Genetic Testing: The Biology
Matt Talbert
The Biology of Genetic Testing |
If you need additional assistance, these excellent resources are available for public use:
GeneTests Public Illustrated Glossary: (GeneTests Illustrated Glossary, 2004) (http://www.genetests.org/servlet/access?qry=ALLTERMS&db=genestar&fcn=term>report2=true&id=8888891&key=omhS2xl5n3sAA)
The National Cancer Institute’s: Understanding Gene Testing (Schindler, Kerrigan, and Kelly, 2003) (http://press2.nci.nih.gov/sciencebehind/genetesting/genetesting01.htm)
The US Department of Health and Human Services: Guide to Understanding Gene Testing (Understanding Gene Testing, 1994) (http://www.accessexcellence.org/AE/AEPC/NIH/index.html)
In this section:
Preimplantation Genetic Diagnosis
This use of genetic testing is usually most applicable with autosomal recessive genetic diseases, genetic diseases that are caused by the inheritance of two mutant alleles for a gene (one from each parent). Someone is deemed a “carrier” of a genetic disease if they are heterozygous for the disorder, meaning that they have one mutant allele and one normal allele. As a carrier, a person typically does not show any outward symptoms and their personal health is generally not affected, though this depends on the disease in question. A physician might suggest that a person undergo carrier screening if a person is a member of a certain ethnic or cultural group for which incidence of a particular genetic disease is high (i.e. Tay-Sachs disease in US Ashkenazi Jews, a disorder which causes fat accumulation in the brain).
Another more obvious situation in which a physician might suggest carrier screening is because the person’s family has a history of a particular genetic disorder. A third reason for carrier screening might be because a recessive genetic disease presently affects the partner. The purpose for carrier screening is normally to make informed reproductive choices as a result of one’s carrier status, this is crucial to the future health of a child of carrier parents. It is crucial to the child because a carrier parent may randomly pass on either their mutant allele or their normal allele, if the child gets two mutant alleles from its parents, it will be affected by the genetic disorder.
In clinics, usually the mother undergoes carrier screening first and if she tests positive, then the father is also tested. If the parents are both determined to be carriers for that mutant allele, then the child is tested as soon as possible during its development. If the developing fetus is diagnosed with the disease, the couple is then given the option to terminate the pregnancy (Godard et al., 2003).
Figure 1) When two carriers of cystic fibrosis have a child, there is a 50% chance that the child will be a carrier, a 25% chance that the child will have cystic fibrosis, and a 25% chance that the child will be normal (Facts, 2004). *Permission Pending*
Performing a genetic test on a person that is already living has two purposes, diagnosis or risk assessment.
Figure 2) When an adult undergoes genetic testing, the DNA is usually extracted from a sample of their blood (What is genetic screening?, 2004). *Permission Pending*
A test that is performed for a diagnostic purpose is usually a genetic test for an abnormality that scientists know, with a degree of certainty, will cause the patient to have a genetic disease (Godard et al., 2003). For example, the genetic test for Huntington’s Disease, a late onset and nearly always fatal genetic disorder, is designed to test for the presence of the telltale excessive repetition of the Huntington’s gene. This genetic abnormality is said to have a high degree of penetrance, meaning that the presence of the gene repeats almost always results in Huntington’s Disease. Therefore, a pre-symptomatic adult that undergoes a Huntington’s Disease diagnostic test and receives a positive result would need to undergo appropriate life planning counseling (Price et al., 1998). Though Huntington’s Disease is not symptomatic until adulthood, many other genetic diagnostic tests are ordered by physicians that suspect a genetic cause for certain symptoms in their patient (Godard et al., 2003).
