The Journal of Allergy and Clinical Immunology
Volume 117, Issue 6 , Pages 1303-1305, June 2006

The Faustian bargain of genetic association studies: Bigger might not be better, or at least it might not be good enough

  • Donata Vercelli, MD

      Affiliations

    • Corresponding Author InformationReprint requests: Donata Vercelli, MD, University of Arizona Health Sciences Center, Respiratory Sciences Center, 1501 N Campbell Ave, Suite 2349, Tucson, AZ 85724-5030.
    • ,
  • Fernando D. Martinez, MD

Received 24 March 2006; accepted 27 March 2006.

Tucson, Ariz

Article Outline

Key word: Genetics

 

Genetics has made a momentous irruption onto the asthma and allergy scene, rapidly becoming a paradigm to reckon with. Meetings focusing on allergic inflammation these days feature talks on genetic determinants of allergy susceptibility, journals are literally inundated with genetic association and linkage studies of allergic phenotypes, and federal agencies are paying more and more attention to genetic research. Importantly, genetic evidence from human populations is often used as a golden standard to validate candidate genes identified in animal models.1, 2, 3

This striking success is likely to be rooted in the hope that genetics might help provide answers to some of the least tractable issues in the field of complex lung diseases, such as the identification of risk factors, the pathophysiology of disease initiation and severity, the rationale for phenotypic diversity, and the pronounced interindividual differences in the response to treatment. Amid the growing interest in genetics as a novel paradigm able to solve old problems, however, some disturbing cracks are appearing that signal a deeper malaise. The most obvious of these signs was provided by an editorial that appeared in Nature Genetics in November 2005 and expressed alarm for the more and more frequent inability of association studies to be replicated.4 Likewise, an editorial published in Thorax acknowledged that genetic association studies of complex diseases have “acquired a bad reputation …because of problems with poor design and variable replication of findings.”5 The first antidote to the lack-of-replication problem, both editorials suggested, is to analyze populations of size adequate for fully powered calculations, with this size being established case by case on the basis of considerations such as expected relative risk and allele frequency of the polymorphism or polymorphisms under consideration.

But bigger is, alas, not necessarily better, or at least it is not necessarily good enough, as eloquently shown by the difficulties Maier and Strachan6 ran into with the work presented in this issue of the Journal. These authors, a well-known group that has made important contributions to the epidemiology of allergic inflammation,7, 8, 9, 10 built on the results of a recent meta-analysis suggesting a significant, albeit small, reduction in the frequency of asthma among children with type 1 diabetes.11 Taking the TH1/TH2 paradigm to its extreme genetic consequences, these investigators asked whether a TH2-mediated disease (allergy) and a TH1-mediated disease (type 1 diabetes) share genetic loci, such that susceptibility alleles for one disease provide protection for the other. The phenotype tested for association was total serum IgE levels. As targets, these authors picked genes known to be associated with allergy (those in the TH2 cytokine pathway: IL13, IL4, and IL4RA, together with FCERIB, IL12B, and TBET), as well as genes unequivocally associated with type 1 diabetes (CTLA4 and PTPN22). Immunoregulatory IL2RA/CD25 was also tested on the grounds that it is expressed in T regulatory cells and might therefore influence susceptibility to both type 1 diabetes and atopic illness. In the perception of the authors (and of most readers, we would guess), the main strength of this work was the size of the population available for analysis: up to 4570 samples from the British 1958 Birth Cohort, which includes all births in England, Wales, and Scotland during 1 week in 1958, and “has been influential in advancing understanding of the natural history and epidemiological features of allergic disease.”12, 13, 14 Most importantly, as the authors point out, “these were the largest sample sizes reported so far for the identification of genetic determinants of circulating IgE levels.”

After such premises, the results of the study are likely to come as an anticlimax. Only one among the 8 genes under study was found to be significantly associated with IgE levels, and this gene was none other than old IL13, to this day perhaps the most replicated genetic determinant of allergy susceptibility in the literature15 and one for which functional data are available to support the genetic evidence.16, 17 Interestingly, even the most IgE-associated IL13 polymorphism (IL13/−1512A>C; minor allele frequency in the study population, 18%) could not explain more than a negligible proportion (0.69%) of the phenotypic variance. No other candidate, not even the type 1 diabetes genes, showed an association, direct or inverse, with the phenotype of interest.

How should we interpret these data? Should we conclude that this study, based as it is on an unprecedentedly large population, definitively proves that none of the genes tested by the British group are implicated in the pathogenesis of IgE dysregulation? Or is it possible that a problematic hypothesis and a large (in fact, too large) study population might have conspired to dictate a confusing outcome?

Back to Article Outline

A problematic hypothesis … 

Understanding the pathogenesis of human complex diseases, such as allergic inflammation, will require a multidisciplinary approach integrating the expertise of clinicians (who have the critical task of phenotyping study participants according to accurate and standardized criteria), biologists (who need to characterize the pathophysiology of the disease in terms of underlying mechanisms and genetic pathways), geneticists (who need to design studies of adequate power and homogenous ethnicity and must choose target genotypes that capture meaningful variation across a locus), and functional genomicists (who sift through the dozens of polymorphisms currently in the databases to identify functional genetic variants). Working hypotheses formulated out of a similar interactive framework are bound for trouble.

