Eliminating the Improbable
2005; Lippincott Williams & Wilkins; Volume: 112; Issue: 14 Linguagem: Inglês
10.1161/circulationaha.105.570069
ISSN1524-4539
Autores Tópico(s)BRCA gene mutations in cancer
ResumoHomeCirculationVol. 112, No. 14Eliminating the Improbable Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBEliminating the ImprobableSherlock Holmes and Standards of Evidence in the Genomic Age David Herrington, MD, MHS David HerringtonDavid Herrington From the Department of Internal Medicine/Cardiology, Wake Forest University School of Medicine, Winston-Salem, NC. Originally published4 Oct 2005https://doi.org/10.1161/CIRCULATIONAHA.105.570069Circulation. 2005;112:2081–2084In 2003, Shearman and colleagues reported that a common variant in the estrogen receptor-alpha gene (ESR1 IVS1 −397 C allele) occurred more frequently in male subjects with myocardial infarction (MI) than in those free of MI.1 The following year, Schuit et al described an association between the common haplotype containing the alternate (T) allele and MI, but only in women.2 In this issue of Circulation, Koch et al3 report no association, in either men or women, between the ESR1 −397 T>C genotype or the common haplotypes containing this allele and MI. What does this collection of heterogeneous results tell us about genetic association studies in general and genetic variation in the estrogen receptor in particular; and how can we use this understanding to improve our ability to understand, treat, and prevent cardiovascular disease?Article p 2138Frequent failure to replicate initially promising genetic associations is a source of consternation and confusion for scientists and journal editors alike. Recent reviews of the literature indicate that 70% to 95% of reported genetic associations were not confirmed in subsequent studies.4,5 It has been argued that false-positive reports of genetic associations distract energy and resources from valid lines of inquiry and erode the credibility of medical research. Some reputable journals now decline to publish genetic associations of complex disorders except in "exceptional circumstances" (http://www.jci.org/misc/jcipoli.pdf), and some scientists have argued that association studies are so unreliable that they should be abandoned altogether.6"The temptation to form premature theories on insufficient evidence is the bane of our profession." —Sherlock Holmes, in "The Valley of Fear"7In their simplest form, genetic association studies determine whether the frequency of an allelic variant is more or less common in cases than it is in appropriately selected controls. Like any case-control study, in ensuring that the cases are representative of all cases and controls are representative of all controls, case definitions are precisely defined and uniformly applied; exposure variables, in this case the genetic variants, are measured with the least amount of error possible that is necessary to avoid biased conclusions. Studies also need adequate power to have a high likelihood of detecting a difference, if one really exists. To interpret any collection of genetic association studies with inconsistent results, these details of study design need to be considered first."It has long been an axiom of mine that the little things are infinitely the most important." —Sherlock Holmes, in "A Case of Identity"8There are, however, additional considerations unique to genetic association studies that also need to be addressed. For instance, allele frequency of many genetic variants differs according to ancestry. Thus, an imbalance in the genetic ancestry of cases versus controls (population stratification) may mask real associations or produce spurious ones.9 Self-reported race/ethnicity is an inadequate means to capture this genetic variability caused by ancestry, especially in admixed populations.10 It is unclear to what extent population stratification may have contributed to inconsistent genetic association findings in the past.11,12 There are now several analytic tools that make use of panels of unlinked genetic markers to estimate and/or adjust for ancestry in tests of association.13,14 These techniques have the added benefit of controlling for unmeasured environmental factors that also differ by genetic ancestry.Another concern has to do with the difference between tests of association with marker alleles (common) versus tests with true causal variants. The likelihood of a marker allele being associated with a genetically mediated trait depends on its correlation (linkage disequilibrium) with a true causal variant. Before the development of rapid sequencing and genotyping technologies, many association studies used sparse sets of markers (sometimes even single nucleotide polymorphisms [SNPs] within a gene) selected more for their ease of detection than their functional potential or ability to represent the genetic diversity in a gene or region. The value of some of these markers in past association studies is highly questionable. To further complicate matters, linkage disequilibrium is not uniform across populations,15 making it possible for a marker allele to be associated with a trait in one population but not another. Fortunately, new efforts to document the linkage disequilibrium architecture of the human genome and in multiple populations16 make it increasingly possible to select sets of informative SNPs that provide comprehensive coverage of an entire gene or region and are expected to be more reliable indicators of the presence or absence of association with traits of interest.Perhaps the biggest concern related to the problem of replication is that the staggering number of gene polymorphisms (≈11 million with allele frequency >1%),17 coupled with the newly developed technical ability to detect them in large numbers of subjects, has produced an irresistible opportunity to examine multiple variants with respect to multiple phenotypes, only to report the most promising and seemingly coherent associations.18 In most cases, such statistically significant associations are merely type I errors. Subsequent attempts to replicate the associations either fail or find that the strength of the association is less dramatic than initially reported—the "winners curse."5,19 Unfortunately, conventional adjustments for multiple independent tests (eg, Bonferroni) are too conservative when the tests are performed on correlated phenotypes (eg, hypertension, left ventricular hypertrophy, heart failure) or when the tested genotypes are commonly inherited