A genetic test that is performed for risk assessment tests for a genetic abnormality, which could substantially increase the risk of having other kinds of diseases. An example of this kind of genetic abnormality is one or more of several mutations in the BRCA1 gene in women. If a patient has a family history of breast cancer, they are already assumed to be at a level of inherent risk, but they then also may be urged to undergo genetic testing for several different mutational variants the BRCA1 gene. Some BRCA1 mutations are estimated to increase a female patient’s risk of breast cancer on average to over 50% by 70 years of age. A positive test result can aid patients with a BRCA1 mutation by steering their physicians towards an appropriate aggressive regimen of prophylactic care, including frequent mammograms and possible early surgical intervention in the form of a mastectomy (Ponder, 1997).
Figure 3) A figure from a paper that displays how BRCA1 mutations and family history might affect the risk of having breast cancer in women (Ponder, 1997).
Prenatal screening is perhaps one of the more widespread forms of genetic testing, which focuses on early detection in the development of a child. Prenatal screening involves obtaining and testing living cells from a developing fetus. The living cells from the fetus are obtained in order to perform genetic tests that inform parents of a child’s disease status. This knowledge enables parents to make reproductive decisions in the form of altered life-planning or elective abortion (Godard et al., 2003). Each of the procedures below is executed by a specialist physician and is often visually guided by ultrasound, an image of the fetus obtained through reflection of high frequency sound waves (GeneTests, 2004).
Chorionic Villus Sampling (CVS) is one prenatal screening technique that can be used during the pregnancy at a gestation time of, on average, 11 weeks (GeneTests, 2004). This is an invasive procedure that involves the use of a long needle syringe, inserted through the mother’s abdomen, or a catheter, inserted through the cervix, to scrape and obtain cells from the sack that surrounds the fetus as it develops. This tissue contains a large amount of fetal DNA and can be analyzed fairly quickly (within 24 hours). CVS possesses a huge advantage over many other methods of prenatal screening because it can be done earliest in the pregnancy, enabling couples to elect abortion of the fetus without as many ethical quandaries (Weatherall, 1991).
Figure 4) A schematic diagram of Chorionic Villus Sampling (CVS). An ultrasound is used as a visual guide while a catheter is inserted through the woman’s cervix to obtain a sample of cells from the membranous sack that surrounds the fetus (Common Tests During Pregnancy, 2004).
Amniocentesis is another prenatal screening technique, normally used in the 15 th to 20 th week of pregnancy (GeneTests, 2004). During this procedure, a long needle is inserted through the mother’s abdomen and into the amniotic sac to withdraw a volume of amniotic fluid. This amniotic fluid contains living cells that have been shed by the fetus and can be cultured for DNA extraction and chromosomal analysis. While not as quick as CVS, this method still affords the couple a measure of time in which to make a reproductive decision upon a positive result for a disease-causing genetic abnormality (Weatherall, 1991).
Figure 5) A schematic diagram of amniocentesis. An ultrasound is used to guide the specialist while a long needle is inserted through the woman’s abdomen in order to draw a portion of amniotic fluid, which contains living fetal cells (Common Tests During Pregnancy, 2004).
Prenatal biopsy is a grouping of techniques that enable DNA analysis from the physical extraction of crude tissue from one of a variety of sources. A placental biopsy can be performed through the use of a needle inserted into the mother’s abdomen under ultrasound guidance starting at 12 weeks of development. Occasionally actual fetal tissue is needed to make a genetic diagnosis. The tissue can be obtained in a procedure such as fetoscopy; it involves the extraction of a small sample of fetal skin under the guidance of an inserted micro-camera. A fetoscopy can be performed after approximately 16 weeks of fetal development (Quintero, 2002).
Figure 6) This is a fetoscopically obtained image of a fetus at 18 weeks. (Diagnosing Fetoscopy, 2000). "COURTESY OF RUBÉN A. QUINTERO, MD at www.fetalmd.com."
