Chapter 6
Genetic Testing

"Diagnosis" means finding the cause of a disorder, not just giving it a name.

—Sydney Walker III, A Dose of Sanity: Mind, Medicine, and Misdiagnosis (1996)

Over the course of the last decade the definitions of health and disease have been transformed by advances in genetics. Genetic testing has enabled researchers and clinicians to detect inherited traits, diagnose heritable conditions, determine and quantify the likelihood that a heritable disease will develop, and identify genetic susceptibility to familial disorders. Many of the strides made in genetic diagnostics are direct results of the Human Genome Project, an international thirteen-year effort begun in 1990 by the U.S. Department of Energy and the National Institutes of Health, which mapped and sequenced the human genome in its entirety. The increasing availability of genetic testing has been one of the most immediate applications of this groundbreaking research.

A genetic test is the analysis of human deoxyribonucleic acid (DNA), ribonucleic acid (RNA), chromosomes, and proteins to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes (standard pictures of the chromosomes in a cell) for the purposes of diagnosis, treatment, and other clinical decision making. Most genetic testing is performed by drawing a blood sample and extracting DNA from white blood cells. Genetic tests may detect mutations at the chromosomal level, such as additional, absent, or rearranged chromosomal material, or even subtler abnormalities such as a substitution in one of the bases that make up the DNA. There is a broad range of techniques that can be used for genetic testing. Genetic tests have diverse purposes, including screening for and diagnosis of genetic disease in newborns, children, and adults; the identification of future health risks; the prediction of drug responses; and the assessment of risks to future children.

There is a difference between genetic tests performed to screen for disease and testing conducted to establish a diagnosis. Diagnostic tests are intended to definitively determine whether a patient has a particular problem. They are generally complex tests and commonly require sophisticated analysis and interpretation. They may be expensive and are generally performed only on people believed to be at risk, such as patients who already have symptoms of a specific disease.

In contrast, screening is performed on healthy, asymptomatic (showing no symptoms of disease) people and often to the entire relevant population. A good screening test is relatively inexpensive, easy to use and interpret, and helps identify which individuals in the population are at higher risk of developing a specific disease. By definition, screening tests identify people who need further testing or those who should take special preventive measures or precautions. For example, people who are found to be especially susceptible to genetic conditions with specific environmental triggers are advised to avoid the environmental factors linked to developing the disease. Examples of genetic tests used to screen relevant populations include those that screen people of Ashkenazi Jewish heritage (the East European Jewish population primarily from Germany, Poland, and Russia, as opposed to the Sephardic Jewish population primarily from Spain, parts of France, Italy, and North Africa) for Tay-Sachs disease, African-Americans for sickle-cell disease, and the fetuses of expectant mothers over age thirty-five for Down syndrome.

QUALITY AND UTILITY OF GENETIC TESTS

Like all diagnostic and screening tests, the quality and utility of genetic tests depend on their reliability, validity, sensitivity, specificity, positive predictive value, and negative predictive value. Reliability of testing refers to the test's ability to be repeated and to produce equivalent results in comparable circumstances. A reliable test is consistent and measures the same way each time it is used with the same patients in the same circumstances. For example, a well-calibrated balance scale is a reliable instrument for measuring body weight.

Validity is the accuracy of the test. It is the degree to which the test correctly identifies the presence of disease, blood level, or other quality or characteristic it is intended to detect. For example, if you put an object you knew weighed ten pounds on a scale and the scale said it weighed ten pounds, then the scale's results are valid. There are two components of validity: sensitivity and specificity.

Sensitivity is the test's ability to identify people who have the disease. Mathematically speaking, it is the percentage of people with the disease who test positive for the disease. Specificity is the test's ability to identify people who do not have the disease—it is the percentage of people without the disease who test negative for the disease. Ideally, diagnostic and screening tests should be highly sensitive and highly specific, thereby accurately classifying all people tested as either positive or negative. In practice, however, sensitivity and specificity are frequently inversely related—most tests with high levels of sensitivity have low specificity, and the reverse is also true.

The likelihood that a test result will be incorrect can be gauged based on the sensitivity and specificity of the test. For example, if a test's sensitivity is 95%, then when one hundred patients with the disease are tested, ninety-five will test positive and five will test false negative—they have the disease but the test has failed to detect it. For example, disorders such as Charcot-Marie-Tooth disease (a group of inherited, slowly progressive disorders that result from progressive damage to nerves; its symptoms include numbness and wasting of muscle tissue in the feet and legs, then in the hands and arms) can arise from mutations in one of many different genes, and because some of these genes have not yet been identified, they will not be detected and a false negative result might be reported. By contrast, if a test is 90% specific, when one hundred healthy, disease-free people are tested, ninety will receive negative test results and ten will be given false-positive results, meaning that they do not have the disease but the test has inaccurately classified them as positive.

The positive predictive value is the percentage of people that actually have the disease of all those with positive test results. The negative predictive value measures the percent of all the people with negative test results who do not have the disease.

PREGNANCY, CHILDBIRTH, AND GENETIC TESTING

There are thousands of genetic diseases, such as sickle-cell anemia, cystic fibrosis, and Tay-Sachs disease, that may be passed from one generation to the next. Many tests have been developed to help screen parents at risk of passing on genetic disease to their children as well as to identify embryos, fetuses, and newborns who suffer from genetic diseases.

Carrier Identification

Carrier identification is the term for genetic testing to determine whether a healthy individual has a gene that may cause disease if passed on to his or her offspring. It is usually performed on people considered to be at higher than average risk, such as those of Ashkenazi Jewish descent, who have a 1 in 27 chance of being Tay-Sachs carriers (in other populations the risk is 1 out of 250), according to the National Tay-Sachs and Allied Diseases Association (2007, http://www.tay-sachs.org/taysachs.php). Testing is necessary because many carriers have just one copy of a gene for an autosomal recessive trait and are unaffected by the trait or disorder. Only someone with two copies of the gene will actually have the disorder. So while it is widely assumed that everyone is an unaffected carrier of at least one autosomal recessive gene, it only presents a problem in terms of inheritance when two parents have the same recessive disorder gene (or both are carriers). In this instance the offspring would each have a one in four chance of receiving a defective copy of the gene from each parent and developing the disorder. Figure 6.1 shows the pattern of inheritance of an autosomal recessive disorder.

Carrier testing is offered to individuals who have family members with a genetic condition, people with family members who are identified carriers, and members of racial and ethnic groups known to be at high risk. Figure 6.2 shows how carrier testing would be used for a family affected by cystic fibrosis and among African-Americans who may carry the gene for sickle-cell anemia.

Preimplantation Genetic Diagnosis

Preimplantation diagnosis is a newer genetic test that enables parents undergoing in vitro fertilization (fertilization that takes place outside the body) to screen an embryo for specific genetic mutations when it is no larger than six or eight cells and before it is implanted in the uterus to grow and develop. Figure 6.3 shows how preimplantation genetic diagnosis is performed.

Prenatal Testing

Prenatal genetic testing enables physicians to diagnose diseases in the fetus. Most genetic tests examine blood or other tissue to detect abnormalities. An example of a blood test is the triple marker screen. This test measures levels of alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated estriol and can identify some birth defects such as Down syndrome and neural tube defects. (Two of the most common neural tube defects are anencephaly—absence of most of the brain—and spina bifida—incomplete development of the back and spine.)

The fetal yolk sac and the fetal liver make AFP, which is continuously processed by the fetus and excreted into the amniotic fluid. A small amount crosses the placenta and can be found in maternal blood. Maternal screening for AFP levels is based on maternal age, fetal gestation, and the number of fetuses the mother is carrying. Elevated levels of AFP are associated with conditions such as spina bifida and low levels are found with Down syndrome. Because AFP levels alone may not always adequately detect disorders, two other blood serum tests have been developed. hCG is a glycoprotein produced by the placenta. Normally, hCG is elevated at the time of implantation, but decreases at about eight weeks of gestation, and then drops again at approximately twelve weeks of gestation. Elevated levels of hCG are found with Down syndrome. The placenta also produces unconjugated estriol. As with AFP, lower unconjugated estriol maternal serum levels are also found with Down syndrome. Triple marker screen results are usually available within several days and women with abnormal results are often advised to undergo additional diagnostic testing such as chorionic villus sampling (CVS), amniocentesis, or percutaneous umbilical blood sampling (withdrawing blood from the umbilical cord).

CVS enables obstetricians and perinatologists (physicians specializing in evaluation and care of high-risk expectant mothers and infants) to assess the progress of pregnancy during the first trimester (the first three months). A physician passes a small, flexible tube called a catheter through the cervix to extract chorionic villi tissue—cells that will become the placenta and are genetically identical to the baby's cells. The chorion develops from trophoblasts, or the same cells as the fetus, and contains the same DNA and chromosomes. The cells obtained via CVS are examined in the laboratory for indications of genetic disorders such as cystic fibrosis, Down syndrome, Tay-Sachs, and thalassemia, and the results of testing are available within seven to fourteen days. Table 6.1 describes CVS and other prenatal diagnostic tests and specifies when during pregnancy they are performed.

Amniocentesis involves taking a sample of the fluid that surrounds the fetus in the uterus for chromosome analysis. An amniocentesis is usually performed at fifteen to twenty weeks of gestation, although it can be done as early as twelve weeks. (See the description of early amniocentesis in Table 6.1.) About twenty milliliters of amniotic fluid is obtained when the physician inserts a hollow needle through the abdominal wall and the wall of the uterus. Fetal karyotyping, DNA analysis, and biochemical testing may be performed on the isolated fetal cells. Like CVS, amniocentesis samples and analyzes cells derived from the baby to enable parents to learn of chromosomal abnormalities, as well as the gender of the unborn child, about two weeks after the test is performed.

Using samples of genetic material obtained from amniocentesis or CVS, physicians can detect disease in an unborn child. Down syndrome (also known as trisomy 21, because it is caused by an extra copy of chromosome 21) is the genetic disease most often identified using this technique. Down syndrome is rarely inherited; most cases result from an error in the formation of the ovum (egg) or sperm, leading to the inclusion of an extra chromosome 21 at conception. As with prenatal diagnosis for most inherited genetic diseases, this use of genetic testing is focused on reproductive decision making.

The most invasive prenatal procedure for genetic testing is periumbilical blood sampling. Table 6.1 describes the technique. Periumbilical blood sampling poses the greatest risk to the unborn child—one in fifty miscarriages occurs as a result of this procedure. It is used when a diagnosis must be made quickly. For example, when an expectant mother is exposed to an infectious agent with the potential to produce birth defects, it may be used to examine fetal blood for the presence of infection.

Until 2006 it was thought that women undergoing CVS were more likely to miscarry than those who had amniocentesis; however, Aaron B. Caugey, Linda M. Hopkins, and Mary E. Norton, in "Chorionic Villus Sampling Compared with Amniocentesis and the Difference in the Rate of Pregnancy Loss" (Obstetrics and Gynecology, September 2006), refute this notion. Caugey, Hopkins, and Norton analyzed the outcomes of nearly 10,000 CVS and 32,000 amniocentesis procedures and found that CVS was no more likely than amniocentesis to lead to pregnancy loss. They attribute previously reported higher rates of miscarriage resulting from CVS to the fact that clinicians were not yet experienced at performing the newer procedure.

Genetic Testing of Newborns

The most common form of genetic testing is the screening of blood taken from newborn infants for genetic abnormalities. The Maternal and Child Health Bureau (September 2004, http://mchb.hrsa.gov/programs/genetics/presentations/comments/meeting2comments.htm) reports that in the United States more than four million newborns are screened every year for specific genetic disorders, such as phenylketonuria (PKU), and other medical conditions that are only indirectly genetically linked, such as congenital hypothyroidism (underactive thyroid gland). PKU is an inherited error of metabolism resulting from a deficiency of an enzyme called phenylalanine hydroxylase. The lack of this enzyme can produce mental retardation, organ damage, and postural problems. Children born with PKU must pay close attention to their diets to lead healthy, normal lives.

Laboratory Techniques for Genetic Prenatal Testing

Genetic testing is performed on chromosomes, genes, or gene products to determine whether a mutation is causing or may cause a specific condition. Direct testing examines the DNA or RNA that makes up a gene. Linkage testing looks for disease-causing gene markers in family members from at least two generations. Biochemical testing assays certain enzymes or proteins, which are the products of genes. Cytogenetic testing examines the chromosomes.

Generally, a blood sample or buccal smear (cells from the mouth) is used for genetic tests. Other tissues used include skin cells from a biopsy, fetal cells, or stored tissue samples. (Table 6.1 lists the tissue samples used in each procedure.) Testing requires highly trained, certified technicians and laboratories because the procedures are complex and varied, the technology is new and evolving, and hereditary conditions are often rare, so many testing techniques require special expertise. In the United States laboratories performing clinical genetic tests must be approved under the Clinical Laboratory Improvement Amendments (CLIA), passed by Congress in 1988 to establish the standards with which all laboratories that test human specimens must comply to receive certification. CLIA standards determine the qualifications of laboratory personnel, categorize the complexity of various tests, and oversee quality improvement and assurance. In 2005 about 600 laboratories in the United States were performing genetic testing to detect and diagnose more than 1,200 conditions. Figure 6.4 shows the tremendous growth of both laboratories and diseases for which testing is available from 1993 to 2005.

The polymerase chain reaction (PCR) technique permits rapid cloning and DNA analysis and allows selective amplification of specific DNA sequences. A polymerase chain reaction can be performed in hours and is a sensitive test that may be used to screen for altered genes, but it is limited by the size and length of the DNA sequences that can be cloned. Figure 6.5 shows how PCR is performed.

Fluorescence in situ hybridization (FISH), in which a fluorescent label is attached to a DNA probe that will bind to the complementary DNA strands, provides a unique opportunity to view specific genetic codes. The FISH technique may be used on cells and fluid obtained by CVS and amniocentesis as well as on maternal blood. With this technique, a single strand of DNA is used to create a probe that attaches at the specific gene location. To separate the double-stranded DNA, heat or chemicals are used to break the chemical bonds of the DNA and obtain a single strand. (See Figure 6.6.)

There are three kinds of chromosome-specific probes: repetitive probes, painting probes, and locus-specific probes. Repetitive probes produce intense signals by creating tandem repeats of base pairs. Painting probes are collections of specific DNA sequences that may extend along either part or all of an individual chromosome. These probe labels are most useful for identifying complex rearrangements of genetic material in structurally abnormal chromosomes. Probes that can hybridize to a single gene locus are called locus-specific probes. They can be used to identify a gene in a particular region of a chromosome. Locus-specific probes are used to identify a deletion or duplication of genetic material. The FISH procedure allows a signal to be visualized that indicates the presence or absence of DNA. The entire process can be completed in fewer than eight hours using five to seven probes. For this reason, FISH is frequently used as a rapid screen for trisomies and genetic disorders.

Although FISH is the most commonly used and most readily available prenatal diagnostic cytogenetic technique, it has limited ability to detect translocations, deletions, and inversions. Microdissection FISH (also used for prenatal diagnosis) is another method that is more sensitive to these alterations. Microdissection FISH constructs probes to define specific regions of the human chromosome. Figure 6.7 shows how the microdissection technique is used to identify structurally abnormal chromosomes.

Newer technologies allow for the identification of all human chromosomes, thereby expanding the FISH application to the entire genome. Multiplex FISH applies combinations of probes to color components of fluorescent dye during metaphase to visualize each chromosome. This type of FISH procedure evolved from the whole-chromosome painting probes and uses a combination of fluorochromes to identify each chromosome. Just five fluorophores are needed to decode the entire complement of human chromosomes. Multicolor spectral karyotyping uses computer imaging and Fourier spectroscopy to increase the analysis of genetic markers. (See Figure 6.8.) Although FISH techniques are considered highly reliable, there are limitations to multiplex and multicolor FISH, in that inversion or subtle deletions may be overlooked. The greatest advant-age of multiplex FISH is its application in cases of dysmorphic infants. Spectral karyotyping techniques can detect cryptic unbalanced translocations that cannot be identified by other techniques.

GENETIC TESTING FOR SICKLE-CELL ANEMIA

Sickle-cell anemia is an autosomal recessive disease that results when hemoglobin S is inherited from both parents. (When hemoglobin S is inherited from only one parent, the individual is a sickle-cell carrier.) Because normal and sickle hemoglobins differ at only one amino acid in the hemoglobin gene, a test called hemoglobin electrophoresis is used to establish the diagnosis.

Genetic testing for sickle-cell anemia involves restriction fragment length polymorphism (DNA sequence variant) that uses specific enzymes to cut the DNA. (See Figure 6.9.) These enzymes cut the DNA at a specific base sequence on the normal gene but not on a gene in which a mutation is present. As a result of this technique, there are longer fragments of sickle hemoglobin. Another technique known as gel electrophoresis sorts the DNA fragments by size. Autoradiography renders the DNA fragments by generating an image after radioactive probes have labeled the DNA fragments that contain the specific gene sequence. The location of the fragments distinguishes carrier status (heterozygous) from sickle-cell anemia (homozygous), or normal blood.

New Techniques Detect Fetal Gene Mutations

In 2005 Ying Li et al. announced success with a technique that identifies fetal gene mutations such as beta-thalassemia from samples of maternal blood and reported their discovery in "Detection of Paternally Inherited Fetal Point Mutations for Beta-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma" (Journal of the American Medical Association, February 16, 2005). The technique relies on the fact that circulatory fetal DNA sequences comprise fewer than 300 base pairs, whereas maternal DNA exceeds 500 base pairs. Li and the other researchers used PCR amplification to select for paternally inherited DNA sequences. Presence of the paternal mutant alleles for beta-thalassemia was then detected by allele-specific PCR.

They tested the new technique on maternal blood samples from expectant mothers whose fetuses were at risk for beta-thalassemia because the father was a carrier for one of four beta-globin gene mutations. The results were verified by comparing the findings from chorionic villus sampling. Li et al. also reported the cost-effectiveness of the new technique. Because it does not require complex machinery and relies on currently available technology, they estimate the cost of a single analysis might be just $8.

Another new technique reported in 2005 was analysis of amniotic fluid using oligonucleotide hybridization and microarray data analysis. (Figure 6.10 shows how microarray technology is performed.) In "Global Gene Expression Analysis of the Living Human Fetus Using Cell-Free Messenger RNA in Amniotic Fluid" (Journal of the American Medical Association, February 16, 2005), Paige B. Larrabee et al. note that they found that gene expression patterns correlated with the gender of the fetus, gestational age, and disease status. Larrabee and her collaborators assert that this technology could assist in advancing human developmental research and in identifying new biomarkers for prenatal assessment.

GENETIC DIAGNOSIS IN CHILDREN AND ADULTS

Genetic testing can also be performed postnatally (after birth) to determine which children and adults are at increased risk of developing specific diseases. By 2006 scientists could perform predictive genetic testing to identify which individuals were at risk for cystic fibrosis, Tay-Sachs disease, Huntington's disease, amyotrophic lateral sclerosis (a degenerative neurological condition commonly known as Lou Gehrig's disease), and several types of cancers, including some cases of breast, colon, and ovarian cancer.

More than 1,200 genetic tests were available in 2006, but public health professionals did not consider it practical to screen for conditions that are rare, have only minor health consequences, or those for which there is still no effective treatment. The most frequently performed genetic tests were those considered most useful in terms of their potential to screen populations for diseases that occur relatively frequently, have serious medical consequences (including death) if untreated, and for which effective treatment is available.

A positive test result (the presence of mutation—a defective or altered gene) from predictive genetic testing does not guarantee that the individual will develop the disease; it simply identifies the individual as genetically susceptible and at an increased risk for developing the disease. For example, a woman who tests positive for the BRCA1 gene has about an 80% chance of developing breast cancer before age sixty. It is also important to note that, like other types of diagnostic medical testing, genetic tests are not 100% predictive—the results rely on the quality of laboratory procedures and accuracy of interpretations. Furthermore, because tests vary in their sensitivity and specificity, there is always the possibility of false-positive and false-negative test results.

Researchers hope that positive test results will encourage people who are at higher than average risk of developing a disease to be especially vigilant about disease prevention and screening for early detection, when many diseases are most successfully treated. There is an expectation that genetic information will increasingly be used in routine population screening to determine individual susceptibility to common disorders such as heart disease, diabetes, and cancer. This type of screening will identify groups at risk so that primary prevention efforts such as diet and exercise or secondary prevention efforts such as early detection can be initiated.

Diagnostic Genetic Testing

Most genetic testing is performed on people who are asymptomatic (people who are apparently healthy). The objective of screening is to determine if people are carriers of a genetic disease or to identify their susceptibility or risk of developing a specific disease or disorder. There is, however, some testing performed on people with symptoms of a disease to clarify or establish the diagnosis and calculate the risk of developing the disease for other family members. This type of testing is known as diagnostic genetic testing or symptomatic genetic testing. It may also assist in directing treatment for symptomatic patients in whom a mutation in a single gene (or in a gene pair) accounts for a disorder. Cystic fibrosis and myotonic dystrophy are examples of disorders that may be confirmed or ruled out by diagnostic genetic testing and other methods (such as the sweat test for cystic fibrosis or a neurological evaluation for myotonic dystrophy).

One issue involved in diagnostic genetic testing is the appropriate frequency of testing in view of rapidly expanding genetic knowledge and identification of genes linked to disease. Physicians frequently see symptomatic patients for whom there is neither a definitive diagnosis nor a genetic test. The as-yet-unanswered question is: Should such people be recalled for genetic testing each time a new test becomes available? Although clinics and physicians who perform genetic testing counsel patients to maintain regular contact so they may learn about the availability of new tests, there is no uniform guideline or recommendation about the frequency of testing.

