The Genetic Testing

The Genetic Testing
By Dr rer. nat , Dr H.c. phil. Theo Giannaros T.U. Karlsruhe, (Kernforschungszentrum-Leopoldshafen) Department of Molecular & Nuclear Biology










Introduction

This article discusses genetic testing; that is, testing that looks at a person's genetic makeup for a variety of reasons. An increasing number of genetic tests are becoming available as a result of recent and rapid advances in biomedical research. It has been said that genetic testing may revolutionize the way many diseases are diagnosed. But genetic testing does not just help a physician diagnose disease. There are a number of different reasons genetic tests are performed. These include the following:
1. clinical genetic testing (diagnosing current or future disease),
2. pharmacogenomics (assessment of therapeutic drug treatment ),
3. identity testing for criminal investigations or forensics studies.
4. parentage testing tissue typing for transplantation,
5. cytogenetics (chromosome analysis),
6. infectious disease testing.
Clinical Genetic Testing
Clinical genetic testing refers to the laboratory analysis of DNA or RNA to aid in the diagnosis of disease. It is very important to understand that clinical genetic testing is quite different than other types of laboratory tests. Genetic testing is unique in that it can provide definitive diagnosis as well as help predict the likelihood of developing a particular disease before symptoms even appear; it can tell if a person is carrying a specific gene that could be passed on to his or her children; it can inform as to whether some treatments will work before a patient starts therapy. These are definite advantages. However, there are also some qualities of genetic testing that should be carefully thought out and perhaps discussed with a genetic counselor before undergoing any test. These aspects are reviewed in the section titled Pros and Cons of Genetic Testing. In an era of patient responsibility, it is important that you be educated in these matters to fully appreciate the value as well as the drawbacks of genetic testing.
Testing Genetic Material
Testing of genetic material is performed on a variety of specimens including blood, urine, saliva, stool, body tissues, bone, or hair. Cells in these samples are isolated and the DNA within them is extracted and examined for possible mutations or alterations. Looking at small portions of the DNA within a gene requires specialized and specific laboratory testing. This is done to pinpoint the exact location of genetic errors. This section will focus on the examination of a person’s genes to look for the one(s) responsible for a particular disease.
There are four basic reasons that genetic material is tested for clinical reasons. Presymptomatic testing identifies the presence of variant genes that cause disease even if the physical abnormalities associated with the disease are not yet present in an individual. Diagnostic genetic testing is performed on a symptomatic individual with symptoms sufficiently suggestive of a genetic disorder. This assists the individual’s physician in making a clear diagnosis.
Testing of genetic material can also be performed as a prenatal screening tool to assess whether two individuals who wish to become parents have an autosomal or X-linked recessive gene that, when combined in a child, will produce a serious disorder in that child. This type of genetic testing is referred to as carrier screening. Fetuses developing in the uterus can also have their genetic material tested to assess their health status if it is thought to be in jeopardy.
To test DNA for medical reasons, some type of cellular material is required. This material can come from blood, urine, saliva, body tissues, bone marrow, hair, etc. The material can be submitted in a tube, on a swab, in a container, or frozen. If the test requires RNA, the same materials can be used. Once received in the laboratory, the cells are removed from the substance they are in, broken apart, and the DNA in the nuclei is isolated and extracted.
The lab professionals who perform and interpret these tests are specially trained physicians and scientists. The extracted DNA is manipulated in different ways in order for the molecular pathologist or genetic technologist to see what might be missing or mutated in such a way as to cause disease. One type of manipulation is “cutting” the DNA into small pieces using special enzymes. These small pieces are much easier to test than the long strands of uncut DNA, and they contain the genes of interest. Another manipulation is to apply the extracted and cut DNA to an agarose gel, apply an electrical field to the gel, and see how the DNA moves on the gel. This can indicate differences in the size of the pieces of the cut DNA which might be caused by specific mutations.
Other manipulations to DNA include amplification, sequencing, or a special procedure called hybridization. When the results of these tests are examined and compared with results from a normal person, it is possible to see differences in the genes that might cause a disease.
Specific Genetic Diseases
There are many diseases that are now thought to be caused by alterations in DNA. These alterations can either be inherited or can occur spontaneously. Some diseases that have a genetic component to them include:
Alzheimer's Disease
Bone Marrow Disorders
Breast Cancer

