Journal of Genetic Counseling, Vol. 8, No. 2, 1999
Complex Genetic Disorders: Evaluating When Genetic Research Findings Are Applicable for Genetic Counseling Practice Chantelle M. Wolpert,1,2 Elizabeth C. Melvin,1 and Marcy C. Speer1
Traditional genetic counseling processes and principles will be extended to a new realm—complex disorders. Although it may seem like a daunting task, understanding the methodologies used to study complex genetic disorders will enable genetic counselors to critically analyze research studies involving complex disorders. In this article, we explain newly evolving methodologies for genetic research, including case-control studies and transmission disequilibrium testing (TDT). Additionally, a framework is provided for evaluating original research findings and replication studies. KEY WORDS: complex disorders; genetic counseling; transmission disequilibrium testing (TDT); genetic analysis methods; genetic research.
INTRODUCTION With the genes for many Mendelian disorders mapped, genetic researchers are now striving to map complex disorders. Complex disorders may be defined as disorders which have a genetic component but do not follow a simple mode of inheritance (i.e., Mendelian inheritance) (Lander and Schork, 1994; Thomson, 1994). Recent success in finding genes associated with Alzheimer disease (PericakVance et al., 1991; Sherrington et al., 1995; Levy-Lahad et al., 1995) and breast cancer (Futreal et al., 1994; Wooster et al., 1995) prove that scientists are beginning to successfully unravel some the major genetic factors increasing susceptibility to 1 Center for Human Genetics, Duke University Medical Center, Durham, North Carolina. Correspondence should be directed to Chantelle M. Wolpert, Center for Human Genetics, Duke University Medical Center, Box 3445-Carl Building, Durham, North Carolina 27710; e-mail:chantell @chg.mc.duke.edu.
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complex disorders. At the same time, the lay press has made the general public more aware of the genetic component of common disorders like heart disease, diabetes, and neurological disorders. Consequently, an emerging population of patients will be asking about their personal risk for developing disorders that may "run in the family." Therefore, it is likely that more referrals for genetic counseling will shift from reproductive issues toward referrals for common adult-onset disorders. In order to meet the anticipated changes in the scope of genetic counseling practice, genetic counselors will need to become familiar with and apply an analytical approach to evaluating a broad range of genetic and epidemiological studies in order to provide the information patients will demand. In an effort to help genetic counselors prepare for this transition, we provide a framework for evaluating and interpreting research for complex disorders and discuss the application of research findings to genetic counseling practice. EVOLVING TERMINOLOGY To understand complex disorders and their accompanying educational and counseling issues, genetic counselors must first be familiar with the relevant terminology. However, because research on complex disorders is in its infancy, the terms and theoretical definitions presented here are likely to be modified over time. The terms polygenic and multifactorial are used to describe disorders displaying patterns of familial recurrence with a genetic component not consistent with Mendelian inheritance (Davidoff et al., 1991). These two definitions imply, respectively, that more than one gene is involved in the inheritance of a disorder and that environmental factors may or may not interact with one or more genes to produce a specific phenotype. A newer term, complex disorder, more realistically reflects the underlying biology and serves as an umbrella term that encompasses the terms polygenic and multifactorial (Pericak-Vance, 1998). A hallmark of complex disorders is that they are common in the population and are frequently of significant public health concern (Taubes, 1995). Some examples of complex disorders are multiple sclerosis, neural tube defects, autistic disorder, coronary artery disease, hypertension, Parkinson disease, and asthma. Disorders that appear to be complex can in fact have both Mendelian and complex mechanistic subsets. Alzheimer disease provides an excellent example. Long considered a familial disorder, genetic studies have now established that there are subtypes of Alzheimer disease, some of which are Mendelian and others complex (Fig. 1). Another distinction made in the genetic literature for complex disorder is between causative and susceptibility alleles. The term causative is typically used with Mendelian disorders. The classic example is a single mutation in a single gene, which causes a particular disorder. The expression of these alleles maybe influenced by other factors, but the presence of the allele confers significant risk.
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Fig. 1. Alzheimer disease (AD) is often categorized by researchers and clinicians by several variables, including age of onset and mode of inheritance, and when possible by the associated gene mutation or genotype. Early-onset AD is defined as age of onset prior to age 60, whereas late-onset AD is defined as onset after age 60. Further distinctions are made by the mode of inheritance. The autosomal dominant genes associated with early-onset AD are amyloid precursor protein (APP) on chromosome 21, presenilin 1 (PSN-1) on chromosome 14, and presenilin 2 (PSN-2) on chromosome 1. Apolipoprotein-E (APOE) is a susceptibility gene on chromosome 19. Another linkage for late-onset AD has been reported for chromosome 12. The only proven environmental factor associated with AD is head trauma in the presence of being heterozygous or homozygous for APOE-4. It is likely that more genetic and environmental subcategories will be defined in the future.