New Genetic Tests Help Patients Choose Optimal Treatment

In August 2005 the Food and Drug Administration (FDA) approved marketing of a new genetic test that will help physicians make personalized drug treatment decisions for some patients ("FDA Clears Genetic Test That Advances Personalized Medicine: Test Helps Determine Safety of Drug Therapy," August 22, 2005, http://www.fda.gov/bbs/topics/NEWS/2005/NEW01220.html). The Invader UGT1A1 Molecular Assay detects variations in a gene that affects how certain drugs are broken down and cleared by the body. Using this information, physicians can determine the optimal drug dosage for each patient and minimize the harsh and potentially life-threatening side effects of drug treatment. The new test joins a growing list of genetic tests used to customize treatment decisions, including the Roche AmpliChip, used to personalize dosage of antidepressants, antipsychotics, beta-blockers, and some chemotherapy drugs, and TRUGENE HIV-1 Genotyping Kit, used to detect variations in the genome of the human immunodeficiency virus (which can cause acquired immunodeficiency syndrome) that make the virus resistant to some antiretroviral drugs.

In 2006 Anil Potti et al. reported in "A Genomic Strategy to Refine Prognosis in Early-Stage Non-Small-Cell Lung Cancer" (New England Journal of Medicine, August 10, 2006) about a new genetic test that predicts with up to 90% accuracy which early-stage lung cancer patients are likely to experience recurrence and therefore would benefit from chemotherapy (drug treatment for cancer). Currently, patients diagnosed with early-stage non-small-cell lung cancer undergo surgery followed by observation, without chemotherapy. About one-third of such patients experience a relapse. Potti and his coauthors describe the test as "a fingerprint unique to the individual patient [that] predicts survival changes."

The National Cancer Institute announced in 2006 another new genetic test that was in clinical trials and that promised to distinguish which breast cancer patients can safely skip chemotherapy (May 23, 2006, http://www.cancer.gov/clinicaltrials/digestpage/TAILORx). The Onco-type DX test examines the surgically removed tumor for twenty-one different genes whose interactions can predict the likelihood of a relapse and calculates the odds of a relapse on a scale from 0 to 100. A score greater than 30 indicates the benefits of chemotherapy, whereas a score lower than 18 suggests forgoing chemotherapy.

Population Screening

Population screening for heritable diseases is one potentially lifesaving application of molecular genetics technology. Prenatal screening has demonstrated benefits and gained widespread use; however, genetic screening has not yet become part of routine medical practice for adults. Geneticists have identified at least seven genes that might be candidates for use as population screening tests in adults in the United States. The genes include HFE, for hereditary hemochromatosis; apolipoprotein E-4, linked to Alzheimer's disease; CYP2D6, linked to ankylosing spondylitis (arthritis of the spine); BRCA1 and BRCA2, genes for hereditary breast and ovarian cancer; familial adenomatous polyposis, associated with precancerous growths in the colon; and factor V Leiden, the most common hereditary blood clotting disorder in the United States. As of 2007, screening for variants in these genes had not entered into routine medical practice because there was considerable controversy about the predictive value of testing for these genes and how to monitor and care for people who test positive for them.

Genetic Susceptibility

Susceptibility testing, also known as predictive testing, determines the likelihood that a healthy person with a family history of a disorder will develop the disease. Testing positive for a specific genetic mutation indicates an increased susceptibility to the disorder but does not establish a diagnosis. For example, a woman may choose to undergo testing to find out whether she has genetic mutations that would indicate likelihood of developing hereditary cancer of the breast or ovary. If she tests positive for the genetic mutation, she may then decide to undergo some form of preventive treatment. Preventive measures may include increased surveillance such as more frequent mammography, chemoprevention—prescription drug therapy intended to reduce risk—or surgical prophylaxis, such as mastectomy and/or oophorectomy (surgical removal of the breasts and ovaries, respectively).

Testing Children for Adult-Onset Disorders

In 2000 the American Academy of Pediatrics Committee on Genetics recommended genetic testing for people under age eighteen only when testing would offer immediate medical benefits or when there is a benefit to another family member and there is no anticipated harm to the person being tested. The committee considered genetic counseling before and after testing as essential components of the process.

The American Academy of Pediatrics Committee on Bioethics and Newborn Screening Task Force recommended the inclusion of tests in the newborn-screening battery based on scientific evidence. The academy advocated informed consent for newborn screening. (To date, most states do not require informed consent.) The Committee on Bioethics did not endorse carrier screening in people under eighteen years of age, except in the case of a pregnant teenager. It also recommended against predictive testing for adult-onset disorders in people under eighteen.

The American College of Medical Genetics, the American Society of Human Genetics (ASHG), and the World Health Organization have also weighed in about genetic testing of asymptomatic children, asserting that decision making should emphasize the child's well-being. One issue involves the value of testing asymptomatic children for genetic mutations associated with adult-onset conditions such as Huntington's disease. Because no treatment can begin until the onset of the disease, and at present there is no treatment to alter the course of the disease, it may be ill advised to test for it. Another concern is testing for carrier status of autosomal recessive or X-linked conditions such as cystic fibrosis or Duchenne muscular dystrophy. Experts caution that children might confuse carrier status with actually having the condition, which in turn might provoke needless anxiety.

There are, however, circumstances in which genetic testing of children may be appropriate and useful. Examples are children with symptoms of suspected hereditary disorders or those at risk for cancers in which inheritance plays a primary role.

ETHICAL CONSIDERATIONS—CHOICES AND CHALLENGES

Rapid advances in genetic research since the 1980s have challenged scientists, health care professionals, ethicists, government regulators, legislators, and consumers to stay abreast of new developments. Understanding the scientific advances and their implications is critical for everyone involved in making informed decisions about the ways in which genetic research and information will affect the lives of current and future generations. American citizens, scientists, ethicists, legislators, and regulators share this responsibility. According to the Human Genome Project Information Web site (February 5, 2003, http://genome.rtc.riken.go.jp/hgmis/elsi/elsi.html), the pivotal importance of these societal decisions was underscored by the allocation of 3% to 5% of the budget of the Human Genome Project for the study of ethical, legal, and social issues related to genetic research. To date, consideration of these issues has not produced simple or universally applicable answers to the many questions posed by the increasing availability of genetic information. Ongoing public discussion and debate is intended to inform, educate, and help people in every walk of life make personal decisions about their health and participate in decisions that concern others.

As researchers learn more about the genes responsible for a variety of illnesses, they can design more tests with increased accuracy and reliability to predict whether an individual is at risk of developing specific diseases. The ethical issues involved in genetic testing have turned out to be far more complicated than originally anticipated. Initially, physicians and researchers believed that a test to determine in advance who would develop or escape a disease would be welcomed by at-risk families, who would be able to plan more realistically about having children, choosing jobs, obtaining insurance, and going about their daily lives. Nevertheless, many people with family histories of a genetic disease have decided that not knowing is better than anticipating a grim future and an agonizing, slow death. They prefer to live with the hope that they will not develop the disease rather than having the certain knowledge that they will.

The discovery of genetic links and the development of tests to predict the likelihood or certainty of developing a disease raise ethical questions for people who carry a defective gene. Should women who are carriers of Huntington's disease or cystic fibrosis have children? Should a fetus with a defective gene be carried to term or aborted?

There are also concerns about privacy and the confidentiality of medical records and the results of genetic testing and possible stigmatization. Some people are reluctant to be tested because they fear they may lose their health, life, and disability insurances, or even their jobs if they are found to be at risk for a disease. Genetic tests are sometimes costly, and some insurers agree to reimburse for testing only if they are informed of the results. The insurance companies feel they cannot risk selling policies to people they know will become disabled or die prematurely.

The fear of discrimination by insurance companies or employers who learn the results of genetic testing is often justified. An insurance carrier may charge a healthy person a higher rate or disqualify an individual based on test results, and an employer might choose not to hire or to deny an affected individual a promotion. The American Society of Medical Genetics and most other medical professional associations agree that people should not be forced to choose between having a genetic test that could provide lifesaving information and avoiding a test to save a job or retain health insurance coverage.

Unanticipated Information and Results of Genetic Testing

Sometimes genetic testing yields unanticipated information, such as paternity, or other unexpected results, such as the presence of a disorder that was not sought directly. Many health professionals consider disclosure of such information, particularly when it does not influence health or medical treatment decision making, as counterproductive and even potentially harmful.

The ASHG is the primary professional organization for human geneticists in the United States. Its 8,000 members include researchers, academicians, clinicians, laboratory practice professionals, genetic counselors, nurses, and others involved in or with a special interest in human genetics. The ASHG recommends that family members not be informed of misattributed paternity unless the test requested was determination of paternity. The ASHG offers comparable guidelines regarding other unexpected results such as associations among diseases. For example, while performing screening for one disease, information about another disease may be discovered. Although the person may have requested screening for the first disorder, the presence of the second disorder may be unanticipated and may lead to stigmatization and discrimination on the part of insurance companies and employers. The ASHG encourages all health professionals to educate, counsel, and obtain informed consents that include cautions regarding unexpected findings before performing genetic testing.

Advertising Genetic Testing Directly to Consumers

Although pharmaceutical drugs have been advertised directly to consumers for more than two decades, direct-to-consumer advertising of genetic tests is a relatively recent phenomenon. Especially controversial are direct-to-consumer DNA tests sold in retail stores and via the Internet that promise personalized nutritional counseling based on genetic makeup. In Nutrigenetic Testing: Tests Purchased from Four Web Sites Mislead Consumers (July 27, 2006, http://www.gao.gov/new.items/d06977t.pdf), Gregory Kutz of the U.S. Government Accountability Office asserts that these tests are of no medical value. He notes that by promising results they cannot deliver, they often deceive people. Although nutritional genomics and nutrigenetic testing, which consider how complex interactions between genes and diet may affect the risk of future illnesses, is a legitimate discipline, Kutz believes the firms offering these services make health claims that are not supported by credible scientific evidence. Kutz decries the current regulatory environment, which provides only limited oversight of firms developing and marketing new types of genetic tests.

FDA Moves to Regulate Genetic Testing

Experts have been urging either the FDA or the Centers for Medicare and Medicaid Services, which regulates clinical labs, to strengthen regulation of diagnostic genetic tests. Andrew Pollack reports in "F.D.A. Seeks to Regulate New Types of Diagnostic Tests" (New York Times, September 6, 2006) that the FDA took its first steps to regulate such tests by issuing in September 2006 a draft guideline that would require FDA approval before the test kits can be marketed. The FDA intends to regulate at least one category of genetic tests: those that measure multiple genes, proteins, or other pieces of clinical information taken from a patient and then analyze the data. The best known of such tests is Oncotype DX, which costs $3,500 and analyzes twenty-one genes in a sample of breast tumor and then computes a score that predicts whether the cancer will recur and whether the patient would benefit from chemotherapy.

Personal Choices and Psychological Consequences of Genetic Testing

The results of genetic tests may be used to make decisions such as whether to have children or end a pregnancy. Results that predict the likelihood that an individual will develop a disease may affect decisions about education, marriage, family, or career choices. The decision to undergo genetic testing and the results of the tests not only affect the individual tested but also his or her family members. For example, when an unaffected patient requests a genetic susceptibility test, another test of an affected relative may be required to accurately calculate probability. Family members may vary in their willingness to share genetic information and their desire to know about genetic risks.

There are psychological consequences of genetic testing and coming to terms with the results. People may be relieved or distressed when they learn the results of a genetic test. The results can change the way they feel about themselves and can influence their relationships with relatives. For example, family members who discover they are carriers for cystic fibrosis may feel isolated or estranged from siblings who have opted not to be tested. By contrast, those who find out they do not have a genetic mutation for Huntington's disease may feel guilty because they were spared and other family members were not, or they may worry about assuming responsibility for family members who develop the disease.

The complexity of genetic testing and the uncertainty of many results pose an additional psychological challenge. The results of predictive genetic tests are often expressed in probabilities rather than certainties, and, even for people with a high probability of developing a disease, there are often conflicting opinions about the most appropriate course of action. For example, a woman who tests positive for BRCA1 or BRCA2 has an increased lifetime risk of developing breast or ovarian cancer or both, but it does not mean that her risk of developing either or both is 100%. Depending on her personal circumstances and the medical advice she receives, she may opt to intensify screening to detect disease; use prescription medication intended to reduce risk, such as tamoxifen; or undergo preventive procedures, such as mastectomy and oophorectomy.

Even a negative test result can be stressful, creating nearly as many questions as it does answers about disease risk. A negative test result for BRCA1 or BRCA2 in a woman with an affected family member (one who has the genetic mutation) means that even though she does not have the genetic mutation, her risk is the same as that of the general population. There are multiple factors associated with the risk of developing breast cancer that are not identified through genetic testing, such as the age at which a woman has her first child. In addition, most breast cancer is not believed to be hereditary, and most women with diagnosed breast cancer do not test positive for BRCA1 or BRCA2 genetic mutations.

LEGISLATION TO PROTECT GENETIC INFORMATION AND PREVENT DISCRIMINATION

By 2007 most state legislatures had taken steps to safeguard genetic information beyond the protections provided for other types of health information. However, absent comprehensive federal legislation, not all people will be protected from discrimination based on genetic information.

Federal Executive Order 13145, "To Prohibit Discrimination in Federal Employment Based on Genetic Information," was signed on February 8, 2000. The executive order prohibits federal government agencies from obtaining genetic information from employees or job applicants and from using genetic information in hiring and promotion decisions. The executive order defines genetic information as information about an individual's genetic tests; information about the genetic tests of an individual's family members; and information about the occurrence of a disease, medical condition, or disorder in family members of the individual.

On October 14, 2003, the U.S. Senate passed the Genetic Information Nondiscrimination Act (S. 1053), which would have prohibited discrimination on the basis of genetic information in health insurance coverage and the workplace. Francis S. Collins, the director of the National Human Genome Research Institute, published a statement (October 14, 2003, http://genome.gov/11009127) expressing his contention that:

No one should lose his job because of the genes he inherited. No one should be denied health insurance because of her DNA. But genetic discrimination affects more than jobs and insurance. It slows the pace of science. We know that many people have refused to participate in genetic research for fear of genetic discrimination. This means that without the kind of legal protections offered by this bill, our clinical research protocols will lack participants, and those who do participate will represent a self-selected group.

The Genetic Information Nondiscrimination Act of 2005 (S. 306) was unanimously passed on February 17, 2005. President George W. Bush expressed his support for the bill in a Statement of Administrative Policy. A virtually identical bill, the Genetic Information Nondiscrimination Act (H.R. 1227), was considered by the U.S. House of Representatives during 2005 but by the close of 2006 it had not made it out of committee.

Health Insurance Portability and Accountability Act of 1996

On August 21, 1996, President Bill Clinton signed the Health Insurance Portability and Accountability Act (HIPAA; PL 104-191). This legislation aims to provide better portability (transfer) of employer-sponsored insurance from one job to another. By preventing job lock—the need to remain in the same position or with the same employer for fear of losing health care coverage—the act was designed to afford American workers greater career mobility and the freedom to pursue job opportunities. Industry observers and policy makers viewed HIPAA as an important first step in the federal initiative to significantly reduce the number of uninsured people in the United States. They also hoped it would provide a measure of protection from genetics-based discrimination.

HIPAA stipulates that American workers who have previous insurance coverage are immediately eligible for new coverage when changing jobs. The law prohibits group health plans from denying new coverage based on past or present poor health and guarantees that employees can retain their health care coverage even after they leave their jobs. New employers can still require a routine waiting period (usually no more than three months) before paying for health benefits, but the new employee who applies for insurance coverage can be continuously covered during the waiting period. HIPPA also prohibits excluding an individual from group coverage because of past or present medical problems, including genetic information.

HIPAA does not, however, prohibit the use of genetic information as a basis for charging a group more for health insurance. It neither limits the collection of genetic information by insurers nor prohibits insurers from requiring an individual to take a genetic test. The act does not limit the disclosure of genetic information by insurers, and it does not apply to individual health insurers unless they are covered by the portability provision.

GENETIC COUNSELING

Generally, patients receive explicit counseling before undergoing genetic testing to ensure that they are able to make informed decisions about choosing to have the tests and the consequences of testing. Most genetic counseling is informative and nondirective—it is intended to offer enough information to allow families or individuals to determine the best courses of action for themselves but avoids making testing recommendations.

Patients undergoing tests to improve their care and treatment have different pretest counseling needs from those choosing susceptibility or predictive testing. In such instances genetic counselors do offer testing recommendations, particularly when a test offers an opportunity to prevent disease. As tests for genetic risk factors increasingly become routine in clinical medical practice, they are likely to be offered without formal pretest counseling. Genetic counselors urge physicians, nurses, and other health care professionals not to discount or rush through the process of obtaining informed consent to conduct a genetic test. They caution that potential psychological, social, and family implications should be acknowledged and addressed in advance of testing, including the potential for discrimination on the basis of genetic-risk status and the possibility that the predictive value of genetic information may be overestimated. The potentially life-changing consequences of genetic testing suggest that all health care professionals involved in the process should not only adhere to thoughtful informed consent procedures for genetic testing but also offer or make available genetic counseling when patients and families receive the results of testing.

CHAPTER 6
GENETIC TESTING

Diagnosis means finding the cause of a disorder, not just giving it a name.

—Sydney Walker III

Over the course of the last decade, the definitions of health and disease have been transformed by advances in genetics. Genetic testing has enabled researchers and clinicians to detect inherited traits, diagnose heritable conditions, determine and quantify the likelihood that a heritable disease will develop, and identify genetic susceptibility to familial disorders. Many of the strides made in genetic diagnostics are direct results of the Human Genome Project, an international thirteen-year effort begun in 1990 by the Department of Energy and the National Institutes of Health, which mapped and sequenced the human genome in its entirety. The increasing availability of genetic testing has been one of the most immediate applications of this groundbreaking research.

A genetic test is the analysis of human deoxyribonucleic acid (DNA), ribonucleic acid (RNA), chromosomes, and proteins in order to detect heritable disease-related genotypes, mutations, phenotypes, or karyotypes (standard pictures of the chromosomes in a cell) for the purposes of diagnosis, treatment, and other clinical decision making. Most genetic testing is performed by drawing a blood sample and extracting DNA from white blood cells. Genetic tests may detect mutations at the chromosomal level, such as additional, absent, or rearranged chromosomal material, or even subtler abnormalities such as a substitution in one of the bases that make up the DNA. There is a broad range of techniques that can be used for genetic testing. Genetic tests have diverse purposes, including screening for and diagnosis of genetic disease in newborns, children, and adults; the identification of future health risks; the prediction of drug responses; and the assessment of risks to future children.

There is a difference between genetic tests performed to screen for disease and testing conducted to establish a diagnosis. Diagnostic tests are intended to definitively determine whether a patient has a particular problem. They are generally complex tests and commonly require sophisticated analysis and interpretation. They may be expensive and are generally performed only on people believed to be "at risk," such as patients who already have symptoms of a specific disease.

In contrast, screening is performed on healthy, asymptomatic (showing no symptoms of disease) people and often to the entire relevant population. A good screening test is relatively inexpensive, easy to use and interpret, and helps identify which individuals in the population are at higher risk of developing a specific disease. By definition, screening tests identify people who need further testing or those who should take special preventive measures or precautions. For example, people who are found to be especially susceptible to genetic conditions with specific environmental triggers are advised to avoid the environmental factors linked to developing the disease. Examples of genetic tests used to screen relevant populations include those that screen people of Ashkenazi Jewish heritage (the east European Jewish population primarily from Germany, Poland, and Russia, as opposed to the Sephardic Jewish population primarily from Spain, parts of France, Italy, and North Africa) for Tay-Sachs disease, African-Americans for sickle-cell disease, and the fetuses of expectant mothers over age thirty-five for Down's syndrome. Table 6.1 lists a few of the more than 900 DNA-based genetic tests that were available as of 2004.

THE QUALITY AND UTILITY OF GENETIC TESTS

Like all diagnostic and screening tests, the quality and utility of genetic tests depend on their reliability, validity, sensitivity, specificity, positive predictive value, and negative predictive value. Reliability of testing refers to the test's ability to be repeated and to produce

equivalent results in comparable circumstances. A reliable test is consistent and measures the same way each time it is used with the same patients in the same circumstances. For example, a well-calibrated balance scale is a reliable instrument for measuring body weight.

Validity is the accuracy of the test. It is the degree to which the test correctly identifies the presence of disease, blood level, or other quality or characteristic it is intended to detect. For example, if you put an object you knew weighed ten pounds on a scale and the scale said it weighed ten pounds, then the scales results are valid. There are two components of validity: sensitivity and specificity.

Sensitivity is the test's ability to identify people who have the disease. Mathematically speaking, it is the percentage of people with the disease who test positive for the disease. Specificity is the test's ability to identify people who do not have the disease—it is the percentage of people without the disease who test negative for the disease. Ideally, diagnostic and screening tests should be highly sensitive and highly specific, thereby accurately classifying all people tested as either positive or negative. In practice, however, sensitivity and specificity are frequently inversely related—most tests with high levels of sensitivity have low specificity, and the reverse is also true.

The likelihood that a test result will be incorrect can be gauged based on the sensitivity and specificity of the test. For example, if a test's sensitivity is 95%, then when one hundred patients with the disease are tested, ninety-five will test positive and five will test "false negative"—they have the disease but the test has failed to detect it. For example, disorders such as Charcot-Marie-Tooth disease can arise from mutations in one of many different genes, and since some of these genes have not yet been identified, they will not be detected and a false negative result might be reported. On the other hand, if a test is 90% specific, when one hundred healthy, disease-free people are tested, ninety will receive negative test results and ten will be given "false-positive" results, meaning that they do not have the disease but the test has inaccurately classified them as positive.

The positive predictive value is the percentage of people that actually have the disease of all those with positive test results. The negative predictive value measures the percent of all the people with negative test results who do not have the disease.

PREGNANCY, CHILDBIRTH, AND
GENETIC TESTING

There are thousands of genetic diseases, such as sickle-cell anemia, cystic fibrosis, and Tay-Sachs disease, that may be passed from one generation to the next. Numerous tests have been developed to help screen parents at risk of passing on genetic disease to their children as well as to identify embryos, fetuses, and newborns that suffer from genetic diseases.

