Epigenetic Influences and Disease | Epigenetics | Dna Methylation

Epigenetic Influences and Disease By: Danielle Simmons, Ph.D. (Write Science Right) © 2008 Nature Education Citation: Simmons, D.

(2008) Epigenetic disease. Nature Education 1(1) influence and

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.

Epigenetics and Disease: Some Examples While epigenetic changes are required for normal development and health. These are chemical processes that add either an acetyl or methyl group. . then it is inactive. and DNA transcription does not occur. methylation of a particular lysine (K9) on a specific histone (H3) that marks silent DNA is widely distributed throughout heterochromatin. RNA might affect geneexpression by causing heterochromatin to form. RNA-Associated Silencing Genes can also be turned off by RNA when it is in the form of antisense transcripts. respectively. if chromatin is condensed (creating a complex calledheterochromatin). it is active. noncoding RNAs. while deacetylation is generally associated with heterochromatin. or RNA interference.. to the amino acid lysine that is located in the histone. or by triggering histone modifications and DNA methylation (Egger et al. There are two main ways histones can be modified: acetylation and methylation. Conversely. histone methylation can be a marker for both active and inactive regions of chromatin. On the other hand. 2004). and the associated DNA can be transcribed. 2004). For example. Acetylation is usually associated with active chromatin. In contrast.If chromatin is not in a compact form. methylation of a different lysine (K4) on the same histone (H3) is a marker for active genes (Egger et al.. This is the type of epigenetic change that is responsible for the inactivated X chromosome of females. they can also be responsible for some disease states.

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. Such disruptions have been associated withcancer. . 2004. As previously mentioned. 1983). This abnormality is the trademark epigenetic change that occurs in tumors and happens early in the development of cancer (Egger et al. Epigenetics and Cancer The first human disease to be linked to epigenetics was cancer. these types of changes may be more common in human cancer than DNA sequence mutations (Figure 2). 2002.Disrupting any of the three systems that contribute to epigenetic alterations can cause abnormal activation or silencing of genes. and mental retardation (Table 1). Jones & Baylin. Robertson. loss of DNA methylation can cause abnormally high gene activation by altering the arrangement of chromatin. DNA methylation occurs at CpG sites. These CpG islands become excessively methylated in cancer cells. On the other hand. Because methylated genes are typically turned off. Researchers found that diseased tissue from patients with colorectal cancerhad less DNA methylation than normal tissue from the same patients (Feinberg & Vogelstein. in 1983. In fact. thereby causing genes that should not be silenced to turn off. and a majority of CpG cytosines are methylated in mammals. 2002).. However. too much methylationcan undo the work of protective tumor suppressor genes. Hypermethylation of CpG islands can cause tumors by shutting off tumor-suppressor genes. syndromes involving chromosomal instabilities.

and RASSF1A. including O6-methylguanine-DNA methyltransferase (MGMT). MLH1 cyclin-dependent kinase inhibitor 2B (CDKN2B). which are repeated sequences of DNA. although epigenetic changes do not alter the sequence of DNA. and gastric cancers (Jones & Baylin. Figure 2: Mechanism of action of nucleoside analogue inhibitors. 2002). Most of these genes normally suppress tumor formation and help repair DNA. ovarian. Too much methylation of the promoter of the DNA repair gene MLH1 can make a microsatelliteunstable and lengthen or shorten it (Figure 2). and they usually consist of repeats of the dinucleotide CA. Hypermethylation can also lead to instability of microsatellites. For example. they can cause mutations. including colorectal.Furthermore. . Microsatellite instability has been linked to many cancers. endometrial. Microsatellites are common in normal individuals. About half of the genes that cause familial or inherited forms of cancer are turned off by methylation. hypermethylation of the promoter of MGMT causes the number of G-to-A mutations to increase (Figure 2).

Nature Publishing Group.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. Once in DNA. Metaphase chromosomes showing the peculiar constriction at the end of the long arm of chromosome X that is characteristic in fragile X (FX) individuals. 415-428 Epigenetics and Mental Retardation Figure 3: The marker X chromosome. © 2002. Jones. et. the fraudulent bases form covalent bonds with DNA methyltransferases (DNMTs). Ribonucleosides such as 5-azacytidine or zebularine are reduced at the diphosphate level by ribonucleotide reductase for incorporation (not shown). A.. Pink circles. P. methylated CpG. unmethylated CpG. Nature Reviews Genetics 3. The black arrowhead marks the marker X chromosome in the upper right hand quadrant.. resulting in the depletion of active enzymes and the demethylation of DNA. . cream circles. al. The fundamental role of epigenetic events in cancer.

individuals with over 200 repeats have a full mutation. Thesyndrome is caused by an abnormality in the FMR1 (fragile X mental retardation 1) gene. Indeed. normally. Angelman. fragile X syndrome occurs in approximately 1 in 4.000 females. . they are not. but because males only have one X chromosome. and they usually show symptoms of the syndrome. Loss of this specific protein causes fragile X syndrome. Coffin-Lowry. the epigenetic change associated with FMR1 methylation is the real syndromeculprit. Both sexes can be affected by this condition. PraderWilli. stopping the FMR1 gene from producing an important protein called fragile X mental retardation protein. particularly in males. Although a lot of attention has been given to the CGG expansion mutation as the cause of fragile X. Other such conditions include Rubenstein-Taybi. one fragile X will impact them more severely. it usually appears as if it is hanging by a thread and easily breakable (Figure 3). ATR-X. 2007). and "autistic-like" behavior (Penagarikano et al. Too many CGGs cause the CpG islands at the promoterregion of the FMR1 gene to become methylated. Thismethylation turns the gene off. People with this syndrome have severe intellectual disabilities. Beckwith-Wiedemann. Fragile X syndrome is not the only disorder associated with mental retardation that involves epigenetic changes. People who do not have fragile X syndrome have 6 to 50 repeats of the trinucleotide CGG in their FMR1 gene. and Rett syndromes (Table 1).Fragile X syndrome is the most frequently inherited mental disability.000 males and 1 in 8. However. delayed verbal development.. Fragile X syndrome gets its name from the way the part of the X chromosomethat contains the gene abnormality looks under a microscope.

2004). it seems reasonable to try to counteract these modifications with epigenetic treatments. epigenetic treatments must be selective to irregular cells. such as cancer. These changes seem an ideal target because they are by nature reversible. activating gene transcription in normal cells could make them cancerous. Despite this possible drawback. involve epigenetic changes. unlike DNA sequence mutations. and valproic acid (Egger et al.Combating Diseases with Epigenetic Therapy Because so many diseases. HDACs are enzymes that remove the acetyl groups from DNA. To be successful.. otherwise. which condenses chromatin and stops transcription. Caution in using epigenetic therapy is necessary because epigenetic processes and changes are so widespread. The most popular of these treatments aim to alter either DNA methylation or histone acetylation. Drugs aimed at histone modifications are called histone deacetylase (HDAC) inhibitors. which inhibits DNA methylation. Blocking this process with HDAC inhibitors turns on gene expression. researchers are finding ways to specifically target abnormal cells with minimal . These medications work by acting like the nucleotide cytosine and incorporating themselves into DNA while it is replicating. After they are incorporated into DNA. Inhibitors of DNA methylation can reactivate genes that have been silenced. the drugs block DNMT enzymes from acting. 2004).. so the treatments could cause the very disorders they are trying to counteract. depsipeptide. SAHA. Two examples of these types of drugs are 5-azacytidine and 5-aza-2′-deoxycytidine (Egger et al. The most common HDAC inhibitors include phenylbutyric acid.

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

This implies that multiple genes may be involved. which results in differences in disease penetrance and expressivity. 2001). signal transduction. These biological interactions are critical for gene regulation. and that multiple genes may interact to increase or decrease disease susceptibility. 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. Greenspan. Together these gene-gene interactions result in an output phenotype. rather. If the effect of the disease-bearing gene is masked or altered by the effects of a second gene. then identifying the genes involved and defining their relationships becomes even more difficult. Epistatic interactions can complicate a scientist's search for the gene responsible for a complex disease. For instance. if more than one epistatic interaction occurs to cause a disease. biochemical networks. In addition. Indeed. 2003. Carlborg & Haley. Certain genes are known to modify the phenotype of other genes.Epistatic gene-gene interactions are perhaps more common than we think. however. while other gene pairs have negative interactions (red lines). some scientists believe that epistasisis ubiquitous in biology and has been ignored for too long in studies of complex traits (Moore. There are. and numerous other physiological and developmental pathways (Moore. As depicted in the schematic in Figure 2. 2004). some genes (depicted as grey hexagons) have positive interactions with one another (blue lines). a number of ways to studyepistasis in populations by adapting . then identifying the first genecan be complicated. 2003. 2003). they constantly interact with one another. Research has shown that genes don't function alone.