Causative alleles may be under the influence of genetic phenomenon such as reduced penetrance but are usually sufficient to cause the disease phenotype. In contrast, a susceptibility allele confers an increased risk for a specific disease phenotype, but this allele is usually not sufficient in itself to cause the disease. Typically, a susceptibility allele is common enough in the general population so that it may be more accurately termed a polymorphism as opposed to a mutation. Different genotypes may reflect different degrees of susceptibility or risk. APOE is an example of a susceptibility allele. An individual who is homozygous for the E4 allele of APOE is more likely to develop Alzheimer disease (AD) than an individual with a different APOE genotype (e.g., 2/2 or 3/3) (Farrer et al., 1997; Saunders et al., 1993; Corder et al., 1993, 1994). Thus, the particular variant of the APOE gene suggests an individual's relative susceptibility to developing AD, but having this genotype does not ensure that an individual will develop symptoms. In clinical practice, there is a blurred distinction between the terms susceptibility and causative loci due, in large part, to confounding factors that affect penetrance, expression, genetic heterogeneity, and age of onset. In fact, susceptibility
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and causative alleles fall on the same spectrum of disease risk resulting from genetic effects. Although the difference between causative and susceptibility alleles can be subtle, it is important to remember that the fundamental characteristic of an allele remains the same: Either it is transmitted to offspring or it is not. We will refer to this critical concept of transmission later. THE IMPORTANCE OF DIAGNOSTIC ACCURACY Accurate diagnosis is the cornerstone of genetic counseling. However, with complex disorders, greater rigor is needed to specify an accurate diagnosis and its likely etiologic cause. The two are deeply intertwined since genetic research findings for complex disorders are likely to apply to a specific subset of a given phenotype. First, the terms used to name and describe complex disorders are often phenotypic descriptions or a summary of physical features. For example, retinitis pigmentosa, once considered a unique disease entity in itself, is now recognized as one physical finding in many disorders. Other examples include the diagnostic terms hypertension and multiple sclerosis. Hypertension is simply a phenotypic description for high blood pressure that can be caused by conditions ranging from the autosomal dominant form of polycystic kidney disease to "essential" or idiopathic hypertension. And relapsing-remitting multiple sclerosis and chronic progressive multiple sclerosis are different subtypes of a myelin disease and are very likely caused by different genetic factors (Semchuk and Love, 1995). Likewise, late-onset Alzheimer disease, defined as age of onset 60 years or older, is associated with a genetic susceptibility gene, APOE, while most early-onset, autosomal dominant cases of AD are usually associated with one of three Mendelian genes (St.George-Hyslop et al., 1987; Sherrington et al., 1995;Levy-Lahad et al., 1995). In this case, accurate genetic counseling and, possibly, recommendations for genetic testing would depend on determining the critical information about whether the disease was classified as early-onset or late-onset AD in a particular individual or family. EVALUATING RESEARCH ON COMPLEX DISORDERS Once an accurate diagnosis has been made, evaluating the genetic literature to provide patient-specific risk information for complex disorders presents a daunting task. Many of the risk data in the recent literature on complex disorders come from research using traditional epidemiological measures, that are now being applied to genetics. In order to meet the needs of their patients, genetic counselors will need to be conversant with several epidemiological methods in order to critically evaluate the ever-increasing volume of genetic studies and provide accurate information to their patients. For this reason, we describe the most common types of studies
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encountered in the genetic research literature: linkage, association, case-control, and linkage disequilibrium studies.
LINKAGE AND ASSOCIATION STUDIES: A CRITICAL CONTRAST Linkage is a physical connection between two loci. The lod score approach is used to detect linkage between families with the same Mendelian disorder and genetic markers. Genes for numerous disorders have been localized by this approach (e.g., Duchenne muscular dystrophy, Huntington disease, neurofibromatosis type 1 and 2), and research successes have translated directly into increased precision in genetic counseling (Collins, 1995). In general, linkage studies require the participation of multiple families. Within those families, both affected and unaffected relatives need to participate in order for this method to be successful. However, association is different from linkage. Association between two traits or loci occurs when alleles at two loci are found together in individuals more frequently than expected by chance alone. Association implies no physical connection between the loci. In contrast to linkage analysis, association studies are usually performed on unrelated individuals, not families (e.g., cases versus controls). In summary, linkage is a characteristic of loci, and association is a characteristic of alleles. CASE-CONTROL STUDIES One common epidemiological study design that has been adopted for genetic research use is the case-control study. Traditionally, a case-control study identifies associations between diseases and exposures, such as chemicals and toxins. A classic example of an early application of this methodology revealed the association between cigarette smoking and lung cancer (Doll and Hill, 1956). In theory, the case-control method translates nicely into the genetic paradigm. Instead of the traditional environmental exposure, the exposure in genetic studies is now defined as the presence of a specific allele at a locus. With a case-control method, genetic researchers are trying to learn how frequently an allele at a locus appears in individuals with the inherited disorder (cases) compared to individuals without the disorder (controls). An association is established when an allele appears more or less frequently in cases than in controls. However, a genetic association does not imply genetic linkage. Adaptation of the case-control method for genetic research is not exempt from difficulties. A significant problem with applying case-control studies to researching genetic disorders is the selection of a control population. In genetic studies, as in epidemiological studies, appropriate controls may be difficult to obtain. For instance, allele frequencies are known to vary dramatically among different ethnic groups and different populations. Therefore, controls and research participants
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(cases) must be matched for ethnicity and for other demographic variables such as age and gender. When evaluating epidemiological-based studies for complex disorders, genetic counselors must therefore take note of the adequacy and/or appropriateness of the control population and how patients compare to the study group. Another difficulty in using case-related studies for genetic research is that while the case-control method can show association between a disease allele and a marker locus, it fails to assess transmission, the critical characteristic which differentiates genetic studies from other types of research. If an allele at a marker locus is important in disease etiology, it must be transmitted to affected offspring from a heterozygous parent (affected or unaffected with disease) more frequently than expected by chance alone or, in other words, more than 50% of the time.
WHEN ASSOCIATION AND LINKAGE OCCUR TOGETHER: LINKAGE DISEQUILIBRIUM Linkage disequilibrium is a combination of two phenomena: association and linkage. This rare phenomenon occurs when two loci are so tightly linked to one another that recombination rarely occurs. Thus, alleles at each of the loci occur together in proportions not consistent with the Hardy-Weinberg equilibrium. In other words, linkage disequilibrium is observed when an allele is so close to the disease locus that evolutionary time has failed to separate the physical association between the two loci via recombination. Linkage differs from linkage disequilibrium. For linkage, the allele associated with the disease in one pedigree need not be the same allele across all linked pedigrees (Fig. 2). However, under linkage disequilibrium, the same allele is associated with the trait across all or most linked pedigrees. One way to test for linkage disequilibrium is with the transmission disequilibrium test (TDT). The TDT takes the adaptation of case control studies for genetic research one step further. The TDT was developed to counter the known difficulties of the case-control study, such as ascertaining appropriate controls and characterizing
Fig. 2. Although both pedigrees are linked to the marker locus, the allele associated with the disease differs between pedigrees. In the pedigree on the left, the disease is associated with marker allele 2, and in the pedigree on the right, the disease is associated with marker allele 3.