Carrier Identification

Carrier identification is the term for genetic testing to determine whether a healthy individual has a gene that may cause disease if passed on to his or her offspring. It is usually performed on people considered to be at higher than average risk, such as those of Ashkenazi Jewish descent, who have a one in thirty chance of being Tay-Sachs carriers (in other populations the risk is about one in three hundred). Testing is necessary because many carriers have just one copy of a gene for an autosomal recessive trait and are unaffected by the trait or disorder. Only someone with two copies of the gene will actually have the disorder. So while it is widely assumed that everyone is an unaffected carrier of at least one autosomal recessive gene, it only presents a problem in terms of inheritance when two parents have the same recessive disorder gene (or both are carriers). In this instance the offspring would each have a one in four chance of receiving a defective copy of the gene from each parent and developing the disorder.

Another example of carrier identification is the test for a deletion in the dystrophin gene, which results in Duchenne muscular dystrophy. Carriers may avoid having an affected child by preventing pregnancy or by undergoing prenatal testing for Duchenne muscular dystrophy, with the option of ending the pregnancy if the fetus is found to be affected.

Using genetic testing to detect carriers poses some challenges. Typically, a carrier has inherited a mutant gene from one parent and a normal gene from another parent. If, however, the carrier harbors a mutation that is only found in germ cells (the sperm or eggs), and only in some of these germ cells, then conventional genetic testing, which is performed on white blood cells, will miss the mutation.

Preimplantation Genetic Diagnosis

Preimplantation genetic diagnosis (PGD) is a newer genetic test that enables parents undergoing in vitro fertilization (fertilization that takes place outside the body) to screen an embryo for specific genetic mutations when it is no larger than six or eight cells and before it is implanted in the uterus to grow and develop. Successful PGD was reported by Yury Verlinsky et al. in "Preimplantation Diagnosis for Sonic Hedgehog Mutation Causing Familial Holoprosencephaly" (New England Journal of Medicine, vol. 348, no. 15, April 10, 2003). The article reports on a couple at risk of transmitting holoprosencephaly, a disorder characterized by face and brain malformations that occurs in about one in every 16,000 live births and is responsible for one in 250 miscarriages. The disorder may cause facial disfigurement, such as a single eye or a malformed nose located above an eye, brain defects, or death. People with the disorder carry a mutation in a gene known as sonic hedgehog (shh), which also plays a role in some cancers.

The couple discovered they were at risk of passing the disease to their offspring when they gave birth to a son and daughter with the disorder. The daughter, who had a severe form of holoprosencephaly, died shortly after birth. The researchers tested the man's sperm cells and found that only some sperm cells carried the shh mutation. Since not all sperm cells were affected, the researchers speculated that the father had not inherited the mutation from his parents; rather, the mutation had arisen in his body after conception. Performing PGD on the couple's potential embryos allowed the researchers to select only those at low risk of disease and enabled the couple to give birth to a healthy girl. The success of PGD prompted the authors to conclude, "PGD can be recommended for any congenital disorder for which the causative gene is known, and when the couples would like to avoid traditional prenatal diagnosis and termination of pregnancy."

Prenatal Testing

Prenatal genetic testing enables physicians to diagnose diseases in the fetus. Most genetic tests examine blood or other tissue to detect abnormalities. An example of a blood test is the triple marker screen. This test measures levels of alpha fetoprotein (AFP), human chorionic gonadotropin (hCG), and unconjugated estriol, and can identify some birth defects such as Down's syndrome and neural tube defects. (Two of the most common neural tube defects are anencephaly—absence of the majority of the brain—and spina bifida—incomplete development of the back and spine.)

The fetal yolk sac and the fetal liver make AFP, which is continuously processed by the fetus and excreted into the amniotic fluid. A small amount crosses the placenta and can be found in maternal blood. Maternal screening for AFP levels is based on maternal age, fetal gestation, and the number of fetuses the mother is carrying. Elevated levels of AFP are associated with conditions such as spina bifida and low levels are found with Down's syndrome. Since AFP levels alone may not always detect disorders adequately, two other blood serum tests have been developed. Human chorionic gonadotropin (hCG) is a glycoprotein produced by the placenta. Normally, hCG is elevated at the time of implantation, but decreases at about eight weeks of gestation, and then drops again at approximately twelve weeks of gestation. Elevated levels of hCG are found with Down's syndrome. The placenta also produces unconjugated estriol. As with AFP, lower unconjugated estriol maternal serum levels also are found with Down's syndrome. Triple marker screen results are usually available within several days and women with abnormal results are often advised to undergo additional diagnostic testing such as chorionic villus sampling (CVS), amniocentesis, or percutaneous umbilical blood sampling (withdrawing blood from the umbilical cord).

CVS enables obstetricians and perinatologists (physicians specializing in evaluation and care of high-risk expectant mothers and infants) to assess the progress of pregnancy during the first trimester (the first three months). A physician passes a small, flexible tube called a catheter through the cervix to extract chorionic villi tissue—cells that will become the placenta and are genetically identical to the baby's cells. The chorion develops from trophoblasts, or the same cells as the fetus, and contains the same DNA and chromosomes. At the end of the eighth week of gestation, the chorionic sac is covered completely by trophoblastic villi, which degenerate by about the eleventh week of pregnancy. During this period, it is considered safe to remove a sample of chorionic villi for testing. The optimal time for CVS is between ten and eleven and a half weeks of gestation. The cells obtained via CVS are examined in the laboratory for indications of genetic disorders such as cystic fibrosis, Down's syndrome, Tay-Sachs, and thalassemia, and the results of testing are available within seven to fourteen days. CVS provides the same diagnostic information as amniocentesis; however, the risks (miscarriage, infection, vaginal bleeding, and birth defects) associated with CVS are slightly higher. Approximately one in every one hundred pregnancies is miscarried as a result of CVS.

Amniocentesis involves taking a sample of the fluid that surrounds the fetus in the uterus for chromosome analysis. An amniocentesis is usually performed at fifteen to eighteen weeks of gestation, although it can be done as early as twelve weeks. A pregnant woman has an abdominal ultrasound (an imaging study) to locate a pocket of amniotic fluid away from the fetus and placenta. About twenty milliliters of amniotic fluid is obtained when the physician inserts a hollow needle through the abdominal wall and the wall of the uterus. Fetal karyotyping, DNA analysis, and biochemical testing may be performed on the isolated fetal cells. Like CVS, amniocentesis samples and analyzes cells derived from the baby to enable parents to learn of chromosomal abnormalities, as well as the gender of the unborn child, about two weeks after the test is performed. The risk of miscarriage—about one in every two hundred pregnancies—resulting from amniocentesis is lower than the risk associated with CVS.

Using samples of genetic material obtained from amniocentesis or CVS, physicians can detect disease in an unborn child. Down's syndrome (also known as trisomy 21, because it is caused by an extra copy of chromosome 21) is the genetic disease most often identified using this technique. Down's syndrome is rarely inherited; most cases result from an error in the formation of the ovum (egg) or sperm, leading to the inclusion of an extra chromosome 21 at conception. As with prenatal diagnosis for most inherited genetic diseases, this use of genetic testing is focused on reproductive decision making.

The most invasive prenatal procedure for genetic testing is percutaneous umbilical blood sampling. Under high-resolution ultrasound, a sample of fetal blood is removed from the umbilical cord. This test can be performed from approximately sixteen weeks gestation to term. Percutaneous umbilical blood sampling poses the greatest risk to the unborn child—one in fifty miscarriages occur as a result of this procedure. It is used when a diagnosis must be made quickly. For example, when an expectant mother is exposed to an infectious agent with the potential to produce birth defects, it may be used to examine fetal blood for the presence of infection.

Genetic Testing of Newborns

The most common form of genetic testing is the screening of blood taken from newborn infants for genetic abnormalities. In the United States about four million newborns are screened every year for specific genetic disorders such as phenylketonuria (PKU) as well as other medical conditions that are only indirectly genetically linked, such as congenital hypothyroidism (underactive thyroid gland). PKU is an inherited error of metabolism resulting from a deficiency of an enzyme called phenylalanine hydroxylase. The lack of this enzyme can produce mental retardation, organ damage, and postural problems. Children born with PKU must pay close attention to their diets in order to lead healthy, normal lives.

TESTING GUIDELINES FOR INFANTS KINDLES DEBATE.

In "Panel to Advise Testing Babies for 29 Diseases" (New York Times, February 21, 2005), Gina Kolata reported that a federal advisory group recommendation advocating screening infants for twenty-nine rare medical conditions generated debate among consumers and health care professionals. The advisory group not only sought to expand newborn screening but also to standardize it from state to state. In 2005 some states screened for just four conditions while others tested newborns for as many as thirty-five diseases.

Advocates of expanded newborn screening asserted that while the twenty-nine conditions occur infrequently, there are effective, potentially lifesaving medical treatments for each condition. Opponents claimed that the efficacy of treatment had not been adequately evaluated or substantiated. They further cautioned that screening detects even those infants with the mildest forms of the disorders and essentially forces parents and health care professionals to consider treatment that may be more harmful than the mild disorder. Critics also wondered about the advisability of labeling infants with diagnoses of diseases from which they will never suffer.

Laboratory Techniques for Genetic Prenatal Testing

Genetic testing is performed on chromosomes, genes, or gene products to determine whether a mutation is causing or may cause a specific condition. Direct testing examines the DNA or the RNA that makes up a gene. Linkage testing looks for disease-causing gene markers in family members from at least two generations. Biochemical testing assays certain enzymes or proteins, which are the products of genes. Cytogenetic testing examines the chromosomes.

Generally, a blood sample or buccal smear (cells from the mouth) is used for genetic tests. Other tissues used include skin cells from a biopsy, fetal cells, or stored tissue samples. Testing requires highly trained, certified technicians and laboratories because the procedures are complex and varied, the technology is new and evolving, and hereditary conditions are often rare, so many testing techniques require special expertise. In the United States laboratories performing clinical genetic tests must be approved under the Clinical Laboratory Improvement Amendments (CLIA), passed by Congress in 1988 to establish the standards with which all laboratories that test human specimens must comply in order to receive certification. CLIA standards determine the qualifications of laboratory personnel, categorize the complexity of various tests, and oversee quality improvement and assurance. By 2005 more than 200 laboratories in the United States were performing genetic testing to detect and diagnose more than 300 conditions.

Fetal nucleated red blood cells are used for prenatal genetic testing because the DNA in these cells can be visualized directly via cell surface markers. The magnetic-activated cell-sorting technique and other separation methods use monoclonal antibodies that attach to specific cells. The most common antibodies studied are the transferrin receptor (CD71), the thrombospondin receptor (CD36), and glycophorin A (GPA). These antibodies attach to fetal cell surface antigens, allowing them to be separated from the maternal blood. The CD71 antibody is the most commonly used for fetal cell selection.

Chromosomes are identified by their size, centromere location, and banding position. Historically, the banding technique most widely used to identify chromosomes was giemsa-trypsin (GTG) banding. This technique arrests cells early in mitosis, which enables laboratory workers to more readily identify chromosomal aberrations. The GTG-banding technique has limitations in that it can achieve a resolution only at the single band level.

The polymerase chain reaction technique permits rapid cloning and DNA analysis and allows selective amplification of specific DNA sequences. A polymerase chain reaction can be performed in hours and is a sensitive test that may be used to screen for altered genes, but it is limited by the size and length of the DNA sequences that can be cloned.

Current DNA technology has made it possible to create fluorescent-labeled DNA probes that hybridize to specific gene loci. A fluorescent label is attached to the DNA probe that will bind to the complementary DNA strands. Fluorescence in situ hybridization (FISH) provides a unique opportunity to view specific genetic codes. The FISH technique may be used on cells and fluid obtained by CVS and amniocentesis as well as maternal blood. With this technique, a single strand of DNA is used to create a probe that attaches at the specific gene location. To separate the double-stranded DNA, heat or chemicals are used to break the chemical bonds of the DNA and obtain a single strand. (See Figure 6.1.)

There are three kinds of chromosome-specific probes: repetitive probes, painting probes, and locus-specific probes. Repetitive probes are the most commonly used. They produce intense signals by creating tandem repeats of base pairs. Painting probes are collections of specific DNA sequences that may extend along either part or all of an individual chromosome. These probe labels are most useful for identifying complex rearrangements of genetic material in structurally abnormal chromosomes. Probes that can hybridize to a single gene locus are called locus-specific probes. They can be used to identify a gene in a particular region of a chromosome. Locus-specific probes are used to identify a deletion or duplication of genetic material. The FISH procedure allows a signal to be visualized that indicates the presence or absence of DNA. The entire process can be completed in fewer than eight hours using five to seven probes. For this reason, FISH is frequently used as a rapid screen for trisomies and genetic disorders.

Although FISH is the most commonly used and most readily available prenatal diagnostic cytogenetic technique, it has limited ability to detect translocations, deletions, and inversions. Another method (known as microdissection FISH), also used for prenatal diagnosis, is more sensitive to these alterations. Microdissection FISH constructs probes to define specific regions of the human chromosome. Figure 6.2 shows how the microdissection technique is used to identify structurally abnormal chromosomes.

Newer technologies allow for the identification of all human chromosomes, thereby expanding the FISH application to the entire genome. Multiplex FISH applies combinations of probes to color components of fluorescent dye during metaphase to visualize each chromosome. This type of FISH procedure evolved from the whole-chromosome painting probes and uses a combination of fluorochromes to identify each chromosome. Just five fluorophores are needed to decode the entire complement of human chromosomes. Multicolor spectral karyotyping uses computer imaging and Fourier spectroscopy to increase the analysis of genetic markers. (See Figure 6.3.) Although FISH techniques are considered highly reliable, there are limitations to multiplex and multicolor FISH, in that inversion or subtle deletions may be overlooked. The greatest advantage of multiplex FISH is its application in cases of dysmorphic infants. Spectral karyotyping techniques can detect cryptic unbalanced translocations that cannot be identified by other techniques.

GENETIC TESTING FOR SICKLE-CELL ANEMIA.

Sickle-cell anemia is an autosomal recessive disease that results when hemoglobin S is inherited from both parents. (When hemoglobin S is inherited from only one parent, the individual is a sickle-cell carrier.) Since normal and sickle hemoglobins differ at only one amino acid in the hemoglobin gene, a test called hemoglobin electrophoresis is used to establish the diagnosis.

Genetic testing for sickle-cell anemia involves restriction fragment length polymorphism (DNA sequence variant) that uses specific enzymes to cut the DNA. (See Figure 6.4.) These enzymes cut the DNA at a specific base sequence on the normal gene but not on a gene in which a mutation is present. As a result of this technique, there are longer fragments of sickle hemoglobin. Another technique known as gel electrophoresis sorts the DNA fragments by size. Autoradiography renders the DNA fragments by generating an image after radioactive probes have labeled the DNA fragments that contain the specific gene sequence. The location of the fragments distinguishes carrier status (heterozygous) from sickle-cell anemia (homozygous), or normal blood.

New Techniques Detect Fetal Gene Mutations

In 2005 an international team of investigators reported success with a technique that identifies fetal gene mutations such as beta-thalassemia from samples of maternal blood. The technique relies on the fact that circulatory fetal DNA sequences comprise fewer than 300 base pairs, while maternal DNA exceeds 500 base pairs. The investigators used PCR amplification to select for paternally inherited DNA sequences. Presence of the paternal mutant alleles for beta-thalassemia was then detected by allele-specific PCR.

They tested the new technique on maternal blood samples from expectant mothers whose fetuses were at risk for beta-thalassemia because the father was a carrier for one of four beta-globin gene mutations. The results were verified by comparing the findings from chorionic villus sampling. The investigators also reported the cost-effectiveness of the new technique. Since it does not require complex machinery and relies on currently available technology, they estimated that the cost of a single analysis might be just $8.00 (Ying Li et al., "Detection of Paternally Inherited Fetal Point Mutations for β-Thalassemia Using Size-Fractionated Cell-Free DNA in Maternal Plasma," Journal of the American Medical Association, vol. 293, no. 7, February 16, 2005).

Another new technique reported in 2005 was analysis of amniotic fluid using oligonucleotide hybridization and microarray data analysis. (Figure 6.5 shows how micro-array technology is performed.) The researchers found that gene expression patterns correlated with the gender of the fetus, gestational age, and disease status. They asserted that this technology could assist in advancing human developmental research, as well as identifying new biomarkers for prenatal assessment (Paige Larrabee et al., "Global Gene Expression Analysis of the Living Human Fetus Using Cell-Free Messenger RNA in Amniotic Fluid," Journal of the American Medical Association, vol. 293, no. 7, February 16, 2005).

GENETIC DIAGNOSIS IN CHILDREN AND
ADULTS

Genetic testing can also be performed postnatally (after birth) to determine which children and adults are at increased risk of developing specific diseases. By 2005 scientists could perform predictive genetic testing to identify which individuals were at risk for cystic fibrosis, Tay-Sachs disease, Huntington's disease, amyotrophic lateral sclerosis (ALS, a degenerative neurological condition commonly known as Lou Gehrig's disease), and several types of cancers, including some cases of breast, colon, and ovarian cancer.

More than 900 genetic tests were available in 2005, but public health professionals did not consider it practical to screen for conditions that are very rare, have only minor health consequences, or those for which there is still no effective treatment. The most frequently performed genetic tests were those considered most useful in terms of their potential to screen populations for diseases that occur relatively frequently, have serious medical consequences (including death) if untreated, and for which effective treatment is available. There are a number of other diseases, such as alcoholism, diabetes, multiple sclerosis, and prostate cancer, for which genetic testing, when it becomes available, will have considerable health impact.

A positive test result (the presence of mutation—a defective or altered gene) from predictive genetic testing does not guarantee that the individual will develop the disease; it simply identifies the individual as genetically susceptible and at an increased risk for developing the disease. For example, a woman who tests positive for the BRCA1 gene has about an 80% chance of developing breast cancer before age sixty-five. It is also important to note that, like other types of diagnostic medical testing, genetic tests are not 100% predictive—the results rely on the quality of laboratory procedures and accuracy of interpretations. Furthermore, since tests vary in their sensitivity and specificity, there is always the possibility of false-positive and false-negative test results.

Researchers hope that positive test results will encourage people who are at higher than average risk of developing a disease to be especially vigilant about disease prevention and screening for early detection, when many diseases are most successfully treated. There is an expectation that genetic information will increasingly be used in routine population screening to determine individual susceptibility to common disorders such as heart disease, diabetes, and cancer. Such screening will identify groups at risk so that primary prevention efforts such as diet and exercise or secondary prevention efforts such as early detection can be initiated.

Diagnostic Genetic Testing

Most genetic testing is performed on people who are asymptomatic (people who are apparently healthy). The objective of these screening tests is to determine if they are carriers of a genetic disease or to identify susceptibility or risk of developing a specific disease or disorder. There is, however, some testing performed on persons with symptoms of a disease in order to clarify or establish the diagnosis and calculate the risk of developing the disease for other family members. This type of testing is known as diagnostic genetic testing or symptomatic genetic testing. It also may assist in directing treatment for symptomatic patients in whom a mutation in a single gene (or in a gene pair) accounts for a disorder. Cystic fibrosis and myotonic dystrophy are examples of disorders that may be confirmed or ruled out by diagnostic genetic testing and other methods (such as the sweat test for cystic fibrosis or a neurological evaluation for myotonic dystrophy).

One issue involved in diagnostic genetic testing is the appropriate frequency of testing in view of rapidly expanding genetic knowledge and identification of genes linked to disease. Physicians frequently see symptomatic patients for whom there is neither a definitive diagnosis nor a genetic test. The as-yet-unanswered question is: Should such people be recalled for genetic testing each time a new test becomes available? Although clinics and physicians who perform genetic testing counsel patients to maintain regular contact so they may learn about the availability of new tests, there is no uniform guideline or recommendation about the frequency of testing.

Population Screening

Population screening for heritable diseases is one potentially lifesaving application of molecular genetics technology. Prenatal screening has demonstrated benefits and gained widespread use; however, genetic screening has not yet become part of routine medical practice for adults. Geneticists have identified at least seven genes that might be candidates for use as population screening tests in adults in the United States. The genes include HFE, for hereditary hemochromatosis; apoE4, linked to Alzheimer's disease; CYP2D6, linked to ankylosing spondylitis (arthritis of the spine); BRCA1 and BRCA2, genes for hereditary breast and ovarian cancer; familial adenomatous polyposis, associated with precancerous growths in the colon; and factor V Leiden, the most common hereditary blood clotting disorder in the United States. As of 2005, screening for variants in these genes had not entered into routine medical practice because there was considerable controversy about the predictive value of testing for these genes and how to monitor and care for people who test positive for them.

In "Genomic Medicine: Population Screening in the Age of Genomic Medicine" (New England Journal of Medicine, vol. 348, no. 1, January 2, 2003), Muin Khoury, Linda McCabe, and Edward McCabe described the principles and practice of genetic screening in the era of genomic medicine. Historically, the decision to institute population screening involved consideration of the consequences of the specific condition to public health, the availability of an effective screening test, the availability of treatment to prevent the disease, and an analysis of the costs and benefits of screening.

Khoury, McCabe, and McCabe observed that each state, as well as the District of Columbia, designated the diseases and methods for screening its newborns, and the only universally conducted screening was for PKU and hypothyroidism. The criteria for adding a screening test are similarly uneven and Khoury, McCabe, and McCabe noted that there was "a lack of research to demonstrate the effectiveness of screening and treatment for a disorder, either before or after the disease is added to the newborn-screening program." They explained that, while new technology such as tandem mass spectrometry, which detects more than twenty disorders, may be used to screen newborns to identify potentially treatable inborn errors of metabolism such as medium chain acyl-coenzyme A (CoA) dehydrogenase deficiency, the test also identifies conditions for which there are no treatments. Furthermore, they cautioned that tests such as tandem mass spectrometry may detect variations of unknown clinical significance, generating unnecessary worry in parents and medical professionals.