Periumbilical Blood Sampling (PUBS) is a final technique that extracts fetal blood to enable DNA analysis through insertion of a needle into the fetal umbilical vein. This test can be performed starting at approximately 18 weeks of fetal development (GeneTests, 2004).
d) Preimplantation Genetic Diagnosis (PGD)
Preimplantation genetic diagnosis is a revolutionary genetic testing technique that can only be performed if a couple has undergone in-vitro fertilization (IVF). During IVF, ova are collected from the mother and fertilized with sperm from the father in a controlled medium. The developing embryo undergoes a few early stages in its development during which genetic material, in the form of a cellular body, can be extracted for genetic testing. After initial fertilization of an oocyte with a sperm, a structure known as a polar body is ejected from the embryo that is maternally haploid. This polar body can be biopsied and used for genetic testing, but it only is useful for genetic diseases that are passed along the maternal family line.
The most used form of PGD biopsy takes place after 3 days of the initiation of IVF when the embryo has developed 8-12 differentiable cells. At this point, it is relatively easy to apply gentle teasing to the embryo to extract one or two of the cells from its exterior. The extracted cells can undergo simple DNA extraction followed by PCR amplification for use in any number of genetic tests that are deemed fit. Each embryo that was created during IVF is sampled and tested, resulting in the ability to preferentially select the healthiest embryo for implantation in the mother’s uterus, one that is unaffected by a genetic disease in question. PGD is extremely expensive and is usually used only in cases of extreme risk, when the parents are of a known detrimental genetic status (Braude et al., 2002).
Figure 7) This is a flow diagram of Preimplantation Genetic Diagnosis (PGD). PGD is a process by which fertilized embryos are screened for genetic disorders and selectively implanted in the mother’s uterus (Preimplantation Genetic Diagnosis, 2004). *Permission Pending*
Newborn screening is an often mandatory public health program, which is performed within a few days of the birth of a child. It is intended to diagnose genetic disease for the purposes of early prophylactics and disease management. The first genetic disease to ever have genetic testing mandated in newborns was PKU or phenylketonuria, a form of mental retardation that is completely avoidable by restrictions of certain dietary elements. Parents that are aware of their child’s PKU status can now aid them in evading any consequences of this disorder through careful lifestyle choices. The genetic material used in newborn screening is usually either a sample of umbilical cord blood or blood obtained through a small heel puncture. The types of newborn genetic tests that are mandated are left up to the discretion of the US state government and are considered based upon their perceived clinical value. Currently in North Carolina, newborn genetic testing is mandatory for PKU, hypothyroidism (mental retardation caused by lack of thyroid hormone), galactosemia (brain and kidney damage as a result of improper metabolism of galactose), and sickle cell anemia (blocked blood vessels caused by irregularly shaped blood cells). In the future, scientists anticipate that even more genetic tests will be added to state newborn testing programs (“What is Newborn Genetic Screening?”, 2001).
Figure 8) This newborn has just had blood drawn for newborn genetic screening through a small puncture in the heel (Newborn Genetic Screening, 2004). *Permission Pending*
How is Genetic Testing Done?
The first thing that is necessary in any genetic test is a sample of DNA from a patient, which can be extracted from almost any living cell. In the laboratory, genetic testing is performed utilizing a variety of tools and methods, which also vary depending on the disease to be tested. It is nearly impossible to address the entire cornucopia of technical methods that are available to scientists in performing a genetic test. Therefore, below you will find a sample of some testing methods that are currently utilized to accomplish a significant multitude of genetic testing.
Public Aid:
One laboratory technique that is referred to below is the polymerase chain reaction ( PCR). If you seek additional clarification as to what PCR is then you may follow this link: (http://www.dnalc.org/Shockwave/pcranwhole.html) (“Polymerase Chain Reaction”, 1994)
Another such laboratory technique that is referred to is the southern blot, a variation of a procedure called gel electrophoresis. For a review of basic concepts you may follow this link: (http://www.dnalc.org/shockwave/southan.html) (“Southern Blotting”, 1994)
Basic Oligonucleotide Probe Tools/Assays
Amplification Refractory Mutation System (ARMS)
The karyotype is one of the more traditional methods of genetic testing, developing in 1956 at the dawn of the Cytogenetics movement. The movement found itself concentrating on the study of chromosomes as a whole. This is precisely what the karyotype seeks to capture in its results, a visual image of the entire set of a person’s chromosomes. An image of the chromosome set is evaluated for diseases related to changes in structure or number of chromosomes like Downs Syndrome, which is produced by Trisomy 21, an extra copy of chromosome 21.
