Predictive Testing in Asymptomatic at-Risk Persons
Predictive testing refers to testing of at-risk asymptomatic relatives for a disease-causing gene alteration known to be present in the family to clarify the relatives’ genetic status. Predictive testing is considered presymptomatic when it is certain that all persons who have the altered gene will become symptomatic; it is considered predispositional when penetrance of the gene is reduced (i.e., fewer than 100% of persons with the altered gene will be affected).
For predictive testing to be useful, specificity and positive predictive value must be high. Cost-effectiveness of predictive testing is realized by reducing morbidity and mortality in patients at high risk through (1) early detection and treatment of those with a disease-causing mutation and (2) removal of persons who do not have the mutation from screening protocols, which can be expensive and invasive.13 The disorders in this category are primarily autosomal dominant cancers [see Table 5].14 The distinctions between presymptomatic testing and predispo-sitional testing may not be meaningful when the test is for a mutation associated with a high risk of a disorder for which the general population is at relatively low risk (e.g., hereditary non-polyposis colon cancer [HNPCC]). Molecular genetic testing may not be required to establish the diagnosis in the proband when the disorder is diagnosed by clinical findings. However, testing of an affected family member is required to identify the family-specific mutation for the purpose of testing asymptomatic at-risk relatives. The interest of at-risk relatives in pursuing testing for certain disorders is largely unknown. Hadley and colleagues determined that 51% of first-degree relatives at risk for HNPCC chose to undergo predispositional testing after education and genetic counseling to clarify the risk to their children. The potential effect on health insurance was the single most common reason to decline testing.15
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Table 5 Autosomal Dominant Cancer Syndromes for Which Molecular Genetic Testing Is Available |
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Von Hippel-Lindau disease |
Multiple endocrine neoplasia type 2 |
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Familial adenomatosis polyposis |
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Multiple endocrine neoplasia type 1 |
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Hereditary nonpolyposis colorectal cancer |
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Breast cancer |
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Retinoblastoma |
Melanoma |
Presymptomatic Testing
FAP is an autosomal dominant disorder in which penetrance of the disease-causing gene mutations is 100%. Persons with an APC gene mutation develop adenomas in the colorectum starting at around 16 years of age; in these individuals, the number of adenomas increases to hundreds or thousands, and colorectal cancer develops at a mean age of 39 years. The mean age at death is 42 years in those who go untreated. Early diagnosis via presymptomatic testing reduces morbidity and increases life expectancy through improved surveillance and timely prophylactic colectomy.16 Testing of the APC gene has been shown to be cost-effective when used to identify individuals with the disease-causing APC mutation among at-risk relatives of persons with FAP.17 For years, the mainstay of FAP testing was protein truncation testing (PTT). However, the mutation-detection rate with PTT was only about 80%; the introduction of other test methods—namely, gene sequencing—has increased the mutation-detection rate to 98%.
Predispositional Testing
Retinoblastoma is an example of a disorder in which pene-trance of disease-causing gene mutations is less than 100%. It is caused by mutations in the RB1 gene and can be inherited in an autosomal dominant manner. On average, penetrance of RB1 gene mutations is 90% (i.e., 90% of persons with a germline disease-causing gene mutation will develop retinoblastoma). Cost-effectiveness and improved outcome through the use of predis-positional gene testing have been demonstrated.
Improved outcome is defined as the preservation of vision in at-risk persons through early detection and treatment of ocular tumors, as well as the reduction of morbidity through early detection of nonocular secondary tumors. Early detection of retinoblastoma, while the tumor is small, allows less aggressive treatments that ablate tumors but preserve vision. Before the availability of molecular genetic testing, recurrence-risk counseling for the parents of a child with retinoblastoma or for an adult with retinoblastoma was empirical; counseling was offered on the basis of a positive or negative family history and the presence of a single tumor or multiple tumors. The surveillance protocol is required whether a child has a 6% risk of retinoblastoma (parent or sibling with unilateral, sporadic retinoblastoma), a 40% risk (parent with bilateral retinoblastoma), or a 90% risk (person known to have a germline RB1 mutation).