Genetic Susceptibility

Susceptibility testing, also known as predictive testing, determines the likelihood that a healthy person with a family history of a disorder will develop the disease. Testing positive for a specific genetic mutation indicates an increased susceptibility to the disorder but does not establish a diagnosis. For example, a woman may choose to undergo testing to find out whether she has genetic mutations that would indicate likelihood of developing hereditary cancer of the breast or ovary (BRCA1 and BRCA2, respectively). If she tests positive for the genetic mutation, she may then decide to undergo some form of preventive treatment. Preventive measures may include increased surveillance such as more frequent mammography, chemoprevention—prescription drug therapy intended to reduce risk—or surgical prophylaxis, such as mastectomy and/or oophorectomy (surgical removal of the breasts and ovaries, respectively).

Khoury, McCabe, and McCabe observed that there are questions about the risks and benefits of identifying and treating asymptomatic people who have been identified through population screening as at high risk for hereditary disorders. They used population testing for mutations in HFE, the gene for hereditary hemochromatosis (HHC), as an example of the complicated range of issues involved in recommending population screening. HHC is an autosomal recessive disorder of iron metabolism that results in iron overload. Without early diagnosis and treatment, HHC is fatal; however, with prompt diagnosis and treatment, affected people can have normal life spans. About one in ten people are carriers, and HHC affects about one in 300 people.

Khoury, McCabe, and McCabe reported that a panel of experts from the Centers for Disease Control and Prevention and the National Human Genome Research Institute determined that population genetic testing for mutations in HFE was not recommended because of uncertainty about the natural history of the disease, age-related disease penetrance, effective care for asymptomatic people identified as carrying mutations, and the psychosocial impact of genetic testing. Concurring with this recommendation, Khoury, McCabe, and McCabe noted that despite the relatively high prevalence of the mutations, studies indicated that the disease penetrance for HFE mutations is low—a large study found that just one of the 152 subjects who were homozygous for the C282Y mutation (i.e., those that had the highest risk of HHC) had symptoms of hereditary hemochromatosis.

Testing Children for Adult-Onset Disorders

In 2000 the American Academy of Pediatrics Committee on Genetics recommended genetic testing for people under age eighteen only when testing would offer immediate medical benefits or when there is a benefit to another family member and there is no anticipated harm to the person being tested. The committee considered genetic counseling before and after testing as essential components of the process.

The American Academy of Pediatrics Committee on Bioethics and Newborn Screening Task Force recommended the inclusion of tests in the newborn-screening battery based on scientific evidence. The academy advocated informed consent for newborn screening. (To date, the majority of states do not require informed consent.) The Committee on Bioethics did not endorse carrier screening in people under eighteen years of age, except in the case of a pregnant teenager. It also recommended against predictive testing for adult-onset disorders in people under eighteen.

The American College of Medical Genetics, the American Society of Human Genetics (ASHG), and the World Health Organization (WHO) have also weighed in about genetic testing of asymptomatic children, asserting that decision making should emphasize the child's well-being. One issue involves the value of testing of asymptomatic children for genetic mutations associated with adult-onset conditions such as Huntington's disease. Since no treatment can be begun until the onset of the disease, and presently there is no treatment to alter the course of the disease, it may be ill advised to test for it. Another concern is testing for carrier status of autosomal-recessive or X-linked conditions such as cystic fibrosis or Duchenne muscular dystrophy. Experts caution that children might confuse carrier-status with actually having the condition, which in turn might provoke needless anxiety.

There are, however, circumstances in which genetic testing of children may be appropriate and useful. Examples are children with symptoms of suspected hereditary disorders or those at risk for cancers in which inheritance plays a primary role. In "Genetic Testing and Screening" (American Journal of Nursing, vol. 102, no. 7, July 2002), Dale Halsey Lea and Janet Williams cited children with a family history of familial adenomatous polyposis (FAP) and those diagnosed with certain childhood cancers, such as multiple endocrine neoplasia, as appropriate candidates for genetic testing. They observed that testing can help to determine planning, surveillance, and treatment for those who are found to have the FAP genetic mutation. Genetic testing for certain childhood cancers may serve to predict risk and improve detection of second malignancies. Lea and Williams concurred that the child must agree to and understand the function of genetic testing and they reiterated that to administer genetic testing to a child requires the consent of both the parents and the child.

ETHICAL CONSIDERATIONS—CHOICES
AND CHALLENGES

Rapid advances in genetic research since the 1980s have challenged scientists, health care professionals, ethicists, government regulators, legislators, and consumers to stay abreast of new developments. Understanding the scientific advances and their implications is critical for everyone involved in making informed decisions about the ways in which genetic research and information will affect the lives of current and future generations. American citizens, scientists, ethicists, legislators, and regulators share this responsibility. The pivotal importance of these societal decisions was underscored by the allocation of 5% of the budget of the Human Genome Project for the study of ethical, legal, and social issues related to genetic research. To date, consideration of these issues has not produced simple or universally applicable answers to the many questions posed by the increasing availability of genetic information. Ongoing public discussion and debate is intended to inform, educate, and help people in every walk of life make personal decisions about their health and participate in decisions that concern others.

As researchers learn more about the genes responsible for a variety of illnesses, they can design more tests with increased accuracy and reliability to predict whether an individual is at risk of developing specific diseases. The ethical issues involved in genetic testing have turned out to be far more complicated than originally anticipated. Initially, physicians and researchers believed that a test to determine in advance who would develop or escape a disease would be welcomed by at-risk families, who would be able to plan more realistically about having children, choosing jobs, obtaining insurance, and going about their daily lives. Nevertheless, many people with family histories of a genetic disease have decided that not knowing is better than anticipating a grim future and an agonizing, slow death. They prefer to live with the hope that they will not develop the disease rather than having the certain knowledge that they will.

The discovery of genetic links and the development of tests to predict the likelihood or certainty of developing a disease raise ethical questions for people who carry a defective gene. Should women who are carriers of Huntington's disease or cystic fibrosis have children? Should a fetus with the defective gene be carried to term or aborted? One health insurance company agreed to pay for prenatal cystic fibrosis testing for a mother who already had one affected child, but the company insisted that if the baby was affected, the mother would have to terminate the pregnancy or it would not cover the child's future medical bills.

There are also concerns about privacy and the confidentiality of medical records and the results of genetic testing and possible stigmatization. Some people are reluctant to be tested because they fear they may lose their health, life, and disability insurances, or even their jobs if they are found to be at risk for a disease. Genetic tests are sometimes costly, and some insurers agree to reimburse for testing only if they are informed of the results. The insurance companies feel they cannot risk selling policies to people they know will become disabled or die prematurely.

The fear of discrimination by insurance companies or employers who learn the results of genetic testing is often justified. An insurance carrier may charge a healthy person a higher rate or disqualify an individual based on test results, and an employer might choose not to hire or to deny an affected individual a promotion. The American Society of Medical Genetics and most other medical professional associations agree that people should not be forced to choose between having a genetic test that could provide lifesaving information and avoiding a test to save a job or retain health insurance coverage.

Unanticipated Information and Results of
Genetic Testing

Sometimes genetic testing yields unanticipated information, such as paternity, or other unexpected results, such as the presence of a disorder that was not sought directly. Many health professionals consider disclosure of such information, particularly when it does not influence health or medical treatment decision making, as counterproductive and even potentially harmful.

The American Society of Human Genetics (ASHG) is the primary professional membership organization for human geneticists in the United States. Its 8,000 members include researchers, academicians, clinicians, laboratory practice professionals, genetic counselors, nurses, and others involved in or with a special interest in human genetics. The ASHG recommends that family members not be informed of misattributed paternity unless the test requested was determination of paternity. The ASHG offers comparable guidelines regarding other unexpected results such as associations among diseases. For example, while performing screening for one disease, information about another disease may be discovered. Although the person may have requested screening for the first disorder, the presence of the second disorder may be unanticipated and may lead to stigmatization and discrimination on the part of insurance companies and employers. The ASHG encourages all health professionals to educate, counsel, and obtain informed consents that include cautions regarding unexpected findings prior to performing genetic testing.

Advertising Genetic Testing Directly to Consumers

Although prescription pharmaceutical drugs have been advertised directly to consumers for more than two decades, direct consumer advertising of genetic tests is a relatively recent phenomenon. In "Limitations of Direct-to-Consumer Advertising for Clinical Genetic Testing" (Journal of the American Medical Association, vol. 288, no. 14, October 9, 2002), Sarah Gollust, Sara Chandros, and Benjamin Wilford considered the potential benefits and risks of direct-to-consumer advertising. Gollust, Chandros, and Wilford observed that such advertisements "describe complex, confusing, and anxiety-producing genetic concepts for the consumer. Drawing on themes of choice, hope, fear, and peace of mind, these advertisements validate patients' worries about their genetic risks and appeal to their desire to assert control over potential outcomes. Direct-to-consumer advertisements will likely become more frequent as increasing numbers of genetic tests become available and testing services seek to compete in this expanding market."

Gollust, Chandros, and Wilford described much of the advertising as containing unsubstantiated, inaccurate, manipulative, and misleading content describing material that is complex and potentially confusing for even the most educated and informed consumers. They also fault advertisers for failing to include risk information to balance their claims of effectiveness and neglecting to mention the potential risk of genetic discrimination. The authors wrote that "even with more suitable content, consumer-directed promotions may be premature. Public understanding of genetics is characterized by misconception and exaggerated expectation, a context advertisers can use to their advantage." They argued that scientific evidence and consensus, rather than commercial interests, should drive increased use of genetic testing.

Gollust, Chandros, and Wilford also lamented the absence of regulatory oversight of such advertising and the fact that, unlike pharmaceuticals that undergo pre-market review by the Food and Drug Administration (FDA), genetic testing services do not yet undergo comparable scrutiny or review. Only tests that are considered diagnostic devices or those that are packaged and sold as kits require premarket FDA approval.

Personal Choices and Psychological Consequences of
Genetic Testing

The results of genetic tests may be used to make decisions such as whether to have children or end a pregnancy. Results that predict the likelihood that an individual will develop a disease may affect decisions about education, marriage, family, or career choices. The decision to undergo genetic testing and the results of the tests not only affect the individual tested, but also his or her family members. For example, when an unaffected patient requests a genetic susceptibility test, another test of an affected relative may be required to accurately calculate probability. Family members may vary in their willingness to share genetic information and their desire to know about genetic risks.

There are psychological consequences of genetic testing and coming to terms with the results. People may be relieved or distressed when they learn the results of a genetic test. The results can change the way they feel about themselves and can influence their relationships with relatives. For example, family members who discover they are carriers for cystic fibrosis may feel isolated or estranged from siblings who have opted not to be tested. On the other hand, those who find out they do not have a genetic mutation for Huntington's disease may feel guilty because they were spared and other family members were not, or they may worry about assuming responsibility for family members who develop the disease.

The complexity of genetic testing and the uncertainty of many results pose an additional psychological challenge. The results of predictive genetic tests are often expressed in probabilities rather than certainties, and, even for people with a high probability of developing a disease, there are often conflicting opinions about the most appropriate course of action. For example, a woman who tests positive for BRCA1 or BRCA2 has an increased lifetime risk of developing breast or ovarian cancer or both, but it does not mean that her risk of developing either or both is 100%. Depending on her personal circumstances and the medical advice she receives, she may opt to intensify screening to detect disease; use prescription medication intended to reduce risk, such as tamoxifin; or undergo preventive procedures such as mastectomy and oophorectomy.

Even a negative test result can be stressful, creating nearly as many questions as it does answers about disease risk. A negative test result for BRCA1 or BRCA2 in a woman with an affected family member (one who has the genetic mutation) means that though she does not have the genetic mutation, her risk is the same as that of the general population. There are multiple factors associated with the risk of developing breast cancer that are not identified through genetic testing, such as the age at which a woman has her first child. In addition, most breast cancer is not believed to be hereditary, and most women with diagnosed breast cancer do not test positive for BRCA1 or BRCA2 genetic mutations.

PSYCHOLOGICAL ISSUES OF PRENATAL, CARRIER, AND PREDICTIVE GENETIC TESTING.

Caryn Lerman et al. examined the psychological issues involved in various types of genetic testing in "Genetic Testing: Psychological Aspects and Implications" (Journal of Consulting and Clinical Psychology, vol. 70, no. 3, June 2002). Although there is little literature describing the psychological impact of prenatal testing, Lerman and her associates concluded that, while many patients experience anxiety before undergoing testing, the studies offer no evidence of lasting significant psychological distress as a result of testing.

Although some studies report psychological distress resulting from carrier testing, as with prenatal testing, there are no significant long-term effects. Lerman et al. attributed this finding to the observation that many people appear highly motivated to seek testing for conditions that are life threatening and for conditions for which treatment is available and so are generally associated with more favorable attitudes regarding the benefits of testing. As expected, the response to carrier testing depends on the results. Many noncarriers report relief and a general sense of well-being about their future child-bearing. In contrast, carriers report feelings of hopelessness about the health of their future children.

While prenatal and carrier testing are primarily used to determine the risk of disease for offspring, predictive genetic testing focuses on the individual's own risk for developing a disease. Intuitively, it might be expected that people would experience the most stress while undergoing testing to predict their likelihood of developing disorders such as Huntington's disease or cancer; however, the few studies conducted show reductions in distress among noncarriers and minimal changes in distress among carriers. Lerman and her co-authors hypothesized that the low levels of psychological distress reported may reflect the comprehensive genetics education and counseling provided to those tested.

An important and somewhat surprising finding is that the results of testing have little impact on health behaviors. Lerman et al. concluded that since genetic testing does not appear to promote changes in health-related behaviors such as increased surveillance, there may be less need for psychological counseling of genetic testing participants and greater need for research and practice to facilitate health protective behaviors in response to genetic risk information.

LEGISLATION TO PROTECT GENETIC
INFORMATION AND PREVENT
DISCRIMINATION

By the end of 2004 forty-one states had enacted legislation banning genetic discrimination in health insurance, including altering rates and coverage based on genetic information, and thirty-one states had enacted legislation on genetic discrimination in the workplace. However, absent comprehensive federal legislation, not all people will be protected from discrimination based on genetic information.

Federal Executive Order 13145 "To Prohibit Discrimination in Federal Employment Based on Genetic Information" was signed on February 8, 2000. The executive order prohibits federal government agencies from obtaining genetic information from employees or job applicants and from using genetic information in hiring and promotion decisions. The executive order defines genetic information as information about an individual's genetic tests; information about the genetic tests of an individual's family members; and information about the occurrence of a disease, medical condition, or disorder in family members of the individual.

President George W. Bush repeatedly expressed strong support for passing genetic nondiscrimination legislation. On June 23, 2001, he devoted his weekly radio address to the need for such legislation, and he has reiterated this position at other public forums. The 107th Congress saw the introduction of five different bills aimed at preventing discrimination on the basis of genetic information, but none were enacted.

On October 14, 2003, the U.S. Senate passed the Genetic Information Nondiscrimination Act of 2003 (S.1053), which would have prohibited discrimination on the basis of genetic information in health insurance coverage and the workplace. Dr. Francis S. Collins, the director of the National Human Genome Research Institute, published a statement expressing his contention that:

No one should lose his job because of the genes he inherited. No one should be denied health insurance because of her DNA. But genetic discrimination affects more than jobs and insurance. It slows the pace of science. We know that many people have refused to participate in genetic research for fear of genetic

The administration favors enactment of legislation to prohibit the improper use of genetic information in health insurance and employment. The administration supports Senate passage of S. 306 (Genetic Information Nondiscrimination Act of 2005) as reported, which would prohibit group health plans and health insurers from denying coverage to a healthy individual or charging that person higher premiums based solely on a genetic predisposition to developing a disease in the future. The legislation also would bar employers from using individuals' genetic information when making hiring, firing, job placement, or promotion decisions.

The mapping of the human genome has led to more information about diseases and a better understanding of our genetic code. Scientists are pursuing new diagnostics, treatments, and cures based on this information, but the potential misuse of this information raises serious moral and legal issues. Concern about unwarranted use of genetic information threatens access to utilization of existing genetic tests as well as the ability to conduct further research. The administration wants to work with Congress to make genetic discrimination illegal and provide individuals with fair, reasonable protections against improper use of their genetic information.

discrimination. This means that without the kind of legal protections offered by this bill, our clinical research protocols will lack participants, and those who do participate will represent a self-selected group.

The 2003 bill, however, was not enacted into law. A virtually identical bill, the Genetic Information Nondiscrimination Act of 2005 (S.1053), was unanimously passed on February 17, 2005. President Bush expressed his support for the bill in a Statement of Administrative Policy. (See Table 6.2.) The House of Representatives was debating similar legislation as of June 1, 2005.

Health Insurance Portability and Accountability
Act of 1996

On August 21, 1996, President Bill Clinton signed the Health Insurance Portability and Accountability Act (HIPAA, PL 104–191). Also known as the Kennedy-Kassebaum Act (for its sponsors, senators Nancy Kassebaum, R-Kansas, and Edward Kennedy, D-Massachusetts), this legislation aims to provide better portability (transfer) of employer-sponsored insurance from one job to another. By preventing "job lock"—the need to remain in the same position or with the same employer for fear of losing health care coverage—the Act was designed to afford American workers greater career mobility and the freedom to pursue job opportunities. Industry observers and policy makers viewed HIPAA as an important first step in the federal initiative to significantly reduce the number of uninsured people in the United States. They also hoped it would provide a measure of protection from genetics-based discrimination.

HIPAA stipulates that American workers who have previous insurance coverage are immediately eligible for new coverage upon changing jobs. The law prohibits group health plans from denying new coverage based on past or present poor health and guarantees that employees can retain their health care coverage even after they leave their jobs. New employers can still require a routine waiting period (usually no more than three months) before paying for health benefits, but the new employee who applies for insurance coverage can be continuously covered during the waiting period.

The following section describes some of the major provisions of HIPAA. In addition to the provisions discussed, the act clarifies current law and stiffens penalties for fraud and abuse. The law applies to both group and individual health insurance policies.

PREEXISTING MEDICAL CONDITIONS.

Prior to the passage of HIPAA, insurers could refuse to cover treatment for a preexisting disease or medical condition—an illness or condition that had been diagnosed or treated before a person enrolled in the health insurance program. Under the new law, if the preexisting condition has been diagnosed or treated within six months of the patient's enrollment in the insurance program, the insurance company can withhold coverage for that condition for no longer than twelve months. If the preexisting condition has not been diagnosed or treated within six months of changing insurance, there is no waiting period beyond the short time an employer may require before providing benefits to a new employee.

This provision, along with the portability provision, means that insured employees are no longer trapped by the fear of losing their health insurance coverage if they lose or leave their jobs. In the past many workers stayed in unsatisfactory jobs because they were ill or had dependents with existing medical conditions. They were afraid to change jobs for fear of losing coverage or having to wait a long time before obtaining new coverage.

For health insurance offered to groups, HIPAA offers the following protections from discrimination:

• It prohibits excluding an individual from group coverage because of past or present medical problems, including genetic information.
• It forbids charging a higher premium to an individual than to others in the group.
• It limits exclusions in group health plans for preexisting conditions to twelve months, and prohibits such exclusions if the individual has been previously covered for that condition for twelve months or more.
• It clearly states that genetic information in the absence of a current diagnosis of illness shall not be considered a preexisting condition.

HIPAA does not, however, prohibit the use of genetic information as a basis for charging a group more for health insurance. It neither limits the collection of genetic information by insurers nor prohibits insurers from requiring an individual to take a genetic test. The act does not limit the disclosure of genetic information by insurers, and it does not apply to individual health insurers unless they are covered by the portability provision.

GENETIC COUNSELING

Generally, patients receive explicit counseling before undergoing genetic testing to ensure that they are able to make informed decisions about choosing to have the tests and the consequences of testing. Most genetic counseling is informative and "nondirective"—it is intended to offer enough information to allow families or individuals to determine the best courses of action for themselves but avoids making testing recommendations. This nondirective approach arose in response to the availability of prenatal testing that is used for reproductive decision making and predictive testing for untreatable conditions such as Huntington's disease, in which the benefit and utility of learning the results of testing are based on deeply held personal values and preferences.

Patients undergoing tests to improve their care and treatment have different pretest counseling needs from those choosing susceptibility or predictive testing. In such instances, genetic counselors do offer testing recommendations, particularly when a test offers an opportunity to prevent disease. As tests for genetic risk factors increasingly become routine in clinical medical practice, they are likely to be offered without formal pretest counseling. Genetic counselors exhort physicians, nurses, and other health care professionals not to discount or rush through the process of obtaining informed consent to conduct a genetic test. They caution that potential psychological, social, and family implications should be acknowledged and addressed in advance of testing, including the potential for discrimination on the basis of genetic-risk status and the possibility that the predictive value of genetic information may be overestimated. The potentially life-changing consequences of genetic testing suggest that all health care professionals involved in the process should not only adhere to thoughtful informed consent procedures for genetic testing, but also offer or make available genetic counseling when patients and families receive the results of testing.

The ASHG describes genetic counselors as health professionals who have obtained graduate (post-baccalaureate) degrees and experience in the areas of medical genetics and counseling. Most genetic counselors enter the field from a variety of disciplines, including biology, genetics, nursing, psychology, public health, and social work. They are members of interdisciplinary health care teams, providing education and support to families who have members with birth defects or genetic disorders and to families and individuals who may be at risk for a variety of inherited conditions. Genetic counselors identify families at risk, research and interpret information about the disorder, analyze inheritance patterns and risks of recurrence, and review available options with individuals and families. They also serve as patient advocates and as consultants to other health care professionals.

Genetic Testing

Definition

A genetic test examines the genetic information contained inside a person's cells, called DNA, to determine if that person has or will develop a certain disease or could pass a disease to his or her offspring. Genetic tests also determine whether or not couples are at a higher risk than the general population for having a child affected with a genetic disorder.