the researchers also noted that while having one or two copies of apolipoprotein E4 increase one's risk of Alzheimer's. in order to evaluate whether epistasis occurred. 1993. 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. Combarros et al. evaluated the likelihood of over 100 published suggestions of epistatic association in sporadic Alzheimer's disease.. the researchers measured both the size and the statistical significance of interactions between pairs of implicated genes. the team instead opted to measure interactions between genes. is a progressive neurodegenerative disorder that causes memory loss and dementia. for instance. In fact.. Saunders et al. Strittmatter et al.. Because the research team realized that studying candidate genes individually had met with little success. 1993). Thus. However. it helps to consider an example of a complex disease.methods used to detect quantitative trait loci (Carlborg & Haley.. Epistasis in Alzheimer’s Disease To better understand how epistasis affects disease development. Alzheimer's disease. not all carriers of apolipoprotein E4 develop the disease. 2008). This suggested that other genes and/or gene-gene interactions were involved in the development of Alzheimer's. 2004). In the early 1990s. but they had never been statistically tested. 1993. Onofre Combarros and his colleagues thus set out to study the role of epistasis in the onset of Alzheimer's disease(Combarros et al. Some of these alleged epistatic effects had been hypothesized to occur between pairs of genes. .

future studies can focus on epistatic interactions between combinations of three or more genes and between additional pairs of genes.. and generally. While it is known that diabetics have insufficient levels of insulin and high blood sugar . inflammation. and other networks. which were grouped into five categories: cholesterol metabolism. Meanwhile. the antagonistic relationships indicate a protective relationship between two genes. β-secretase.Eventually. now that there is a foundation for understanding epistatic interactions between pairs of genes in sporadic Alzheimer's disease. Only in rare cases does the disease appear to be monogenic. 2003). multiple genes seem to be involved (Florez et al. Thus. Evidence for Epistasis in Other Diseases Diabetes is another complex disease that is influenced by both epistatic and environmental factors. Indeed. The strongest interactions involved the pairing of apolipoprotein E4 with three different genes: alpha(1)antichymotrypsin. 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. but in pathways that affect one another. and that these genes are not acting alone. beta-amyloid production. Some interactions were synergistic. it is clear that epistatic interactions are involved in complex diseases like Alzheimer's disease. and butyrylcholinesterase K (Combarros et al. Combarros et al. The synergistic interactions indicate that the pair of involved genes together increase the risk of Alzheimer's disease.. confirmed 27 different significant epistatic interactions using this method. while others were antagonistic. 2008). oxidative stress.

Evidence also exists that epistasis is involved with other complex diseases.. with transcription level used as the measured phenotype. bladder cancer. For instance.. highthroughput experimental tools are available to measure molecular and biochemical data. networks. in patients with type II diabetes (Cox et al. Understanding the causes and genetic basis behind these diseases proved elusive when using single-gene studies. Making Sense of the Complex It is now becoming possible to identify gene relationships. Today. 2008). including cardiovascular disease. Vieira. However. cleft lip and/or palate. there may be more progress toward understanding the manifestation of these complex human diseases. the specific factors underlying disease susceptibility are still being researched. it is hoped that they can eventually be mapped and identified. 1999. and other types of cancer (Combarros et al. as well as between loci on chromosomes 1 and 10. and epistatic interactions on a systems level. For example. interactions have been detected between loci on chromosomes 2 and 15. hypertension. 2006). DNA microarrays allow scientists to gather hundreds of thousands of data points from cells. and schizophrenia and other neurological disorders. While we do not know the identities of these genes now. Wiltshire et al.levels. Once we identify and understand epistatic . autism. Then. as well as sporadic breast cancer. now that there is a greater focus on epistatic interactions. computational and bioinformatics methods can be used to sift and sort though the massive amounts of biological data to search for epistatic interactions. 2008..

we can apply this knowledge to better diagnose and treat complex diseases. .relationships using techniques such as these.

The Use of Animal Models in Studying Genetic Disease: Transgenesis and Induced Mutation By: Danielle Simmons. worms. and plant species as model organisms for their studies. D. Right) © 2008 Nature Education Ph. Instead.g.D.g. fungal. (Write Science Citation: Simmons. But what do these models reveal about us? Except in the case of highly controlled and regulated clinical trials. and other animals. they use various animal. (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.. mitosis) anddiseases (e. geneticists and scientists do not use humans for their experimental investigations because of the obvious risk to life.. Some suchspecies are described in Table 1. Table 1: Models Used to Study Genetic Principles and Human Diseases Model Organism Common Research Applications Name Saccharomyces Yeast cerevisiae Used for biological studies of cell processes (e. cancer . scientists are creating models of human genetic disease using mice. bacterial. flies.

Pisum sativum Pea plant Used by Gregor Mendel to patterns of inheritance describe Drosophila Fruit fly melanogaster Employed in a wide variety of studies ranging from early genemapping via linkage and recombination studies. to large scalemutant screens to identify genes related to specific biological functions Caenorhabditis Roundworm simple nervous (nematode) elegans the aging process Valuable for studying the development systems and Danio rerio Zebra fish Used for mapping and identifying genes involved in organdevelopment House mouse Mus musculus Commonly used to study genetic principles and human disease Rattus norvegicus Brown rat Commonly used to study genetic principles and human disease .

and baker's yeast. and it is easier for scientists to use sufficiently large numbers of animals (rather than people) to attain significant results. they are frequently selected because of their similarity to humans in terms of genetics. In addition. and extensive research has been conducted using rats. to test hypotheses about how a disease develops. scientists cannot conduct research on just one animal or human.When animal models are employed in the study of human disease. Rodents are the most common type of mammal employed in experimental studies. Methods of Inducing Human Disease in Other Organisms Despite their genomic similarities to humans. Also. Therefore. the majority of genetic studies. Among these rodents. high reproductive rates. most model organisms typically do not contract the same genetic . have employed mice. Other common experimental organisms include fruit flies. and relatively lowcost of use. especially those involving disease. and then observing the consequences of that change. but also because of their availability. the conditions associated with an experiment must be closely controlled. anatomy. mice. guinea pigs. not only because their genomes are so similar to that of humans. and physiology. an adequate number of subjects must be used to statistically test the results of the experiment. to obtain scientifically valid research. and hamsters. zebra fish. gerbils. animal models are often preferable for experimental disease research because of their unlimited supply and ease of manipulation. For example. ease of handling. This often means manipulating only one variable while keeping others constant.

2000). mutation-driven method uses radiation and chemicals to cause mutations. In attempting to engineer a genetic mouse model for a human disorder. Large-Scale Mutation Screens As the name might imply. hypomorphic. Thus. . depending on the exact type of mutation involved in the disease under study. the directed. single-gene knock-outs and knock-ins. it is important to know what kind of mutation causes the disease (for example. so scientists must alter their genomes to induce human disease states. disease-driven approach can employ any one of a number of techniques. the animals are screened in an attempt to determine which ones show phenotypes that are similar to human diseases.diseases as people. Common directed techniques include transgenesis. Scientists approach this task in two main ways: one that is directed and disease driven. Then. is the disease null. instead of being driven by the disease mutation. On the other hand. indirect approaches attempt to randomly make mutations in animal models' genomes. for example. conditional gene modifications. and the other that is nondirected and mutation driven (Hardouin & Nagy. One common technique associated with this method is the large-scale mutation screen. these methods are based on screening the phenotypes. The nondirected. so that the same kind of mutationcan be introduced into the corresponding mouse gene. or dominant negative?). and chromosomal rearrangements.

Transgenic animals are generated by adding foreign genetic information to the nucleus of embryonic cells. they weren't always expressed. 2000). transgenesis is a directed approach. Also. at that time. the methods for transgenesis were not optimal.. More recently. 1997a). 2000).Two of the most effective ways to generate mutations are by exposing organisms to X-rays or to the chemical N-ethyl-Nnitrosourea (ENU). small transgenes were inserted into random sites in the genome. respectively). however. whereas ENU treatment is linked to mutations within single genes. the foreign DNA was incorporated into only a small percentage of embryos and was inconsistently passed to the next generation. ENU can produce mutations with many different types of effects. These larger transgenes are more likely to . and depending on their location. 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. 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. These types of models are particularly useful for identifying new genes and pathways that contribute to disease. thereby inhibiting gene expression. 2007). The first mouse gene transfers were performed in 1980 (Hardouin & Nagy. and it is frequently employed in screens in model organisms such as zebra fish. Transgenesis As opposed to the use of X-rays and ENU. X-rays often cause large deletion and translocation mutations that involve multiple genes (Bedell et al. For instance. such as loss and gain offunction (Rosenthal & Brown. such as point mutations (Hardouin & Nagy.