Sequencing of the RB1 gene detects mutations in over 80% of patients with bilateral or hereditary retinoblastoma.19 Although it is both labor intensive and expensive, gene sequencing is required to establish the molecular diagnosis in a proband. Because of extensive allelic heterogeneity, gene sequencing is the gold standard for detection of RB1 gene mutations. Thus, an adult proband who has had retinoblastoma can undergo RB1 sequence analysis in hopes of identifying the disease-causing mutation so that molecular genetic testing can be used in the management of his or her at-risk offspring. When a germline RB1 mutation is identified in the proband, the offspring can be tested prenatally or at birth to determine the genetic status and whether there is a need for frequent ophthalmologic examinations. When no mutation is identified in the adult, the risk of recurrence is determined empirically, and all offspring must be evaluated regularly by an ophthalmologist. Cost-effectiveness results from not subjecting at-risk children to unnecessary and expensive screening protocols after they test negative for an RB1 germline mutation known to be in their family.13
Counseling-Only Paradigm of Genetic Testing
In the counseling-only paradigm of genetic testing, genetic tests provide persons with information pertaining to disease risk for the purpose of personal decision making, which may include reproductive planning. Issues of test sensitivity, specificity, positive predictive value, and recurrence risk are as relevant in the genetic-counseling paradigm as they are in the medical model, but cost-effectiveness cannot be assessed when testing is used only for personal decision making.
Predictive Testing
Predictive testing used for presymptomatic and predisposi-tional diagnosis of persons at risk for disorders for which no medical interventions exist falls into the genetic-counseling paradigm.
Presymptomatic Diagnosis
Huntington disease is an example of a disorder for which no medical intervention exists. Huntington disease is caused by a CAG trinucleotide repeat expansion in the HD gene. When the CAG expansion is greater than 41 repeats, the penetrance is 100%—that is, all persons with an allele that size will eventually develop Huntington disease. Clarification of genetic status in persons at risk for Huntington disease allows those who have inherited the altered gene and those who have not inherited the altered gene to make informed personal and social decisions. Such decisions may include matters of lifestyle, employment, personal finance, and family planning.
Offering genetic testing to persons at risk for an untreatable, debilitating, fatal disorder requires careful forethought. The molecular diagnosis must always be confirmed in a symptomatic relative before testing can be offered to asymptomatic family members who are at risk. Because no medical intervention can be offered, anticipating and addressing the patient’s psycho-emotional needs are paramount. In a position paper, the National Society of Genetic Counselors emphasized that pretest education and genetic counseling are necessary and that posttest follow-up care must be in place at the time of genetic testing.20 Informing the patient of normal results requires as much preparation and counseling as the relaying of abnormal results. The pretest counseling with the patient must address the positive predictive value of the test, particularly as relating to age at onset and severity of the disease. Greater repeat length is usually associated with earlier onset and more severe disease, but repeat length is not always a predictor of disease onset or severity. Furthermore, patients with an intermediate (or moderately abnormal) number of trinucleotide repeats (i.e., 36 to 40 CAG repeats) may have an indeterminate genetic risk.21 Trinucleotide repeat sizes in this range are considered to have reduced pene-trance because they can cause disease symptoms but do not always do so within a normal life expectancy.2 Thus, the patient who is prepared to hear a negative or a positive result may be in the same uncertain position after testing as before.
Confidentiality of test results and possible discrimination in employment and health insurance coverage22 may be issues for an asymptomatic person who has undergone genetic testing. Predictive genetic testing of asymptomatic at-risk individuals younger than 18 years is strongly discouraged in the genetic-counseling paradigm because of concerns that children will be inappropriately labeled at a time when they cannot be expected to use this information for personal planning or reproductive decision making.23 Diagnostic testing of symptomatic at-risk children is always appropriate.
Predispositional Testing
Predispositional testing for a disorder may not be appropriate when the disorder is highly prevalent in the general population and when the efficacy of measures to reduce risk in persons with disease-predisposing mutations is unknown. Predisposi-tional testing for breast cancer through molecular genetic testing of the genes BRCA1 and BRCA2 can be considered in this category, because the efficacy of measures to reduce cancer risk for individuals with BRCA1 or BRCA2 cancer-predisposing mutations is unknown.24 Furthermore, the high prevalence of breast cancer in the general population means that the rigorous screening for early breast cancer identification recommended for all women cannot be relaxed even when an at-risk woman does not have the BRCA1 or BRCA2 cancer-predisposing mutation identified in a relative.