Ovarian Cancer
Colon Cancer
Cystic Fibrosis

Down Syndrome
Hemochromotosis
Leukemia

Lupus
Lymphoma
Osteoarthritis

Pre-senilin Mutation Sickle Cell Anemia
Thalassemia

Several things can go wrong with the genes that make up the DNA, resulting in these and other diseases. The section below discusses what can happen to DNA, and specifically to genes, that might lead to a disease.
Genetic Variation and Mutation
All genetic variations or polymorphisms originate from the process of mutation. Genetic variations occur sometimes during the process of somatic cell division (mitosis). Other genetic variations can occur during meiosis, the cycle of division that a sperm cell or an ovum goes through. Some variations are passed along through the generations, adding more and more changes over the years. Sometimes these mutations lead to disease, other times there is no noticeable effect. Genetic variations can be classified into different categories: stable genetic variations, unstable genetic variations, silent genetic variations, and other types.
Stable genetic variations are caused by specific changes in single nucleotides. These changes are called single nucleotide polymorphisms or SNPs and can include
1. substitutions, in which one nucleotide is replaced by another,
2. deletions, in which a single nucleotide is lost, and
3. insertions, in which one or more nucleotides are inserted into a gene.
If the SNP causes a new amino acid to be made, it is called a “missense mutation.” An example of this is in sickle cell anemia, in which one nucleotide is substituted for another. The genetic variation in the gene causes a different amino acid to be added to a protein, resulting in a protein that doesn’t do its job properly and causes cells to form sickle shapes and not carry oxygen.
Unstable genetic variations occur when a nucleotide sequence repeats itself over and over. This is called a “repeat” and is usually normal; however, if the number of repeats increases too greatly, it is called an “expanded repeat” and has been found to be the cause of many genetic disorders. An example of a disease caused by an expanded repeat is Huntington disease, a severe disorder of a part of the brain that is marked by dementia, hydrocephalus, and unusual movements.
Silent genetic variations are those mutations or changes in a gene that do not change the protein product of the gene. These mutations rarely result in a disease.
Other types of variations occur when an entire gene is duplicated somewhere in a person’s genome. When this occurs, extra copies of the gene are present and makes extra protein product. This is seen in a disorder that effects peripheral nerves and is called Charcot-Marie-Tooth disease type 1. Some variations occur in a special part of the gene that controls when DNA is copied to RNA. When the timing of protein production is thrown off, it results in decreased protein production. Other variations include a defect in a gene that makes a protein that serves to repair broken DNA in our cells. This variation can result in many types of diseases, including colorectal cancer and a skin disease called xeroderma pigmentosum.
Testing for Products of Genetic Expression
Many inherited disorders are identified indirectly by examining abnormalities in the genetic end products (proteins or metabolites) that are present in abnormal forms or quantities. As a reminder, genes code for the production of thousands of proteins and, if there is an error in the code, changes can occur in the production of those proteins. So, rather than detecting the problem in the gene, some types of testing look for unusual findings related to the pertinent proteins, such as their absence.
An example of testing for genetic products includes those widely used to screen newborns for a variety of disorders. For example, newborns are tested for phenylketonuria (PKU), an inherited autosomal recessive metabolic disorder caused by a variation in a gene that makes a special enzyme that breaks down phenylalanine, an amino acid. When too much of this substance builds up in blood, it can lead to mental retardation if not treated early in life with a special, restricted diet. The test uses a blood sample from a baby’s heel to look for the presence of extra phenylalanine, rather than looking for the mutated gene itself. Other examples include blood tests for congenital hypothyroidism, diagnosed by low blood levels or absence of thyroid hormone, and congenital adrenal hyperplasia, a genetic disease that causes the hormone cortisol to be decreased in blood.

