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 Organism

 

Common Name

 

Research Applications

 

Saccharomyces cerevisiae

Yeast

 

Used for biological studies of cell   processes (e.g.,   mitosis )  anddiseases

 (e.g.,   cancer )

 

Pisum sativum

 

Pea plant

 

Used by Gregor Mendel to describe patterns of inheritance

 

Drosophila melanogaster

 

Fruit fly

 

Employed in a wide variety of studies ranging from early genemappingvia linkage and recombination studies, to large scalemutant screens to identify genes related to specific biological functions

 

Caenorhabditis elegans

 

Roundworm (nematode)

 

Valuable for studying the development of simple nervous systems and the aging process

 

Danio rerio

 

Zebra fish

 

Used for mapping and identifying genes involved in organdevelopment

 

Mus musculus

 

House mouse

 

Commonly used to study genetic principles and human disease

 

Rattus norvegicus

 

Brown rat

 

Commonly used to study genetic principles 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

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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)

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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)

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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

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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

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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 Therapy Clinical Trials Worldwide website .

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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.