chromosome was common among those hybrid cell lines
to which the disease-associated DNA probe could bind. Using this
method, researchers were able to map 1,148 human genes by
1991 (McKusick, 1991).
Chromosomes Are a Useful Tool for Mapping Disease Genes
Thanks to the completion of the Human Genome Project, and due
to our knowledge of chromosomes at the base-pair level,
somatic cell hybrids are no longer the first option
for mapping genes. However, the ability to identify chromosomes
and the location of defects associated with them remains a valuable
tool. Identifying a chromosomal breakpoint that is associated
with a clinical manifestation (e.g., mental retardation or cancer) can
often provide the location of the gene that is likely disrupted and
causing the defect. Although these chromosomal defects are
extremely rare, they are highly treasured among human genetics
researchers. One such advance was the cytological identification and
characterization of the Philadelphia chromosome, one of the most
famous chromosomal translocations, which is associated with
chronic myelogenous leukemia.
Through the study of rare cases involving chromosomal defects, a
number of genes have been linked to disease phenotypes. For
example, Felix Mitelman's Catalog of Chromosome Aberrations
in Cancer, which was first published almost two decades ago,
contains over 7,100 references encompassing some 100,000
aberrations related to cancer. In addition, in the case of the
Philadelphia chromosome, cytological observations were met head-
on by thedevelopment of one of the most highly
praised cancer drugs of the past decade, Gleevec. Thus, even
though techniques like G-banding provide data at relatively low
resolution, a great deal of valuable information can be gleaned from
these data with regard to human genetics, chromosomal biology, and
the role of chromosome structure in human disease.
Gene-Based Therapeutic Approaches
By: Heidi Chial, Ph.D. (Write Science Right) © 2008 Nature
Education
Citation: Chial, H. (2008) Gene-based therapeutic
approaches. Nature Education 1(1)
Can we take genetic “pills” for disease-related mutations? No, not
yet, but our knowledge of the human genome sequence has enabled
the development of other gene-based therapeutic approaches.
Human somatic cells contain 23 pairs of chromosomes, and
we inherit one set of 23 chromosomes from each of our parents.
Chromosomes are built using four different types
of DNA bases: adenine (A), cytosine (C), guanine (G),
and thymine (T). The human genome is defined by more than 3
billion base pairs of DNA that make up our 46 chromosomes;
these DNA base pairs are organized in a specific order and serve as
the blueprint that distinguishes humans from other species. Studies
of the human genome have shown that between 20,000 and 25,000
genes reside along our chromosomes. Although redundancy is built
into the human genome, our gene repertoire is exquisitely regulated
in order to maintain cell function by copying to RNA, which in
turn determines the formation of proteins. Thus, even a single base-
pair mutation in one of our genes can lead to an altered protein and
consequently disease.
Although the complexities of human biology suggest an equally
complex genome, the human genome is actually quite similar to
that of the mouse. The mouse genome is built of 2.7
billion base pairs of DNA, and current estimates suggest that the
mouse genome contains at least 28,972 genes, of which at least
16,927 have homologues (called orthologues) in humans. In fact,
846 human genetic diseases have been reproduced in mouse strains
(called mouse genotypic models) that carry a mutation in the
related mouse gene.
So, what distinguishes humans from other organisms? What makes
us human is a combination of the following:
The ability of a single gene product (protein) to perform multiple
functions due to overlapping roles in different pathways
The ability of our genes to undergo different types
of RNA splicing to produce gene variants with alternative
functions
The important functions carried out by
chromosomal DNA segments located between genes
Epigenetic regulation
Roll Call: From Genes to Disease
Because we inherit one set of 23 chromosomes from each of our
parents, our somatic cells contain two copies of each of these 20,000
to 25,000 genes. Most of the human genome is nearly identical
from one person to another. In fact, on average, a randomly chosen
1,200-base-pair segment of human chromosomal DNA contains
only one base pair that varies between two unrelated individuals.