The dilemma posed by the indeterminate role of BRCA1 and BRCA2 molecular genetic testing in reducing morbidity from breast cancer may turn out to be a recurring issue in genetic testing for common diseases. Breast cancer, like such other common disorders as coronary artery disease, diabetes mellitus, and Alzheimer disease, is regarded as a complex disorder. Complex disorders have multiple etiologies, including heritable single genes, multiple genes with an additive effect that interact with often undefined environmental influences, and acquired environmental or genetic changes. With regard to the overall incidence and morbidity of common diseases, the contribution of single heritable genes is relatively small. For example, breast cancer affects one in nine women, yet only 5% to 10% of cases of breast cancer are attributed to mutations in single genes, including BRCA1 and BRCA2. For a woman whose relatives have a known BRCA1 mutation but who has tested negative for the mutation known to be in the family, the chance of breast cancer developing is still one in nine. She therefore has the same need for close surveillance as women in the general population. Furthermore, detection of a BRCA1 or BRCA2 mutation may not alter the surveillance protocol for breast cancer that is recommended for all women, but it may increase the utilization of mammography, breast self-examination, and oophorectomy.25 The options for breast cancer prevention (e.g., bilateral mastectomy), however, might be considered in a different light for women with a BRCA1 or BRCA2 mutation. Such a prevention strategy should be undertaken with caution because the positive predictive value of a BRCA1 or BRCA2 mutation for the development of breast cancer may not be fully understood and may be biased upward as a result of higher risks and different disease spectra in the high-risk families studied initially.
Serious issues surround testing for BRCA1 and BRCA2 mutations, including appropriate pretest counseling for at-risk women26; appropriate interpretation of positive and negative test results [see Table 1]; the high probability of missense mutations, which are considered indeterminate test results; recommendations for surveillance; and consideration of prophylactic mastectomy [see 12:VIIBreast Cancer].
Carrier Testing
Carrier testing is used primarily to identify carriers of autoso-mal recessive gene mutations and X-linked gene mutations. There are no health-related issues for carriers of an autosomal recessive gene mutation, because all are expected to be asymptomatic. Health-related issues are a concern for a subset of female carriers of an X-linked gene mutation.
Autosomal Recessive Disorder
In cases of autosomal recessive disorders, testing may be used for diagnosis in a symptomatic person, for evaluation of at-risk asymptomatic persons, and for detection of carriers and affected fetuses. Although the gene causing an autosomal recessive disorder may be well characterized, allelic heterogeneity may reduce the sensitivity of molecular genetic testing below levels acceptable for diagnostic use, because it may not be technically feasible to identify all possible disease-causing mutations. In other cases, carrier detection and prenatal testing may be possible only through molecular genetic testing, which may provide information for reproductive decision making that would not otherwise be accessible [see Table 6].
Cystic fibrosis (CF) is an example of such an autosomal recessive disorder. Although discovery of disease-causing mutations in the CFTR gene has led to new tests for CF and redefinition of the disease spectrum, the traditional diagnostic criteria for classic CF are still valid.27 The diagnosis of CF is established when the amount of sweat chloride is greater than 60 mEq/L in the presence of one or more characteristic clinical findings (e.g., typical gastrointestinal or sinopulmonary disease or obstructive azoospermia) or when the family history is positive for the disease. In questionable cases, CFTR molecular genetic testing can be helpful in establishing the diagnosis; however, genotyping alone rarely establishes the diagnosis of CF. Some individuals may have classic CF without a detectable CFTR disease-causing mutation because of allelic heterogeneity. Allelic heterogeneity in CF is extensive, with over 1,000 known disease-causing mutations. The American College of Medical Genetics (ACMG) has recommended a panel of 25 mutations for routine testing in clinical laboratories.28 With the use of this panel, mutation detection rates vary by ethnicity; in white Europeans, 2% of patients with CF have no detectable abnormal alleles and 26% have only one detectable abnormal allele.
When two disease-causing alleles are identified in the proband, both parents can be tested to determine which parent carries which allele. Then, relatives of the mother can be tested for the presence of her disease-causing allele, and relatives of the father can be tested for the presence of his disease-causing allele. Any relative who is found to be a carrier of a disease-causing al-lele has the option of having his or her spouse tested with the clinically available panel of 25 common disease-causing alleles.