this is a process that physically rearranges two strands of DNA for the exchange of genetic material. 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. 2000). This is because ES cells can contribute to all celllineages when injected into blastocysts. knock-in mice are produced by inserting a transgene into an exact location where it is overexpressed. Homologous recombinationcreates the mutations.contain regulatory sequences necessary for normal gene expression and are usually more comparable to the endogenous gene (Bedell et al. which creates less expression and loss of function. The use of embryonic stem (ES) cells is required for this technology.. As a result. the use of transgenic mice has dramatically increased in the past two decades. and most of these genes have been related to disease (Hardouin & Nagy. and then it is conveyed to the next generation through breeding. including null or point mutations and complex chromosomal rearrangements such as large deletions. translocations. or . and they can be genetically modified and selected for the desired gene changes.000 genes have been knocked out of mice. Both knock-out and knock-in animals are created in the same way: a specific mutation is inserted into the endogenous gene. Many types of mutations can be introduced into a model gene in this way. These methods are particularly useful if a single gene is shown to be the primary cause of a disease. 1997a). more than 3. Over the years. Knock-out mice carry a gene that has been inactivated.

the mutations can profoundly affect development and cause early death. knock-in. which is implanted into a foster mother. These effects would preclude using animal models to study adult diseases. 1997a).inversions (Bedell et al. phenotypes to human patients and are therefore good models for human disease.. To do this. Thankfully. including adulthood. The vector contains a drug-resistant marker genethat allows only the targeted ES cells to survive when exposed to the drug. and when genes are altered to model such diseases. that encodes a certain component of a protein that will be deleted (Bedell et al. 1997a). The mice containing the Cre recombinase under the control of tissue-specific . mice with two different types of genetic alterations are needed: one that contains a conditional vector. Thus. new technology has made it possible to generate mutations in specific tissues and at different stages ofdevelopment. or part of a gene. Many knock-out and knock-in mice have similar. 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 mutant ES cells can be selected and injected into the host mouseembryo. and the resulting generation of offspring has the recombinase effector gene.. which is like an "on switch" for the mutation. Conditional Gene Modifications One drawback to using transgenic. if not identical. and one that contains specific sites (called loxP) inserted on either side of a whole gene. The resulting offspring are chimeras and have multiple populations of genetically distinct cells. Chimericoffspring are then crossed. and knock-out mice to study human diseases is that many disorders occur late in life.

duplications. and international initiatives have been created to accommodate their demand (Rosenthal & Brown. and translocations. recombinationoccurs at the loxP sites. over 1. such as radiation. inversions. 2001). Mouse models of these disorders can be created using indirect approaches. and most of these mutants are models for inherited genetic diseases . but their usefulness is restricted because pathological endpoints are unpredictable and undefined (Yu & Bradley.000 mutant strains exist. Mouse Models Exist for Many Human Genetic Diseases. When Cre is expressed. 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.or inducible regulatory elements are crossed to the mice with the desired loxP sites. but Are They Effective? Human genetic diseases affect a wide range of tissues throughout the body and are caused by numerous types of mutations. which delete the intervening sequences. and the resulting mutation is induced in specific regions and times. These conditionalmutant models are becoming increasingly popular. These mutations can include chromosome deletions. Creating mouse models for all these disorders is understandably a daunting task for scientists. Nevertheless. 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. 2007). as well as nested chromosomedeletions.

However. In . diabetes. obesity. Perhaps using nonhuman primates might alleviate some of these discrepancies because their physiology is closer to that of humans. psychiatric disturbances (including anxiety and depression). 1997b).. hypertension. osteoporosis. if any. many diseases are still incurable. despite promising results with certain preclinical treatments in animal models. blindness. glaucoma. For example. These diseases include several types of cancer. Nonetheless.(Hardouin & Nagy. and they recreate some aspects of the particular disease. these models replicate many of the corresponding human diseasephenotypes. metabolic and hormonal disorders. heart disease. whereas mice do not. This statement is particularly true for neurodegenerative diseases. 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. Most available animal models are made in mice. skin pigmentation diseases. As a result. Huntington's disease patients show dyskinesia (involuntary movements). 2000). neurodegenerative disorders (such as Huntington's or Alzheimer's disease). most of which involve cognitive deficits. deafness. the same treatments do not always translate to human clinical trials. 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. 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. replicate all the symptoms. few. 2007). and birth defects (such as cleft palate and anencephaly) (Rosenthal & Brown. and in most cases.

. 2008). Nature Education 1(1) Most diseases are caused by mutations in more than one gene.fact. studies of monogenic diseases provide an invaluable . due in large part to knowledge of the human genome sequence. and microarray data. 2002). some researchers have pursued this possibility despite the technical difficulties and additional costs to perform transgenesis in primates (Wolfgang & Golos. Rare Genetic Disorders: Learning About Genetic Disease through Gene Mapping. 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. and Microarray Data By: Heidi Chial. H. 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.D. Although polygenic diseases are more common than single-gene disorders. the generation of widespread markers of genetic variation. For example. Indeed. or single-gene disorders provide? Researchers have made dramatic inroads into the study of polygenic and other complex human diseases. SNPs. (2008) Rare genetic disorders: Learning about genetic disease through gene mapping. and the development of new technologies that allow investigators to associate disease phenotypes with genetic loci. So what clues can monogenic. SNPs. Ph. (Write Science Right) © 2008 Nature Education Citation: Chial.

Mendel Revisited: Monogenic Diseases The human genome contains an estimated total of 20. In single-gene diseases.000 genes that serve as blueprints for building all of our proteins (International Human Genome Sequencing Consortium. OMIM also reported 1. more questions than answers remain regarding the identity of single genes and their role in human disease. 2008. Victor A. However. a mutation in just one of these genes is responsible for disease. OMIM. OMIM reported 387 human genes of known sequence with a known phenotype. OMIM reported 2. Furthermore. thereby contributing a great deal to our understanding of all forms of genetic disease. Trends in Gene Discovery . As of June 15.084 phenotypes for which a Mendelian basis is suspected but has not been fully established. McKusick that is focused on inherited genetic diseases in humans. Online Mendelian Inheritance in Man.310 human phenotypes with a known molecular basis.621 confirmed Mendelian phenotypes for which the molecular basis is not known. is a regularly updated. and 2. Table 1 includes some examples of single-gene diseases. or that may exhibit overlap with other characterized phenotypes. As you can see. Single-gene diseases run in families and can be dominant or recessive.opportunity to learn about underlying molecular mechanisms. 2004). and autosomal or sexlinked.000-25. online database established in 1997 by Dr. Pedigree analyses of large families with many affected members are very useful for determining the inheritance pattern of single-gene diseases.

which involve many genes. researchers face difficulties in identifying families with the disease and in obtaining sufficient numbers of DNA samples for comparison to unaffected family members. studies of monogenic diseases contribute a great deal to knowledge of polygenic forms of human disease (Antonarakis & Beckmann. many of the 1. With the sequence of the human genome available. There are several reasons for this movement toward polygenic diseases. showing the precise location of every gene and determining areas of the genome that can differ from one person to the next (termed "polymorphic"). and pharmaceutical companies are often less likely to invest financial resources in research efforts focused on rare diseases. As a result. researchers have been able to generate maps of each chromosome. 2006).621 monogenic disorders without known genes are very rare.After the human genome was sequenced. However. For instance. Also. the gene was not identified until 1993. researchers began to shift their focus from monogenic diseases to polygenic diseases. although efforts to isolate the gene associated with Huntington's disease began in the late 1970s. Of special interest to researchers are single nucleotide polymorphisms (SNPs). biotechnology companies. 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. which are . funding agencies. For one. slow-going techniques for mapping and isolating disease-associated genes.

and converted into singlestranded DNA. A laser is then used to scan each grid position to determine which SNP variants are represented. . fluorescently labeled genomic DNA fragments are then incubated with the SNP chip. 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.000 base pairs. SNP profiles can be compared between affected and unaffected family members (or unaffected.single base pair polymorphic regions. The single-stranded. A SNP chip contains short. By using SNP chips. SNPs occur throughout the human genome at an average rate of one SNP per every 1. 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 diseaseassociated genes. researchers can obtain a SNP profile of an individual that spans the entire genome. unrelated individuals) to determine which SNPs segregate with a disease (or are associated with thedisease). single-stranded DNA molecules called oligonucleotides that correspond to known SNP variants. The DNA isolated from the blood sample is broken into fragments. and only those DNAfragments that are a perfect match will bind to their complementary SNP oligonucleotide on the SNP chip grid. labeled with a fluorescent dye.

then convert it into singlestranded complementary DNA (cDNA) and label it with a fluorescent dye. The single-stranded. but their grids contain single-stranded DNA fragments that correspond to protein-encoding genes.Because the SNP sequences have already been mapped to specific chromosomal locations. Bioinformaticists can easily examine a region of a chromosome and determine which segments correspond to protein-encoding genes. Gene chips are similar in concept to SNP chips. Researchers can now use gene chips to simultaneously examine the expression (mRNA) levels of all human protein-encoding genes from a given cell population. In order to study gene expression. researchers can also immediately map the disease-associated gene to a specific region of a given human chromosome. 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. Furthermore. respectively. researchers can often predict how geneencoded proteins may function within a cell. fluorescently labeled cDNA is then incubated with the gene chip. The Bioinformatics Era: Genomics and Proteomics Bioinformatics is the genome-inspired field of biology that analyzes genomic information to predict gene and protein function. allowing hybridization between the cDNA molecules and theircomplementary sequences on . Large-scale studies of genes and proteins are referred to as genomics and proteomics. researchers first isolate mRNA from a tissue of interest.