The vast majority of these DNA variants are not deleterious, but
some specific alterations in the human DNA sequence are
associated with disease.
Indeed, recent estimates suggest that mutations in at least 383 human
genes lead to known phenotypes, and that more than 2,336 human
phenotypes are understood at the molecular level. However, the
molecular basis of 1,630 confirmed Mendelian phenotypes is not
known, and an additional 2,081 phenotypes are suspected to
exhibit Mendelian inheritance (Online Mendelian Inheritance in
Man, 2008). Although genetic "pills" do not yet exist to target the
vast majority of disease-related mutations, our knowledge of the
human genome sequence has opened new doors to
the development of gene-based therapeutic approaches
(Brinkman et al., 2006; O'Connor & Crystal, 2006).
Treating Disease Before Knowing the Genes Involved
Long before the human genome was sequenced, doctors were
already treating many hereditary forms of human disease with
surprising success. Many of these diseases were metabolic disorders,
and doctors were able to determine whether a metabolic product was
accumulating to harmful levels, or whether a key intermediary in a
metabolic pathway was missing, even though the disease-
associated gene was not yet known. In some cases,
such diseasephenotypes can be kept in check by dietary
modification or by providing a missing protein. In other cases,
surgical approaches can be used to repair or replace an organ or
tissue damaged by disease.
Metabolic Manipulation
Physicians have developed approaches to regulate the metabolic
pathways associated with a number of disorders,
including phenylketonuria (PKU),sickle-cell anemia, hereditary
angioedema, familial hypercholesterolemia, thalassemia, and
many others. Often, this form of "metabolic manipulation" can be
accomplished by modifying a patient's diet. For example, patients
with PKU accumulate high levels of a protein building block, called
phenylalanine, in their bloodstream. Doctors are able to diagnose
PKU using a simple heel-prick blood test to detect high levels of
phenylalanine by three days of age. Once diagnosed, infants with
PKU are fed a diet low in protein and phenylalanine, which helps
prevent PKU-associated cognitive decline.
In other cases, metabolic manipulation involves the use of small
molecules or drugs to target the activity of proteins linked
to disease. For instance, familial hypercholesterolemia is associated
with high levels of "bad" (LDL) cholesterol and early heart disease;
in this case, treatment includes both dietary modifications (a diet
low in cholesterol) and the administration of a class of drugs called
statins. These medications inhibit the activity of an enzyme (HMG
CoA reductase) involved in a rate-limiting step of cholesterol
biosynthesis.
Hemoglobin is the iron-containing protein that carries oxygen in our
red blood cells. Humans express different forms of hemoglobin
during development. Hemoglobin is built of four protein subunits:
two ά-subunits and a second pair of subunits, which vary by age. The
main adult form of hemoglobin consists of two ά-subunits and two
β-subunits (ά2β2), whereas the fetal form of hemoglobin consists of
two ά-subunits and two γ-subunits (ά2γ2). Sickle-cell anemia is due
to a mutation in the gene encoding the β-subunit of hemoglobin
(called HBB), which leads to an abnormal structure of the β-
subunit protein chain and the sickle-cell phenotype (Weatherall,
2003). In the 1980s, researchers discovered that treatment with
a drug called hydroxyurea, which increases levels of the γ-subunit
of fetal hemoglobin, leads to a 50% reduction in the painful sickle-
cell crises associated with sickle-cell anemia (Platt et al., 1984).
Hydroxyurea is still used to treat sickle-cell anemia today.
Protein Augmentation
In another approach called protein augmentation, physicians treat
patients by providing them with a purified form of the missing,
defective, or depletedprotein. This protein-add-back approach has
been used to successfully treat patients suffering from a wide range
of diseases, including various membrane transport disorders (cystic
fibrosis), coagulation disorders (hemophilia A, hemophilia B, and
Von Willebrand disease), emphysema (ά1-antitrypsin deficiency),
immune deficiency (severe combined immune deficiency), endocrine
disorders (growth hormone deficiency, congenital leptin deficiency,
andcongenital neurogenic diabetes insipidus), and lysosomal
storage disorders (Gaucher's disease type I, Fabry disease,
mucopolysaccharidosis I, mucopolysaccharidosis II,
mucopolysaccharidosis VI, and Pompe's disease).