The procedure for producing a karyotype involves first obtaining a sample of blood (or other tissue) and then centrifuging it to isolate the white blood cells. These cells are then exposed to chemicals called lectins that stimulate them to begin mitosis, causing the DNA structure of the chromosome to become more compact and easier to visualize. After a sufficient number of cells have been produced, their division is arrested through the addition of chemicals known as colchicines. Then, the white blood cells are placed in a hypotonic solution, which causes them to absorb water and swell. After a sufficient degree of swelling has taken place, the cells are gently mounted on a slide and stained. The cells are photographed and a geneticist uses the image to arrange the chromosomes into pairs (Therman and Susman, 1993).
Figure 9) This is an image of the final result of a karyotype. The red circle indicates an abnormality that is associated with Klinefelter’s Syndrome, an extra X chromosome where there should only be one (Klinefelter Syndrome, 2004). *Permission Pending*
Basic Oligonucleotide Probe Assays/Tools:
One of the most significant innovations in genetic testing has been the discovery, generation, and subsequent use of custom-made DNA oligonucleotide probes, which make use of the ability of a DNA strand to pair with its compliment. These probes, usually very small segments of DNA that have been amplified through a procedure such as PCR ( polymerase chain reaction), attempt to seek out and bind to their complimentary section on DNA that is being tested to usually either confirm or deny the presence of a particular gene. There are many uses for the concept of the oligonucleotide probe; it will certainly be a recurring theme in some of the other methods that are discussed (Polak and McGee, 1990).
Chromosomal Fluorescence In-Situ Hybridization (FISH) entered its preliminary stages in approximately 1969 through the independent efforts of several different research groups. FISH refers to using fluorescently labeled DNA probes to seek out a particular segment of a chromosome and bind to it, while emitting a signal that responds to ultraviolet light. This is achieved by first performing a procedure similar to a karyotype (see above) that results in cells that can be mounted on a slide. Next, the cells to be tested are treated with a detergent, which punches a series of small holes in the membrane of the cell that are big enough for the DNA probes to slip through. The oligonucleotide probes that are allowed to hybridize with the chromosomes of the cell are previously labeled for visualization purposes with either a fluorescent reporter molecule or some form of radioactivity (though fluorescence seems to be the more convenient option). After the hybridization of probes and chromosomal DNA is complete, imaging of the chromosomes occurs. During imaging of the labeled chromosomes, the regions where the DNA probe has bound emit a glow due to energetic excitement from the ultraviolet light emitted in the imaging microscope. This test can be used in genetic testing to aid in diagnosing a multitude of illnesses which result from changes in chromosome structure or at the molecular level (Polak and McGee, 1990).
Figure 10) An image of chromosomal Fluorescence In-Situ Hybridization (FISH). These chromosomes have been probed with a specific oligonucleotide sequence, as indicated by the yellow glowing. This glowing coincides with presence and the location of a genetic abnormality that the DNA probe was designed to detect when FISH is used for genetic testing (Annual Project Report, 1999).
The allele specific oligonucleotide screen is a very common concept in genetic testing with applications ranging from dot blot arrays to gel electrophoresis. This method is clinically limited almost solely to genetic disorders for which the molecular causes are known. In a dot blot array, nylon membranes are specifically treated and then receive a transfer of PCR-amplified genomic DNA from a patient. Next, the clinic hybridizes radioactively labeled oligonucleotide probes with the DNA that are specifically constructed to bind a particular disease-causing region or mutation. After hybridization is complete, the excess and unbound oligonucleotide probes are washed away and the test proceeds to imaging. The results of the screening indicate the degree of hybridization that has taken place between the DNA on the nylon fiber and the probe that was added. If the probe was designed to bind a known cystic fibrosis mutation and there is significant hybridization, indicated quantitatively through automated scanning and sometimes simply qualitatively by the darkness of a dot, then that person would be considered positive for the disease mutation (Mutation Detection Systems, 2001).