The TH1/TH2 genetic paradigm tested by Maier et al,6 although strongly supported in mouse strains exhibiting polarized patterns of TH cell differentiation and TH cell–dependent phenotypes, demands more nuances in human subjects. Human immunologists know from everyday experience that the contraposition between TH1 and TH2 responses is an oversimplification because human T-cell polarization can be occasionally pronounced but is never complete. Therefore the expectation that genes involved in susceptibility to TH1 inflammation might protect against TH2-dependent high IgE levels or vice versa might have been unrealistic. In fact, the inability of the British study to find genetic evidence in support of a rigid TH1/TH2 dichotomy will provide one more argument in favor of a less mechanic and more articulate view of human immunologic diseases.

Back to Article Outline

… And a large, possibly too large, study population 

According to the more dynamic paradigm that is currently replacing the TH1/TH2 dichotomy, both arms of the T-cell response are modulated and balanced by regulatory populations of T cells18, 19 and, as more recently suggested, even B cells. Of note, the function of these regulatory populations is strongly influenced by environmental factors acting through the innate immune interface.20 In fact, the most updated and functionally oriented version of the hygiene hypothesis attributes the current, rapid, and steep increase in the incidence of both TH1- and TH2-like diseases to the elimination of environmental, pathogen-derived signals that drove the evolution of our immune system and maintained an active regulatory interface.21, 22

The effects of natural variation at this regulatory interface are often tricky and always complex because they are inevitably intertwined with those of the relevant environmental exposures. In polygenic diseases, such as asthma and allergy, the effect of variation on patterns of gene expression is difficult to decipher unless the appropriate exposures are taken into consideration, but what these exposures are is not always clear. A good example is provided by the conflicting results obtained for CD14/−260C>T (also known as CD14/−159), a functional promoter polymorphism.23 The ability of CD14/−260C>T to influence IgE levels and asthma in opposite ways only became evident once the relevant environmental exposures (cat/dog versus stable animals in a European population24 and low versus high endotoxin in subjects of African descent25) were taken into consideration. The striking conclusion reached independently by both these studies was that the same polymorphism can be associated with either disease or protection, depending on the environment to which the subjects are exposed. A genome screen for asthma and bronchial hyperresponsiveness also recently published in the Journal provides another case in point. In 200 Dutch families ascertained through a parent with asthma, strong evidence for linkage was obtained for chromosome 5q but only for the families in which the children were exposed to passive smoking.26 Interestingly, figuring out what could count as a biologically meaningful environment might require some imagination. For instance, recent work strongly suggests that sex could be considered as an environmental factor that can lead to different effects of the same variation in men and women.27, 28 It is not unreasonable to surmise that as-yet undetected gene-environment interactions might confound the picture more often than expected, contributing to the lamented problems of replication frequently encountered by genetic association studies.

Because twin studies on the heritability of total IgE levels have clearly shown 40% of the variance in the phenotype is due to environmental factors,9 it is possible, nay likely, that the current results obtained in the British 1958 Birth Cohort have been affected by the lack of an environmental component to the analysis. Paradoxically, the very size of the study population might have carried along differences in lifestyle, socioeconomic status, and exposure among study participants recruited from different parts of the United Kingdom. Because the association between genetic variations, environment, and polygenic diseases is not always linear and unidirectional,27, 29, 30 lumping together groups of individuals faced with different environmental pressures is likely to drastically dilute the recognizable role of genetic determinants to the point of erasing them. Thus the inclusion of thousands of subjects might have resulted in a Faustian bargain in which sample size failed to compensate for heterogeneity of environment, and the lack of environmental data might have made it impossible to tease out distinct environmental effects on specific genotypes.

If this is true, one might be tempted to conclude that the genetic epidemiology of complex lung diseases is caught in a vicious circle. Genetic dysregulation in allergy and asthma hits multiple steps in key pathways, with small effects. The smaller the effects, the larger a study population needs to be for those effects to become appreciable, but the larger the population, the more difficult it is to control for environmental factors. A solution is to adjust study design to our deepened understanding of the complexity of the interactions underlying disease pathogenesis. Indeed, experience tells us that although large populations recruited without paying attention to environmental determinants might not be terribly informative, targeted recruitment efforts collecting a wealth of relevant environmental data might provide exciting breakthroughs, even when more limited in size.25, 31, 32, 33, 34 In the end, hypothesis-driven genetic epidemiology might be a more effective and more interesting partner for disease-oriented biologic research. Furthermore, dissecting the effect of the environmental context might foster preventive and therapeutic strategies predicated on innocuous surrogates or antagonists of the exposures found to be associated with the phenotypes of interest in susceptible individuals.