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Fig. 3. A'2 x 2 table used for scoring results of the transmission disequilibrium test. The letters a, b, c, and d are used to designate the different categories.
transmission (Spielman, McGinnis, and Ewens, 1993), the fundamental characteristic of genetic traits. With the TDT, the unit of sampling is the nuclear family (the affected individual and his or her parents), whereas within the case-control study, the unit of sampling is the affected individual and an appropriate control only. After identification of a potential genetic association through a case-control study, studies of nuclear families using the TDT can clarify whether the association is truly due to linkage between the loci or is a spurious result. The TDT is a modified case-control study, which involves determining which of the parents' alleles were transmitted to the affected child and which were not. For a two-allele marker, these counts are scored in a 2 x 2 table (Fig. 3). A statistically significant result indicates that a specific allele is transmitted from heterozygous parent to offspring more frequently than expected by chance, and thus it identifies evidence for linkage disequilibrium (allelic association and linkage). The TDT is an effective and elegant methodological tool from a variety of angles. First, the issue of selecting appropriate controls is avoided, since the parental nontransmitted alleles are used as internal controls and, by definition, are appropriately matched. Second, this approach allows the inclusion of sporadic cases and their parents in genetic linkage studies instead of the sole use of families with multiple incidences of the disease, so-called multiplex pedigrees, thereby dramatically expanding the applications of genetic studies. Third, investigation of these families with a sporadic or single incidence of a disorder (so-called singleton families) and no family history of the disorder allows the researcher to begin to answer the question of whether cases in multiplex pedigrees are genetically different from sporadic cases. This answer has major public health benefits since common complex disorders are often found more frequently in sporadic cases than in familial cases. Case control and association studies, particularly TDT studies, are likely to be reported more frequently in the genetic literature attempting to characterize the genetic effects of complex disease. Therefore, it is important for genetic counselors to be aware of these studies and their methodology in order to analyze research on the complex disorders. Variations of the TDT are now being investigated that allow sampling patients and their unaffected siblings when parents are not available (Boehnke and Langefeld, 1998; Monks etal., 1997). This methodological advance is important in the study of late-onset diseases, such as glaucoma and dementia.
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EVALUATING THE RESEARCH FINDINGS Even with some knowledge of new methodologies used in case-control studies for complex diseases, genetic health professionals are still faced with the task of determining the credibility and clinical application of a research finding. Unfortunately, no standard guide or criterion that measures the validity of a genetic research finding is available. However, a general framework can be utilized. When investigating the validity of a genetic research finding, it is wise to first find the report of the original finding. The next step is to search for articles that contain information about replication studies, both positive and negative. We initially place heavy emphasis on evaluating replication studies since the clinical applicability of a research finding will be determined by multiple confirmations.
REPLICATION OF RESEARCH FINDINGS Replication is the gold standard for determining the credibility of research findings. Simply put, researchers try to replicate the analysis or experiment that produced the original research finding. When another scientific team performs the replication experiments, this is called external replication. External replication is welcomed because it provides an outside assessment of the validity and generalizability of the findings. However, external replication necessitates another scientific team with the expertise, interest, and resources to replicate the study in a timely manner. Unfortunately, external replication is not always possible. For instance, other researchers may not have a large enough data set to perform the same analyses as the original researchers. Or differences in phenotypic assignment between data sets may hamper comparisons. Sometimes, if another data set is available, the original research team may do their own replication analysis. This is called internal replication. Internal replication has the advantage that the conditions used in the first experiment can readily be re-created in a second experiment. Internal replication may or may not be done before the original research finding is announced. There are two possible outcomes of replication studies. Specifically, research findings can be confirmed or disputed. Nonconfirmations may result in a dispute of the original findings, or the overall interpretations can be inconclusive. Each outcome portends different action on the part of the genetic counselor.
CONFIRMATION IN REPLICATION Sometimes, multiple external groups replicate an original research finding. For instance, the association of the APOE-4 allele with an increase in risk for Alzheimer disease in a dose-dependent manner has been confirmed in more than
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150 studies. Genetic counselors can feel comfortable that these research findings are accurate. Still, confirmation of research findings does not always allow a clear understanding of the relevance of the finding(s) for patients and their families. Often, much work remains to understand what relevance this finding has for patients and their families. Unfortunately, it is rare to find such consistent and overwhelming replication. However, extensive confirmation of results is the first step toward the development of clinical recommendations.
FAILURE TO REPLICATE A STUDY If replication studies do exist, results may be confusing and conflicting. For instance, some studies may support one conclusion while identically designed studies with diametrically opposed outcomes support other conclusions. Dispute of the original research finding may indicate that the finding is unique to the population studied in the original research. Alternatively, the reported genetic effect may be small or the result of a flawed study. This scenario is probably encountered frequently by genetic counselors. When there are conflicting studies, a preponderance of the evidence (such as three studies confirming and one disputing the original finding) does not prove that one finding is right while the alternative is wrong. When one study disputes the original research finding, other studies must be done which will support either the original research finding or the alternative finding. All the studies need to be reviewed in context and examined to see how closely the parameters in each study matched. It may not be possible to draw a conclusion until further research is done to resolve conflicting results. As a result, no clinical recommendations may be available but may be forthcoming. However, genetic counselors can educate patients and their families about the conflicting research evidence.
RESEARCH FINDINGS CAN DEVELOP INTO CLINICAL RECOMMENDATIONS Although clinical recommendations may not already exist, genetic counselors can look forward to, and participate in, the development of such recommendations. As peer-reviewed evidence accumulates for or against a research finding, a panel of experts will often review the reports associated with the finding and make a clinical recommendation. An example of a research finding that evolved into a clinical recommendation is that of preconceptional and prenatal supplementation of 0.4 mg of folic acid to reduce the incidence of neural tube defects. Currently, at least five organizations now recommend the preconceptional use of folic acid (Baty et al., 1996; Centers for Disease Control and Prevention, 1992; Crandall et al., 1995; MRC Vitamin Study Research Group, 1991). Clinical recommendations,
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while seemingly simplistic, are often the result of years of research, multiple replications of a specific research finding, and careful review by an interdisciplinary team of professionals. When a credible organization or consensus panel issues a clinical recommendation, it is usually appropriate to make the recommendation to a patient. Another appropriate sign is when multiple credible organizations make the same clinical recommendation. Concurring clinical recommendations can increase the genetic counselor's confidence in making the recommendation and, in turn, increase a patient's confidence in acting on that recommendation. Finally, even when a research finding leads to the availability of genetic testing, the genetic counselor may feel more comfortable waiting for a clinical recommendation for this test before widely offering the genetic test to patients and their families. Several considerations should be explored: Although it may be feasible to obtain genetic testing, is the test appropriate given our knowledge about the disorder? Is the test useful and valuable for families? Will the results of the genetic test provide enough reliable information for a patient or family to make medical management decisions? Genetic research findings, which may result in development of genetic tests or therapeutic recommendations, do not have a standard professional board which deems them ready for public medical use. In contrast, the Food and Drug Administration evaluates the efficacy and safety of potential therapeutic agents, such as medications. Nonetheless, genetic counselors and other professionals can play a role or participate in the discourse concerning the appropriateness and readiness of genetic testing for complex disorders for public use even before clinical recommendation can be made.