Purpose

Some families or ethnic groups have a higher incidence of a certain disease than the population as a whole. For example, individuals from Eastern European, Ashkenazi Jewish descent are at higher risk for carrying genes for rare conditions that occur much less frequently in populations from other parts of the world. Before having a child, a couple from such a family or ethnic group may want to know if their child would be at risk of having that disease. Genetic testing for this type of purpose is called genetic screening.

During pregnancy, a baby's cells can be studied for certain genetic disorders or chromosomal problems such as Down syndrome. Chromosome testing is most commonly offered when the mother is 35 years or older at the time of delivery. When there is a family medical history of a genetic disease or there are individuals in a family affected with developmental and physical delays, genetic testing also may be offered during pregnancy. Genetic testing during pregnancy is called prenatal diagnosis.

Prior to becoming pregnant, couples who are having difficulty conceiving a child or who have suffered multiple miscarriages may be tested to see if a genetic cause can be identified.

A genetic disease may be diagnosed at birth by doing a physical evaluation of the baby and observing characteristics of the disorder. Genetic testing can help to confirm the diagnosis made by the physical evaluation. In addition, genetic testing is used routinely on all newborns to screen for certain genetic diseases that can affect a newborn baby's health shortly after birth.

There are several genetic diseases and conditions in which the symptoms do not occur until adulthood. One such example is Huntington's disease. This is a serious disorder affecting the way in which individuals walk, talk and function on a daily basis. Genetic testing may be able to determine if someone at risk will in fact develop the disease.

Genetic testing may take on new emphasis in the near future as genetic research continues to advance. In April 2003, the Human Genome Project announced completion of mapping the entire human genetic makeup. The project identified more than 1,400 disease genes and completed study of the ethical, legal, and social issues raised by this expanded knowledge of human genetics. As knowledge expands and scientists discover more methods to identify and treat various diseases, people will face more difficult decisions about their own genetic information. In fact, the amount of genetic testing was increasing internationally in 2003, especially for rare diseases.

Some genetic defects may make a person more susceptible to certain types of cancer. Testing for these defects can help predict a person's risk. Other types of genetic tests help diagnose and predict and monitor the course of certain kinds of cancer, particularly leukemia and lymphoma.

Precautions

Because genetic testing is not always accurate and because there are many concerns surrounding insurance and employment discrimination for the individual receiving a genetic test, genetic counseling should always be performed prior to genetic testing. A genetic counselor is an individual with a master's degree in genetic counseling. A medical geneticist is a physician specializing and board certified in genetics.

A genetic counselor reviews the person's family history and medical records and the reason for the test. The counselor explains the likelihood that the test will detect all possible causes of the disease in question (known as the sensitivity of the test), and the likelihood that the disease will develop if the test is positive (known as the positive predictive value of the test).

Learning about the disease in question, the benefits and risks of both a positive and a negative result, and what treatment choices are available if the result is positive, will help prepare the person undergoing testing. During the genetic counseling session, the individual interested in genetic testing will be asked to consider how the test results will affect his or her life, family, and future decisions.

After this discussion, the person should have the opportunity to indicate in writing that he or she gave informed consent to have the test performed, verifying that the counselor provided complete and understandable information.

Description

Genes and chromosomes

Deoxyribonucleic acid (DNA) is a long molecule made up of two strands of genetic material coiled around each other in a unique double helix structure. This structure was discovered in 1953 by Francis Crick and James Watson.

DNA is found in the nucleus, or center, of most cells (Some cells, such as a red blood cell, don't have a nucleus). Each person's DNA is a unique blueprint, giving instructions for a person's physical traits, such as eye color, hair texture, height, and susceptibility to disease. DNA is organized into structures called chromosomes.

The instructions are contained in DNA's long strands as a code spelled out by pairs of bases, which are four chemicals that make up DNA. The bases occur as pairs because a base on one strand lines up with and is bound to a corresponding base on the other strand. The order of these bases form DNA's code. The order of the bases on a DNA strand is important to ensuring that we are not affected with any genetic diseases. When the bases are out of order, or missing, our cells often do not produce important proteins which can lead to a genetic disorder. While our genes are found in every cell of our body, not every gene is functioning all of the time. Some genes are turned on during critical points in development and then remain silent for the rest of our lives. Other genes remain active all of our lives so that our cells can produce important proteins that help us digest food properly or fight off the common cold.

The specific order of the base pairs on a strand of DNA is important in order for the correct protein to be produced. A grouping of three base pairs on the DNA strand is called a codon. Each codon, or three base pairs, comes together to spell a word. A string of many codons together can be thought of as a series of words all coming together to make a sentence. This sentence is what instructs our cells to make a protein that helps our bodies function properly.

Our DNA strands, containing a hundred to several thousand copies of genes, are found on structures called chromosomes. Each cell typically has 46 chromosomes arranged into 23 pairs. Each parent contributes one chromosome to each pair. The first 22 pairs are called autosomal chromosomes, or non-sex chromosomes, and are assigned a number from 1-22. The last pair are the sex chromosomes and include the X and Y chromosomes. If a child receives an X chromosome from each parent, the child is female. If a child receives an X from the mother, and a Y from the father, the child is male.

Just as each parent contributes one chromosome to each pair, so each parent contributes one gene from each chromosome. The pair of genes produces a specific trait in the child. In autosomal dominant conditions, it takes only one copy of a gene to influence a specific trait. The stronger gene is called dominant; the weaker gene, recessive. Two copies of a recessive gene are needed to control a trait while only one copy of a dominant gene is needed. Our sex chromosomes, the X and the Y, also contain important genes. Some genetic diseases are caused by missing or altered genes on one of the sex chromosomes. Males are most often affected by sex chromosome diseases when they inherit an X chromosome with missing or mutated genes from their mother.

TYPES OF GENETIC MUTATIONS. Genetic disease results from a change, or mutation, in a chromosome or in one or several base pairs on a gene. Some of us inherit these mutations from our parents, called hereditary or germline mutations, while other mutations can occur spontaneously, or for the first time in an affected child. For many of the adult on-set diseases, genetic mutations can occur over the lifetime of the individual. This is called acquired or somatic mutations and these occur while the cells are making copies of themselves or dividing in two. There may be some environmental effects, such as radiation or other chemicals, which can contribute to these types of mutations as well.

There are a variety of different types of mutations that can occur in our genetic code to cause a disease. And for each genetic disease, there may be more than one type of mutation to cause the disease. For some genetic diseases, the same mutation occurs in every individual affected with the disease. For example, the most common form of dwarfism, called achondroplasia, occurs because of a single base pair substitution. This same mutation occurs in all individuals affected with the disease. Other genetic diseases are caused by different types of genetic mutations that may occur anywhere along the length of a gene. For example, cystic fibrosis, the most common genetic disease in the caucasian population is caused by over hundreds of different mutations along the gene. Individual families may carry the same mutation as each other, but not as the rest of the population affected with the same genetic disease.

Some genetic diseases occur as a result of a larger mutation which can occur when the chromosome itself is either rearranged or altered or when a baby is born with more than the expected number of chromosomes. There are only a few types of chromosome rearrangements which are possibly hereditary, or passed on from the mother or the father. The majority of chromosome alterations where the baby is born with too many chromosomes or missing a chromosome, occur sporadically or for the first time with a new baby.

The type of mutation that causes a genetic disease will determine the type of genetic test to be performed. In some situations, more than one type of genetic test will be performed to arrive at a diagnosis. The cost of genetic tests vary: chromosome studies can cost hundreds of dollars and certain gene studies, thousands. Insurance coverage also varies with the company and the policy. It may take several days or several weeks to complete a test. Research testing where the exact location of a gene has not yet been identified, can take several months to years for results.

Types of Genetic Testing

Direct DNA mutation analysis

Direct DNA sequencing examines the direct base pair sequence of a gene for specific gene mutations. Some genes contain more than 100,000 bases and a mutation of any one base can make the gene nonfunctional and cause disease. The more mutations possible, the less likely it is for a test to detect all of them. This test usually is done on white blood cells from a person's blood but also can be performed on other tissues. There are different ways in which to perform direct DNA mutation analysis. When the specific genetic mutation is known, it is possible to perform a complete analysis of the genetic code, also called direct sequencing. There are several different lab techniques used to test for a direct mutation. One common approach begins by using chemicals to separate DNA from the rest of the cell. Next, the two strands of DNA are separated by heating. Special enzymes (called restriction enzymes) are added to the single strands of DNA and then act like scissors, cutting the strands in specific places. The DNA fragments are then sorted by size through a process called electrophoresis. A special piece of DNA, called a probe, is added to the fragments. The probe is designed to bind to specific mutated portions of the gene. When bound to the probe, the mutated portions appear on x-ray film with a distinct banding pattern.

Indirect DNA Testing

Family linkage studies are done to study a disease when the exact type and location of the genetic alteration is not known, but the general location on the chromosome has been identified. These studies are possible when a chromosome marker has been found associated with a disease. Chromosomes contain certain regions that vary in appearance between individuals. These regions are called polymorphisms and do not cause a genetic disease to occur. If a polymorphism is always present in family members with the same genetic disease, and absent in family members without the disease, it is likely that the gene responsible for the disease is near that polymorphism. The gene mutation can be indirectly detected in family members by looking for the polymorphism.

To look for the polymorphism, DNA is isolated from cells in the same way it is for direct DNA mutation analysis. A probe is added that will detect the large polymorphism on the chromosome. When bound to the probe, this region will appear on x-ray film with a distinct banding pattern. The pattern of banding of a person being tested for the disease is compared to the pattern from a family member affected by the disease.

Linkage studies have disadvantages not found in direct DNA mutation analysis. These studies require multiple family members to participate in the testing. If key family members choose not to participate, the incomplete family history may make testing other members useless. The indirect method of detecting a mutated gene also causes more opportunity for error.

Chromosome analysis

Various genetic syndromes are caused by structural chromosome abnormalities. To analyze a person's chromosomes, his or her cells are allowed to grow and multiply in the laboratory until they reach a certain stage of growth. The length of growing time varies with the type of cells. Cells from blood and bone marrow take one to two days; fetal cells from amniotic fluid take seven to 10 days.

When the cells are ready, they are placed on a microscope slide using a technique to make them burst open, spreading their chromosomes. The slides are stained: the stain creates a banding pattern unique to each chromosome. Under a microscope, the chromosomes are counted, identified, and analyzed based on their size, shape, and stained appearance.

A karyotype is the final step in the chromosome analysis. After the chromosomes are counted, a photograph is taken of the chromosomes from one or more cells as seen through the microscope. Then the chromosomes are cut out and arranged side-by-side with their partner in ascending numerical order, from largest to smallest. The karyotype is done either manually or using a computer attached to the microscope. Chromosome analysis also is called cytogenetics.

Applications for Genetic Testing

Newborn screening

Genetic testing is used most often for newborn screening. Every year, millions of newborn babies have their blood samples tested for potentially serious genetic diseases.

Carrier testing

An individual who has a gene associated with a disease but never exhibits any symptoms of the disease is called a carrier. A carrier is a person who is not affected by the mutated gene he or she possesses, but can pass the gene to an offspring. Genetic tests have been developed that tell prospective parents whether or not they are carriers of certain diseases. If one or both parents are a carrier, the risk of passing the disease to a child can be predicted.

To predict the risk, it is necessary to know if the gene in question is autosomal or sex-linked. If the gene is carried on any one of chromosomes 1-22, the resulting disease is called an autosomal disease. If the gene is carried on the X or Y chromosome, it is called a sex-linked disease.

Sex-linked diseases, such as the bleeding condition hemophilia, are usually carried on the X chromosome. A woman who carries a disease-associated mutated gene on one of her X chromosomes, has a 50% chance of passing the gene to her son. A son who inherits that gene will develop the disease because he does not have another normal copy of the gene on a second X chromosome to compensate for the mutated copy. A daughter who inherits the disease associated mutated gene from her mother on one of her X chromosomes will be at risk for having a son affected with the disease.

The risk of passing an autosomal disease to a child depends on whether the gene is dominant or recessive. A prospective parent carrying a dominant gene has a 50% chance of passing the gene to a child. A child needs to receive only one copy of the mutated gene to be affected by the disease.

If the gene is recessive, a child needs to receive two copies of the mutated gene, one from each parent, to be affected by the disease. When both prospective parents are carriers, their child has a 25% chance of inheriting two copies of the mutated gene and being affected by the disease; a 50% chance of inheriting one copy of the mutated gene, and being a carrier of the disease but not affected; and a 25% chance of inheriting two normal genes. When only one prospective parent is a carrier, a child has a 50% chance of inheriting one mutated gene and being an unaffected carrier of the disease, and a 50% chance of inheriting two normal genes.

Cystic fibrosis is a disease that affects the lungs and pancreas and is discovered in early childhood. It is the most common autosomal recessive genetic disease found in the caucasian population: one in 25 people of Northern European ancestry are carriers of a mutated cystic fibrosis gene. The gene, located on chromosome 7, was identified in 1989.

The gene mutation for cystic fibrosis is detected by a direct DNA test. More than 600 mutations of the cystic fibrosis gene have been found; each of these mutations causes the same disease. Tests are available for the most common mutations. Tests that check for 86 of the most common mutations in the Caucasian population will detect 90% of carriers for cystic fibrosis. (The percentage of mutations detected varies according to the individual's ethnic background). If a person tests negative, it is likely, but not guaranteed that he or she does not have the gene. Both prospective parents must be carriers of the gene to have a child with cystic fibrosis.

Tay-Sachs disease, also autosomal recessive, affects children primarily of Ashkenazi Jewish descent. Children with this disease die between the ages of two and five. This disease was previously detected by looking for a missing enzyme. The mutated gene has now been identified and can be detected using direct DNA mutation analysis.

Presymptomatic testing

Not all genetic diseases show their effect immediately at birth or early in childhood. Although the gene mutation is present at birth, some diseases do not appear until adulthood. If a specific mutated gene responsible for a late-onset disease has been identified, a person from an affected family can be tested before symptoms appear.

Huntington's disease is one example of a late-onset autosomal dominant disease. Its symptoms of mental confusion and abnormal body movements do not appear until middle to late adulthood. The chromosome location of the gene responsible for Huntington's chorea was located in 1983 after studying the DNA from a large Venezuelan family affected by the disease. Ten years later the gene was identified. A test now is available to detect the presence of the expanded base pair sequence responsible for causing the disease. The presence of this expanded sequence means the person will develop the disease.

The specific genetic cause of Alzheimer's disease, another late onset disease, is not as clear. Although many cases appear to be inherited in an autosomal dominant pattern, many other cases exist as single incidents in a family. Like Huntington's, symptoms of mental deterioration first appear in adulthood. Genetic research has found an association between this disease and genes on four different chromosomes. The validity of looking for these genes in a person without symptoms or without family history of the disease is still being studied.

CANCER SUSCEPTIBILITY TESTING. Cancer can result from an inherited (germline) mutated gene or a gene that mutated sometime during a person's lifetime (acquired mutation). Some genes, called tumor suppressor genes, produce proteins that protect the body from cancer. If one of these genes develops a mutation, it is unable to produce the protective protein. If the second copy of the gene is normal, its action may be sufficient to continue production, but if that gene later also develops a mutation, the person is vulnerable to cancer. Other genes, called oncogenes, are involved in the normal growth of cells. A mutation in an oncogene can cause too much growth, the beginning of cancer.

Direct DNA tests currently are available to look for gene mutations identified and linked to several kinds of cancer. People with a family history of these cancers are those most likely to be tested. If one of these mutated genes is found, the person is more susceptible to developing the cancer. The likelihood that the person will develop the cancer, even with the mutated gene, is not always known because other genetic and environmental factors also are involved in the development of cancer.

Cancer susceptibility tests are most useful when a positive test result can be followed with clear treatment options. In families with familial polyposis of the colon, testing a child for a mutated APC gene can reveal whether or not the child needs frequent monitoring for the disease. In 2003, reports showed that genetic testing for high-risk colon cancer patients has improved risk assessment. In families with potentially fatal familial medullary thyroid cancer or multiple endocrine neoplasia type 2, finding a mutated RET gene in a child provides the opportunity for that child to have preventive removal of the thyroid gland. In the same way, MSH1 and MSH2 mutations can reveal which members in an affected family are vulnerable to familiar colorectal cancer and would benefit from aggressive monitoring.

In 1994, a mutation linked to early-onset familial breast and ovarian cancer was identified. BRCA1 is located on chromosome 17. Women with a mutated form of this gene have an increased risk of developing breast and ovarian cancer. A second related gene, BRCA2, was later discovered. Located on chromosome 13, it also carries increased risk of breast and ovarian cancer. Although both genes are rare in the general population, they are slightly more common in women of Ashkenazi Jewish descent.

When a woman is found to have a mutation of one of these genes, the likelihood that she will get breast or ovarian cancer increases, but not to 100%. Other genetic and environmental factors influence the outcome.

Testing for these genes is most valuable in families where a mutation has already been found. BRCA1 and BRCA2 are large genes; BRCA1 includes 100,000 bases. More than 120 mutations to this gene have been discovered, but a mutation could occur in any one of the bases. Studies show tests for these genes may miss 30% of existing mutations. The rate of missed mutations, the unknown disease likelihood in spite of a positive result, and the lack of a clear preventive response to a positive result, make the value of this test for the general population uncertain.

Prenatal and postnatal chromosome analysis

Chromosome analysis can be done on fetal cells primarily when the mother is age 35 or older at the time of delivery, experienced multiple miscarriages, or reports a family history of a genetic abnormality. Prenatal testing is done on the fetal cells from a chorionic villus sampling (from the baby's developing placenta) at 9-12 weeks or from the amniotic fluid (the fluid surrounding the baby) at 15-22 weeks of pregnancy. Cells from amniotic fluid grow for seven to 10 days before they are ready to be analyzed. Chorionic villi cells have the potential to grow faster and can be analyzed sooner.

Chromosome analysis using blood cells is done on a child who is born with or later develops signs of mental retardation or physical malformation. In the older child, chromosome analysis may be done to investigate developmental delays.

Extra or missing chromosomes cause mental and physical abnormalities. A child born with an extra chromosome 21 (trisomy 21) has Down syndrome. An extra chromosome 13 or 18 also produce well known syndromes. A missing X chromosome causes Turner syndrome and an extra X in a male causes Klinefelter syndrome. Other abnormalities are caused by extra or missing pieces of chromosomes. Fragile X syndrome is a sex-linked disease, causing mental retardation in males.

Chromosome material also may be rearranged, such as the end of chromosome 1 moved to the end of chromosome 3. This is called a chromosomal translocation. If no material is added or deleted in the exchange, the person may not be affected. Such an exchange, however, can cause infertility or abnormalities if passed to children.

Evaluation of a man and woman's infertility or repeated miscarriages will include blood studies of both to check for a chromosome translocation. Many chromosome abnormalities are incompatible with life; babies with these abnormalities often miscarrry during the first trimester. Cells from a baby that died before birth can be studied to look for chromosome abnormalities that may have caused the death.

Cancer diagnosis and prognosis

Certain cancers, particularly leukemia and lymphoma, are associated with changes in chromosomes: extra or missing complete chromosomes, extra or missing portions of chromosomes, or exchanges of material (translocations) between chromosomes. Studies show that the locations of the chromosome breaks are at locations of tumor suppressor genes or oncogenes.

Chromosome analysis on cells from blood, bone marrow, or solid tumor helps diagnose certain kinds of leukemia and lymphoma and often helps predict how well the person will respond to treatment. After treatment has begun, periodic monitoring of these chromosome changes in the blood and bone marrow gives the physician information as to the effectiveness of the treatment.

A well-known chromosome rearrangement is found in chronic myelogenous leukemia. This leukemia is associated with an exchange of material between chromosomes 9 and 22. The resulting smaller chromosome 22 is called the Philadelphia chromosome.

Preparation

Most tests for genetic diseases of children and adults are done on blood. To collect the 5-10 mL of blood needed, a healthcare worker draws blood from a vein in the inner elbow region. Collection of the sample takes only a few minutes.

Prenatal testing is done either on amniotic fluid or a chorionic villus sampling. To collect amniotic fluid, a physician performs a procedure called amniocentesis. An ultrasound is done to find the baby's position and an area filled with amniotic fluid. The physician inserts a needle through the woman's skin and the wall of her uterus and withdraws 5-10 mL of amniotic fluid. Placental tissue for a chorionic villus sampling is taken through the cervix. Each procedure takes approximately 30 minutes. A 2003 study comparing the two tests reported that chorionic villus sampling resulted in fewer cases of pregnancy loss, amniotic fluid leakage, and birth defects.

Bone marrow is used for chromosome analysis in a person with leukemia or lymphoma. The person is given local anesthesia. Then the physician inserts a needle through the skin and into the bone (usually the sternum or hip bone). One-half to 2 mL of bone marrow is withdrawn. This procedure takes approximately 30 minutes.

Aftercare

After blood collection the person can feel discomfort or bruising at the puncture site or may become dizzy or faint. Pressure to the puncture site until the bleeding stops reduces bruising. Warm packs to the puncture site relieve discomfort.

Chorionic villus sampling, amniocentesis and bone marrow procedures are done under a physician's supervision. The person is asked to rest after the procedure and is watched for weakness and signs of bleeding.

Risks

Collection of amniotic fluid and chorionic villus sampling, have the risk of miscarriage, infection, and bleeding; the risks are higher for the chorionic villus sampling. Because of the potential risks for miscarriage, 0.5% following the amniocentesis and 1% following the chorionic villus sampling procedure, both of these prenatal tests are offered to couples, but not required. A woman should tell her physician immediately if she has cramping, bleeding, fluid loss, an increased temperature, or a change in the baby's movement following either of these procedures.

After bone marrow collection, the puncture site may become tender and the person's temperature may rise. These are signs of a possible infection.

Genetic testing involves other nonphysical risks. Many people fear the possible loss of privacy about personal health information. Results of genetic tests may be reported to insurance companies and affect a person's insurability. Some people pay out-of-pocket for genetic tests to avoid this possibility. Laws have been proposed to deal with this problem. Other family members may be affected by the results of a person's genetic test. Privacy of the person tested and the family members affected is a consideration when deciding to have a test and to share the results.