a number of databases have been established. By comparing gene expression profiles from normal and diseased individuals. researchers can determine the proteomic profile associated with a given form of human disease. 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. scientists can also determine changes in gene expression associated with human disease. and a laser is used to scan the chip to determine the levels of each protein represented by the antibodies on the grid. generate a tremendous amount of data. researchers have developed chip-based methods for the simultaneous examination of thousands of proteins within a given cell population. which simultaneously examine thousands of genes and proteins. Furthermore. Databases As you can imagine. genomic and proteomic approaches. In this way. In these approaches. In order to make meaningful connections among worldwide scientific discoveries. scientists are publishing new data at a very fast pace. More recently. The protein chip is incubated with a fluorescently labeled protein sample from a given individual. From Man to Mouse: Using Genetic Model Organisms to Understand Single-Gene Diseases in Humans . and they can see which proteins show altered expression.the gene chip grid. which provide a number of useful links to other databases. the protein chip grid contains adhered antibodies that recognize and bind to specific human proteins. Examples of useful databases include OMIM and Entrez Gene.

the entire sequence of the mouse genome is known. and even yeast. cell division. Today. 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. . Moreover. Although genetically engineered mice are not perfect models of human disease. worms. in which the mutant mice carry the expanded CAG repeat within the Huntington's disease-associated gene. frogs. Many human genes are also found in mice. researchers have developed a mouse model of Huntington's disease. Similar to that of humans. several of the same basic cellular processes are shared among humans and these organisms.Due to the remarkable level of homology between genomes across the evolutionary tree. researchers can generate mice with a mutation or deletion of a disease-associated gene. flies. They can carry out detailed phenotypic analyses of the mutant mice and learn how the corresponding gene may function in humans. growth regulation. Although this discussion focuses on mouse models. Many of the genes found in humans are also present in these other types of organisms. including metabolism. and using mice as a modelorganism for genetic studies has contributed to our understanding of human disease. 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. and more. they can offer valuable insights into the function of disease-associated genes. For example.

TGFB1. can exhibit strikingly . 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. In addition. One such example is the discovery of trinucleotide repeat expansions and their association with several forms of neurodegenerativedisease. and microbial infections (NOS1). For example. cystic fibrosis was considered a single-gene disease associated with mutations in the cystic fibrosisassociated gene. 2006). for many years. identical twins with the same mutation in the gene associated with Duchenne muscular dystrophy. fragile X syndrome (FMR1 gene). the initial discovery of the CFTR gene was followed by the identification of several additional genes that contribute to cystic fibrosis. called DMD. and spinocerebellar ataxias (SCA1. Friedreich's ataxia (FRDAgene). SCA3. bowel obstruction at birth/meconium ileus (CFM1). SCA2. However. For instance. pulmonary phenotypes (TNF.Studies of single-gene diseases in humans have led to many completely unexpected findings. myotonic dystrophy (DMPK gene). several modifier genes have also been identified that can modulate the phenotypes associated with mutations in CFTR (Guggino & Stanton. Cystic fibrosis-associated phenotypes due to mutations in the CFTR gene are in turn modulated by mutations in the following genes: gastrointestinal phenotypes (MUC1). studies of monogenic disease transmission in identical twins have uncovered various nongenetic mechanisms associated with disease. including Huntington's disease (HTT gene). CFTR. and MBL2).

2004). and even occasional hypodontia. 2004). 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. patterns of X Finally. researchers isolated tooth agenesis. 2007). which is characterized by lower lip pits.different disease phenotypes due to different chromosome inactivation (Abbadi et al.000 births. 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. orofacial clefts.. a complex birth defect suggested to be caused by as many as three to 14 genes (Schliekelman & Slatkin. Consider the example of Van der Woude syndrome. 1994)... and they showed that IRF6 variation contributes to this condition as well (Vieira et al.. 2002). such as isolated cleft lip with or without cleft palate (frequencyof 1:700 births) and isolated tooth agenesis (frequency of 1:100 births). In related studies. monogenic syndromes can sometimes serve as models for complex diseases. This disorder is caused by dominant mutations in the IRF6 (interferon regulatory factor 6) gene (Kondo et al. Scientists have proposed that IRF6 variation may also contribute to isolated cleft lip with or without cleft palate (Zucchero et al.. Figure 1) and also contribute to much more common defects. 2002). another complex phenotype commonly found in the generalpopulation and that is present in a subset of cases of Van der Woude syndrome. that have more complex genetic etiologies. From Simple Beginnings to Complex Endings .

among other things: Precisely label the chromosomal location of any gene using different colored dots Examine cells from any type of tissue. with the help of databases and reference systems.Armed with knowledge of the human genome sequence and an arsenal of new molecular tools for gene discovery. even tumor cells Identify cells that have lost or gained a specific chromosome. 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. Find out what techniques scientists are using to dissect these chromosomes at the molecular level. our collective knowledge of single-gene diseases. Cytogenetic Methods and Disease: Flow Cytometry. (Write Science Right) © 2008 Nature Education Citation: Chial. and FISH. Cytogenetic approaches to studying chromosomes and their relationship to human disease have improved greatly over the past several decades. today's gene hunters are prepared to greatly expand our knowledge of disease-associated genes. CGH. Modern cytogenetic approaches enable researchers to do the following. (2008) Cytogenetic methods and disease: Flow cytometry. Ph. Nature Education 1(1) Some diseases involve regions of chromosomes that have been flipped or damaged. H.D. or lost or gained a copy of a given gene or genes . CGH. and FISH By: Heidi Chial. undergone a translocation event involving a specific set of chromosomes. Most certainly.

researchers developed methods to visualize chromosome structure and organization. 1959). 1956). Early cytogenetic studies showed that an extra or missing copy of certain human chromosomes could lead to disease. 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. As the field of human cytogenetics emerged. For example. Scientists quickly realized that not all chromosomes are created equal: specifically. Since then. Researchers also embarked on numerous studies to determine the relationship between human disease and chromosomes.. several abnormalities in sex chromosome number were linked to disease: Turner's syndrome was shown to be associated with the presence of . Trask. 2005. 2002).Determine whether specific regions of chromosomes have been lost or gained without ever looking at the chromosomes under a microscope Clearly. in 1959. an extra copy of chromosome 21 was shown to be associated with Down syndrome (also called trisomy 21) (Lejeune et al. the field of cytogenetics has developed into a vital tool for studying and diagnosing human disease. In the same year. they differ in their length and in the position of their centromere. 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.

and David Hungerford.X) (Ford et al. which is largely responsible for CML-associated phenotypes. uncovered a link between chronic myelogenous leukemia (CML) and abnormal chromosome structure. a graduate student at the Institute for Cancer Research in Philadelphia. 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. which they named the "Philadelphia chromosome. a collaborative study between Peter Nowell. 1973). 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." that was unique to CML cells (Nowell & Hungerford.a single X chromosome and no Y chromosome (45. 1985). a new faculty member at the University of Pennsylvania. 1959). 1959). The ABL1 half of the encoded fusion protein exhibits high tyrosine kinase activity. in 1985. Using . in 1968. Both Turner's syndrome and Klinefelter's syndromeaffect sexual differentiation in affected invidivuals. Thus. resulting in a gene fusion product called BCR-ABL (Heisterkamp et al. Then. researchers turned their attention to the role of chromosome structure. the researchers discovered the presence of a small chromosome. 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.XXY) (Jacobs & Strong.. The Philadelphia Chromosome Soon after discovering the link between chromosome number and disease. Later. Specifically. in 1960. 1960)..

. Studies of RB1-associated retinoblastoma led to the establishment of the two-hit hypothesis. 1963). and he noted . the first official tumor suppressor gene ever identified. early studies of cells from patients with retinoblastoma. Using Cytogenetic Approaches to Map Genes In addition to providing associations between chromosomal abnormalities and disease. in 1968. An important breakthrough occurred in 1963. when cri du chat syndrome. other scientists were examining the link between chromosomal deletions and disease. 1963). This region was later determined to harbor the RB1 gene. which is a cornerstone of cancer biology (Knudson. Imatinib treatment of CML has been heralded as one of the biggest success stories in cancer treatment over the past decade.. Roger Donahue used new methods to study metaphase chromosomes in his own blood cells. a congenital disorder that is associated with severe mental retardation and a cat-like cry in affected infants. scientists developed a drug called imatinib mesylate (also called STI571 and Gleevec) to inhibit ABL1 kinase activity (Druker. For example. a childhood form of retinal cancer. also showed deletion of a specific chromosomal region (Lele et al. Cri du Chat Syndrome and Retinoblastoma During the period in which Nowell and Hungerford discovered the Philadelphia chromosome. That same year. 2002). 1971). cytogenetic approaches have also allowed researchers to map genes to particular chromosomes. was found to result from a loss of part of the short arm of chromosome 5 (Lejeune et al.this information.