CONCLUSION This is an exciting time for genetic counselors. The emergence of new statistical and molecular methodologies will extend the role of genetic counselors into personalized risk assessment in common complex disorders. Although it may seem like a daunting task, an understanding of the methodologies, such as case-control studies and TDT, and a framework for evaluating original research findings and replication studies will enable genetic counselors to critically analyze complex disorders studies for use in patient risk analysis.
ACKNOWLEDGMENTS This work was presented at the National Society of Genetic Counseling's 16th Annual Education Conference in Baltimore in 1997. The authors wish to thank Chad Haynes for assistance with figure design and Roberta Parker for
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expert secretarial assistance. The authors gratefully acknowledge support from grants of the National Institute of Neurological Disorders and Stroke (NINDS) NS26630, NS36768, and HD33400, and grants from the March of Dimes and from the American Syringomyelia Alliance Project. REFERENCES Baty BJ, Cohen L, Phelps L, Speer MC, Stengel P, Williamson-Kruse L (1996) Folic acid and the prevention of neural tube defects: A position paper: National Society of Genetic Counselors. J Gen Couns 5:139-145. Boehnke M, Langefeld CD (1998) Genetic association mapping based on discordant sib pairs: The discordant alleles test (DAT). Am J Hum Genet 62:950-961. Centers for Disease Control and Prevention (1992) Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. Morbidity and Mortality Weekly 41:1-7. Collins FS (1995) Positional cloning moves from periditional to traditional. Nat Genet 9:347-350. Corder EH, Saunders AM, Risch N, Strittmatter WJ, Schmechel DE, Gaskell PC, Rimmler JB, Locke PA, Conneally PM, Schmader KE, Small GW, Roses AD, Haines JL, Pericak-Vance MA (1994) Apolipoprotein E type 2 allele decreases the risk of late onset Alzheimer disease. Nat Gene 7:180-184. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261:921-923. Crandall BF, Corson VL, Goldberg JD, Knight G, Salafsky IS (1995) Folic acid and pregnancy. Am J Med Genet 55:134-135. Davidoff AM, Thompson CV, Grimm JK, Shorter NA, Filston HC, Oakes WJ (1991) Occult spinal dysraphism in patients with anal agenesis. J Pediatr Surg 26:1001-1005. Doll R, Hill AB (1956) Lung cancer and other causes of death in relation to smoking: A second report on the mortality of British doctors. Br Med J 2:1071. Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, Myers RH, Pericak-Vance MA, Risch N, van Duijn CM. APOE, Alzheimer Disease Meta Analysis Consortium (1997) Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. JAMA 278:1349-1356. Futreal PA, Liu Q, Shattuck-Eidens D, Cochran C, Harshman K, Tavtigian S, Bennett LM, HaugenStrano A, Swensen J, Miki Y, Eddington K, McClure M, Frye C, Weaver-Feldhaus J, Ding W, Gholami Z, Soderkvist P, Terry L, Jhanwar S, Berchuck A, Iglehart JD, Marks J, Ballinger DG, Barrett JC, Skolnick MH, Kamb A, Wiseman R (1994) BRCA 1 mutations in primary breast and ovarian carcinomas. Science 266:120-126. Lander ES, Schork NJ (1994) Genetic dissection of complex traits. Science 265:2037-2048. Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH, Yu CE, Jondro PD, Schmidt SD, Wang K, Crowley AC, Fu Y-H, Guenette SY, Galas D, Nemens E, Wijsman EM, Bird TD, Schellenberg GD, Tanzi RE (1995) Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 269:973-977. Monks SA, Martin ER, Weir BS, Kaplan NL (1997) A sibship test of linkage in the absence of parental information. Am J Hum Genet 61:A286 (Abstract). MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet 338:131-137. Pericak-Vance MA (1998) Overview of linkage analysis in complex traits. Unit 1.9. In: Protocols in Human Genetics. Pericak-Vance MA, Bebout JL, Gaskell PC, Yamaoka LH, Hung W-Y, Alberts MJ, Walker AP, Bartlett RJ, Haynes CS, Welsh KA, Earl NL, Heyman A, Clark CM, Roses AD (1991) Linkage studies in familial Alzheimer's disease: Evidence for chromosome 19 linkage. Am J Hum Genet 48:10341050.
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St. George-Hyslop PH, Tanzi RE, Polinsky RJ, Haines JL, Watkins PC, Myers RH, Feldman RG, Pollen D, Drachman D (1987) The genetic defect causing familial Alzheimer's disease maps on chromosome 21. Science 235:885-890. Saunders AM, Strittmatter WJ, Breitner JC, Schmechel D, St. George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella JF, Crapper-MacLachlan DR, Growden J, Alberts MJ, Hulette C, Crain B, Goldgaber D, Roses AD (1993) Association of apolipoprotein E allele 4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43:1467-1472. Semchuk KM, Love EJ (1995) Effects of agricultural work and other proxy-derived case-control data on Parkinson's disease risk estimates. Am J Epidemiol 141:747-754. Sherrington R, Rogaev E, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J-F, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Polinsky RJ, Wasco W, DaSilva HAR, Haines JL, Pericak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St. George-Hyslop PH (1995) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375:754-760. Spielman RS, McGinnis RE, Ewens WJ (1993) Transmission test for linkage disequilibrium: The insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 52:506516. Taubes G (1995) Epidemiology faces its limits. Science 269:164-169. Thomson G (1994) Identifying complex disease genes: Progress and paradigms. Nat Genet 8:189-194. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G (1995) Identification of the breast cancer suspectibility gene BRCA2. Nature 378:789-792.
Complex Genetic Disorders: Evaluating When Genetic Research Findings Are Applicable for Genetic Counseling Practice Chantelle M. Wolpert,1,2 Elizabeth C. Melvin,1 and Marcy C. Speer1
Traditional genetic counseling processes and principles will be extended to a new realm—complex disorders. Although it may seem like a daunting task, understanding the methodologies used to study complex genetic disorders will enable genetic counselors to critically analyze research studies involving complex disorders. In this article, we explain newly evolving methodologies for genetic research, including case-control studies and transmission disequilibrium testing (TDT). Additionally, a framework is provided for evaluating original research findings and replication studies. KEY WORDS: complex disorders; genetic counseling; transmission disequilibrium testing (TDT); genetic analysis methods; genetic research.