For example, the gastrointestinal symptoms that accompany cystic
fibrosis can be corrected by protein augmentation through the
administration of pancreatic enzymes. Similarly, individuals with
growth hormone deficiency can be treated with purified growth
hormone to restore normal growth. In addition, symptoms associated
with Pompe's disease, including reduced heart, lung, and
skeletal function, can be improved by treatment with acid ά-
glucosidase enzyme.
Protein augmentation requires that the protein be added to the
outside of cells (i.e., the extracellular space). Therefore, this
approach works best for replacing proteins that are normally present
in the extracellular space. Protein augmentation approaches are
often less effective if the missing protein is normally located inside
of a cell, or if it is normally targeted to a specific intracellular
organelle. Indeed, it can be difficult to ensure that a protein added to
the outside of a cell will be taken up by the cell and targeted
correctly to its normal location or organelle.
In some cases, the success of protein augmentation depends on how
well the protein is delivered to the organ(s) in which its function is
required. The brain is a particularly difficult organ to target, because
the access of proteins is limited by a membrane structure called the
blood-brain barrier (BBB), which inhibits the passage of proteins
and other chemicals from the bloodstream into the brain. The eye
also presents challenges to drug access from the bloodstream.
However, therapeutic agents can be delivered directly to the eye; this
is not possible with the brain.
Surgical Approaches
Although more invasive, organ transplantation is also used to treat
certain genetic diseases that affect particular organs. Unless the
organ donor and the organ recipient are monozygotic twins, the
chromosomal DNA sequence of the donor will be different from
that of the recipient. Despite these differences, organ transplantation
remains a viable therapy that continues to be used widely to this day.
In still other cases, surgery can be used to repair an organ or tissue
that has been targeted by disease. For example, cleft lip, with or
without cleft palate, is the fourth most common birth defect in the
U.S., and it affects 1 in every 700 babies born each year. Cleft lip
and/or cleft palate arise early during pregnancy while a baby is
developing in the uterus; the malformations occur when there is not
enough tissue in the lip or mouth area to permit joining of the tissue,
or when the fusion of the lateral structures of the face does not occur
properly. Cleft lip occurs when the two sides of the upper lip are
separated, and cleft palate occurs when either the hard palate or the
soft palate is separated. Surgical approaches can be used to
effectively repair both cleft lip and cleft palate defects.
Putting the Human Genome to Work: Using Genes to Treat Disease
The identification of disease-causing mutations can be a rich source
of opportunity for identifying new disease treatments. After
identifying a disease-associated mutation, scientists can study how
the function of the corresponding gene product (protein) is altered.
With this information in hand, three main gene-based therapeutic
approaches are currently being pursued:
Gene-transfer approaches, in which a wild-type copy of the
mutated gene is delivered
RNA modification therapy, in which the mRNA encoded by
a mutant gene is targeted
Stem cell therapy, in which human stem cells are used to repair
disease-damaged tissue
Gene Transfer
.