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Table 6 Autosomal Recessive Disorders for Which Genetic Molecular Testing Permits Carrier Detection* |
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Cystic fibrosis |
Congenital disorders of |
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Phenylketonuria |
glycosylation |
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21-Hydroxylase deficiency |
p-Thalassemia |
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Tay-Sachs disease |
Canavan disease |
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a-Thalassemia |
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* Partial list of disorders.
Couples in which both partners are carriers of disease-causing alleles have a 25% chance of having a child who inherits two CFTR disease-causing mutations; however, the clinical manifestations and severity of the disease cannot necessarily be predicted by the specific mutations present. When the spouse has no identifiable disease-causing mutations, carrier risk can be calculated using Bayesian analysis.29
The use of DNA-based testing is sensitive in high-risk families. Its use in preconceptual counseling for carrier detection is more complicated because of its low sensitivity; however, such testing was endorsed by the American College of Obstetrics and Gynecology (ACOG), the ACMG, and the National Human Genome Research Institute in 2001.
X-Linked Disorders
In X-linked disorders, molecular genetic testing may be used to diagnose symptomatic males and the occasional symptomatic female and to detect carrier females and affected male fetuses. As in autosomal recessive disorders, molecular genetic testing is often the only option for carrier detection and prenatal testing. Certain factors make the testing for carriers of X-linked disorders more complicated; these include the high frequency of new gene mutations in males who are the only affected family member, as well as the possibility of germline mosaicism in the mother of a male who is the only affected family member. New gene mutations are borne by only a single egg or a single sperm. Germline mosaicism is the presence in some germline cells (eggs or sperm) of a mutation that is not found in other germline cells or somatic cells. Germline mosaicism for an X-linked disorder is surmised to be present in the mother of two or more affected males when there is no evidence of their disease-causing mutation present in her leukocytes.
DMD is an example of an X-linked disorder in which these issues must be considered in genetic counseling. Carrier detection in DMD can be problematic because a significant number of cases of DMD in males are simplex cases (i.e., single occurrences in a family). The following three equally probable possibilities exist for males with DMD who have a negative family history:
1. The affected boy has a new (de novo) gene mutation. In this case, his mother does not carry a disease-causing allele, and her female relatives are not at risk to be carriers of the altered allele.
2. The mother carries a de novo mutation, which places her daughters but not her sisters at risk for being carriers of the altered allele.
3. The maternal grandmother carries a de novo mutation, which places all her daughters at risk for being carriers of the altered allele.
Thus, in families in which DMD occurs in one male only, recurrence-risk counseling depends on establishing which, if any, of the women are carriers of a disease-causing mutation. The following testing and recurrence-risk counseling paradigm is used:
• DNA testing is performed on the male proband with DMD to identify the causative DMD gene mutation. When a DMD gene mutation is identified, a blood sample from the proband’s mother is tested for the same mutation.
• If she has the same mutation as her son, she is counseled regarding the 50% risk of other sons being affected and the 50% risk of daughters being carriers; it is appropriate to test the proband’s maternal grandmother for the same disease-causing mutation.
• If the proband’s mother tests negative for his mutation, two possibilities exist: the son has a new gene mutation or the mother has germline mosaicism, which occurs in about 20% of women in this situation.
• If the son has a new gene mutation, the mother is not at increased risk for having other affected sons, and other women in the family are not at increased risk for being carriers.
• If the mother has germline mosaicism, she is at risk for having carrier daughters and additional affected sons. Her sisters, however, are not at increased risk for being carriers.
Prenatal Testing
Prenatal testing is used to evaluate a fetus at high risk for a genetic disorder on the basis of family history or to evaluate a fetus at no known increased risk but who is suspected of having a genetic disorder because of suggestive findings during the pregnancy.
Positive Family History
Testing of fetuses using molecular genetic testing can be offered to couples at risk for having a child with an autosomal dominant, autosomal recessive, or X-linked disorder for which the specific gene mutation (or mutations) has been identified in the family. Genetic counseling must be offered to provide the family an opportunity to review their reproductive options. Molecular genetic testing can be performed on tissues obtained by chorionic villus sampling at 9 to 11 weeks’ gestation or from amniocentesis at 16 to 18 weeks’ gestation to provide timely information should pregnancy termination be considered.