INTRODUCTION With the genes for many Mendelian disorders mapped, genetic researchers are now striving to map complex disorders. Complex disorders may be defined as disorders which have a genetic component but do not follow a simple mode of inheritance (i.e., Mendelian inheritance) (Lander and Schork, 1994; Thomson, 1994). Recent success in finding genes associated with Alzheimer disease (PericakVance et al., 1991; Sherrington et al., 1995; Levy-Lahad et al., 1995) and breast cancer (Futreal et al., 1994; Wooster et al., 1995) prove that scientists are beginning to successfully unravel some the major genetic factors increasing susceptibility to 1 Center for Human Genetics, Duke University Medical Center, Durham, North Carolina. Correspondence should be directed to Chantelle M. Wolpert, Center for Human Genetics, Duke University Medical Center, Box 3445-Carl Building, Durham, North Carolina 27710; e-mail:chantell @chg.mc.duke.edu.
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complex disorders. At the same time, the lay press has made the general public more aware of the genetic component of common disorders like heart disease, diabetes, and neurological disorders. Consequently, an emerging population of patients will be asking about their personal risk for developing disorders that may "run in the family." Therefore, it is likely that more referrals for genetic counseling will shift from reproductive issues toward referrals for common adult-onset disorders. In order to meet the anticipated changes in the scope of genetic counseling practice, genetic counselors will need to become familiar with and apply an analytical approach to evaluating a broad range of genetic and epidemiological studies in order to provide the information patients will demand. In an effort to help genetic counselors prepare for this transition, we provide a framework for evaluating and interpreting research for complex disorders and discuss the application of research findings to genetic counseling practice. EVOLVING TERMINOLOGY To understand complex disorders and their accompanying educational and counseling issues, genetic counselors must first be familiar with the relevant terminology. However, because research on complex disorders is in its infancy, the terms and theoretical definitions presented here are likely to be modified over time. The terms polygenic and multifactorial are used to describe disorders displaying patterns of familial recurrence with a genetic component not consistent with Mendelian inheritance (Davidoff et al., 1991). These two definitions imply, respectively, that more than one gene is involved in the inheritance of a disorder and that environmental factors may or may not interact with one or more genes to produce a specific phenotype. A newer term, complex disorder, more realistically reflects the underlying biology and serves as an umbrella term that encompasses the terms polygenic and multifactorial (Pericak-Vance, 1998). A hallmark of complex disorders is that they are common in the population and are frequently of significant public health concern (Taubes, 1995). Some examples of complex disorders are multiple sclerosis, neural tube defects, autistic disorder, coronary artery disease, hypertension, Parkinson disease, and asthma. Disorders that appear to be complex can in fact have both Mendelian and complex mechanistic subsets. Alzheimer disease provides an excellent example. Long considered a familial disorder, genetic studies have now established that there are subtypes of Alzheimer disease, some of which are Mendelian and others complex (Fig. 1). Another distinction made in the genetic literature for complex disorder is between causative and susceptibility alleles. The term causative is typically used with Mendelian disorders. The classic example is a single mutation in a single gene, which causes a particular disorder. The expression of these alleles maybe influenced by other factors, but the presence of the allele confers significant risk.
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Fig. 1. Alzheimer disease (AD) is often categorized by researchers and clinicians by several variables, including age of onset and mode of inheritance, and when possible by the associated gene mutation or genotype. Early-onset AD is defined as age of onset prior to age 60, whereas late-onset AD is defined as onset after age 60. Further distinctions are made by the mode of inheritance. The autosomal dominant genes associated with early-onset AD are amyloid precursor protein (APP) on chromosome 21, presenilin 1 (PSN-1) on chromosome 14, and presenilin 2 (PSN-2) on chromosome 1. Apolipoprotein-E (APOE) is a susceptibility gene on chromosome 19. Another linkage for late-onset AD has been reported for chromosome 12. The only proven environmental factor associated with AD is head trauma in the presence of being heterozygous or homozygous for APOE-4. It is likely that more genetic and environmental subcategories will be defined in the future.
Causative alleles may be under the influence of genetic phenomenon such as reduced penetrance but are usually sufficient to cause the disease phenotype. In contrast, a susceptibility allele confers an increased risk for a specific disease phenotype, but this allele is usually not sufficient in itself to cause the disease. Typically, a susceptibility allele is common enough in the general population so that it may be more accurately termed a polymorphism as opposed to a mutation. Different genotypes may reflect different degrees of susceptibility or risk. APOE is an example of a susceptibility allele. An individual who is homozygous for the E4 allele of APOE is more likely to develop Alzheimer disease (AD) than an individual with a different APOE genotype (e.g., 2/2 or 3/3) (Farrer et al., 1997; Saunders et al., 1993; Corder et al., 1993, 1994). Thus, the particular variant of the APOE gene suggests an individual's relative susceptibility to developing AD, but having this genotype does not ensure that an individual will develop symptoms. In clinical practice, there is a blurred distinction between the terms susceptibility and causative loci due, in large part, to confounding factors that affect penetrance, expression, genetic heterogeneity, and age of onset. In fact, susceptibility
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and causative alleles fall on the same spectrum of disease risk resulting from genetic effects. Although the difference between causative and susceptibility alleles can be subtle, it is important to remember that the fundamental characteristic of an allele remains the same: Either it is transmitted to offspring or it is not. We will refer to this critical concept of transmission later. THE IMPORTANCE OF DIAGNOSTIC ACCURACY Accurate diagnosis is the cornerstone of genetic counseling. However, with complex disorders, greater rigor is needed to specify an accurate diagnosis and its likely etiologic cause. The two are deeply intertwined since genetic research findings for complex disorders are likely to apply to a specific subset of a given phenotype. First, the terms used to name and describe complex disorders are often phenotypic descriptions or a summary of physical features. For example, retinitis pigmentosa, once considered a unique disease entity in itself, is now recognized as one physical finding in many disorders. Other examples include the diagnostic terms hypertension and multiple sclerosis. Hypertension is simply a phenotypic description for high blood pressure that can be caused by conditions ranging from the autosomal dominant form of polycystic kidney disease to "essential" or idiopathic hypertension. And relapsing-remitting multiple sclerosis and chronic progressive multiple sclerosis are different subtypes of a myelin disease and are very likely caused by different genetic factors (Semchuk and Love, 1995). Likewise, late-onset Alzheimer disease, defined as age of onset 60 years or older, is associated with a genetic susceptibility gene, APOE, while most early-onset, autosomal dominant cases of AD are usually associated with one of three Mendelian genes (St.George-Hyslop et al., 1987; Sherrington et al., 1995;Levy-Lahad et al., 1995). In this case, accurate genetic counseling and, possibly, recommendations for genetic testing would depend on determining the critical information about whether the disease was classified as early-onset or late-onset AD in a particular individual or family. EVALUATING RESEARCH ON COMPLEX DISORDERS Once an accurate diagnosis has been made, evaluating the genetic literature to provide patient-specific risk information for complex disorders presents a daunting task. Many of the risk data in the recent literature on complex disorders come from research using traditional epidemiological measures, that are now being applied to genetics. In order to meet the needs of their patients, genetic counselors will need to be conversant with several epidemiological methods in order to critically evaluate the ever-increasing volume of genetic studies and provide accurate information to their patients. For this reason, we describe the most common types of studies
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encountered in the genetic research literature: linkage, association, case-control, and linkage disequilibrium studies.