A positive result carries a psychological burden, especially if the test indicates the person will develop a disease, such as Huntington's chorea. The news that a person may be susceptible to a specific kind of cancer, while it may encourage positive preventive measures, also may negatively shadow many decisions and activities.

A genetic test result may also be inconclusive, meaning no definitive result can be given to the individual or family. This may cause the individual to feel more anxious and frustrated and experience psychological difficulties.

Prior to undergoing genetic testing, individuals need to learn from the genetic counselor the likelihood that the test could miss a mutation or abnormality.

Normal results

A normal result for chromosome analysis is 46, XX or 46, XY. This means there are 46 chromosomes (including two X chromosomes for a female or one X and one Y for a male) with no structural abnormalities. A normal result for a direct DNA mutation analysis or linkage study is no gene mutation found.

There can be some benefits from genetic testing when the individual tested is not found to carry a genetic mutation. Those who learn with certainty they are no longer at risk for a genetic disease may choose not to undergo preventive therapies and may feel less anxious and relieved.

Abnormal results

An abnormal chromosome analysis report will include the total number of chromosomes and will identify the abnormality found. Tests for gene mutations will report the mutations found.

There are many ethical issues to consider with an abnormal prenatal test result. Many of the diseases tested for during a pregnancy cannot be treated or cured. In addition, some diseases tested for during pregnancy may have a late-onset of symptoms or have minimal effects on the affected individual.

Before making decisions based on an abnormal test result, the person should meet again with a genetic counselor to fully understand the meaning of the results, learn what options are available based on the test result, and the risks and benefits of each of those options.

KEY TERMS

Autosomal disease— A disease caused by a gene located on a chromosome other than a sex chromosome (autosomal chromosome).

Carrier— A person who possesses a gene for an abnormal trait without showing signs of the disorder. The person may pass the abnormal gene on to offspring.

Chromosome— A microscopic thread-like structure found within each cell of the body that consists of a complex of proteins and DNA. Humans have 46 chromosomes arranged into 23 pairs. Changes in either the total number of chromosomes or their shape and size (structure) may lead to physical or mental abnormalities.

Deoxyribonucleic acid (DNA)— The genetic material in cells that holds the inherited instructions for growth, development, and cellular functioning.

Dominant gene— A gene, whose presence as a single copy, controls the expression of a trait.

Enzyme— A protein that catalyzes a biochemical reaction or change without changing its own structure or function.

Gene— A building block of inheritance, which contains the instructions for the production of a particular protein, and is made up of a molecular sequence found on a section of DNA. Each gene is found on a precise location on a chromosome.

Karyotype— A standard arrangement of photographic or computer-generated images of chromosome pairs from a cell in ascending numerical order, from largest to smallest.

Mutation— A permanent change in the genetic material that may alter a trait or characteristic of an individual, or manifest as disease, and can be transmitted to offspring.

Positive predictive value (PPV)— The probability that a person with a positive test result has, or will get, the disease.

Recessive gene— A type of gene that is not expressed as a trait unless inherited by both parents.

Sensitivity— The proportion of people with a disease who are correctly diagnosed (test positive based on diagnostic criteria). The higher the sensitivity of a test or diagnostic criteria, the lower the rate of 'false negatives,' people who have a disease but are not identified through the test.

Sex-linked disorder— A disorder caused by a gene located on a sex chromosome, usually the X chromosome.

Resources

PERIODICALS

"Best Early Test." Fit Pregnancy (October-November, 2003): 37.

Bodenhorn, Nancy, and Gerald Lawson. "Genetic Counseling: Implications for Community Counselors." Journal of Counseling and Development (Fall 2003): 497-495.

"Genetic Testing for High-risk Colon Cancer Patients has Improved Risk Assessment." Genomics & Genetics Weekly (August 1, 2003): 18.

"Genetic Testing Increasing Internationally." Health & Medicine Week (September 29, 2003): 283.

Wechsler, Jill. "From Genome Exploration to Drug Development." Pharmaceutical Technology Europe (June 2003): 18-23.

Yan, Hai. "Genetic Testing-Present and Future." Science (September 15, 2000): 1890-1892.

ORGANIZATIONS

Alliance of Genetic Support Groups. 4301 Connecticut Ave. NW, Suite 404, Washington, DC 20008. (202) 966-5557. Fax: (202) 966-8553. 〈http://www.geneticalliance.org〉.

American College of Medical Genetics. 9650 Rockville Pike, Bethesda, MD 20814-3998. (301) 571-1825. 〈http://www.faseb.org/genetics/acmg/acmgmenu.htm〉.

American Society of Human Genetics. 9650 Rockville Pike, Bethesda, MD 20814-3998. (301) 571-1825. 〈http://www.faseb.org/genetics/ashg/ashgmenu.htm〉.

Centers for Disease Control. GDP Office, 4770 Buford Highway NE, Atlanta, GA 30341-3724. (770) 488-3235. 〈http://www.cdc.gov/genetics〉.

March of Dimes Birth Defects Foundation. 1275 Mamaroneck Ave., White Plains, NY 10605. (888) 663-4637. [email protected]. 〈http://www.modimes.org〉.

National Human Genome Research Institute. The National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. (301) 496-2433. 〈http://www.nhgri.nih.gov〉.

National Society of Genetic Counselors. 233 Canterbury Dr., Wallingford, PA 19086-6617. (610) 872-1192. 〈http://www.nsgc.org/GeneticCounselingYou.asp〉.

OTHER

Blazing a Genetic Trail. Online genetic tutorial. 〈http://www.hhmi.org/GeneticTrail/〉.

The Gene Letter. Online newsletter. 〈http://www.geneletter.org〉.

Online Mendelian Inheritance in Man. Online genetic testing information sponsored by National Center for Biotechnology Information. 〈http://www.ncbi.nlm.-nih.gov/Omim/〉.

Understanding Gene Testing. Online brochure produced by the U.S. Department of Health and Human Services. 〈http://www.gene.com/ae/AE/AEPC/NIH/index.html〉.

Genetic Testing

Definition

A genetic test examines the genetic information contained inside a person's cells, called DNA, to determine if that person has or will develop a certain disease or could transmit a disease to a child. Genetic tests also determine whether or not couples are at a higher risk than the general population for having a child affected with a genetic disorder.

Purpose

Some families or ethnic groups have a higher incidence of a certain disease than do the population as a whole. For example, individuals of Eastern European, Ashkenazi Jewish descent are at higher risk for carrying genes for rare conditions such as Tay-Sachs disease (a lipid storage disease) that occur much less frequently in populations from other parts of the world. Before having a child, a couple from such a family or ethnic group may want to know if their child would be at risk of having that disease. Genetic testing for this type of purpose is called genetic screening.

During pregnancy, a baby's cells can be studied for certain genetic disorders or chromosomal problems such as Down syndrome. Chromosome testing is most commonly offered when a mother is 35 years or older at the time of delivery. When there is a family medical history of a genetic disease or there are individuals in a family affected with developmental and physical delays, genetic testing may also be offered during pregnancy. Genetic testing during pregnancy is called prenatal diagnosis.

Prior to becoming pregnant, couples who are having difficulty conceiving a child or who have had multiple miscarriages may be tested to see if a genetic cause can be identified.

A genetic disease may be diagnosed at birth by performing a physical evaluation of the baby and observing characteristics of the disorder. Genetic testing can help to confirm the diagnosis made by a physical evaluation. In addition, biochemical tests (e.g., blood phenylalanine measurement) are performed routinely on all newborns to screen for certain genetic diseases that can affect a newborn baby's health shortly after birth.

There are several genetic diseases and conditions in which the symptoms do not occur until adulthood. One such example is Huntington's disease. This is a serious disorder affecting the way in which individuals walk, talk and function on a daily basis. Genetic testing may be able to determine if someone at risk for the disease will in fact develop the disease.

Some genetic abnormalities may make a person more susceptible to certain types of cancer. Testing for these abnormalities can help predict a person's risk. Other types of genetic tests help diagnose, predict, and monitor the course of certain kinds of cancer, particularly leukemia, lymphoma, and breast cancer.

Precautions

Genetic tests are performed on cells derived from blood, bone marrow, amniotic fluid, or tissues. The health care worker collecting the specimen should observe universal precautions for the prevention of transmission of bloodborne pathogens. Because genetic testing is not always accurate and because there are many concerns surrounding insurance and employment discrimination for an individual receiving a genetic test, genetic counseling should always be performed prior to genetic testing. A genetic counselor is an individual with a master's degree in genetic counseling. A medical geneticist is a physician specializing and board certified in genetics.

A genetic counselor reviews a person's family history and medical records and the reason for the test. The counselor explains the likelihood that the test will detect all possible causes of the disease in question (known as the sensitivity of the test), and the likelihood that the disease will develop if the test is positive (known as the positive predictive value of the test).

Learning about the disease in question, the benefits and risks of both a positive and a negative result, and what treatment choices are available if the result is positive, will help prepare a person undergoing testing. During the genetic counseling session, an individual interested in genetic testing will be asked to consider how the test results may affect his or her life, family, and future decisions.

After this discussion, persons should have the opportunity to indicate, in writing, that they gave informed consent to have the test performed, verifying that the counselor provided complete and understandable information.

Description

Genes and chromosomes

Deoxyribonucleic acid (DNA) is a long molecule made up of two strands of genetic material coiled around each other in a unique double helix structure. Francis Crick and James Watson discovered this structure in 1953. Each strand of DNA consists of a backbone of deoxyribose sugars linked together by phosphate groups. Each sugar is bound (with a covalent bond) to one of four bases, adenine, guanine, thymine or cytosine. The two strands are held together by hydrogen bonds between the bases. Adenine pairs with thymine and guanine pairs with cytosine.

Most of the DNA is found in the nucleus of the cell. Each person's DNA is a unique blueprint, giving instructions for that person's physical traits, such as eye color, hair texture, height, and susceptibility to disease. DNA is organized into structures called chromosomes.

The instructions are contained as a code spelled out by the order of the four bases in each of the strands of DNA. When a base pair is out of order, or is missing, then cells may not produce an important protein or may produce an abnormal protein resulting in a genetic disorder. While genes are found in every nucleated cell of the human body, not every gene is functioning all of the time. Some genes are turned on during critical points in development and then remain silent for the rest of a cell's life. Other genes remain active throughout life so that cells can produce important proteins that help humans digest food properly or fight off the common cold.

In the human genome there are 3.1 billion bases and approximately 100,000 genes. Approximately 95% of DNA consists of non-coding regions called introns. The remaining 5% consists of coding regions called exons, which are the structural units that form genes. Within the exon, the specific order of the base pairs on a strand of DNA dictates the order of amino acids that comprise the protein made when the gene is transcribed and translated. A grouping of three sequential base pairs within the exon is called a codon. Each codon, or triplet, codes for an amino acid. These are added in tandem to form a protein. A string of many codons together can be thought of as a series of words all coming together to make a sentence. This sentence provides the instructions for cells to make a protein that is needed in order for the body to function properly.

Human DNA strands containing a hundred to several thousand genes are found on structures called chromosomes. Each cell typically has 46 chromosomes arranged into 23 pairs. Each parent contributes one chromosome to each pair. The first 22 pairs are called autosomal chromosomes, or non-sex chromosomes and are assigned a number from one to 22. The last pair are the sex chromosomes and include the X and the Y chromosomes. If a child receives an X chromosome from each parent, the child is female. If a child receives an X from the mother, and a Y from the father, the child is male.

Just as each parent contributes one chromosome to each pair, so each parent contributes one gene from each chromosome. The pair of genes produces a specific trait in the child. In autosomal dominant conditions, it takes only one copy of a gene to influence a specific trait. The stronger gene is called dominant; the weaker gene is said to be recessive. Two copies of a recessive gene are needed to express a trait while only one copy of a dominant gene is needed. Some genes, (for example, those coding for blood groups) exhibit codominant expression. In this case, both genes are active and produce traits. Human sex chromosomes, the X and the Y also contain important genes. Some genetic diseases are caused by missing or altered genes on one of the sex chromosomes. Males are most often affected by sex chromosome diseases when they inherit an X chromosome with missing or mutated genes from their mothers.

Types of genetic mutations

Genetic disease results from a change, or mutation, in a chromosome or in one of several base pairs on a gene. Some people inherit such mutations from their parents. These are called hereditary or germline mutations. Other mutations can occur spontaneously, or for the first time in an affected child. For many of the adult on-set diseases, genetic mutations can occur over the lifetime of an individual. These are called acquired or somatic mutations and occur while cells are making copies of themselves or dividing in two. Environmental effects, such as radiation or other chemicals, can also contribute to these types of mutations.

There are a variety of different types of mutations that can occur in the genetic code to cause disease. For each genetic disease, there may be more than one type of mutation to cause the disease. For some genetic diseases, the same mutation occurs in every individual affected with the disease. For example, the most common form of dwarfism, called achondroplasia, is caused by a single base pair substitution. This same mutation occurs in all individuals affected with the disease. Other genetic diseases are caused by different types of genetic mutations that may occur anywhere along the length of a gene. For example, cystic fibrosis, the most common genetic disease in the Caucasian population, is caused by any of several hundred different gene mutations. Individual families may carry the same mutation as each other, but not as the rest of the population affected with the same genetic disease.

Some genetic diseases occur as a result of a larger mutation that can occur when a chromosome itself is either rearranged or altered or when a baby is born with more than the expected number of chromosomes. There are only a few types of chromosome rearrangements that are possibly hereditary, or passed on from the mother or the father. The majority of chromosome alterations, where a baby is born with too many chromosomes or missing a chromosome, occur sporadically or for the first time with a new baby.

The type of mutation that causes a genetic disease will determine the type of genetic test to be performed. In some situations, more than one type of genetic test will be performed to arrive at a diagnosis. The cost of genetic tests vary. Chromosome studies can cost hundreds of dollars and some gene studies, thousands. Insurance coverage also varies among companies and the policies. It may take several days or several weeks to complete a test. Research testing, when the exact location of a gene has not yet been identified, can require several months to years for results.

Types of genetic testing

DIRECT DNA MUTATION ANALYSIS. Direct DNA sequencing examines the base pair sequence of a gene for specific mutations. Some genes contain more than 100,000 bases and a mutation of any one base can make the gene nonfunctional and cause disease. As the number of possible mutations increases, the less likely a test is able to detect all of them. DNA sequencing is a research tool that is used to identify the order of bases in cloned genes. Base sequencing identifies the specific mutation. Once this is known a DNA probe can be made that recognizes the mutation. DNA probes typically contain 20-60 bases to insure that they bind only to the specific mutation site. DNA testing for disease genes is usually performed on white blood cells but can also be performed on other tissues.

There are several different lab techniques used to test for a direct mutation. One early approach begins by using chemicals to separate DNA from the rest of the cell. Special enzymes (called restriction enzymes) are added to the DNA. The enzymes then function like scissors and cut the strands in specific places. Next, the DNA fragments are separated by size using a process called electrophoresis. The fragments are treated chemically to separate them into single-stranded fragments and transferred to a nylon filter, a process called Southern blotting. The DNA probe is added to the fragments. The probe is designed to bind to the specific mutated portion of the gene. The probe is labeled with a radioactive isotope. When the probe hybridizes with the target sequence containing the mutation, it will render this piece of DNA on the filter radioactive. The radioactivity will expose a piece of x-ray film layered over the nylon filter, and the mutation will appear as a band in the expected location.

When only small quantities of DNA are available, as in prenatal diagnosis, the target DNA from the fetal cells must first be amplified. This is accomplished by a method known as the polymerase chain reaction. This procedure can copy a specific sequence of DNA that frames the gene to be tested. Up to one million copies can be made in as little as two hours.

INDIRECT DNA TESTING. Family linkage studies are performed to study a disease when the exact type and location of the genetic alteration is not known but the general location on the chromosome has been identified. These studies are possible when a chromosome marker has been found to be associated with a disease. Chromosomes contain certain regions that vary in appearance between individuals. These regions are called polymorphisms and do not cause a genetic disease to occur. If a polymorphism is always present in family members with the same genetic disease, and absent in family members without the disease, it is likely that the gene responsible for the disease is near that polymorphism. The gene mutation can be indirectly detected in family members by looking for the polymorphism.

To look for the polymorphism, DNA is isolated from cells in the same way it is for direct DNA mutation analysis. A restriction enzyme known to cut the DNA at the site where the polymorphism occurs is added. If the polymorphism is present, the restriction enzyme will not recognize the site and will not cut the DNA there. This results in a larger size fragment of DNA. If sufficient DNA is present, the fragments can be separated by electrophoresis, and the DNA bands are stained with ethidium bromide to visualize the position of the bands. If the amount of DNA is small, the double stranded fragments can be separated, and a DNA probe can be used to determine whether the polymorphism is present. The pattern of banding of a person being tested for the disease is compared to the pattern from a family member affected by the disease.

Linkage studies have disadvantages not found in direct DNA mutation analysis. These studies require multiple family members to participate in the testing. If key family members choose not to participate, the incomplete family history may make testing other members useless. The indirect method of detecting a mutated gene also causes more opportunity for error, and many disease genes are not associated with polymorphisms.

CHROMOSOME ANALYSIS. Various genetic syndromes are caused by structural chromosome abnormalities. To analyze a person's chromosomes, cells are allowed to grow and multiply in the laboratory until they reach a certain stage of growth. The length of growing time varies with the type of cells. Cells from blood and bone marrow take one to two days, fetal cells from amniotic fluid require seven to 10 days.

When the cells are ready, they are placed on a microscope slide using a technique to make them swell, allowing easier visualization of chromosomes. The slides are stained. The stain creates a banding pattern unique to each chromosome. Under a microscope, the chromosomes are counted, identified, and analyzed based on their size, shape, and stained appearance.

A karyotype is the final step in the chromosome analysis. After the chromosomes are counted, a photograph is taken of the chromosomes from one or more cells as seen through a microscope. Then the chromosomes are cut out and arranged side-by-side with their partner in ascending numerical order, from largest to smallest. The karyotype is done either manually or using a computer attached to the microscope. Chromosome analysis is also called cytogenetics.

Applications for genetic testing

NEWBORN SCREENING. Genetic testing is used most often for newborn screening. Every year, millions of newborn babies have their blood samples tested for potentially serious genetic diseases. Phenylketonuria is the genetic disease test most commonly performed.

CARRIER TESTING. An individual who has a gene associated with a disease but never exhibits any symptoms of the disease is called a carrier. A carrier is a person who is not affected by a possessed mutated gene, but can pass the gene to an offspring. Genetic tests have been developed that tell prospective parents whether or not they are carriers of certain diseases. If one or both parents are carriers, the risk of passing the disease to a child can be predicted.

To predict the risk, it is necessary to know if the gene in question is autosomal or sex-linked. If the gene is carried on any one of chromosomes one through 22, the resulting disease is called an autosomal disease. If the gene is carried on the X or Y chromosome, it is called a sex-linked disease.

Sex-linked diseases, such as the bleeding condition hemophilia, are usually carried on the X chromosome. A woman who carries a disease-associated mutated gene on one of her X chromosomes has a 50% chance of passing that gene to her son. A son who inherits that gene will develop the disease because he does not have another normal copy of the gene on a second X chromosome to compensate for the abnormal copy. A daughter who inherits the disease associated abnormal gene from her mother, on one of her X chromosomes will become a carrier and be at risk for having a son affected with the disease.

The risk of passing an autosomal disease to a child depends on whether the gene is dominant or recessive. A prospective parent carrying a dominant gene, has a 50% chance of passing the gene to a child. A child needs to receive only one copy of the mutated gene to be affected by the disease.

If the gene is recessive, a child needs to receive two copies of the mutated gene, one from each parent, to be affected by the disease. When both prospective parents are carriers, their child has a 25% chance of inheriting two copies of the mutated gene and being affected by the disease; a 50% chance of inheriting one copy of the mutated gene and being a carrier of the disease; and a 25% chance of inheriting two normal genes. When only one prospective parent is a carrier, a child has a 50% chance of inheriting one mutated gene and being an unaffected carrier of the disease, and a 50% chance of inheriting two normal genes.

Cystic fibrosis is a disease that affects the lungs and pancreas and is discovered in early childhood. It is the most common autosomal recessive genetic disease found in the Caucasian population: one in 25 people of Northern European ancestry are carriers of a mutated cystic fibrosis gene. The gene, located on chromosome seven, was identified in 1989.

The gene mutation for cystic fibrosis is detected by a direct DNA test. Over 600 mutations of the cystic fibrosis gene have been identified. Each of these mutations cause the same disease. Tests are available for the most common mutations. Tests that check for the 86 most common mutations in the Caucasian population will detect 90% of carriers for cystic fibrosis. The percentage of mutations detected varies according to an individual's ethnic background. When persons test negative, it is likely, but not guaranteed that they do not have the gene. Both prospective parents must be carriers of the gene to conceive a child with cystic fibrosis.

Tay-Sachs disease, also autosomal recessive, affects children primarily of Ashkenazi Jewish descent. Children with this disease die between the ages of two and five. This disease was previously detected by looking for a missing enzyme. The mutated gene has now been identified and can be detected using direct DNA mutation analysis.

PRESYMPTOMATIC TESTING. Not all genetic diseases show their effect immediately at birth or early in childhood. Although the gene mutation is present at birth, some diseases do not appear until adulthood. If a specific mutated gene responsible for a late-onset disease has been identified, a person from an affected family can be tested before symptoms appear.

Huntington's disease is one example of a late-onset autosomal dominant disease. Its symptoms of mental confusion and abnormal body movements do not appear until middle to late adulthood. The chromosome location of the gene responsible for Huntington's chorea was located in 1983 after studying the DNA from a large Venezuelan family affected by the disease. Ten years later, the gene was identified. A test is now available to detect the presence of the expanded base pair sequence responsible for causing the disease. The presence of this expanded sequence means a person will develop the disease.