Findings Suggestive of a Genetic Disorder
Prenatal molecular genetic testing can be a part of the diagnostic evaluation of a fetus not known to be at increased risk for a genetic disorder that is being evaluated further because of abnormalities detected during routine monitoring of the pregnancy. When such findings are detected early in the pregnancy, DNA-based diagnosis may be undertaken if pregnancy termination is being considered. When findings are not apparent until the third trimester, diagnosis may be initiated for the purpose of perinatal management. For example, ultrasound findings of intestinal obstruction with hyperechoic meconium would warrant CFTR molecular genetic testing because of the association of CFTR with cystic fibrosis. Such testing can be performed on DNA extracted from amniocytes obtained from amniocentesis after 16 weeks’ gestation, when timing is not an issue, or from white cells obtained by percutaneous umbilical blood sampling (PUBS) when results are needed urgently.
Genetic Consultation
Genetic consultation is as essential to the care of the patient with a genetic disorder as the testing itself; it is required for persons considering either the medical paradigm or the counseling-only paradigm of genetic testing. A positive genetic test result always raises the consideration of referral for genetic counsel-ing.30-33 Genetic counseling is the process of helping patients understand the nature and cause of the inherited disorder, of outlining the advantages and disadvantages of genetic testing to allow them to make informed medical and personal decisions, and of offering necessary psychosocial support and referral.30,31,34 Genetic evaluation is the process of information gathering regarding a patient or family with a known or suspected genetic disorder. Genetic evaluation and genetic counseling are integral to genetic testing.
Genetic evaluation involves the gathering of information before a clinic visit and during the initial portion of the visit, which usually lasts 1 hour. The following information is obtained from the patient or family: the reason for referral; a family history, including the history of first- and second-degree relatives of the consultand; additional directed family history based on the known or suspected diagnosis and information provided by the patient or other family members; medical records of affected relatives; prenatal and perinatal history; past medical history; and information on growth, development, education, and employment. In addition, family functioning is assessed, potential ethical issues are identified, and a physical examination is performed on the patient and other family members as needed.
Once the gathering of information is complete, genetic counseling is provided. Discussion with the patient or family includes a summary of information obtained; the possible diagnosis and the degree of certainty of that diagnosis, determined on the basis of available information; recommended tests and evaluations necessary to establish the diagnosis or for management of the patient; the sensitivity and positive predictive value of such tests; the natural history of the disorder, including prognosis; inheritance pattern, including pene-trance and variable expressivity (i.e., the variation in the type and severity of a genetic disorder between affected individuals, even within the same family); and recurrence risk for affected persons and for at-risk persons, including reproductive options and options for prenatal diagnosis. Medical management and referrals to appropriate medical specialists are discussed. Psychosocial issues discussed include anticipatory guidance of the patient and family, the availability of community support services, and the availability of regional or national disease-specific or umbrella organizations, many of which can be identified through the Genetic Alliance (www.geneticalliance.org). Genetic-counseling issues for the extended family are addressed. Geographically dispersed family members are referred to local genetic services that can be identified through the GeneTests Clinic Directory, which is available on the GeneTests Web site; the Clinic Directory can be searched by location within the United States, disease specialty, and services offered. Clinic visits and genetic-counseling sessions are documented with detailed summaries suitable for distribution to the family and health care providers. Summary letters are often sent to the family. Short-term follow-up is planned for conveying outstanding test results or other information; long-term follow-up at 2- to 5-year intervals is planned for routine management and updating of genetic-counseling issues.