LINKAGE AND ASSOCIATION STUDIES: A CRITICAL CONTRAST Linkage is a physical connection between two loci. The lod score approach is used to detect linkage between families with the same Mendelian disorder and genetic markers. Genes for numerous disorders have been localized by this approach (e.g., Duchenne muscular dystrophy, Huntington disease, neurofibromatosis type 1 and 2), and research successes have translated directly into increased precision in genetic counseling (Collins, 1995). In general, linkage studies require the participation of multiple families. Within those families, both affected and unaffected relatives need to participate in order for this method to be successful. However, association is different from linkage. Association between two traits or loci occurs when alleles at two loci are found together in individuals more frequently than expected by chance alone. Association implies no physical connection between the loci. In contrast to linkage analysis, association studies are usually performed on unrelated individuals, not families (e.g., cases versus controls). In summary, linkage is a characteristic of loci, and association is a characteristic of alleles. CASE-CONTROL STUDIES One common epidemiological study design that has been adopted for genetic research use is the case-control study. Traditionally, a case-control study identifies associations between diseases and exposures, such as chemicals and toxins. A classic example of an early application of this methodology revealed the association between cigarette smoking and lung cancer (Doll and Hill, 1956). In theory, the case-control method translates nicely into the genetic paradigm. Instead of the traditional environmental exposure, the exposure in genetic studies is now defined as the presence of a specific allele at a locus. With a case-control method, genetic researchers are trying to learn how frequently an allele at a locus appears in individuals with the inherited disorder (cases) compared to individuals without the disorder (controls). An association is established when an allele appears more or less frequently in cases than in controls. However, a genetic association does not imply genetic linkage. Adaptation of the case-control method for genetic research is not exempt from difficulties. A significant problem with applying case-control studies to researching genetic disorders is the selection of a control population. In genetic studies, as in epidemiological studies, appropriate controls may be difficult to obtain. For instance, allele frequencies are known to vary dramatically among different ethnic groups and different populations. Therefore, controls and research participants
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(cases) must be matched for ethnicity and for other demographic variables such as age and gender. When evaluating epidemiological-based studies for complex disorders, genetic counselors must therefore take note of the adequacy and/or appropriateness of the control population and how patients compare to the study group. Another difficulty in using case-related studies for genetic research is that while the case-control method can show association between a disease allele and a marker locus, it fails to assess transmission, the critical characteristic which differentiates genetic studies from other types of research. If an allele at a marker locus is important in disease etiology, it must be transmitted to affected offspring from a heterozygous parent (affected or unaffected with disease) more frequently than expected by chance alone or, in other words, more than 50% of the time.
WHEN ASSOCIATION AND LINKAGE OCCUR TOGETHER: LINKAGE DISEQUILIBRIUM Linkage disequilibrium is a combination of two phenomena: association and linkage. This rare phenomenon occurs when two loci are so tightly linked to one another that recombination rarely occurs. Thus, alleles at each of the loci occur together in proportions not consistent with the Hardy-Weinberg equilibrium. In other words, linkage disequilibrium is observed when an allele is so close to the disease locus that evolutionary time has failed to separate the physical association between the two loci via recombination. Linkage differs from linkage disequilibrium. For linkage, the allele associated with the disease in one pedigree need not be the same allele across all linked pedigrees (Fig. 2). However, under linkage disequilibrium, the same allele is associated with the trait across all or most linked pedigrees. One way to test for linkage disequilibrium is with the transmission disequilibrium test (TDT). The TDT takes the adaptation of case control studies for genetic research one step further. The TDT was developed to counter the known difficulties of the case-control study, such as ascertaining appropriate controls and characterizing
Fig. 2. Although both pedigrees are linked to the marker locus, the allele associated with the disease differs between pedigrees. In the pedigree on the left, the disease is associated with marker allele 2, and in the pedigree on the right, the disease is associated with marker allele 3.
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Fig. 3. A'2 x 2 table used for scoring results of the transmission disequilibrium test. The letters a, b, c, and d are used to designate the different categories.
transmission (Spielman, McGinnis, and Ewens, 1993), the fundamental characteristic of genetic traits. With the TDT, the unit of sampling is the nuclear family (the affected individual and his or her parents), whereas within the case-control study, the unit of sampling is the affected individual and an appropriate control only. After identification of a potential genetic association through a case-control study, studies of nuclear families using the TDT can clarify whether the association is truly due to linkage between the loci or is a spurious result. The TDT is a modified case-control study, which involves determining which of the parents' alleles were transmitted to the affected child and which were not. For a two-allele marker, these counts are scored in a 2 x 2 table (Fig. 3). A statistically significant result indicates that a specific allele is transmitted from heterozygous parent to offspring more frequently than expected by chance, and thus it identifies evidence for linkage disequilibrium (allelic association and linkage). The TDT is an effective and elegant methodological tool from a variety of angles. First, the issue of selecting appropriate controls is avoided, since the parental nontransmitted alleles are used as internal controls and, by definition, are appropriately matched. Second, this approach allows the inclusion of sporadic cases and their parents in genetic linkage studies instead of the sole use of families with multiple incidences of the disease, so-called multiplex pedigrees, thereby dramatically expanding the applications of genetic studies. Third, investigation of these families with a sporadic or single incidence of a disorder (so-called singleton families) and no family history of the disorder allows the researcher to begin to answer the question of whether cases in multiplex pedigrees are genetically different from sporadic cases. This answer has major public health benefits since common complex disorders are often found more frequently in sporadic cases than in familial cases. Case control and association studies, particularly TDT studies, are likely to be reported more frequently in the genetic literature attempting to characterize the genetic effects of complex disease. Therefore, it is important for genetic counselors to be aware of these studies and their methodology in order to analyze research on the complex disorders. Variations of the TDT are now being investigated that allow sampling patients and their unaffected siblings when parents are not available (Boehnke and Langefeld, 1998; Monks etal., 1997). This methodological advance is important in the study of late-onset diseases, such as glaucoma and dementia.