Pharmacogenomics
There have been cases regarding individuals who are given a certain therapeutic drug to treat symptoms or to keep symptoms from occurring in which the individual has a very violent reaction to the drug or feels no affect whatsoever. Many times this happens because of the genetic makeup of the individual. The study of this phenomenon is called “pharmacogenomics” or “pharmacogenetics.”
As an example, a woman had a surgery to remove a tumor and was given codeine as a pain reliever. Although she was doing well after the surgery, as soon as she began treatment with codeine she developed a full-body rash, difficulty breathing, and an irregular heartbeat. When she was taken off the codeine, her reaction disappeared. Upon further study, it was found that she lacked the enzyme in her blood that metabolized (broke it down into different components) the codeine into morphine and other substances, so she was essentially being overdosed with codeine. The lack of the enzyme was directly related to a variation in the gene that produced it. This genetic variation is a polymorphism between normal individuals and those who carry it. Sometimes these polymorphisms can cause a very serious reaction in an individual that could lead to death.
In some cases, individuals “hypermetabolize” drugs. This occurs when there is too much of an enzyme present that breaks down the helpful drug too quickly, leading to a lack of response to the drug. This can happen when too many copies of the gene are made and too much enzyme is produced. In other cases, the special receptor that the drug binds to on cells or tissues is missing, again because of a variation in the gene that makes the receptor protein. When there is no receptor to bind the drug, the drug may not have any affect on the cells or tissues that it should.
Genetic testing to determine the polymorphisms that play a role in our response to a drug is typical of basic genetic analyses. DNA is removed from cells, manipulated to find a specific area on a specific chromosome, and compared to “normal” DNA. In this way, genetic variations can be seen that may play a role in the over- or under-responsiveness to a therapeutic drug. This testing can also determine an individual’s resistance or sensitivity to the effectiveness of certain drugs used in viral therapy (HIV or hepatitis C drugs, for example).
There are many, many enzymes in our blood that act to metabolize or break down specific drugs, allowing them to be excreted in urine or by other means. At present, there are no testing programs in place that can give us an overall picture of our specific genetic variations that may cause us to be unresponsive or over-responsive to a therapeutic drug.

Identity Testing
Identity testing is sometimes referred to as “DNA testing”, a term most frequently used in relation to criminal investigations. "DNA testing" is an unfortunate misnomer as all types of genetic analysis, whether for disease or identification or for tissue typing, involves assessment of DNA or RNA.
Identity testing focuses on the identification of an individual through the analysis of either nuclear or mitochondrial DNA extracted from some material: blood, tissue, hair, bone, etc. Any material that contains cells with nuclei can be used for nuclear DNA extraction and eventual identity testing. Mitochondrial DNA, which is “extra-nuclear,” is used when a sample is severely degraded or if only hair shafts with no attached cells are available.
Increasingly, identity testing is used to identify a suspect in a criminal investigation by comparing the DNA found at a crime scene to that of the suspected individual. If suspected individual is convicted of the crime, his or her DNA polymorphisms are put into a data bank system that is accessible by law enforcement officials. This system is referred to as CODIS, or “Combined DNA Index System.” This system has helped to solve many crimes and also to clear those wrongfully accused of a crime.
Other uses of identity testing are to identify individuals whose identity cannot be distinguished by other means, as with decomposed bodies. In this type of genetic testing, specific parts of DNA are examined for polymorphisms (differences) that are unique to the individual. These parts of the DNA strand are referred to as microsatellites or minisatellites and are composed of repeated subunits of the DNA strand. Sometimes these repeated units are called short tandem repeats (STRs) or variable number of tandem repeats (VNTRs). In forensics, these unique sequences are given the name “DNA fingerprint.”
Other types of identity testing include the determination of an individual’s parent or parents, often called “parentage testing”, and identifying organ donors by using genetic testing for tissue transplantation, called "tissue typing".

Parentage Testing
The primary goal of parentage testing is to identify the biological parent of a given child. It is done to determine an individual’s parent or parents in, for example, cases of adoption or alleged paternity. This determination must be looked at very carefully and must identify the alleged parent with at least 99% certainty.
Many different types of laboratory tests can be done to assess parentage, including examination of red blood cell antigens (blood typing), examination of polymorphic serum protein genes, and assessment of short tandem repeats (see above). The DNA testing techniques used are similar to those used in identity testing for a criminal investigation, that is, extracting DNA from cells and manipulating it in such a way as to be able to examine the individual uniqueness of it.
If, after testing multiple systems, the parent in dispute is not excluded as a possible parent, a mathematical estimate of the possibility that the tested person could be the biological parent must be calculated. This mathematical testing combines the results of the genetic tests with other “non-genetic events” (location of the alleged parent at the time of conception, phenotype of the parent and child, etc.) and results in a “parentage index.” This index is a percentage of the likelihood of parentage. Results of these tests are admissible as evidence in court.