Gene-transfer approaches are based upon the simple concept that if
a disease is due to a recessive loss-of-functionmutation in a
single gene, then adding back the wild-type gene should restore
normal function and alleviate the diseasephenotype. So far, two
strategies have been used to deliver wild-type genes (O'Connor &
Crystal, 2006):
Ex vivo approaches, which require the use of a cell population that
can be removed from a patient, genetically altered to express
the wild-type gene, and then delivered back into the patient
In vivo approaches, which deliver the wild-type gene directly into a
patient's affected cell population, tissue, or organ
Although the concept is simple, gene-transfer approaches have
presented many clinical challenges. For example, both ex vivo and in
vivo approaches require two key components: a gene expression
cassette, consisting of a wild-type gene and some
extra DNA sequences that permit the gene's expression in human
cells, and a delivery system to introduce the wild-type gene into
cells. The gene delivery system can be a cell membrane–like
lipid carrier, or it can be derived from a virus. Table 1 shows some
examples of gene-transfer clinical trials and the gene delivery
systems employed, including delivery of the factor VIII (F8) gene to
fibroblasts for the treatment of hemophilia A, delivery of
the CFTR gene to the nasal and airway epithelium for
the treatment of cystic fibrosis, delivery of the ornithine
transcarbamylase (OTC) gene to the liver for the treatment of
ornithine transcarbamylase deficiency, delivery of the
glucocerebrosidase (GBA)gene to blood and bone marrow cells for
the treatment of Gaucher's disease, and delivery of the ά1-
antitrypsin (SERPINA1) gene for the treatment of ά1-antitrypsin
deficiency. Additional information about clinical trials
using gene delivery approaches to treat human disease can be
found on the Gene TherapyClinical Trials Worldwide website.
.
Figure 1a shows a typical gene expression cassette. The wild-
type gene (called a transgene) has a promoter sequence attached
to its 5′ end, which allows it to be recognized by
the transcription machinery of the cell, and a polyadenylation
signal at its 3′ end, which leads to the production of a poly-A tail and
improved stability of the resulting mRNA. Current approaches
typically use the cDNA corresponding to the wild-type gene,
which does not contain introns. Figures 1b through 1f show some of
the different methods used for gene delivery. Each gene delivery
system has its own preferred type of expression cassette and its own
maximal capacity in terms of the size of the transgene it can carry,
as shown.
In order to successfully deliver the gene of interest, both lipid
carriers and viruses must facilitate cell entry by breaking through
the target cell's plasma membrane, and they must allow the
expression cassette to enter the cell nucleus so that it can be
transcribed into mRNA. Once in the nucleus, the expression
cassette can either undergo random insertion into
thecell's genome, or it can be maintained extrachromosomally
within the nucleus, depending upon the delivery system. If
chromosomally integrated, the transgene will be expressed
continuously; however, the random nature
of chromosomeintegration can lead to problems, such as cancer, if
the gene is inserted in such a way that it disrupts the function of
anothergene. On the other hand, if the expression vector is
maintained extrachromosomally, expression of the transgene can be
lost over time due to cell division.
Researchers must also strike a balance in terms of the expression
levels of the transgene. Specifically, expression must be sustained
at high enough levels to rescue disease-related phenotypes, but it
must also be maintained at low enough levels to prevent an immune
response by the patient.
Targeting RNA to Treat Dominant Genetic Diseases

Gene-transfer approaches are particularly useful when a disease-

associated mutation encodes a protein with decreasedfunction,
called a loss-of-function mutation; in this case, normal
cellular function is restored when a wild-type copy of thegene is
introduced, because loss-of-function mutations are
usually recessive. However, human disease can also be associated
with dominant mutations in genes that encode hyperactive proteins,
called gain-of-function mutations. Furthermore, human disease can
be associated with dominant mutations in which
the mutant proteins interfere with thefunction of wild-
type proteins, called dominant negative mutations.
In the case of a dominant mutation, introduction of the
corresponding wild-type gene is usually not sufficient to rescue the
disease-associated phenotype(s); rather, researchers would prefer to
"turn off" expression of the mutant gene, or to inhibit
the function of the mutant protein it encodes. To achieve this goal,
researchers have turned their attention to RNA-based approaches,
which target the RNA(either pre-mRNA or mRNA) transcribed
from the dominant negative gene and effectively inhibit expression
of the mutant protein.
Figure 2 shows examples of RNA-based strategies for
the treatment of disease. Five approaches have been used
experimentally to modify RNA levels: antisense oligonucleotides
(ASO), RNA interference (RNAi), trans-splicing,
segmental trans-splicing, and ribozymes.