Maximo Drets and Margery Shaw established methods to stain metaphase chromosomes using a dye called Giemsa.Gbanding patterns can be used to detect chromosomal translocations. which are easily obtained from blood samples but are more difficult to retrieve from solid tissue samples. 1971). Using his extended familypedigree and conducting biochemical tests to determine blood group markers. G-banding methods continue to be widely used today. and they have made key advances in gene discovery possible.that one of his copies of chromosome 1 had a region near the centromere that was loosely structured and uncoiled. Furthermore. Rowley used G-banding patterns to determine that a translocation event involving chromosomes 9 and 22 was responsible for CML (Rowley. For instance. called G-bands.. deletions. for each of the 24 different human chromosomes (Drets & Shaw. For instance. as previously mentioned. Shortly after the Duffy blood group locus was mapped. 1973). which can lead to lower resolution in mapping. and insertions. 1968). Gbanding requires metaphase chromosomes. which produces a signature banding pattern. though such approaches have certain drawbacks. Donahue employed cytogenetic techniques to map the Duffy blood grouplocus to chromosome 1 (Donahue et al. metaphase chromosomes are highly condensed. 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 .

researchers used the Sendai virus to induce fusion between a human cell and a mouse cell. Such techniques involve using mitotic cellsuspensions and disrupting . researchers adapted these techniques to isolate individual human chromosomes as shown in Figure 1. Yet another advance in cytogenetic techniques involved the process known as flow cytometry. based on their size and the intensity of their fluorescence signal. With this technique. 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.more detail. Although flow cytometry and FACS were initially used to isolate populations of intact cells. Using Flow Cytometry to Sort Chromosomes . 1965). Harris & Watkins. Fluorescently labeled cells can also be sorted into separate tubes. as well as sparse numbers of human chromosomes (Ephrussi & Weiss. which was originally used to study distinct cell populations within a mixture of different cell types. In an effort to meet this need. resulting in a human-mouse somatic cell hybrid that contained the complete mouse genome. a fluorescent dye is used to specifically label the cell population of interest. An extensive series of humanmouse hybrid cell lines that carried known combinations of human chromosomes was thus developed. using diffraction plates in a process called fluorescenceactivated cell sorting (FACS). 1965. and this series greatly facilitated the mapping of human genes to specific chromosomes prior to the advent of the Human Genome Project.

and the second dye. The first dye. and with the exception of four chromosomes (9. This led to the generation of chromosome-specific collections of DNAfragments. called chromomycin A. 11. binds to G-C base pairs. The resulting DNA fragments were ligated into DNA plasmid vectors. connects the smallest (21) to the largest (1 and 2) in Figure 1. which are the largest. which allowed them to be propagated in bacteria or yeast host cells. and 12). leading to higher levels of Hoechst and chromomycin staining. Chromosomes 1 and 2. researchers used restriction enzymes to cut pooled chromosome populations into smallerDNA fragments. respectively. each of increasing size.. all of the human chromosomes can be resolved based on their laser light-scattering properties. shows the lowest Hoechst and chromomycin staining intensity.the cell membranes to release the condensed chromosomes that are labeled using two different types of fluorescent dyes (Carrano et al. FACS is used to isolate individual fluorescently labeled chromosomes in the solution. the A-T and G-C contents of a given chromosome are quite similar. 10. In addition to isolating pools of individual chromosomes. called Hoechst 33269. . chromosome 21. Fluorescence In Situ Hybridization (FISH) . 1979). which is the smallest. show the highest Hoechst and chromomycin staining. Larger chromosomes contain a higher number of A-T or GC base pairs. called libraries. this approach can be used to determine changes in chromosome size and number. In further chromosomal studies. binds to A-T base pairs. As illustrated in Figure 1. In general. and that is why a diagonal line of chromosomes.

FISH allows a higher level of resolution than standard Gbanding approaches. Figure 2a demonstrates an example of a standard FISH experiment. FISH involves the use of fluorescently labeled DNA probes that are capable of hybridizing to complementary chromosomal regions. Furthermore. is used to label a metaphase chromosome spread. Probes to different genes or DNA sequences can even be used simultaneously. two red fluorescent dots can be observed. In this case. corresponding to the maternal and paternal copies of chromosome 1. This technique allows researchers to view the chromosomal location of a particular gene or DNA sequence through a microscope. 1985). 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 .which served as a platform for the Human Genome Project. Thus. The first single-copy human gene to be mapped using FISH was thyroglobulin in 1985(Landegent et al. Researchers also wanted to study regions of individual chromosomes within the nucleus of intact cells. in which the red fluorescent DNA probe. corresponding to a 150 kilobase pair region of chromosome 1.. the net result is a fluorescent dot at the chromosomal location where the labeled probe binds. they used a cytogenetic method called fluorescence in situ hybridization (FISH) to map DNA sequences to specific regions of human chromosomes. the ability to isolate collections of DNA fragments that span individual chromosomes led to the development of chromosome-specific staining methods.

FISH can be carried out using nondividing cells. they found that Angelman syndrome patients inherited the deleted copy of chromosome 15 from their mother. This is important.000 times less compact in nonmitotic (interphase) cells. because DNA packing is approximately 10.. which allows investigators to examine nonmitotic cells. 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. FISH analyses have also contributed greatly to our understanding of Angelman syndrome and Prader-Willi syndrome (Knoll et al. 1989). 1993). respectively. Furthermore. the net result is a rainbow-colored chromosome. FISH can be used to identify genes with increased copy number or to detect gene loss. FISH has been very useful in the characterization and diagnosis of disease. However. allowing researchers to achieve a higher level of resolution. For instance.the length of a human chromosomewere each labeled with a different color.. For example. whereas Prader-Willi syndrome patients inherited the deleted copy of chromosome 15 from their father. as shown by more or fewer than two fluorescent "dots" in a somaticcell. Researchers found that Angelman syndrome and PraderWilli syndrome were both associated with the same deletion in chromosome 15 (from region q11 to q13). this is due to chromosomal imprinting. the neurological disorder Charcot-MarieTooth type 1A is associated with a duplication of one .

is carried out using isolated chromosomes that are free from nuclear architecture and exist as long. 1991). Wiegant et al. An extremely high-resolution form of FISH. but not by metaphase FISH (Lupski et al. 1993.. Spectral Karyotyping (SKY) and Multiplex-FISH (M-FISH) . Chromosomespecific 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. 1992). researchers can resolve generearrangements and duplications with incredible precision. The labeled DNA probes are then pooled and used in hybridization experiments with metaphase chromosome spreads.. which permits the simultaneous tracking of all human chromosomes. The ability to isolate individual human chromosomes using flow cytometry. as shown in Figure 3b. called fiber-FISH. stretched-out DNA fibers (Parra & Windle. This powerful approach. The labeled DNA probe sets bind to their complementary chromosomes. has . has allowed cytogeneticists to develop 24color probe sets that are used to label each human chromosome with a distinct color (Figure 3). the maternal and paternal copy of each chromosome will be labeled with the same colors. By using DNA fibers as a template for FISH. combined with knowledge of the human genomesequence. In a somatic cell.million base pairs that can be resolved by interphase FISH. allowing each individual chromosome to be labeled with a specific fluorescent color along its entire length.

SKY/M-FISH techniques have allowed researchers to detect small chromosomal rearrangements in individuals with seemingly normal karyotypes. Speicher et al. the second color reports the identity the other chromosome involved in the translocation. 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. researchers developed a technique called comparative genome hybridization (CGH) (Figure 4). 1996.been called spectral karyotyping (SKY) or multiplex-FISH (MFISH) (Schrock et al. 1996). and the fragmented experimental DNA sample is labeled with red fluorescence. As you can see in the bladder cancer cell depicted in Figure 3c.. The fragmented control DNA sample is labeled with green fluorescence. SKY analysis can be used to detect interchromosomal rearrangements (indicated by arrows) and aneuploidy (abnormal chromosome number). Because each chromosome has its own color. In order to address this need. The two DNAsamples are pooled and used together as DNA probes in hybridization experiments with . chromosomal translocations are easily detected when a chromosome shows a region with a different color.. These techniques are probably the most significant development in molecular cytogenetics in the past decade. and also to determine more precisely the cytogenetic aberrations in individuals with complex aberrant karyotypes. This approach involves the isolation and fragmentation of genomic DNA from a control subject and an experimental subject.