The southern blot is another application of ASO, utilizing a scientifically renowned process called gel electrophoresis. First, DNA from a patient is digested into fragments after exposure to proteins called restriction enzymes. After the DNA has been broken into fragments of varying size by the restriction enzymes, they are placed in an indentation in a small square made of agarose gel. Next, electrical current is passed through the gel, which causes the DNA to be moved out of the well that it was placed in and across the gel surface. The DNA, which then appears as smears of varying size on the gel, is transferred to a nylon membrane and hybridized with a radioactively labeled oligonucleotide probe. After radioactive imaging, a dark band appears where the oligonucleotide probe was able to find its compliment in the patient’s DNA. If the patient’s DNA possesses a dark band that corresponds to the presence of a probe, which is complimentary to a mutation associated with a genetic disorder, they are likely positive for that disorder (“Southern Blotting”, 1994).
Figure 11) This southern blot image indicates the results of a test for hepatitis-related genetic abnormalities to serve as an example for how to read a southern blot. The presence of the dark band under the “HB” column indicates the mutational changes brought about in a hepatitis-infected patient. Each subsequent column labeled with numbers indicates an individual patient that is undergoing testing. The presence of a dark band parallel to the HB column’s hepatitis band indicates that the patient possesses genetic abnormalities that coincide with the structure and profile of hepatitis changes brought about by hepatitis and are thus considered infected (Mason et al., 1998). *Permission Pending*
Amplification Refractory Mutation System (ARMS)
This method makes use of the workings of the polymerase chain reaction ( PCR) to achieve its means through differential amplification of a person’s alleles for a particular trait. Cystic fibrosis is a disease that is caused by inheritance of two mutant alleles, one from each parent, and could be tested with this method. During PCR, the process is partially driven by the use of short segments of DNA called primers, which attach to complimentary segments of the DNA to be amplified, and signal for polymerase enzymes to extend the DNA chain. If a specifically engineered primer does not match with the DNA that it is exposed to, then any hybridization that occurs between the primer and the DNA will not result in amplification due to the nature of Taq polymerase. Thus, in an ARMS test, one primer is specifically designed to bind to the mutant cystic fibrosis allele and another primer is specifically designed to bind to the normal cystic fibrosis allele. After the test has been performed, a person that is homozygous for the cystic fibrosis allele will have exclusive amplification of the mutant strain (indicates cystic fibrosis), a person that is homozygous for the normal allele will have amplification of only the wild strain (indicates normal status), and a person that is heterozygous (a carrier) for cystic fibrosis will have amplification of both the mutant and wild strains (Mutation Detection Systems, 2001).
One of the most certain ways of detecting a mutation in a genetic test is through direct sequencing of a suspected region of a patient’s DNA. That segment of the patient’s DNA undergoes PCR, which results in the amplification of DNA through assembly of chains of basic nucleotides by polymerase enzymes. During DNA sequencing, nucleotides that have been fluorescently labeled, called dideoxy nucleotides, are utilized in strain assembly along with the normal nucleotides. The structure of the dideoxy nucleotides is such that after one of them is inserted in a strain of DNA that is being assembled by the normal nucleotides, the assembly stops and leaves the DNA in a segmented state. The different segments are sorted by size through the use of a special machine that is designed to do so by measure of dideoxy fluorescence. The process results in a figure called an electropherogram, which uses a series of peaks that represent each individual nucleotide in the DNA strain. Scientists may then examine the actual DNA sequence for the abnormality in question (GeneTests Illustrated Glossary, 2004).
Figure 12) This is an image of an electropherogram, the end result of DNA sequencing. Each letter represents one nucleotide and corresponds to the peak below it (DNA Sequencing, 2000).