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EVALUATING THE RESEARCH FINDINGS Even with some knowledge of new methodologies used in case-control studies for complex diseases, genetic health professionals are still faced with the task of determining the credibility and clinical application of a research finding. Unfortunately, no standard guide or criterion that measures the validity of a genetic research finding is available. However, a general framework can be utilized. When investigating the validity of a genetic research finding, it is wise to first find the report of the original finding. The next step is to search for articles that contain information about replication studies, both positive and negative. We initially place heavy emphasis on evaluating replication studies since the clinical applicability of a research finding will be determined by multiple confirmations.
REPLICATION OF RESEARCH FINDINGS Replication is the gold standard for determining the credibility of research findings. Simply put, researchers try to replicate the analysis or experiment that produced the original research finding. When another scientific team performs the replication experiments, this is called external replication. External replication is welcomed because it provides an outside assessment of the validity and generalizability of the findings. However, external replication necessitates another scientific team with the expertise, interest, and resources to replicate the study in a timely manner. Unfortunately, external replication is not always possible. For instance, other researchers may not have a large enough data set to perform the same analyses as the original researchers. Or differences in phenotypic assignment between data sets may hamper comparisons. Sometimes, if another data set is available, the original research team may do their own replication analysis. This is called internal replication. Internal replication has the advantage that the conditions used in the first experiment can readily be re-created in a second experiment. Internal replication may or may not be done before the original research finding is announced. There are two possible outcomes of replication studies. Specifically, research findings can be confirmed or disputed. Nonconfirmations may result in a dispute of the original findings, or the overall interpretations can be inconclusive. Each outcome portends different action on the part of the genetic counselor.
CONFIRMATION IN REPLICATION Sometimes, multiple external groups replicate an original research finding. For instance, the association of the APOE-4 allele with an increase in risk for Alzheimer disease in a dose-dependent manner has been confirmed in more than
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150 studies. Genetic counselors can feel comfortable that these research findings are accurate. Still, confirmation of research findings does not always allow a clear understanding of the relevance of the finding(s) for patients and their families. Often, much work remains to understand what relevance this finding has for patients and their families. Unfortunately, it is rare to find such consistent and overwhelming replication. However, extensive confirmation of results is the first step toward the development of clinical recommendations.
FAILURE TO REPLICATE A STUDY If replication studies do exist, results may be confusing and conflicting. For instance, some studies may support one conclusion while identically designed studies with diametrically opposed outcomes support other conclusions. Dispute of the original research finding may indicate that the finding is unique to the population studied in the original research. Alternatively, the reported genetic effect may be small or the result of a flawed study. This scenario is probably encountered frequently by genetic counselors. When there are conflicting studies, a preponderance of the evidence (such as three studies confirming and one disputing the original finding) does not prove that one finding is right while the alternative is wrong. When one study disputes the original research finding, other studies must be done which will support either the original research finding or the alternative finding. All the studies need to be reviewed in context and examined to see how closely the parameters in each study matched. It may not be possible to draw a conclusion until further research is done to resolve conflicting results. As a result, no clinical recommendations may be available but may be forthcoming. However, genetic counselors can educate patients and their families about the conflicting research evidence.
RESEARCH FINDINGS CAN DEVELOP INTO CLINICAL RECOMMENDATIONS Although clinical recommendations may not already exist, genetic counselors can look forward to, and participate in, the development of such recommendations. As peer-reviewed evidence accumulates for or against a research finding, a panel of experts will often review the reports associated with the finding and make a clinical recommendation. An example of a research finding that evolved into a clinical recommendation is that of preconceptional and prenatal supplementation of 0.4 mg of folic acid to reduce the incidence of neural tube defects. Currently, at least five organizations now recommend the preconceptional use of folic acid (Baty et al., 1996; Centers for Disease Control and Prevention, 1992; Crandall et al., 1995; MRC Vitamin Study Research Group, 1991). Clinical recommendations,
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while seemingly simplistic, are often the result of years of research, multiple replications of a specific research finding, and careful review by an interdisciplinary team of professionals. When a credible organization or consensus panel issues a clinical recommendation, it is usually appropriate to make the recommendation to a patient. Another appropriate sign is when multiple credible organizations make the same clinical recommendation. Concurring clinical recommendations can increase the genetic counselor's confidence in making the recommendation and, in turn, increase a patient's confidence in acting on that recommendation. Finally, even when a research finding leads to the availability of genetic testing, the genetic counselor may feel more comfortable waiting for a clinical recommendation for this test before widely offering the genetic test to patients and their families. Several considerations should be explored: Although it may be feasible to obtain genetic testing, is the test appropriate given our knowledge about the disorder? Is the test useful and valuable for families? Will the results of the genetic test provide enough reliable information for a patient or family to make medical management decisions? Genetic research findings, which may result in development of genetic tests or therapeutic recommendations, do not have a standard professional board which deems them ready for public medical use. In contrast, the Food and Drug Administration evaluates the efficacy and safety of potential therapeutic agents, such as medications. Nonetheless, genetic counselors and other professionals can play a role or participate in the discourse concerning the appropriateness and readiness of genetic testing for complex disorders for public use even before clinical recommendation can be made.
CONCLUSION This is an exciting time for genetic counselors. The emergence of new statistical and molecular methodologies will extend the role of genetic counselors into personalized risk assessment in common complex disorders. Although it may seem like a daunting task, an understanding of the methodologies, such as case-control studies and TDT, and a framework for evaluating original research findings and replication studies will enable genetic counselors to critically analyze complex disorders studies for use in patient risk analysis.