ASO strategies use short single-stranded DNA (ssDNA) molecules,
usually between 18 and 30 bases long, which
are complementary to the mRNA to be targeted (Figure 2a). The
ssDNA binds to the target mRNA, and the resulting DNA-
RNA hybrid molecule is then degraded by the
intracellular enzymeribonuclease H (RNase H).
RNAi involves the use of double-stranded RNA molecules
(dsRNA), typically 22 base pairs long, corresponding to a region of
the target gene (Figure 2b). The dsRNA is processed within
the cell in such a way that it becomes part of an RNA-
induced silencing complex (RISC) that recognizes and degrades the
corresponding target mRNA.
Trans-splicing is a gene-transfer approach that targets a pre-mRNA
containing a disease-associated mutation within one of its exons
(Figure 2c). In this case, the transgene is used to replace
the exon carrying the disease-associated mutation (exon C* in
Figure 2c) with a wild-type copy of the exon.
Thetransgene contains a hybridization domain, which
is complementary to a region of the 5′ flanking intron between the
donor and branch-point sites for RNAsplicing, followed by
the splicing branch point, the splice acceptor site, the wild-
type exon sequence, and the rest of the gene. Trans-splicing leads
to the production of a wild-type copy of the mature mRNA and
thus a corresponding wild-type protein.
Segmental trans-splicing is an approach used to get around the size
limitations associated with gene-transfer methods that involve
vectors. (Sometimes, a given cDNA is too large to be carried within
a single viral vector.) In this case, the gene is divided into two
smaller pieces, which are delivered together using two separate
gene-transfer vectors (Figure 2d). The vector carrying the second
half of the gene includes
a hybridization domain complementary to anintron located at
the 3′ end of the first half of the gene, similar to that described
for trans-splicing. In this case, trans-splicing leads to the production
of a mature mRNA encoding the full length of the wild-
type protein of interest.
Ribozymes are RNA molecules with inherent catalytic activity that
recognize a particular mRNA and cleave it (Figure 2e). Ribozymes
containing ahybridization domain followed by
a ribozyme nucleolytic motif that recognizes a target mRNA and
the corresponding wild-type gene sequence can be used to
selectively cleave a target mRNA that contains a mutation after
the ribozyme cleavage site. Once the target gene is cleaved, the
ribozyme-derivedhybridization motif binds,
and RNA splicing leads to the formation of a wild-type copy of
the mature mRNA.
Many of these RNA-based strategies have been developed in recent
years, and numerous questions remain regarding the cellular
mechanisms involved inmRNA targeting. Furthermore, researchers
must exercise caution with respect to the specificity of any given
mRNA-targeting approach, whether ASO, RNAi, trans-splicing,
or ribozyme based, to ensure that only the mRNA of interest is
targeted.
Stem Cell Therapy
On the genetic horizon, the modern-day equivalent of organ
transplantation is likely to be the use of stem cell therapy. Unlike
organs, which are built of specialized mature cells with tissue-
specific characteristics and a very limited ability to divide, stem
cells are immature cells that have not yet specialized and that have
the capacity to divide and mature into a wide variety of tissue types.
Stem cells naturally occur in two forms: embryonic stem cells and
somatic stem cells. Embryonic stem cells are derived from a
specific group of cells within an embryo. They are capable of
unrestricted cell divisions (i.e., they are immortal) and are
pluripotent, which means that they are able to become nearly
anycell type imaginable as long as they are provided with the
appropriate environment. Ethical concerns regarding the use of
human embryos as a source ofstem cells, as well as technical
difficulties in obtaining and culturing these cells, have hampered the
clinical use of embryonic stem cells.
Somatic stem cells are derived from a specific group of cells within
an adult tissue that serve to renew the tissue cell populations over
time. Unlike pluripotent cells, somatic stem cells are more
restricted in terms of the type of cells they can become after they
divide; their fate depends upon the tissue type from which they are
derived. Figure 3 shows the differences between embryonic stem
cells and somatic stem cells in terms of how they are derived and
their ability to differentiate.