Difficulties Encountered in DNA-Based Testing for Inherited Disorders
Lack of Awareness of Test Availability
For many inherited disorders, molecular genetic testing is not available, because the causative gene (or genes) is not known. In other instances, the gene is known but test sensitivity is less than that of clinical evaluation, and testing is done only in a research context. For rarer disorders, the gene may be known and clinical testing may be theoretically possible, but clinical laboratories do not offer the test because the cost of low-volume, highly complex testing is prohibitive. For other inherited diseases, the causative gene or genes are known, research testing is currently available, and clinical testing is expected to be available in the near future.35,36 The rapid transition of testing from research laboratory to clinical practice makes it difficult even for those who are familiar with genetic testing to keep abreast of new developments. For those not familiar with genetic testing and its applications to patient care, the task is even more daunting.37
GeneTests is a genetic-testing information resource, funded by the National Institutes of Health (NIH) and maintained at the University of Washington in Seattle, that is designed to facilitate awareness of test availability and use.38 The Web site (http:// www.genetests.org) includes a laboratory directory that serves to help health care providers identify clinical and research laboratories offering testing of heritable disorders. As of July 2005, the Laboratory Directory contained listings of about 1,100 diseases for which clinical (~ 800) and research-only (~ 300) testing was available from approximately 575 laboratories. Clinical laboratories are defined as those that examine human specimens and report results for the purpose of diagnosis, prevention, or treatment in the care of individual patients; such laboratories must be licensed according to the Clinical Laboratory Improvement Amendments (http:\\www.cms.hhs.gov/clia). The Laboratory Directory can be searched by disease name, gene symbol, protein name, clinical features, the laboratory director’s name, and the laboratory’s geographic location.
Complexity of testing Methodologies And Interpretation OF Test Results
Physicians may not be familiar with the use and limitations of molecular genetic tests in patient care. For example, Giardiello and colleagues determined that almost 20% of clinicians ordering APC molecular genetic testing for FAP used the wrong testing strategy.31 These investigators also determined that 34% of clinicians ordering APC molecular genetic testing were unable to identify and interpret false negative results. Several genetic concepts that are intrinsic to the correct use of testing may be confusing. These concepts include (1) locus heterogeneity, in which the identical phenotype can be caused by a single mutation in one of two or more genes (e.g., mutation in either the TSC1 or TSC2 gene can cause tuberous sclerosis complex), which means that negative testing of the gene at only one locus does not rule out the disease; (2) allelic heterogeneity, in which multiple disease-causing mutations at a locus reduce the sensitivity of molecular testing below an acceptable level for use in diagnosis but not for recurrence-risk counseling; and (3) redefinition of phenotypes on the basis of molecular genetic findings (e.g., trinucleotide repeat diseases, CF, and the dystrophinopathies, including DMD, BMD, and X-linked dilated cardiomyopathy).
In a survey of genetic counselors in the United States, Mc-Govern and colleagues determined that genetic counselors contacted laboratory-testing personnel for 58% of tests ordered regarding details of test ordering or interpretation of test results.39 Only 72% felt that laboratory reports contain enough information to explain results to patients. There are concerns that non-geneticist health care providers face even greater challenges in using genetic-testing laboratories.
The GeneReviews portion of the GeneTests Web site contains current information on the use of genetic testing in diagnosis, management, and genetic counseling for specific inherited disorders. Entries on over 300 diseases (as of July 2005) provide expert-authored, peer-reviewed information for health care professionals. A context-sensitive illustrated glossary familiarizes non-geneticists with genetic counseling and genetic testing terms.
Confusion Between Testing Paradigms
The intertwining of genetic testing for medical management and testing for personal decision making may lead to confusion about medical necessity, privacy, and discrimination. Just as the application of testing to patient care is context specific, consideration of these social issues is also context specific.
Underutilization of Genetic Services
Giardiello and colleagues determined that only 18% of patients undergoing predictive testing for FAP, an autosomal dominant disorder that has 100% penetrance and that is associated with a 100% risk of cancer by 40 years of age, received genetic counseling.31 A survey of 600 primary care physicians in Oregon revealed that 20% of internists did not know of any genetic services available to them for consultation.40 Furthermore, most felt they did not need to refer patients for genetic consultation, preferring to offer risk-assessment and recurrence-risk counseling themselves, even though they were unfamiliar with the specific disorders and genetic-counseling concepts.40 The need for primary care physicians to understand and use genetic services has been emphasized.33,37,41,42 Possible explanations of un-derutilization of genetic services are the so-called therapeutic gap between diagnosis and prediction of diseases and the ability to treat or prevent them43; real or perceived restrictive reimbursement policies of health care payers; and concern about ethical and social issues that would seem to create "genetic excep-tionalism" (the justification, provided by genetic testing, for special consideration regarding issues of informed consent and privacy),44 which would move genetic testing beyond the purview of traditional medical care.45 The change in the use of some genetic tests from a diagnostic role to a screening role (e.g., testing for factor V Leiden) may shift the emphasis of testing away from the evaluation of at-risk family members and genetic counseling to a broader role related to population-based health care.