Another late onset condition, Alzheimer's disease, is not as well understood as Huntington's disease. The specific genetic cause of Alzheimer's disease is not as clear. Although many cases appear to be inherited in an autosomal dominant pattern, many cases exist as single incidents in a family. Like Huntington's disease, symptoms of mental deterioration first appear in adulthood. Genetic research has found an association between this disease and genes on four different chromosomes. The validity of looking for these genes in a person without symptoms or without family history of the disease is still being studied.

CANCER SUSCEPTIBILITY TESTING. Cancer can result from an inherited (germline) mutated gene or a gene that mutated sometime during a person's lifetime (acquired mutation). Some genes, called tumor suppressor genes, produce proteins that protect the body from cancer. If one of these genes develops a mutation, it is unable to produce the protective protein. If the second copy of the gene is normal, its action may be sufficient to continue production, but if that gene later develops a mutation, the person is vulnerable to cancer. Other genes, called oncogenes, are involved in the normal growth of cells. A mutation in an oncogene can cause too much growth, the beginning of cancer.

Direct DNA tests are currently available to look for gene mutations identified and linked to several kinds of cancer. People with a family history of these cancers are those most likely to be tested. If one of these mutated genes is found, the person is more susceptible to developing the cancer. The likelihood that the person will develop the cancer, even with the mutated gene, is not always known because other genetic and environmental factors are also involved in the development of cancer.

Cancer susceptibility tests are most useful when a positive test result can be followed with clear treatment options. In families with familial polyposis of the colon, testing a child for a mutated APC gene can reveal whether or not the child needs frequent monitoring for the disease. In families with potentially fatal familial medullary thyroid cancer or multiple endocrine neoplasia type two, finding a mutated RET gene in a child provides the opportunity for that child to have preventive removal of the thyroid gland. In the same way, MSH1 and MSH2 mutations can reveal which members in an affected family are vulnerable to familiar colorectal cancer and would benefit from aggressive monitoring.

In 1994, a mutation linked to early-onset familial breast and ovarian cancer was identified. BRCA1 is located on chromosome 17. Women with a mutated form of this gene have an increased risk of developing breast and ovarian cancer. A second related gene, BRCA2, was later discovered. Located on chromosome 13, it also carries increased risk of breast and ovarian cancer. Although both genes are rare in the general population, they are slightly more common in women of Ashkenazi Jewish descent.

When a woman is found to have a mutation of one of these genes, the likelihood that she will get breast or ovarian cancer increases, but not to 100%. Other genetic and environmental factors may also influence the outcome.

Testing for these genes is most valuable in families where a mutation has already been found. BRCA1 and BRCA2 are large genes; BRCA1 includes 100,000 bases. More than 120 mutations to this gene have been discovered, but a mutation could occur in any one of the bases. Studies show tests for these genes may miss 30% of existing mutations. The rate of missed mutations, the unknown disease likelihood in spite of a positive result, and the lack of a clear preventive response to a positive result, make the value of this test for the general population uncertain.

PRENATAL AND POSTNATAL CHROMOSOME ANALYSIS. Chromosome analysis is usually performed on fetal cells when a mother will be age 35 or older at the time of delivery, has experienced multiple miscarriages, or reports a family history of a genetic abnormality. Prenatal testing is done on the fetal cells from a chorionic villus sampling (from the baby's developing placenta) at nine to 12 weeks of gestation or from the amniotic fluid (the fluid surrounding the baby) at 15-18 weeks of pregnancy. Cells from amniotic fluid usually must grow for seven to 10 days before they are ready to be analyzed. Chorionic villi cells have the potential to grow faster and can be analyzed sooner.

Chromosome analysis using blood cells is performed for a child who is born with or later develops signs of mental retardation or physical malformation. In an older child, chromosome analysis may be requested to investigate developmental delays.

Extra or missing chromosomes cause mental and physical abnormalities. A child born with an extra chromosome 21 (trisomy 21) has Down syndrome. An extra chromosome 13 or 18 also produces well-known syndromes. A missing X chromosome causes Turner syndrome and an extra X in a male causes Klinefelter syndrome. Other abnormalities are caused by extra or missing pieces of chromosomes. Fragile X syndrome is a sex-linked disease causing mental retardation in males.

Chromosome material may also be rearranged, such as the end of chromosome one moved to the end of chromosome three. This is called a chromosomal translocation. If no material is added or deleted in the exchange, a person may not be affected. Such an exchange, however, can cause infertility or abnormalities if passed to children.

Evaluation of a couple's infertility or repeated miscarriages will include blood studies of both to check for a chromosome translocation. Many chromosome abnormalities are incompatible with life. Fetuses with these abnormalities often spontaneously abort during the first trimester. Cells from a fetus that died before birth can be studied to look for chromosome abnormalities that may have caused the death.

CANCER DIAGNOSIS AND PROGNOSIS. Certain cancers, particularly leukemia and lymphoma, are associated with changes in chromosomes: extra or missing complete chromosomes, extra or missing portions of chromosomes, or exchanges of material (translocations) between chromosomes. Studies show that the locations of the chromosome breaks are at locations of tumor suppressor genes or oncogenes.

Chromosome analysis on cells from blood, bone marrow, or a solid tumor help to diagnose certain kinds of leukemia and lymphoma and often help predict how well a person will respond to treatment. After treatment has begun, periodic monitoring of these chromosome changes in the blood and bone marrow gives a physician information as to the effectiveness of the treatment.

A well-known chromosome rearrangement is found in chronic myelogenous leukemia. This leukemia is associated with an exchange of material between chromosomes 9 and 22. The resulting smaller chromosome 22 is called the Philadelphia chromosome.

Preparation

Most tests for genetic diseases of children and adults are done using blood. To collect the 5-10 mL of blood needed, a health care worker draws blood from a vein in the inner elbow region. Collection of the sample takes only a few minutes.

Prenatal testing is done either on amniotic fluid or a chorionic villus sampling. To collect amniotic fluid, a physician performs a procedure called amniocentesis. An ultrasound is done to find the baby's position and an area filled with amniotic fluid. The physician inserts a needle through the woman's skin and the wall of her uterus and withdraws 5-10 mL of amniotic fluid. Placental tissue for a chorionic villus sampling is taken through the cervix. Each procedure takes approximately 30 minutes.

Bone marrow is used for chromosome analysis in a person with leukemia or lymphoma. The person is given local anesthesia. Then the physician inserts a needle through the skin and into the bone (usually the hip bone). A small sample of bone marrow is withdrawn. This procedure takes approximately 30 minutes.

Aftercare

After blood collection, a person may feel discomfort or bruising at the puncture site or may become dizzy or faint. Pressure to the puncture site until the bleeding stops reduces bruising. Warm packs to the puncture site relieve discomfort.

The chorionic villi sampling, amniocentesis, and bone marrow procedures are all done under a physician's supervision. A person to be tested is asked to rest after the procedure and is watched for weakness and signs of bleeding.

Complications

Collection of amniotic fluid and chorionic villi share the risks of miscarriage, infection, and bleeding. The risks are higher for the chorionic villi sampling. Because of the potential risks for miscarriage, 0.5% following the amniocentesis and 1% following chorionic villi sampling procedure, both of these prenatal tests are offered to couples, but are not required. A woman should tell her physician immediately if she has cramping, bleeding, fluid loss, an increased temperature, or a change in the baby's movement following either of these procedures.

After bone marrow collection, the puncture site may become tender and a person's temperature may rise. These are signs of a possible infection.

Genetic testing involves other nonphysical risks. Many people fear the possible loss of privacy about personal health information. Results of genetic tests may be reported to insurance companies and affect a person's insurability. Some people pay out-of-pocket for genetic tests to avoid this possibility. Laws have been proposed to deal with this problem. Other family members may be affected by the results of a person's genetic test. Privacy of the person tested and the family members affected is a consideration when deciding to have a test and to share the results.

A positive result carries a psychological burden, especially if the test indicates a person will develop a disease, such as Huntington's chorea. The news that a person may be susceptible to a specific kind of cancer, while it may encourage positive preventive measures, may also negatively shadow many future decisions and activities.

A genetic test result may also be inconclusive, meaning no definitive result can be given to the individual or family. This may cause an individual to feel more anxious and frustrated and experience psychological difficulties.

Prior to undergoing genetic testing, genetic counselors should inform individuals to be tested about the likelihood that the test could miss a mutation or abnormality.

Results

Normal results

A normal result for chromosome analysis is 46, XX or 46, XY. This means there are 46 chromosomes (including two X chromosomes for a female or one X and one Y for a male) with no structural abnormalities. A normal result for a direct DNA mutation analysis or linkage study is the absence of gene mutations.

There can be some benefits from genetic testing when an individual tested is not found to carry a genetic mutation. Those who learn with great certainty they are no longer at risk for a genetic disease may choose not to undergo prophylactic therapies and may feel less anxious and relieved.

Abnormal results

An abnormal chromosome analysis report will include the total number of chromosomes and will identify the abnormality found. Tests for gene mutations will report the mutations found.

There are many ethical issues to consider with an abnormal prenatal test result. Many of the diseases tested for during a pregnancy cannot be treated or cured. In addition, some diseases tested for during pregnancy may have a late-onset of symptoms or have minimal effects on an affected individual.

Before making decisions based on an abnormal test result, a person should meet again with a genetic counselor to fully understand the meaning of the results, learn what options are available based on the test result, and what are the risks and benefits of each of those options.

Health care team roles

A family physician or obstetrician often makes an initial recommendation for genetic counseling. A physician specially training in the technique will perform bone marrow aspiration, amniocentesis, or chorionic villus sampling. A nurse or phlebotomist usually collects blood samples. A cytogenetic technologist or clinical laboratory scientist/medical technologist will perform the DNA test depending upon the type of testing requested. A pathologist or geneticist processes and interprets findings of tests. Genetic counselors interpret test results and discuss options. Members of the clergy often assist people who have been tested to make decisions based on test results.

KEY TERMS

Autosomal disease— A disease caused by a gene located on chromosomes 1 through 22.

Carrier— A person who has a disease-causing gene but does not show symptoms of that disease.

Chromosome— Structures made up of DNA, on which the genes are located.

DNA (Deoxyribonucleic acid)— A long molecule made up of two strands of material coiled around each other in a unique double helix. DNA contains the blueprint for all of a person's traits.

Dominant gene— A gene, whose presence as a single copy, controls the expression of a trait.

Enzyme— A protein produced in a cell.

Gene— A grouping of base pairs that give instruction for a specific trait.

Karyotype— Visual comparison of chromosomes arranged side-by-side with their partner in ascending numerical order, from largest to smallest.

Mutation— Any change in the sequence of DNA.

Positive predictive value (PPV)— The probability that a person with a positive test result has, or will develop a disease or condition.

Recessive gene— A gene that must be present in both copies of the gene pair to allow the expression of a trait.

Sensitivity— The likelihood that a negative test means a person will not have the disease or a mutation.

Sex-linked disorder— A disorder caused by a gene located on a sex chromosome, usually the X chromosome.

Resources

BOOKS

Beaudet, Arthur L. "Genetics and disease." In Harrison's Principles of Internal Medicine, 14th ed., edited by Anthony S. Fauci, et al. New York: McGraw-Hill, 1998, 365-395.

Gelehrter, Thomas D., Francis S. Collins, and David Ginsburg. Principles of Medical Genetics, 2nd ed. Baltimore: Williams and Wilkins, 1998.

Grody, Wayne W., and Walter W. Noll. "Molecular Diagnosis of Genetic Diseases. In Clinical Diagnosis and Management by Laboratory Methods, 19th ed. edited by John B. Henry. Philadelphia: Saunders, 1996, pp.1374-1389.

Needleman, Robert D. "Fetal growth and development." In Nelson Textbook of Pediatrics, 16th ed. edited by Richard E. Behrman et al., Philadelphia: Saunders, 2000, pp.27-30.

PERIODICALS

Brunger, J.W., Matthews, A.L., Smith, R.H., Robin, N.H. "Genetic testing and genetic counseling for deafness: the future is here." Laryngoscope 111, no. 4 Pt 1 (2001): 715-718.

D'Souza, G., McCann, C.L., Hedrick, J., Fairley, C., Nagel, H.L., Kushner, J.D., Kessel, R. "Tay-Sachs disease carrier screening: a 21-year experience." Genetic Testing 4, no. 3 (2001): 257-263.

Gilbert, F. "Chromosome 10." Genetic Testing 5, no.1 (2001): 69-82.

Hakimian, R. "Disclosure of Huntington's disease to family members: the dilemma of known but unknowing parties." Genetic Testing 4, no. 4 (2000): 359-364.

Harris, R.A., Washington, A.E., Feeny, D., Kuppermann, M. "Decision analysis of prenatal testing for chromosomal disorders: what do the preferences of pregnant women tell us?" Genetic Testing 5, no. 1 (2001): 23-32.

Wallerstein, R., Starkman, A., Jansen, V. "Carrier screening for Gaucher disease in couples of mixed ethnicity." Genetic Testing 5, no.1 (2001): 61-64.

ORGANIZATIONS

American Board of Genetic Counseling. 9650 Rockville Pike, Bethesda, MD 20814-3998. (301) 571-1825. Fax: (301) 571-1895. 〈http://www.faseb.org/genetics/abgc/abgcmenu.htm〉.

American College of Obstetricians and Gynecologists. 409 12th St., S.W., PO Box 96920, Washington, D.C. 20090-6920. 〈http://www.acog.org/〉.

American Infertility Association. 666 Fifth Avenue, Suite 278, New York, NY 10103. (718) 621-5083. 〈http://www.americaninfertility.org/〉. [email protected].

American Public Health Association. 800 I Street, NW, Washington, DC 20001-3710. (202) 777-2742. Fax: (202) 777-2534. 〈http://www.apha.org/〉. [email protected].

American Society for Reproductive Medicine. 1209 Montgomery Highway, Birmingham, AL 35216-2809. (205) 978-5000. 〈http://www.asrm.com〉.

OTHER

American College of Physicians. 〈http://www.acponline.org/journals/annals/15jun97/currgene.htm〉.

Lawrence Livermore Laboratory. 〈http://www.lbl.gov/Education/ELSI/Frames/genetic-testing-f.html〉.

National Cancer Institute. 〈http://rex.nci.nih.gov/PREV_AND_ERLYDETC/PREVED_MAIN_DOC.html〉 and 〈http://cancernet.nci.nih.gov〉 and 〈http://cancernet.nci.nih.gov/genetics/breast.htm〉.

National Human Genome Research Institute. 〈http://www.nhgri.nih.gov/ELSI/TFGT_final/〉.

National Institute of Health. 〈http://odp.od.nih.gov/consensus/cons/106/106_intro.htm〉 and 〈http://rex.nci.nih.gov/NCI_PUB_INDEX/GENBRST/INDEX.HTM〉 and 〈http://www.med.jhu.edu/tfgtelsi/promoting/prinrec.html〉.

National Library of Medicine. 〈http://www.nlm.nih.gov/medlineplus/genetictestingcounseling.html〉 and 〈http://www.nlm.nih.gov/pubs/cbm/cystic_fibrosis.html〉.

Genetic testing

Definition

A genetic test examines the genetic information contained inside a person's cells, called DNA , to determine if that person has or will develop a certain disease or could pass a disease to his or her offspring. Genetic tests also determine whether or not couples are at a higher risk than the general population for having a child affected with a genetic disorder.

Purpose

Some families or ethnic groups have a higher incidence of a certain disease than does the population as a whole. For example, individuals from Eastern European, Ashkenazi Jewish descent are at higher risk for carrying genes for rare conditions that occur much less frequently in populations from other parts of the world. Before having a child, a couple from such a family or ethnic group may want to know if their child would be at risk of having that disease. Genetic testing for this type of purpose is called genetic screening.

During pregnancy, the baby's cells can be studied for certain genetic disorders or chromosomal problems such as Down syndrome . Chromosome testing is most commonly offered when the mother is 35 years or older at the time of delivery. When there is a family medical history of a genetic disease or there are individuals in a family affected with developmental and physical delays, genetic testing may also be offered during pregnancy. Genetic testing during pregnancy is called prenatal diagnosis.

Prior to becoming pregnant, couples who are having difficulty conceiving a child or who have suffered multiple miscarriages may be tested to see if a genetic cause can be identified.

A genetic disease may be diagnosed at birth by doing a physical evaluation of the baby and observing characteristics of the disorder. Genetic testing can help to confirm the diagnosis made by the physical evaluation. In addition, genetic testing is used routinely on all newborns to screen for certain genetic diseases which can affect a newborn baby's health shortly after birth.

There are several genetic diseases and conditions in which the symptoms do not occur until adulthood. One such example is Huntington's disease. This is a serious disorder affecting the way in which individuals walk, talk, and function on a daily basis. Genetic testing may be able to determine if someone at risk for the disease will in fact develop the disease.

Some genetic defects may make a person more susceptible to certain types of cancer . Testing for these defects can help predict a person's risk. Other types of genetic tests help diagnose and predict and monitor the course of certain kinds of cancer, particularly leukemia and lymphoma.

Precautions

Because genetic testing is not always accurate and because there are many concerns surrounding insurance and employment discrimination for the individual receiving a genetic test, genetic counseling should always be performed prior to genetic testing. A genetic counselor is an individual with a master's degree in genetic counseling. A medical geneticist is a physician specializing and board certified in genetics.

A genetic counselor reviews the person's family history and medical records and the reason for the test. The counselor explains the likelihood that the test will detect all possible causes of the disease in question (known as the sensitivity of the test), and the likelihood that the disease will develop if the test is positive (known as the positive predictive value of the test).

Learning about the disease in question, the benefits and risks of both a positive and a negative result, and what treatment choices are available if the result is positive, will help prepare the person undergoing testing. During the genetic counseling session, the individual interested in genetic testing will be asked to consider how the test results will affect his or her life, family, and future decisions.

After this discussion, the person should have the opportunity to indicate in writing that he or she gave informed consent to have the test performed, verifying that the counselor provided complete and understandable information.

Background

Genes and chromosomes

Deoxyribonucleic acid (DNA) is a long molecule made up of two strands of genetic material coiled around each other in a unique double helix structure. This structure was discovered in 1953 by Francis Crick and James Watson.

DNA is found in the nucleus, or center, of most cells (some cells, such as a red blood cell, don't have a nucleus). Each person's DNA is a unique blueprint, giving instructions for a person's physical traits, such as eye color, hair texture, height, and susceptibility to disease. DNA is organized into structures called chromosomes .

The instructions are contained in DNA's long strands as a code spelled out by pairs of bases, which are four chemicals that make up DNA. The bases occur as pairs because a base on one strand lines up with and is bound to a corresponding base on the other strand. The order of these bases form DNA's code. The order of the bases on a DNA strand is important to ensuring that a person is not affected with any genetic disorders. When the bases are out of order or missing, cells often may not produce important proteins; this can lead to a genetic disorder. While genes are found in every cell of the body, not every gene is functioning all of the time. Some genes are turned on during critical points in development and then remain silent for the rest of the individual's life. Other genes always remain active so that cells can produce important proteins such as those that help digest food properly or fight off the common cold.

The specific order of the base pairs on a strand of DNA is important in order for the correct protein to be produced. A grouping of three base pairs on the DNA strand is called a codon. Each codon, or three base pairs, comes together to spell a word. A string of many codons together can be thought of as a series of words all coming together to make a sentence. This sentence is what instructs cells to make a protein that helps bodies function properly.

DNA strands containing a hundred to several thousand copies of genes are found on structures called chromosomes. Each cell typically has 46 chromosomes arranged into 23 pairs. Each parent contributes one chromosome to each pair. The first 22 pairs are called autosomal chromosomes, or non-sex chromosomes and are assigned a number from 1–22. The last pair are the sex chromosomes and include the X and the Y chromosomes. If a child receives an X chromosome from each parent, the child is female. If a child receives an X from the mother, and a Y from the father, the child is male.

Just as each parent contributes one chromosome to each pair, so each parent contributes one gene from each chromosome. The pair of genes produces a specific trait in the child. In autosomal dominant conditions, it takes only one copy of a gene to influence a specific trait. The stronger gene is called dominant; the weaker gene is called recessive. Two copies of a recessive gene are needed to control a trait, while only one copy of a dominant gene is needed. Our sex chromosomes, the X and the Y, also contain important genes. Some genetic diseases are caused by missing or altered genes on one of the sex chromosomes. Males are most often affected by sex chromosome diseases when they inherit an X chromosome with missing or mutated genes from their mother.

Types of genetic mutations

Genetic disease results from a change, or mutation, in a chromosome or in one or several base pairs on a gene. Some of us inherit these mutations from our parents, called hereditary or germline mutations, while other mutations can occur spontaneously, or for the first time in an affected child. For many of the adult on-set diseases, genetic mutations can occur over the lifetime of the individual. This is called acquired or somatic mutations, and these occur while the cells are making copies of themselves or dividing in two. There may be some environmental effects, such as radiation or other chemicals, that can contribute to these types of mutations as well.

There are a variety of different types of mutations that can occur in the genetic code to cause a disease. And for each genetic disease, there may be more than one type of mutation to cause the disease. For some genetic diseases, the same mutation occurs in every individual affected with the disease. For example, the most common form of dwarfism, called achondroplasia , occurs because of a single base pair substitution. This same mutation occurs in all individuals affected with the disease. Other genetic diseases are caused by different types of genetic mutations that may occur anywhere along the length of a gene. For example, cystic fibrosis , the most common genetic disease in the caucasian population, is caused by hundreds of different mutations along the gene. Individual families may carry the same mutation as each other, but not as the rest of the population affected with the same genetic disease.

Some genetic diseases occur as a result of a larger mutation that can occur when the chromosome itself is either rearranged or altered or when a baby is born with more than the expected number of chromosomes. There are only a few types of chromosome rearrangements that are possibly hereditary, or passed on from the mother or the father. The majority of chromosome alterations occur sporadically or for the first time with a new baby.