One of the most exciting breakthroughs in stem cell research

occurred recently and has been reproduced in labs across the world:
the ability to convert somatic cells into pluripotent stem
cells (Takahashi et al., 2007; Yu et al., 2007; Lowry et al., 2008;
Park et al., 2008b). Scientists have identified a set of four genes
(OCT4/POU5F1, SOX2, KLF4, and c-MYC/MYC) that
encode transcription factors that, when expressed at the same time,
can convert skin cells (dermal fibroblasts) taken from an adult
human into induced pluripotent stem cells (iPS cells) that are
phenotypically indistinguishable from human embryonic stem
cells in terms of their gene expression, cell surface markers, and
cellular morphology. Like human embryonic stem cells, the iPS
cells are immortal, are pluripotent, and express genes characteristic
of all three embryonic germ cell layers (endoderm, ectoderm, and
mesoderm) when induced to differentiate. Both opponents and
proponents of human stem cell research have warmly welcomed the
promise of somatic cell–derived iPS cell lines.
The ability to produce iPS cell lines from somatic cells was heralded
as an invaluable tool for understanding the underlying mechanisms
associated withdisease and for developing novel approaches to
the treatment of human disease. Recently, a team of researchers at
Harvard University applied this technique and established a panel of
human disease–specific iPS cell lines (Park et al., 2008a); in this
case, the investigators expressed three (OCT4/POU5F1, SOX2,
and KLF4), four (OCT4/POU5F1, SOX2, KLF4, and c-MYC/MYC),
or five (OCT4/POU5F1, SOX2, KLF4, c-
MYC/MYC andNANOG) transcription factor genes.
To generate disease-specific iPS cell lines, researchers collected
skin cells and bone marrow–derived mesenchymal cells from
patients with one of ten different diseases, including adenosine
deaminase deficiency-related severe combined immunodeficiency
(ADA-SCID), Shwachman-Bodian-Diamondsyndrome (SBDS),
Gaucher's disease (GD) type III, Duchenne muscular dystrophy
(DMD), Becker muscular dystrophy (BMD),
Parkinson's disease (PD), Huntington's disease (HD), juvenile-
onset type 1 diabetes mellitus (JDM), Down syndrome/trisomy
21 (DS), and Lesch-Nyhan syndrome (carrier). Similar to the
original iPS cell lines, the disease-specific iPS cell lines were
immortal, pluripotent, and capable of expressing genes
corresponding to all three embryonic cell layers when induced to
differentiate (Park et al., 2008a).
Table 2 summarizes the panel of disease-specific iPS cell lines and
provides details about the corresponding mutated somatic cell types
from which they were derived, as well as the age and sex of the
somatic cell donor (Park et al., 2008a). These cell lines are freely
accessible to researchers worldwide, and they will serve as a strong
foundation for future studies aimed at the eradication of these
devastating diseases. Due to the viral-based methods used to deliver
the transcription factor genes into the somatic cells, the resulting
iPS cell lines cannot currently be used to treat human patients.
Nevertheless, thesecell lines are an invaluable resource for future
investigation. With these disease-specific iPS cell lines in hand,
researchers will be able to carry out experiments to better
understand disease pathology and to develop effective gene-transfer
techniques, RNA-based therapies, and drug-screening approaches to
target disease phenotypes.
Table 2: iPS Cells Derived from Somatic Cells of Patients with
Genetic Disease.Reproduced from Park et al., 2008a
Looking Ahead: Gene-Inspired Drug Design and Multimodal
Therapies
Researchers' ever-increasing knowledge of human genes and their
disease-associated mutations has inspired new approaches
to drug design and discovery. By understanding the underlying
molecular mechanisms linked to disease, investigators can better
target the activities of the enzymes, cellsurface receptors, secreted
proteins, intracellular signaling proteins, and transcription factors
that regulate disease-associated phenotypes. As gene-based
therapeutics continue to evolve, multimodal approaches to
human disease will emerge. The future of genetic medicine will
require collaboration and multidisciplinary approaches, which will
most certainly be accompanied by unexpected, life-changing
discoveries.