Tissue Typing for Transplantation
In the past, it was difficult to tell exactly whether an organ or tissue, such as a kidney, lung or bone marrow, was an exact match for the transplant between a donor and recipient. If it was not, a serious rejection reaction could sometimes occur between the recipient patient and the transplanted organ.
Basic laboratory testing for tissue transplantation involves mixing the white blood cells (leukocytes) from the donor (or the donor tissue) and the recipient together and observing whether an immune response occurs. Proliferation of a specific population of leukocytes signals the onset of an immune response and the likely rejection of the tissue by the recipient’s body. Although this technique is still commonly used, analysis of the DNA in both the donor and the recipient (tissue typing) is used to diminish the likelihood of rejection in the case of tissue transplantation. In bone marrow transplants, DNA testing is done to determine whether the leukocytes and their precursors repopulating a recipient’s bone marrow are his own or those of the donor.
A very specific set of genes is examined when DNA testing is used for tissue typing. On chromosome 6, a large set of genes called the “Major Histocompability Complex,” or MHC, resides. These genes are very polymorphic (different) between individuals, and they code for the production of specific glycoprotein antigens located on the surface of many cells. It is these antigens that “recognize” our own organs and tissues from those of another individual. These antigens have the ability to begin an immune system response that results in organ or tissue rejection if the tissue looks foreign.
A distinct region within the MHC on chromosome 6 is used in the DNA analysis of tissue that could be used for transplantation. This region called the human leukocyte antigen, or HLA-D, region, and sets of genes located there are further subdivided into HLA-DR, HLA-DQ, and HLA-DP depending on the type of glycoprotein antigen they code for. Polymorphisms in these genes are carefully compared between donor and recipient to determine the appropriateness of the transplant.
The exact techniques used to test DNA for tissue typing are similar to those mentioned in the sections above. DNA is extracted from donor and recipient cells, then manipulated and fragmented in such a way as to isolate a specific region on a chromosome and within a gene. The fragments are subjected to further analysis that allows for comparison of the polymorphisms in the HLA-DP between the donor’s tissue and the recipient’s blood. This careful analysis of genetic material results in fewer rejection reactions and the chance for a successful transplant.

Cytogenetics (Chromosome Analysis)
Everyone has 23 pairs of chromosomes, 22 pairs of autosomes and one pair of sex chromosomes. The science that relates to the study of these chromosomes is referred to as “cytogenetics.” Persons who look at chromosome preparations on slides are “cytogenetic technologists” or “cytogeneticists.” A trained cytogeneticist examines the number, shape and staining pattern of these structures using special technologies. In this way, extra chromosomes, missing chromosomes, or rearranged chromosomes can be detected.
Studies of chromosomes begin with the extraction of whole chromosomes from the nuclei of cells. These chromosomes are then placed on glass slides, stained with special stains, and examined under a microscope. Sometimes, pictures are taken of the chromosomes on the slides, and the picture is cut into pieces so the chromosome pairs can be matched. Each chromosome pair is assigned special number (from 1 to 22, then X and Y) that is based on their staining pattern and size.
There are many disorders that can be diagnosed by examining a person’s whole chromosomes. Down syndrome, in which an individual has an extra chromosome 21, can be determined by cytogenetic studies. When there are three chromosomes in one group instead of a pair, it is referred to as a “trisomy.” Missing chromosomes can also be detected, as in the case of Turner’s syndrome, in which a female has only a single X chromosome. When there is only one chromosome instead of a pair, it is referred to as a “monosomy.”
Abnormalities in chromosome structure are also observed with cytogenetic staining techniques. The Fragile X syndrome, the most common inherited cause of mental retardation, takes its name from the appearance of the stained X chromosome under a microscope. There is a site near the end of this chromosome that does not stain, indicating its fragility. The gene in the fragile region is important in making a special protein needed by developing brain cells.
Sometimes, pieces of a chromosome will break off and attach to another chromosome somewhere in a person’s genome. When this happens, it is referred to as a “translocation.” An example of a disease caused by a translocation would be chronic myelogenous leukemia (CML) in which a part of chromosome 9 breaks off and attaches itself to chromosome 22. Another example would be Burkitt’s lymphoma, in which a piece of chromosome 8 attaches to chromosome 14. These chromosomal translocations cause disease because the broken piece usually attaches to the new chromosome near a special gene that then becomes activated and produces tumor cells. Translocations can sometimes be seen under the microscope if a special stain is used.
A special technique called “fluorescent in situ hybridization” or FISH can be used to view changes in chromosomes that result from genetic variations. A mutated gene segment in a chromosome can be made to “light up” or fluoresce when it is bound by a special probe. Genetic changes in some cancers can be detected using this method. For instance, FISH is one of the methods used to determine increased expression of the gene HER2/neu. There are many other applications of FISH technology as well, such as chromosome microdeletions, in which a certain part of a chromosome is completely missing. In this case, the chromosome segment will not fluoresce compared to a normal set of chromosomes.