The type of mutation that causes a genetic disease will determine the type of genetic test to be performed. In some situations, more than one type of genetic test will be performed to arrive at a diagnosis. The cost of genetic tests vary: chromosome studies can cost hundreds of dollars and certain gene studies can cost thousands. Insurance coverage also varies with the company and the policy. It may take several days or several weeks to complete a test. Research testing where the exact location of a gene has not yet been identified can take several months or years for results.

Types of genetic testing

Direct DNA mutation analysis

Direct DNA sequencing examines the direct base pair sequence of a gene for specific gene mutations. Some genes contain more than 100,000 bases, and a mutation of any one base can make the gene nonfunctional and cause disease. The more mutations possible, the less likely it is for a test to detect all of them. This test is usually done on white blood cells from a person's blood, but can also be performed on other tissues. There are different ways in which to perform direct DNA mutation analysis. When the specific genetic mutation is known, it is possible to perform a complete analysis of the genetic code, also called direct sequencing. There are several different lab techniques used to test for a direct mutation. One common approach begins by using chemicals to separate DNA from the rest of the cell. Next, the two strands of DNA are separated by heating. Special enzymes (called restriction enzymes) are added to the single strands of DNA; they act like scissors and cut the strands in specific places. The DNA fragments are then sorted by size through a process called electrophoresis. A special piece of DNA, called a probe, is added to the fragments. The probe is designed to bind to specific mutated portions of the gene. When bound to the probe, the mutated portions appear on x-ray film with a distinct banding pattern.

Indirect DNA Testing

Family linkage studies are done to study a disease when the exact type and location of the genetic alteration is not known, but the general location on the chromosome has been identified. These studies are possible when a chromosome marker has been found associated with a disease. Chromosomes contain certain regions that vary in appearance between individuals. These regions are called polymorphisms and do not cause a genetic disease to occur. If a polymorphism is always present in family members with the same genetic disease, and absent in family members without the disease, it is likely that the gene responsible for the disease is near that polymorphism. The gene mutation can be indirectly detected in family members by looking for the polymorphism.

To look for the polymorphism, DNA is isolated from cells in the same way it is for direct DNA mutation analysis. A probe is added that will detect the large polymorphism on the chromosome. When bound to the probe, this region will appear on x-ray film with a distinct banding pattern. The pattern of banding of a person being tested for the disease is compared to the pattern from a family member affected by the disease.

Linkage studies have disadvantages not found in direct DNA mutation analysis. These studies require multiple family members to participate in the testing. If key family members choose not to participate, the incomplete family history may make testing other members useless. The indirect method of detecting a mutated gene also causes more opportunity for error.

Chromosome analysis

Various genetic syndromes are caused by structural chromosome abnormalities. To analyze a person's chromosomes, his or her cells are allowed to grow and multiply in the laboratory until they reach a certain stage of growth. The length of growing time varies with the type of cells. Cells from blood and bone marrow take 1–2 days; fetal cells from amniotic fluid take 7–10 days.

When the cells are ready, they are placed on a microscope slide using a technique to make them burst open, spreading their chromosomes. The slides are stained: the stain creates a banding pattern unique to each chromosome. Under a microscope, the chromosomes are counted, identified, and analyzed based on their size, shape, and stained appearance.

A karyotype is the final step in the chromosome analysis. After the chromosomes are counted, a photograph is taken of the chromosomes from one or more cells as seen through the microscope. Then the chromosomes are cut out and arranged side-by-side with their partner in ascending numerical order, from largest to smallest. The karyotype is done either manually or using a computer attached to the microscope. Chromosome analysis is also called cytogenetics.

Applications for genetic testing

Newborn screening

Genetic testing is used most often for newborn screening. Every year, millions of newborn babies have their blood samples tested for potentially serious genetic diseases.

Carrier testing

An individual who has a gene associated with a disease but never exhibits any symptoms of the disease is called a carrier. A carrier is a person who is not affected by the mutated gene he or she possesses, but can pass the gene to an offspring. Genetic tests have been developed that tell prospective parents whether or not they are carriers of certain diseases. If one or both parents are a carrier, the risk of passing the disease to a child can be predicted.

To predict the risk, it is necessary to know if the gene in question is autosomal or sex-linked. If the gene is carried on any one of chromosomes 1–22, the resulting disease is called an autosomal disease. If the gene is carried on the X or Y chromosome, it is called a sex-linked disease.

Sex-linked diseases, such as the bleeding condition hemophilia , are usually carried on the X chromosome. A woman who carries a disease-associated gene on one of her X chromosomes has a 50% chance of passing that gene to her son. A son who inherits that gene will develop the disease because he does not have another normal copy of the gene on a second X chromosome to compensate for the abnormal copy. A daughter who inherits the disease-associated gene from her mother will be at risk for having a son affected with the disease.

The risk of passing an autosomal disease to a child depends on whether the gene is dominant or recessive. A prospective parent carrying a dominant gene has a 50% chance of passing the gene to a child. A child needs to receive only one copy of the mutated gene to be affected by the disease.

If the gene is recessive, a child needs to receive two copies of the mutated gene, one from each parent, to be affected by the disease. When both parents are carriers, their child has a 25% chance of inheriting two copies of the mutated gene and being affected by the disease; a 50% chance of inheriting one copy of the mutated gene, and being a carrier of the disease but not affected; and a 25% chance of inheriting two normal genes. When only one parent is a carrier, a child has a 50% chance of inheriting one mutated gene and being an unaffected carrier of the disease, and a 50% chance of inheriting two normal genes.

Cystic fibrosis is a disease that affects the lungs and pancreas and is discovered in early childhood. It is the most common autosomal recessive genetic disease found in the caucasian population: one in 25 people of Northern European ancestry are carriers of a mutated cystic fibrosis gene. The gene, located on chromosome 7, was identified in 1989.

The gene mutation for cystic fibrosis is detected by a direct DNA test. Over 600 mutations of the cystic fibrosis gene have been found; each of these mutations cause the same disease. Tests are available for the most common mutations. Tests that check for the 86 of the most common mutations in the Caucasian population will detect 90% of carriers for cystic fibrosis. (The percentage of mutations detected varies according to the individual's ethnic background). If a person tests negative, it is likely, but not guaranteed that he or she does not have the gene. Both parents must be carriers of the gene to have a child with cystic fibrosis.

Tay-Sachs disease , also autosomal recessive, affects children primarily of Ashkenazi Jewish descent. Children with this disease die between the ages of two and five. This disease was previously detected by looking for a missing enzyme. The mutated gene has now been identified and can be detected using direct DNA mutation analysis.

Presymptomatic testing

Not all genetic diseases show their effect immediately at birth or early in childhood. Although the gene mutation is present at birth, some diseases do not appear until adulthood. If a specific mutated gene responsible for a late-onset disease has been identified, a person from an affected family can be tested before symptoms appear.

Huntington disease is one example of a late-onset autosomal dominant disease. Its symptoms of mental confusion and abnormal body movements do not appear until middle to late adulthood. The chromosome location of the gene responsible for Huntington's chorea was located in 1983 after studying the DNA from a large Venezuelan family affected by the disease. Ten years later, the gene was identified. A test is now available to detect the presence of the expanded base pair sequence responsible for causing the disease. The presence of this expanded sequence means the person will develop the disease.

Another late onset disease, Alzheimer's, does not have as well a understood genetic cause as Huntington's disease. The specific genetic cause of Alzheimer disease is not as clear. Although many cases appear to be inherited in an autosomal dominant pattern, many cases exist as single incidents in a family. Like Huntington's, symptoms of mental deterioration first appear in adulthood. Genetic research has found an association between this disease and genes on four different chromosomes. The validity of looking for these genes in a person without symptoms or without family history of the disease is still being studied.

CANCER SUSCEPTIBILITY TESTING Cancer can result from an inherited (germline) mutated gene or a gene that mutated sometime during a person's lifetime (acquired mutation). Some genes, called tumor suppressor genes, produce proteins that protect the body from cancer. If one of these genes develops a mutation, it is unable to produce the protective protein. If the second copy of the gene is normal, its action may be sufficient to continue production, but if that gene later also develops a mutation, the person is vulnerable to cancer. Other genes, called oncogenes, are involved in the normal growth of cells. A mutation in an oncogene can cause too much growth, which is the beginning of cancer.

Direct DNA tests are currently available to look for gene mutations identified and linked to several kinds of cancer. People with a family history of these cancers are those most likely to be tested. If one of these mutated genes is found, the person is more susceptible to developing the cancer. The likelihood that the person will develop the cancer, even with the mutated gene, is not always known because other genetic and environmental factors are also involved in the development of cancer.

Cancer susceptibility tests are most useful when a positive test result can be followed with clear treatment options. In families with familial polyposis of the colon, testing a child for a mutated APC gene can reveal whether or not the child needs frequent monitoring for the disease. In families with potentially fatal familial medullary thyroid cancer or multiple endocrine neoplasia type 2, finding a mutated RET gene in a child provides the opportunity for that child to have preventive removal of the thyroid gland. In the same way, MSH1 and MSH2 mutations can reveal which members in an affected family are vulnerable to familiar colorectal cancer and would benefit from aggressive monitoring.

In 1994, a mutation linked to early-onset familial breast and ovarian cancer was identified. BRCA1 is located on chromosome 17. Women with a mutated form of this gene have an increased risk of developing breast and ovarian cancer. A second related gene, BRCA2, was later discovered. Located on chromosome 13, it also carries increased risk of breast and ovarian cancer. Although both genes are rare in the general population, they are slightly more common in women of Ashkenazi Jewish descent.

When a woman is found to have a mutation in one of these genes, the likelihood that she will get breast or ovarian cancer increases, but not to 100%. Other genetic and environmental factors influence the outcome.

Testing for these genes is most valuable in families where a mutation has already been found. BRCA1 and BRCA2 are large genes; BRCA1 includes 100,000 bases. More than 120 mutations to this gene have been discovered, but a mutation could occur in any one of the bases. Studies show tests for these genes may miss 30% of existing mutations. The rate of missed mutations, the unknown disease likelihood in spite of a positive result, and the lack of a clear preventive response to a positive result make the value of this test for the general population uncertain.

Prenatal and postnatal chromosome analysis

Chromosome analysis is performed on fetal cells primarily when the mother is age 35 or older at the time of delivery, has experienced multiple miscarriages, or reports a family history of a genetic abnormality. Prenatal testing is done on the fetal cells from a chorionic villus sampling (from the baby's developing placenta) at 10–12 weeks or from the amniotic fluid (the fluid surrounding the baby) at 16–18 weeks of pregnancy. Cells from amniotic fluid grow for seven to 10 days before they are ready to be analyzed. Chorionic villi cells have the potential to grow faster and can be analyzed sooner.

Chromosome analysis using blood cells is done on a child who is born with or later develops signs of mental retardation or physical malformation. In the older child, chromosome analysis may be done to investigate developmental delays.

Extra or missing chromosomes cause mental and physical abnormalities. A child born with an extra chromosome 21 (trisomy 21) has Down syndrome. An extra chromosome 13 or 18 also produce well known syndromes. A missing X chromosome causes Turner syndrome and an extra X in a male causes Klinefelter syndrome . Other abnormalities are caused by extra or missing pieces of chromosomes. Fragile X syndrome is a sex-linked disease that causes mental retardation in males.

Chromosome material may also be rearranged, such as the end of chromosome 1 moving to the end of chromosome 3. This is called a chromosomal translocation. If no material is added or deleted in the exchange, the person may not be affected. Such an exchange, however, can cause infertility or abnormalities if passed to children.

Evaluation of a man and woman's infertility or repeated miscarriages will include blood studies of both to check for a chromosome translocation. Many chromosome abnormalities are incompatible with life; babies with these abnormalities often miscarrry during the first trimester. Cells from a baby that died before birth can be studied to look for chromosome abnormalities that may have caused the death.

Cancer diagnosis and prognosis

Certain cancers, particularly leukemia and lymphoma, are associated with changes in chromosomes: extra or missing complete chromosomes, extra or missing portions of chromosomes, or exchanges of material (translocations) between chromosomes. Studies show that the locations of the chromosome breaks are at locations of tumor suppressor genes or oncogenes.

Chromosome analysis on cells from blood, bone marrow, or solid tumor helps diagnose certain kinds of leukemia and lymphoma and often helps predict how well the person will respond to treatment. After treatment has begun, periodic monitoring of these chromosome changes in the blood and bone marrow gives the physician information as to the effectiveness of the treatment.

A well-known chromosome rearrangement is found in chronic myelogenous leukemia. This leukemia is associated with an exchange of material between chromosomes 9 and 22. The resulting smaller chromosome 22 is called the Philadelphia chromosome.

Preparation

Most tests for genetic diseases of children and adults are done on blood. To collect the 5–10 mL of blood needed, a healthcare worker draws blood from a vein in the inner elbow region. Collection of the sample takes only a few minutes.

Prenatal testing is done either on amniotic fluid or a chorionic villus sampling. To collect amniotic fluid, a physician performs a procedure called amniocentesis . An ultrasound is done to find the baby's position and an area filled with amniotic fluid. The physician inserts a needle through the woman's skin and the wall of her uterus and withdraws 5–10 mL of amniotic fluid. Placental tissue for a chorionic villus sampling is taken through the cervix. Each procedure takes approximately 30 minutes.

Bone marrow is used for chromosome analysis in a person with leukemia or lymphoma. The person is given local anesthesia. Then the physician inserts a needle through the skin and into the bone (usually the sternum or hip bone). One-half to 2 mL of bone marrow is withdrawn. This procedure takes approximately 30 minutes.

Aftercare

After blood collection the person can feel discomfort or bruising at the puncture site or may become dizzy or faint. Pressure to the puncture site until the bleeding stops reduces bruising. Warm packs to the puncture site relieve discomfort.

The chorionic villus sampling, amniocentesis, and bone marrow procedures are all done under a physician's supervision. The person is asked to rest after the procedure and is watched for weakness and signs of bleeding.

Risks

Collection of amniotic fluid and chorionic villus sampling, have the risk of miscarriage, infection, and bleeding; the risks are higher for the chorionic villus sampling. Because of the potential risks for miscarriage, 0.5% following the amniocentesis and 1% following the chorionic villus sampling procedure, both of these prenatal tests are offered to couples, but not required. A woman should tell her physician immediately if she has cramping, bleeding, fluid loss, an increased temperature, or a change in the baby's movement following either of these procedures.

After bone marrow collection, the puncture site may become tender and the person's temperature may rise. These are signs of a possible infection.

Genetic testing involves other nonphysical risks. Many people fear the possible loss of privacy about personal health information. Results of genetic tests may be reported to insurance companies and affect a person's insurability. Some people pay out-of-pocket for genetic tests to avoid this possibility. Laws have been proposed to deal with this problem. Other family members may be affected by the results of a person's genetic test. Privacy of the person tested and the family members affected is a consideration when deciding to have a test and to share the results.

A positive result carries a psychological burden, especially if the test indicates the person will develop a disease, such as Huntington's chorea. The news that a person may be susceptible to a specific kind of cancer, while it may encourage positive preventive measures, may also negatively shadow many decisions and activities.

A genetic test result may also be inconclusive meaning no definitive result can be given to the individual or family. This may cause the individual to feel more anxious and frustrated and experience psychological difficulties.

Prior to undergoing genetic testing, individuals need to learn from the genetic counselor the likelihood that the test could miss a mutation or abnormality.

Normal results

A normal result for chromosome analysis is 46, XX or 46, XY. This means there are 46 chromosomes (including two X chromosomes for a female or one X and one Y for a male) with no structural abnormalities. A normal result for a direct DNA mutation analysis or linkage study is no gene mutation found.

There can be some benefits from genetic testing when the individual tested is not found to carry a genetic mutation. Those who learn with great certainty they are no longer at risk for a genetic disease may choose not to undergo prophylactic therapies and may feel less anxious and relieved.

Abnormal results

An abnormal chromosome analysis report will include the total number of chromosomes and will identify the abnormality found. Tests for gene mutations will report the mutations found.

There are many ethical issues to consider with an abnormal prenatal test result. Many of the diseases tested for during a pregnancy cannot be treated or cured. In addition, some diseases tested for during pregnancy may have a late-onset of symptoms or have minimal effects on the affected individual.

Before making decisions based on an abnormal test result, the person should meet again with a genetic counselor to fully understand the meaning of the results, learn what options are available based on the test result, and what are the risks and benefits of each of those options.

Resources

BOOKS

Berg, Paul, and Maxine Singer. Dealing with Genes: TheLanguage of Heredity. Mill Valley, CA: University Science Books, 1992.

Farkas, Daniel H. DNA Simplified: The Hitchhiker's Guide to DNA. Washington, DC: American Association of Clinical Chemistry Press, 1996.

Gelehrter, Thomas D., Francis S. Collins, and David Ginsburg. Principles of Medical Genetics. 2nd ed. Baltimore: Williams and Wilkins, 1998.

Grody, Wayne W., and Walter W. Noll. "Molecular Diagnosis of Genetic Diseases." In Clinical Diagnosis and Management by Laboratory Methods, edited by John B. Henry. 19th ed. Philadelphia: W. B. Saunders Company, 1996, pp. 1374-1389.

Holtzman, Neil A., and Michael S. Watson, eds. Promoting Safe and Effective Genetic Testing in the United States. Final Report of the Task Force on Genetic Testing. National Institutes of Health-Department of Energy Working Group on Ethical, Legal, and Social Implications of Human Genome Research, 1997.

Motulsky, Arno G., Richard A. King, and Jerome I. Rotter. The Genetic Basis of Common Diseases. New York: Oxford University Press, 1992.

Mueller, Robert F., and Ian D. Young. Emery's Elements of Medical Genetics. 9th ed. New York and Edinburgh: Churchill Livingstone, 1995.

Watson, James D. The Double Helix. New York: Atheneum, 1968.

PERIODICALS

Auxter, Sue. "Genetic Information—What Should be Regulated?" Clinical Laboratory News (December 1997): 9-11.

Biesecker, Barbara Bowles. "Genetic Susceptibility Testing for Breast and Ovarian Cancer: A Progress Report." Journal of the American Medical Women's Association (Winter 1997): 22-27.

Fink, Leslie, and Francis S. Collins. "The Human Genome Project: View From the National Institutes of Health." Journal of the American Medical Women's Association (Winter 1997): 4-7, 15.

Holtzman, Neil A., et al. "Predictive Genetic Testing: From Basic Research to Clinical Practice." Science (October 24, 1997): 602-605.

Karnes, Pamela S. "Ordering and Interpreting DNA Tests." Mayo Clinical Proceedings (December 1996): 1192-1195.

Malone, Kathleen E, et al. "BRCA1 Mutations and Breast Cancer in the General Population." Journal of the American Medical Association (March 25, 1998): 922-929.

McKinnon, Wendy C., et al. "Predisposition Genetic Testing for Late-Onset Disorders in Adults: A Position Paper of the National Society of Genetic Counselors." Journal of the American Medical Association (October 15, 1997): 1217-1221.

Newman, Beth, et al. "Frequency of Breast Cancer Attributable to BRCA1 in a Population-Based Series of American Women." Journal of the American Medical Association (March 25, 1998): 915-921.

Ponder, Bruce. "Genetic Testing for Cancer Risk." Science (November 7, 1997): 1050-1054.

Roses, Allen. "Genetic Testing for Alzheimer Disease. Practical and Ethical Issues." Archives of Neurology (October 1997): 1226-1229.

Whittaker, Lori. "Clinical Applications of Genetic Testing: Implications for the Family Physician." American Family Physician (May 1996): 2077-2084.

Wisecarver, James. "The ABCs of DNA." Laboratory Medicine (January 1997): 48-52.

Yablonsky, Terri. "Genetic Testing Helps Patients and Researchers Predict the Future." Laboratory Medicine (May 1997): 316-321.

Yablonsky, Terri. "Unlocking the Secrets to Disease. Genetic Tests Usher in a New Era in Medicine." Laboratory Medicine (April 1997): 252-256.

Yan, Hai. "Genetic Testing-Present and Future." Science (September 15, 2000): 1890-1892.

ORGANIZATIONS

Alliance of Genetic Support Groups. 4301 Connecticut Ave. NW, Suite 404, Washington, DC 20008. (202) 966-5557. Fax: (202) 966-8553. <http://www.geneticalliance.org>.

American College of Medical Genetics. 9650 Rockville Pike, Bethesda, MD 20814-3998. (301) 571-1825. <http://www.faseb.org/genetics/acmg/acmgmenu.htm>

American Society of Human Genetics. 9650 Rockville Pike, Bethesda, MD 20814-3998. (301) 571-1825. <http://www.faseb.org/genetics/ashg/ashgmenu.htm>.

Centers for Disease Control. GDP Office, 4770 Buford Highway NE, Atlanta, GA 30341-3724. (770) 488-3235. <http://www.cdc.gov/genetics>.

March of Dimes Birth Defects Foundation. 1275 Manaroneck Ave., White Plains, NY 10605. (888) 663-4637. [email protected]. <http://www.modimes.org>.

National Human Genome Research Institute. The National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892. (301) 496-2433. <http://www.nhgri.nih.gov>.

National Society of Genetic Counselors. 233 Canterbury Dr., Wallingford, PA 19086-6617. (610) 872-1192. <http://www.nsgc.org/GeneticCounselingYou.asp>.

OTHER

Blazing a Genetic Trail. Online genetic tutorial. <http://www.hhmi.org/GeneticTrail/>.

The Gene Letter. Online newsletter. <http://www.geneletter.org>.

Online Mendelian Inheritance in Man. Online genetic testing information sponsored by National Center for Biotechnology Information. <http://www.ncbi.nlm.nih.gov/Omim/>.

Understanding Gene Testing. Online brochure produced by the U.S. Department of Health and Human Services. <http://www.gene.com/ae/AE/AEPC/NIH/index.html>.

Katherine S. Hunt, MS