If a chromosomal region was amplified in the experimental group. a more recent microarray-based CGH method does not require the use of metaphase chromosomes (Pinkel et al. corresponds to a known DNA sequence that has been mapped to a specific chromosomal region. 1998). 1999). which leads to limited resolution. Using this approach. resulting in an orange/yellow color. which is located in a specific position on the chip. unaltered chromosome regions would show equal binding of the green and red probes and a resulting orange/yellow color. researchers have discovered that the gene encoding the catalytic subunit of phosphatidylinositol 3-kinase (PIK3CA) was amplified in ovarian cancer (Shayesteh et al. If a chromosomal region was deleted in the experimental group. that region will appear more green under the microscope. As described for standard CGH experiments. and red for the experimental group) are used in hybridization experiments with the CGH microarray platform. For unaltered chromosomal regions. Researchers can then scan along the length of the chromosomes to identify genomic alterations. which can be scanned using an automated approach. This method uses arrays containing thousands of base pair fragments of the human genome adhered to a microchip. Each individual DNA fragment. Standard CGH methods are very labor intensive and require the use of metaphase chromosomes. The green and red probes then compete to bind to the chromosomes.. the green and red probes should bind equally. whereas ..normal chromosomes. the corresponding chromosomal region will appear more red under the microscope. The same color-coded probes (green for the control group. However. thus identifying PIK3CA as an oncogene associated with ovarian cancer.

amplified and deleted chromosomal regions in the experimental group would appear red and green. abnormal chromosomes can actually cause genetic diseases. (Write Science Right) © 2008 Nature Education Citation: Chial. The future of molecularcytogenetics is bright. What methods have scientists invented to study these abnormalities? To be able to map the location of disease-associated genes within a genome. The information derived from a single array-CGH experiment is equal to that derived from thousands of FISH experiments. 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. Ph. By using the arrays.D. Nature Education 1(1) Since genes are packed into chromosomes. researchers can very precisely determine the chromosomal regions and genes that are amplified or missing. The powerful combination of cytogenetics and the human genome sequence has permitted new views of human genetic disease. respectively. scientists first needed to determine the landscape of human chromosomes. and this field will most certainly continue to uncover new and unexpected insights. H. . (2008) Cytogenetic methods in diagnosing genetic disorders. Cytogenetic Methods in Diagnosing Genetic Disorders By: Heidi Chial.

G-Bands Distinguish Individual Human Chromosomes . The short arm of a human chromosome is referred to as the p arm. while the long arm is called the q arm. Researchers were also aided in this endeavor by the fact that human chromosomes are not symmetrical. Human Chromosome Anatomy Human chromosomes come in 24 varieties: 22 different autosomes and two different sex chromosomes (X and Y). and the two arms that extend from the centromere are often different lengths. By combining Giemsa staining with somatic cell hybrids. when Tjio and Levan provided the first accurate count of the number of chromosomes in a human cell: 46. researchers were only able to distinguish these different types of chromosomes from each other based on their length and the position of their centromere. somatic cells contain 23 pairs of chromosomes. which yields signature banding patterns for each of the 24 types of human chromosomes -is still in use today. one of the earliest methods used to study human chromosomes . In fact.Giemsa staining. which contain a mixture of human and mouse chromosomes. scientists were eventually able to map diseaseassociated genes to individual chromosomes. which is the "waistline" of the chromosome where the two sister chromatids are attached following DNA replication. The ends of chromosomes are called telomeres.but the first of these discoveries wasn't made until 1956. Moreover. Before various staining techniques were developed. including 22 autosomal pairs and one pair of sex chromosomes. This knowledge led researchers in new directions that facilitated gene mapping and discovery.

which allowed them to be flame dried and adhered to a glass microscope slide. karyotyped them by cutting out individual photographs of each chromosome. 1971). which disrupts microtubules and causes cells to arrest in metaphase with duplicated chromosomes consisting of two sister chromatids. 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. Next. Here. The cells were then exposed to hypotonic conditions to permeabilize them. after which Giemsa staining yielded distinct and reproducible Gbanding patterns. the duo photographed the chromosomes in each of the 15 cells. and labeled the back of the . 1971).. the cells were fixed in a mixture of methanol and acetic acid. Drets and Shaw then photographed and generated G-banding maps for each human chromosome. 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. Furthermore. In 1971. After that. They found that each pair of chromosomes within a cell showed a similar G-banding pattern (Figure 1). the chromosomes from the cells were treated with sodium hydroxide and increasing concentrations of sodium citrate. Drets and Shaw took cultures of human lymphocytes and fibroblasts that were growing and undergoing mitosis and exposed them to a drug called colchicine. they found that the G-banding patterns were the same for a given chromosome when they compared different cells. In their experiments.

D14. Next. and whether each cell contained two chromosomes with each G-banding pattern.photos to designate which cell each chromosome came from. and found that each cell contained two copies apiece of the D13. which they termed D13. was examined in more detail. they noted three distinct types of G-banding patterns. 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. One group of three chromosomes. The duo then determined the Gbanding patterns within each of the 15 cells. and D15. called group D. Figure 2: Analysis of banding patterns of D-group chromosomes among 15 male cells. and D15 . they put the chromosomes into groups according to their length and centromere position without looking at the banding patterns. D14. Immediately. 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.

Indeed. As shown in Figure 2. 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. scientists could determine the approximate chromosomal location of a diseaseassociated gene. Human-Mouse Somatic Cell Hybrids Also in 1971. centromereposition. 1976). and G-banding pattern are taken into account. Collectively. Furthermore. 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 & . however. each of the 15 cells examined (numbered 1-15) typically showed two chromosomes with each of the three group D G-banding patterns (D13. and D15). Sometimes. researchers found that banding techniques made it possible to determine which chromosomes were involved in various types of structural rearrangements. a fact that highlights the real-world difficulties associated with studying chromosomes. not all of the group D chromosomes could be identified. Knudson et al. D14. By determining which part of a chromosome was deleted or translocated. the results of these experiments demonstrate that each autosomeand sex chromosome can be clearly distinguished when its length.chromosomes. the ability to determine the nature of chromosomal translocations and deletions contributed immensely to the discovery and mapping of human disease-associated genes. For example. or they were not present in the photograph..

. the hybrid cells contained two complete sets of mouse chromosomes.148 human genes by 1991 (McKusick. defined number of human chromosomes. in this case. 1971). Using this method. these efforts to create human-mouse somatic cell hybrids may seem more like science fiction than true science. researchers found that if they could identify a DNAprobe linked to a human disease. human-mouse somatic cell hybrids played a key role in the mapping and eventual cloning of the Huntington's disease-associated gene. researchers were able to map 1. Indeed. They could then determine which human chromosome was common among those hybrid cell lines to which the disease-associated DNA probe could bind. At first glance. the hybrid cells retained only between 1 and 15 of the original human chromosomes. 1991). as shown by a persistence of 10 or more human chromosomes after 150 cell divisions. Occasionally. they could use this probe in Southern blot experiments with chromosomal DNA from a series of well-defined human-mouse somatic cell hybrids. In the years that followed. HTT. the human chromosomes were retained at higher levels. 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. They found that the resulting hybridcells rapidly lost many of the human chromosomes but retained the mouse chromosomes. after 20 cell divisions.Grandchamp. various researchers were able to generate a series of human-mouse somatic cell hybrid lines that stably maintained a small. For instance. Indeed. However.

a number of genes have been linked to disease phenotypes. However. chromosomal biology.100 references encompassing some 100. Gleevec. and the role of chromosome structure in human disease.. which was first published almost two decades ago. For example. in the case of the Philadelphia chromosome. . One such advance was the cytological identification and characterization of the Philadelphia chromosome. In addition. Through the study of rare cases involving chromosomal defects. one of the most famous chromosomal translocations. cytological observations were met headon by thedevelopment of one of the most highly praised cancer drugs of the past decade.Chromosomes Are a Useful Tool for Mapping Disease Genes Thanks to the completion of the Human Genome Project. even though techniques like G-banding provide data at relatively low resolution. Felix Mitelman's Catalog of Chromosome Aberrations in Cancer. Although these chromosomal defects are extremely rare. a great deal of valuable information can be gleaned from these data with regard to human genetics. they are highly treasured among human genetics researchers. the ability to identify chromosomes and the location of defects associated with them remains a valuable tool.g. which is associated with chronic myelogenous leukemia. contains over 7. somatic cell hybrids are no longer the first option for mapping genes.000 aberrations related to cancer. mental retardation or cancer) can often provide the location of the gene that is likely disrupted and causing the defect. Identifying a chromosomal breakpoint that is associated with a clinical manifestation (e. Thus. and due to our knowledge of chromosomes at the base-pair level.

Although the complexities of human biology suggest an equally complex genome.Gene-Based Therapeutic Approaches By: Heidi Chial. but our knowledge of the human genome sequence has enabled the development of other gene-based therapeutic approaches. our gene repertoire is exquisitely regulated in order to maintain cell function by copying to RNA.D. guanine (G). Studies of the human genome have shown that between 20. even a single basepair mutation in one of our genes can lead to an altered protein and consequently disease. Nature Education 1(1) therapeutic Can we take genetic “pills” for disease-related mutations? No. Human somatic cells contain 23 pairs of chromosomes. Ph. these DNA base pairs are organized in a specific order and serve as the blueprint that distinguishes humans from other species. (Write Science Right) © 2008 Nature Education Citation: Chial. Thus. H. the human genome is actually quite similar to . cytosine (C). and thymine (T). Although redundancy is built into the human genome. Chromosomes are built using four different types of DNA bases: adenine (A). which in turn determines the formation of proteins. not yet.000 and 25. (2008) Gene-based approaches.000 genes reside along our chromosomes. The human genome is defined by more than 3 billion base pairs of DNA that make up our 46 chromosomes. and we inherit one set of 23 chromosomes from each of our parents.