Linkage analysis is a technique that is employed when the exact disease-causing genetic abnormality cannot be pinpointed. Members of the extended family have portions of their DNA sequenced that are at or near an area that is suspected of disease-involvement to look for benign polymorphisms. Once an area within the suspected region that possesses signature polymorphisms is found, scientists attempt to use family data to see which family members have the genetic disorder and out of those, which have inherited those signature polymorphisms. Once a sequence possessing signature polymorphisms is correlated with the disorder, the sequence is termed a disease haplotype and can be used to make future predictions within a family based on inheritance of said haplotype (GeneTests Illustrated Glossary, 2004).
A DNA Microarray refers to a glass slide onto which oligonucleotides have been annealed in localized spots, each spot representing a particular sequence of oligonucleotide. One of the more remarkable recent uses of DNA microarray analysis involved testing for mutations of the BRCA1 gene, a gene that is strongly linked to occurrence of breast or ovarian cancer. The thousands of oligonucleotide variants on the surface of the microarray represent all of the mutational possibilities in the BRCA1 gene. DNA is taken from a patient that is going to undergo testing for a mutation in the BRCA1 gene and is converted into fluorescently labeled RNA. The RNA from the patient is then incubated with the microarray, during which the RNA seeks out its complimentary DNA oligonucleotide strands on the vast microarray matrix. The mutation or mutations that the patient has in their BRCA1 gene are manifested in the form of high levels of hybridization of their RNA strands with a particular mutant oligonucleotide spot. Geneticists can examine the levels of hybridization and diagnose not only if a patient is positive for a BRCA1 mutation, but also what kind of mutation is present. The results of this method can be confirmed through more specific methods such as an allele specific oligonucleotide assay (ASO) or in-Situ hybridization (ISH) (Hacia et al., 1996).
Figure 13) This is an example of a final result of a microarray analysis. The lighter colored dots indicate places where differential hybridization has taken place (Computational and Molecular Population Genetics Lab, 2003) *Permission Pending*.
Specific Mutation Scanning Techniques:
One of the most common forms of mutation scanning, SSCP begins with a disease-related region of a patient’s DNA. The DNA region in question is split into sections and each individual section is amplified separately by PCR to produce copious amounts of DNA exclusive to each region segment. The patient’s DNA, along with the DNA of a known non-mutant patient, is loaded onto a gel medium and exposed to an electric current. The current causes the DNA of each patient to uncouple from its complimentary strand and migrate as a single strand. Since a single stranded DNA molecule is extremely vulnerable to even small changes in sequence, a patient’s DNA segment that possesses a mutation will migrate differently than a normal person’s DNA in the electric current. After imaging, a geneticist can compare the banding pattern of each region of the normal person’s DNA with the DNA of a patient that is being tested for a mutation and subsequently confirm both the presence and approximate location of a patient’s mutation (Mutation Detection Systems, 2001).
Figure 14) A self-explanatory partial view of GeneTests glossary entry for SSCP (GeneTests Illustrated Glossary, 2004). *Permission Pending*
This form of mutation scanning involves gel electrophoresis of a suspected disease-related region of patient DNA in the presence of a harsh gradient of temperature and denaturant. DNA possesses different “melting points” for strand separation, depending on what nucleotides are present. As gel electrophoresis occurs, DNA is driven through this gradient whose varying harshness eventually causes it to separate at a certain point. When the DNA separates and becomes single stranded, the chemical structure of the DNA causes it to become trapped and stop moving with the electric current. After imaging, geneticists can compare the melting pattern of the DNA of a patient that is suspected of being afflicted with a genetic disorder with the DNA of a person who is not to confirm mutational status (Mutation Detection Systems, 2001).
Questions or Comments: Email Dr. Verna Case
Davidson College Biology Department
Davidson College
This web page was produced as an assignment for an undergraduate course at Davidson College.
The Biology of Genetic Testing |