ACKNOWLEDGMENTS This work was presented at the National Society of Genetic Counseling's 16th Annual Education Conference in Baltimore in 1997. The authors wish to thank Chad Haynes for assistance with figure design and Roberta Parker for
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expert secretarial assistance. The authors gratefully acknowledge support from grants of the National Institute of Neurological Disorders and Stroke (NINDS) NS26630, NS36768, and HD33400, and grants from the March of Dimes and from the American Syringomyelia Alliance Project. REFERENCES Baty BJ, Cohen L, Phelps L, Speer MC, Stengel P, Williamson-Kruse L (1996) Folic acid and the prevention of neural tube defects: A position paper: National Society of Genetic Counselors. J Gen Couns 5:139-145. Boehnke M, Langefeld CD (1998) Genetic association mapping based on discordant sib pairs: The discordant alleles test (DAT). Am J Hum Genet 62:950-961. Centers for Disease Control and Prevention (1992) Recommendations for the use of folic acid to reduce the number of cases of spina bifida and other neural tube defects. Morbidity and Mortality Weekly 41:1-7. Collins FS (1995) Positional cloning moves from periditional to traditional. Nat Genet 9:347-350. Corder EH, Saunders AM, Risch N, Strittmatter WJ, Schmechel DE, Gaskell PC, Rimmler JB, Locke PA, Conneally PM, Schmader KE, Small GW, Roses AD, Haines JL, Pericak-Vance MA (1994) Apolipoprotein E type 2 allele decreases the risk of late onset Alzheimer disease. Nat Gene 7:180-184. Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses AD, Haines JL, Pericak-Vance MA (1993) Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families. Science 261:921-923. Crandall BF, Corson VL, Goldberg JD, Knight G, Salafsky IS (1995) Folic acid and pregnancy. Am J Med Genet 55:134-135. Davidoff AM, Thompson CV, Grimm JK, Shorter NA, Filston HC, Oakes WJ (1991) Occult spinal dysraphism in patients with anal agenesis. J Pediatr Surg 26:1001-1005. Doll R, Hill AB (1956) Lung cancer and other causes of death in relation to smoking: A second report on the mortality of British doctors. Br Med J 2:1071. Farrer LA, Cupples LA, Haines JL, Hyman B, Kukull WA, Mayeux R, Myers RH, Pericak-Vance MA, Risch N, van Duijn CM. APOE, Alzheimer Disease Meta Analysis Consortium (1997) Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. JAMA 278:1349-1356. Futreal PA, Liu Q, Shattuck-Eidens D, Cochran C, Harshman K, Tavtigian S, Bennett LM, HaugenStrano A, Swensen J, Miki Y, Eddington K, McClure M, Frye C, Weaver-Feldhaus J, Ding W, Gholami Z, Soderkvist P, Terry L, Jhanwar S, Berchuck A, Iglehart JD, Marks J, Ballinger DG, Barrett JC, Skolnick MH, Kamb A, Wiseman R (1994) BRCA 1 mutations in primary breast and ovarian carcinomas. Science 266:120-126. Lander ES, Schork NJ (1994) Genetic dissection of complex traits. Science 265:2037-2048. Levy-Lahad E, Wasco W, Poorkaj P, Romano DM, Oshima J, Pettingell WH, Yu CE, Jondro PD, Schmidt SD, Wang K, Crowley AC, Fu Y-H, Guenette SY, Galas D, Nemens E, Wijsman EM, Bird TD, Schellenberg GD, Tanzi RE (1995) Candidate gene for the chromosome 1 familial Alzheimer's disease locus. Science 269:973-977. Monks SA, Martin ER, Weir BS, Kaplan NL (1997) A sibship test of linkage in the absence of parental information. Am J Hum Genet 61:A286 (Abstract). MRC Vitamin Study Research Group (1991) Prevention of neural tube defects: Results of the Medical Research Council Vitamin Study. Lancet 338:131-137. Pericak-Vance MA (1998) Overview of linkage analysis in complex traits. Unit 1.9. In: Protocols in Human Genetics. Pericak-Vance MA, Bebout JL, Gaskell PC, Yamaoka LH, Hung W-Y, Alberts MJ, Walker AP, Bartlett RJ, Haynes CS, Welsh KA, Earl NL, Heyman A, Clark CM, Roses AD (1991) Linkage studies in familial Alzheimer's disease: Evidence for chromosome 19 linkage. Am J Hum Genet 48:10341050.
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St. George-Hyslop PH, Tanzi RE, Polinsky RJ, Haines JL, Watkins PC, Myers RH, Feldman RG, Pollen D, Drachman D (1987) The genetic defect causing familial Alzheimer's disease maps on chromosome 21. Science 235:885-890. Saunders AM, Strittmatter WJ, Breitner JC, Schmechel D, St. George-Hyslop PH, Pericak-Vance MA, Joo SH, Rosi BL, Gusella JF, Crapper-MacLachlan DR, Growden J, Alberts MJ, Hulette C, Crain B, Goldgaber D, Roses AD (1993) Association of apolipoprotein E allele 4 with late-onset familial and sporadic Alzheimer's disease. Neurology 43:1467-1472. Semchuk KM, Love EJ (1995) Effects of agricultural work and other proxy-derived case-control data on Parkinson's disease risk estimates. Am J Epidemiol 141:747-754. Sherrington R, Rogaev E, Liang Y, Rogaeva EA, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, Tsuda T, Mar L, Foncin J-F, Bruni AC, Montesi MP, Sorbi S, Rainero I, Pinessi L, Nee L, Chumakov I, Pollen D, Brookes A, Sanseau P, Polinsky RJ, Wasco W, DaSilva HAR, Haines JL, Pericak-Vance MA, Tanzi RE, Roses AD, Fraser PE, Rommens JM, St. George-Hyslop PH (1995) Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature 375:754-760. Spielman RS, McGinnis RE, Ewens WJ (1993) Transmission test for linkage disequilibrium: The insulin gene region and insulin-dependent diabetes mellitus (IDDM). Am J Hum Genet 52:506516. Taubes G (1995) Epidemiology faces its limits. Science 269:164-169. Thomson G (1994) Identifying complex disease genes: Progress and paradigms. Nat Genet 8:189-194. Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J, Collins N, Gregory S, Gumbs C, Micklem G (1995) Identification of the breast cancer suspectibility gene BRCA2. Nature 378:789-792.