Most of the human genome is nearly identical from one person to another. and current estimates suggest that the mouse genome contains at least 28. 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 of RNA splicing to produce gene functions undergo different types variants with alternative out by The important functions carried 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.972 genes.that of the mouse.7 billion base pairs of DNA.200-base-pair segment of human chromosomal DNA contains only one base pair that varies between two unrelated individuals.927 have homologues (called orthologues) in humans. In fact. a randomly chosen 1. 846 human genetic diseases have been reproduced in mouse strains (called mouse genotypic models) that carry a mutation in the related mouse gene.000 genes.000 to 25. The mouse genome is built of 2. but . of which at least 16. So. on average. The vast majority of these DNA variants are not deleterious. In fact. our somatic cells contain two copies of each of these 20.

336 human phenotypes are understood at the molecular level. Although genetic "pills" do not yet exist to target the vast majority of disease-related mutations. and an additional 2. O'Connor & Crystal. such diseasephenotypes can be kept in check by dietary modification or by providing a missing protein. However. Indeed. 2006.630 confirmed Mendelian phenotypes is not known. In some cases. surgical approaches can be used to repair or replace an organ or tissue damaged by disease. 2008).081 phenotypes are suspected to exhibit Mendelian inheritance (Online Mendelian Inheritance in Man. our knowledge of the human genome sequence has opened new doors to the development of gene-based therapeutic approaches (Brinkman et al. Metabolic Manipulation . even though the diseaseassociated gene was not yet known. In other cases.some specific alterations in the human DNA sequence are associated with disease. 2006). and that more than 2. the molecular basis of 1. doctors were already treating many hereditary forms of human disease with surprising success. or whether a key intermediary in a metabolic pathway was missing. recent estimates suggest that mutations in at least 383 human genes lead to known phenotypes.. and doctors were able to determine whether a metabolic product was accumulating to harmful levels. Many of these diseases were metabolic disorders. Treating Disease Before Knowing the Genes Involved Long before the human genome was sequenced.

familial hypercholesterolemia is associated with high levels of "bad" (LDL) cholesterol and early heart disease. thalassemia. familial hypercholesterolemia. patients with PKU accumulate high levels of a protein building block. in this case. Humans express different forms of hemoglobin during development. in their bloodstream. Often.sickle-cell anemia. called phenylalanine. Hemoglobin is the iron-containing protein that carries oxygen in our red blood cells. and many others. In other cases. this form of "metabolic manipulation" can be accomplished by modifying a patient's diet. These medications inhibit the activity of an enzyme (HMG CoA reductase) involved in a rate-limiting step of cholesterol biosynthesis. Hemoglobin is built of four protein subunits: two ά-subunits and a second pair of subunits. Once diagnosed. Sickle-cell anemia is due to a mutation in the gene encoding the β-subunit of hemoglobin . which helps prevent PKU-associated cognitive decline. metabolic manipulation involves the use of small molecules or drugs to target the activity of proteins linked to disease. treatment includes both dietary modifications (a diet low in cholesterol) and the administration of a class of drugs called statins. whereas the fetal form of hemoglobin consists of two ά-subunits and two γ-subunits (ά2γ2). hereditary angioedema. infants with PKU are fed a diet low in protein and phenylalanine. which vary by age. For example. The main adult form of hemoglobin consists of two ά-subunits and two β-subunits (ά2β2). Doctors are able to diagnose PKU using a simple heel-prick blood test to detect high levels of phenylalanine by three days of age. For instance.Physicians have developed approaches to regulate the metabolic pathways associated with a number of disorders. including phenylketonuria (PKU).

mucopolysaccharidosis II. symptoms associated with Pompe's disease. mucopolysaccharidosis I.(called HBB). 2003). and Von Willebrand disease). the gastrointestinal symptoms that accompany cystic fibrosis can be corrected by protein augmentation through the administration of pancreatic enzymes. hemophilia B. coagulation disorders (hemophilia A. 1984). This protein-add-back approach has been used to successfully treat patients suffering from a wide range of diseases. defective. Hydroxyurea is still used to treat sickle-cell anemia today. lung. including various membrane transport disorders (cystic fibrosis). Protein Augmentation In another approach called protein augmentation. immune deficiency (severe combined immune deficiency). or depletedprotein. including reduced heart.. and . andcongenital neurogenic diabetes insipidus). mucopolysaccharidosis VI. In the 1980s. which increases levels of the γ-subunit of fetal hemoglobin. physicians treat patients by providing them with a purified form of the missing. and Pompe's disease). For example. leads to a 50% reduction in the painful sicklecell crises associated with sickle-cell anemia (Platt et al. and lysosomal storage disorders (Gaucher's disease type I. individuals with growth hormone deficiency can be treated with purified growth hormone to restore normal growth. Similarly. congenital leptin deficiency. In addition. Fabry disease. endocrine disorders (growth hormone deficiency. researchers discovered that treatment with a drug called hydroxyurea. which leads to an abnormal structure of the βsubunit protein chain and the sickle-cell phenotype (Weatherall. emphysema (ά1-antitrypsin deficiency).

skeletal function, can be improved by treatment with acid άglucosidase enzyme.

Protein augmentation requires that the protein be added to the
outside of cells (i.e., the extracellular space). Therefore, this approach works best for replacing proteins that are normally present in the extracellular space. Protein augmentation approaches are often less effective if the missing protein is normally located inside of a cell, or if it is normally targeted to a specific intracellular organelle. Indeed, it can be difficult to ensure that a protein added to the outside of a cell will be taken up by the cell and targeted correctly to its normal location or organelle. In some cases, the success of protein augmentation depends on how well the protein is delivered to the organ(s) in which its function is required. The brain is a particularly difficult organ to target, because the access of proteins is limited by a membrane structure called the blood-brain barrier (BBB), which inhibits the passage of proteins and other chemicals from the bloodstream into the brain. The eye also presents challenges to drug access from the bloodstream. However, therapeutic agents can be delivered directly to the eye; this is not possible with the brain. Surgical Approaches Although more invasive, organ transplantation is also used to treat certain genetic diseases that affect particular organs. Unless the organ donor and the organ recipient are monozygotic twins, the chromosomal DNA sequence of the donor will be different from that of the recipient. Despite these differences, organ transplantation remains a viable therapy that continues to be used widely to this day.

In still other cases, surgery can be used to repair an organ or tissue that has been targeted by disease. For example, cleft lip, with or without cleft palate, is the fourth most common birth defect in the U.S., and it affects 1 in every 700 babies born each year. Cleft lip and/or cleft palate arise early during pregnancy while a baby is developing in the uterus; the malformations occur when there is not enough tissue in the lip or mouth area to permit joining of the tissue, or when the fusion of the lateral structures of the face does not occur properly. Cleft lip occurs when the two sides of the upper lip are separated, and cleft palate occurs when either the hard palate or the soft palate is separated. Surgical approaches can be used to effectively repair both cleft lip and cleft palate defects. Putting the Human Genome to Work: Using Genes to Treat Disease The identification of disease-causing mutations can be a rich source of opportunity for identifying new disease treatments. After identifying a disease-associated mutation, scientists can study how the function of the corresponding gene product (protein) is altered. With this information in hand, three main gene-based therapeutic approaches are currently being pursued: Gene-transfer approaches, in which a wild-type copy of the mutated gene is delivered

RNA modification therapy, in which the mRNA encoded by a mutant gene is targeted
Stem cell therapy, in which human stem cells are used to repair disease-damaged tissue Gene Transfer

. Gene-transfer approaches are based upon the simple concept that if a disease is due to a recessive loss-of-functionmutation in a single gene, then adding back the wild-type gene should restore normal function and alleviate the diseasephenotype. So far, two strategies have been used to deliver wild-type genes (O'Connor & Crystal, 2006): Ex vivo approaches, which require the use of a cell population that can be removed from a patient, genetically altered to express the wild-type gene, and then delivered back into the patient In vivo approaches, which deliver the wild-type gene directly into a patient's affected cell population, tissue, or organ Although the concept is simple, gene-transfer approaches have presented many clinical challenges. For example, both ex vivo and in vivo approaches require two key components: a gene expression cassette, consisting of a wild-type gene and some extra DNA sequences that permit the gene's expression in human cells, and a delivery system to introduce the wild-type gene into cells. The gene delivery system can be a cell membrane–like lipid carrier, or it can be derived from a virus. Table 1 shows some examples of gene-transfer clinical trials and the gene delivery systems employed, including delivery of the factor VIII (F8) gene to fibroblasts for the treatment of hemophilia A, delivery of the CFTR gene to the nasal and airway epithelium for the treatment of cystic fibrosis, delivery of the ornithine transcarbamylase (OTC) gene to the liver for the treatment of ornithine transcarbamylase deficiency, delivery of the