Infectious Disease Testing
When we hear the term “infectious disease”, we usually think of something that can infect us and cause a disease process to begin. That “something” can be a bacteria, virus, parasite, or fungus obtained from many different sources (other infected individuals, poor hygiene, transfusion with infected blood, shared needles between drug users, etc.). Disease-causing bacteria and viruses are known as infectious agents, and some of them can be quickly identified by using genetic testing techniques; however, common infectious agents, such as certain bacteria and viruses, are much less expensive to identify using standard laboratory methods that don’t involve genetic testing techniques.
Bacteria are one-celled organisms that contain their own DNA and in some cases can cause serious disease. Even those bacteria that harmlessly live inside our bodies and are involved in beneficial chemical processes can become mutated under unusual conditions and cause us to be very sick. By isolating the DNA from bacteria, breaking it into small pieces and amplifying them, the bacteria can be identified very quickly. Some of the bacteria that can be quickly identified using these genetic testing techniques include: Chlamydia trachomitis, which is an organism that causes a sexually-transmitted disease; Neisseria gonorrhea, which causes gonorrhea, Borrelia burgdorferi which causes Lyme Disease, Legionella pneumophilia which causes Legionnaire’s disease, Mycoplasma pneumoniae which leads to “walking pneumonia,” Mycopbacterium tuberculosis which can cause tuberculosis, and Bordetella pertussis which causes whooping cough. Specimens that might contain these bacteria include urine, blood, sputum, cerebrospinal fluid, and others.
Viruses are unusual organisms that sometimes insert their DNA into a host’s genome. The viral RNA or DNA utilizes the host’s cells to produce proteins and make more viruses. Viruses such as hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are examples of RNA viruses.
Other disease-causing viruses that contain DNA instead of RNA include Herpes simplex virus, cytomegalovirus, Epstein-Barr virus, parvovirus, and varicella-zoster viruses. All of these viruses can be identified by first removing the suspected viral DNA or RNA from a patient specimen and then using it to provide a “fingerprint” of the suspected virus. Specimens usually include blood, cerebrospinal fluid, sputum, other body fluids, amniotic fluid, tissue, or bone marrow. Much of the testing on donor blood that will be used in a blood transfusion utilizes genetic testing to inspect the blood for viral contamination.
Determining how many copies of a virus’ RNA are present in an individual’s blood is another use of infectious disease genetic testing techniques. The number of copies present is typically referred to as the “viral load” or “viral burden”. This testing is usually done after a drug therapy is initiated to assess whether it is working or not to remove or decrease the viral RNA load. The most common viral load tests are for HCV or HIV, and the tests require a sample of blood.
A parasite is a complex multi-cellular organism. Parasites usually infect an individual through the saliva of a biting insect, such as a mosquito, or through infected material. An example of a parasite that can be identified using genetic tests is Toxoplasma gondii which can cause encephalitis or congenital infections that lead to severe damage of a fetus (fetal toxoplasmosis).