together (ie, in the same haplotype block), leaving open the door of ambiguity about the real level of significance for any individual finding. Multivariate modeling, cluster analysis, haplotype analysis, and permutation tests in the setting of correlated phenotypes or genotypes can be used to reduce the chance of a type I error, but they still may not completely overcome the problem, especially if all of the comparisons performed are not completely described in the published reports.Another way to look at this problem, strangely enough, is based on a concept well known to clinicians but frequently forgotten by investigators: The prior probability of a positive test greatly influences the credibility of a test result. Often, investigators errantly assume the P value equals the likelihood that the observed difference is false. (The P value is actually the likelihood that an observed difference occurred because of chance alone when, in fact, there is no real difference.) In fact, for any level of statistical significance (typically P=0.05), the likelihood of a false-positive result is determined by the prior probability of a positive test and, to a lesser extent, the power to detect a difference, if one really exists (Figure). This is just as true for exercise stress tests as it is for tests of association in genetic case-control studies. The problem with genetic association studies, and especially genome-wide association studies, is that the prior probability of an association being true is generally quite low. Consider, for example, a trait with 5 causal variants. If there are 50 plausible candidate genes and an average of 20 independent variants per gene, there are 1000 possible associations to consider. The prior probability that any of the possible associations are real is only 5/1000=0.005. Even in a study with perfect power, 90% of the observed associations with a P 3000 cases compared with only 59 subjects with MI in the report by Sherman et al and 285 in the study by Schuit et al.1,2 On the basis of this fact alone, it would appear that the Koch et al study3 should provide more reliable evidence concerning the presence or absence of an association than either of the other studies. The confidence limits for the association between the −397 T>C variant and MI in the Koch et al study3 indicate that a positive or negative association as extreme as observed in the other 2 studies is highly unlikely; however, odds ratios as small as 0.9 or as large as 1.2 are still possible. These may seem like small effects, but they are in fact consistent with the common disease/common variant model of gene effects25 and are in line with the apparent association between the same variants and risk of fracture.24It is important to note that none of the studies provides a comprehensive evaluation of the genetic variation in the ESR1 gene as it relates to risk for MI. The 2 SNPs included in all 3 studies only provide information about common haplotypes for one of the roughly 20 haplotype blocks that exist in the ESR1 gene in white subjects of European descent (D.H., unpublished data). In addition, there are no established functional effects of either of the SNPs, although there is suggestive evidence of a potential effect of the −397 T>C on a transcription factor binding site.26 Thus, more information is needed about other potentially functional SNPs in linkage disequilibrium with the SNPs included in these studies, as well as SNPs in other regions of the ESR1 gene before making claims with confidence about genetic variation in the ESR1 gene and risk for MI.These studies also provide an opportunity to comment on the publication process for genetic association studies. If we attempt to constrain genetic epidemiology, or at least reported genetic epidemiology to a minimum of false-positive results, then we run the risk of replacing irrational exuberance over genotyping with imprudent pessimism concerning potentially valid genetic associations. With careful attention to study design and use of contemporary methods to address population stratification, linkage disequilibrium, and multiple testing, there is good reason to expect more consistent and informative results from genetic association studies in the future. It is important to acknowledge that most of science is in fact a recursive process of identifying patterns in observable events followed by testing to confirm whether the pattern is reproducible. The medical literature is the necessary forum for this recursive process, even if it means occasionally publishing false-positive or true negative results. The emphasis should be on studies that are performed with methodological rigor and accompanied by a realistic assessment of the strength of evidence for or against a putative association. To that end, the editors of Circulation should be commended for publishing an important negative study that adds to our still-incomplete fund of knowledge about the cardiovascular effects of the estrogen receptor and its genetic variants."Any truth is better than indefinite doubt."Sherlock Holmes, in "The Yellow Face"27The opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.FootnotesCorrespondence to Dr David Herrington, Dept of Internal Medicine/Cardiology, Wake Forest University School of Medicine, Winston-Salem NC, 27157-1040. E-mail [email protected] References 1 Shearman AM, Cupples LA, Demissie S, Peter I, Schmid CH, Karas RH, Mendelsohn ME, Housman DE, Levy D. Association between estrogen receptor alpha gene variation and cardiovascular disease. JAMA. 2003; 290: 2263–2270.CrossrefMedlineGoogle Scholar2 Schuit SC, Oei HH, Witteman JC, Geurts van Kessel CH, van Meurs JB, Nijhuis RL, van Leeuwen JP, de Jong FH, Zillikens MC, Hofman A, Pols HA, Uitterlinden AG. Estrogen receptor alpha gene polymorphisms and risk of myocardial infarction. JAMA. 2004; 291: 2969–2977.CrossrefMedlineGoogle Scholar3 Koch W, Hoppmann P, Pfeufer A, Mueller JC, Schömig A, Kastrati A. No replication of association between estrogen receptor α gene polymorphisms and susceptibility to myocardial infarction in a large sample of patients of European descent. Circulation. 2005; 112: 2138–2142.LinkGoogle Scholar4 Hirschhorn JN, Lohmueller K, Byrne E, Hirschhorn K. A comprehensive review of genetic association studies. Genet Med. 2002; 4: 45–61.CrossrefMedlineGoogle Scholar5 Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN. Meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease. 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