Once in the nucleus. which does not contain introns.glucocerebrosidase (GBA)gene to blood and bone marrow cells for the treatment of Gaucher's disease. Additional information about clinical trials using gene delivery approaches to treat human disease can be found on the Gene TherapyClinical Trials Worldwide website. both lipid carriers and viruses must facilitate cell entry by breaking through the target cell's plasma membrane. Current approaches typically use the cDNA corresponding to the wild-type gene. If . Figure 1a shows a typical gene expression cassette. as shown. depending upon the delivery system. 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. and delivery of the ά1antitrypsin (SERPINA1) gene for the treatment of ά1-antitrypsin deficiency. the expression cassette can either undergo random insertion into thecell's genome. . The wildtype gene (called a transgene) has a promoter sequence attached to its 5′ end. or it can be maintained extrachromosomally within the nucleus. and they must allow the expression cassette to enter the cell nucleus so that it can be transcribed into mRNA. In order to successfully deliver the gene of interest. 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.

called a loss-of-function mutation. expression must be sustained at high enough levels to rescue disease-related phenotypes. Researchers must also strike a balance in terms of the expression levels of the transgene. However. called gain-of-function mutations. if the gene is inserted in such a way that it disrupts the function of anothergene. Specifically. human disease can also be associated with dominant mutations in genes that encode hyperactive proteins.chromosomally integrated. the transgene will be expressed continuously. expression of the transgene can be lost over time due to cell division. but it must also be maintained at low enough levels to prevent an immune response by the patient. Furthermore. normal cellular function is restored when a wild-type copy of thegene is introduced. Targeting RNA to Treat Dominant Genetic Diseases Gene-transfer approaches are particularly useful when a diseaseassociated mutation encodes a protein with decreasedfunction. such as cancer. because loss-of-function mutations are usually recessive. however. the random nature of chromosomeintegration can lead to problems. in this case. if the expression vector is maintained extrachromosomally. . called dominant negative mutations. human disease can be associated with dominant mutations in which the mutant proteins interfere with thefunction of wildtype proteins. On the other hand.

. rather. and the resulting DNARNA hybrid molecule is then degraded by the intracellular enzymeribonuclease H (RNase H). RNA interference (RNAi). typically 22 base pairs long. RNAi involves the use of double-stranded RNA molecules (dsRNA). and ribozymes. Five approaches have been used experimentally to modify RNA levels: antisense oligonucleotides (ASO). usually between 18 and 30 bases long. researchers would prefer to "turn off" expression of the mutant gene. Figure 2 shows examples of RNA-based strategies for the treatment of disease. introduction of the corresponding wild-type gene is usually not sufficient to rescue the disease-associated phenotype(s). which are complementary to the mRNA to be targeted (Figure 2a). The ssDNA binds to the target mRNA. segmental trans-splicing. trans-splicing. To achieve this goal. corresponding to a region of the target gene (Figure 2b).In the case of a dominant mutation. or to inhibit the function of the mutant protein it encodes. The dsRNA is processed within the cell in such a way that it becomes part of an RNAinduced silencing complex (RISC) that recognizes and degrades the corresponding target mRNA. ASO strategies use short single-stranded DNA (ssDNA) molecules. 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.

trans-splicing leads to the production of a mature mRNA encoding the full length of the wildtype protein of interest. 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. similar to that described for trans-splicing. (Sometimes. which is complementary to a region of the 5′ flanking intron between the donor and branch-point sites for RNAsplicing. the wildtype exon sequence. a given cDNA is too large to be carried within a single viral vector. Segmental trans-splicing is an approach used to get around the size limitations associated with gene-transfer methods that involve vectors. Trans-splicing leads to the production of a wild-type copy of the mature mRNA and thus a corresponding wild-type protein.Trans-splicing is a gene-transfer approach that targets a pre-mRNA containing a disease-associated mutation within one of its exons (Figure 2c). the splice acceptor site. and the rest of the gene. Ribozymes containing ahybridization domain followed by . 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. followed by the splicing branch point. Ribozymes are RNA molecules with inherent catalytic activity that recognize a particular mRNA and cleave it (Figure 2e). the gene is divided into two smaller pieces. In this case.) In this case. which are delivered together using two separate gene-transfer vectors (Figure 2d). In this case. Thetransgene contains a hybridization domain.

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. Many of these RNA-based strategies have been developed in recent years. and RNA splicing leads to the formation of a wild-type copy of the mature mRNA. Stem cells naturally occur in two forms: embryonic stem cells and somatic stem cells. Unlike organs. whether ASO.e. Stem Cell Therapy On the genetic horizon. Embryonic stem cells are derived from a specific group of cells within an embryo. Furthermore. which means that they are able to become nearly anycell type imaginable as long as they are provided with the . which are built of specialized mature cells with tissuespecific characteristics and a very limited ability to divide. Once the target gene is cleaved. 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. the modern-day equivalent of organ transplantation is likely to be the use of stem cell therapy. researchers must exercise caution with respect to the specificity of any given mRNA-targeting approach. or ribozyme based. RNAi. They are capable of unrestricted cell divisions (i. they are immortal) and are pluripotent. and numerous questions remain regarding the cellular mechanisms involved inmRNA targeting.. the ribozyme-derivedhybridization motif binds. trans-splicing. to ensure that only the mRNA of interest is targeted.

SOX2. 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. ectoderm.. KLF4. Scientists have identified a set of four genes (OCT4/POU5F1. 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. their fate depends upon the tissue type from which they are derived. somatic stem cells are more restricted in terms of the type of cells they can become after they divide. Park et al. Yu et al. and c-MYC/MYC) that encode transcription factors that.appropriate environment. when expressed at the same time. 2007. 2008. as well as technical difficulties in obtaining and culturing these cells. the iPS cells are immortal. Like human embryonic stem cells... 2008b). Unlike pluripotent cells. 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. are pluripotent. cell surface markers.. have hampered the clinical use of embryonic stem cells. and express genes characteristic of all three embryonic germ cell layers (endoderm. and cellular morphology. 2007. Lowry et al. Ethical concerns regarding the use of human embryos as a source ofstem cells. 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. and .

Duchenne muscular dystrophy (DMD). four (OCT4/POU5F1. . pluripotent. SOX2. KLF4. and c-MYC/MYC). 2008a). and Lesch-Nyhan syndrome (carrier). Huntington's disease (HD). 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. SOX2. juvenileonset type 1 diabetes mellitus (JDM). Down syndrome/trisomy 21 (DS). researchers collected skin cells and bone marrow–derived mesenchymal cells from patients with one of ten different diseases. Recently. the disease-specific iPS cell lines were immortal. or five (OCT4/POU5F1.. Becker muscular dystrophy (BMD). KLF4. including adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID). Both opponents and proponents of human stem cell research have warmly welcomed the promise of somatic cell–derived iPS cell lines. a team of researchers at Harvard University applied this technique and established a panel of human disease–specific iPS cell lines (Park et al.. To generate disease-specific iPS cell lines. Gaucher's disease (GD) type III. Shwachman-Bodian-Diamondsyndrome (SBDS). SOX2. the investigators expressed three (OCT4/POU5F1. Parkinson's disease (PD). and capable of expressing genes corresponding to all three embryonic cell layers when induced to differentiate (Park et al. and KLF4). cMYC/MYC andNANOG) transcription factor genes.mesoderm) when induced to differentiate. Similar to the original iPS cell lines. in this case. 2008a).

multimodal approaches to . 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. and drug-screening approaches to target disease phenotypes. as well as the age and sex of the somatic cell donor (Park et al. intracellular signaling proteins. the resulting iPS cell lines cannot currently be used to treat human patients. and transcription factors that regulate disease-associated phenotypes. investigators can better target the activities of the enzymes. As gene-based therapeutics continue to evolve.. cellsurface receptors. and they will serve as a strong foundation for future studies aimed at the eradication of these devastating diseases. These cell lines are freely accessible to researchers worldwide. RNA-based therapies. secreted proteins. Due to the viral-based methods used to deliver the transcription factor genes into the somatic cells. researchers will be able to carry out experiments to better understand disease pathology and to develop effective gene-transfer techniques. By understanding the underlying molecular mechanisms linked to disease. 2008a). Nevertheless.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. With these disease-specific iPS cell lines in hand. Table 2: iPS Cells Derived from Somatic Cells of Patients with Genetic Disease.Reproduced from Park et al. thesecell lines are an invaluable resource for future investigation..

. life-changing discoveries. The future of genetic medicine will require collaboration and multidisciplinary approaches. which will most certainly be accompanied by unexpected.human disease will emerge.

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