Back to Article Outline

References 

  1. Zimmermann N, King N, Laporte J, Yang M, Mishra A, Pope SM, et al. Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest. 2003;111:1863–1874
  2. Vercelli D. Arginase: marker, effector, or candidate gene for asthma?. J Clin Invest. 2003;111:1815–1817
  3. Li H, Romieu I, Sienra-Monge JJ, Ramirez-Aguilar M, Estela del Rio-Navarro B, Kistner EO, et al. Genetic polymorphisms in arginase I and II and childhood asthma and atopy. J Allergy Clin Immunol. 2006;117:119–126
  4. Anonymous . Framework for a fully powered risk engine. Nat Genet. 2005;37:1153
  5. Hall IP, Blakey JD. Genetic association studies in Thorax. Thorax. 2005;60:357–359
  6. Maier LM, Howson JMM, Walker N, Spickett GP, Jones RW, Ring SM, et al. Association of IL13 with total IgE: Evidence against an inverse association of atopy and diabetes. J Allergy Clin Immunol. 2006;117:1306–1313
  7. Strachan D, Sibbald B, Weiland S, Ait-Khaled N, Anabwani G, Anderson HR, et al. Worldwide variations in prevalence of symptoms of allergic rhinoconjunctivitis in children: the International Study of Asthma and Allergies in Childhood (ISAAC). Pediatr Allergy Immunol. 1997;8:161–176
  8. Strachan DP, Cook DG. Parental smoking and allergic sensitisation in children. Thorax. 1998;53:117–123
  9. Strachan DP, Wong HJ, Spector TD. Concordance and interrelationship of atopic diseases and markers of allergic sensitization among adult female twins. J Allergy Clin Immunol. 2001;108:901–907
  10. Weiland SK, Bjorksten B, Brunekreef B, Cookson WO, von Mutius E, Strachan DP. Phase II of the International Study of Asthma and Allergies in Childhood (ISAAC II): rationale and methods. Eur Respir J. 2004;24:406–412
  11. Cardwell CR, Shields MD, Carson DJ, Patterson CC. A meta-analysis of the association between childhood type 1 diabetes and atopic disease. Diabetes Care. 2003;26:2568–2574
  12. Strachan DP. Hay fever, hygiene, and household size. BMJ. 1989;299:1259–1260
  13. Strachan DP, Butland BK, Anderson HR. Incidence and prognosis of asthma and wheezing illness from early childhood to age 33 in a national British cohort. BMJ. 1996;312:1195–1199
  14. Williams HC, Strachan DP. The natural history of childhood eczema: observations from the British 1958 birth cohort study. Br J Dermatol. 1998;139:834–839
  15. Ober C, Hoffjan S. Asthma genetics 2006: the long and winding road to gene discovery. Genes Immun. 2006;7:95–100
  16. van der Pouw Kraan TCTM, van Veen A, Boeije LCM, van Tuyl SAP, de Groot ER, Stapel SO, et al. An IL-13 promoter polymorphism associated with increased risk of allergic asthma. Genes Immun. 1999;1:61–65
  17. Vladich FD, Brazille SM, Stern D, Peck ML, Ghittoni R, Vercelli D. IL-13 R130Q, a common variant associated with allergy and asthma, enhances effector mechanisms essential for human allergic inflammation. J Clin Invest. 2005;115:747–754
  18. Chatila TA. Role of regulatory T cells in human diseases. J Allergy Clin Immunol. 2005;116:949–959
  19. Seroogy CM, Gern JE. The role of T regulatory cells in asthma. J Allergy Clin Immunol. 2005;116:996–999
  20. Vercelli D. Innate immunity: sensing the environment and regulating the regulators. Curr Opin Allergy Clin Immunol. 2003;3:343–346
  21. Wills-Karp M, Santeliz J, Karp CL. The germless theory of allergic disease: revisiting the hygiene hypothesis. Nat Rev Immunol. 2001;1:69–75
  22. Vercelli D. Genetics, epigenetics and the environment: switching, buffering, releasing. J Allergy Clin Immunol. 2004;113:381–386
  23. LeVan TD, Bloom JW, Bailey TJ, Karp CL, Halonen M, Martinez FD, et al. A common single nucleotide polymorphism in the CD14 promoter decreases the affinity of Sp protein binding and enhances transcriptional activity. J Immunol. 2001;167:5838–5844
  24. Eder W, Klimecki W, Yu L, von Mutius E, Riedler J, Braun-Fahrländer C, et al. Opposite effects of CD14/-260 on serum IgE levels in children raised in different environments. J Allergy Clin Immunol. 2005;116:601–607
  25. Zambelli-Weiner A, Ehrlich E, Stockton ML, Grant AV, Zhang S, Levett PN, et al. Evaluation of the CD14/-260 polymorphism and house dust endotoxin exposure in the Barbados Asthma Genetics Study. J Allergy Clin Immunol. 2005;115:1203–1209
  26. Meyers DA, Postma DS, Stine OC, Koppelman GH, Ampleford EJ, Jongepier H, et al. Genome screen for asthma and bronchial hyperresponsiveness: interactions with passive smoke exposure. J Allergy Clin Immunol. 2005;115:1169–1175
  27. Ober C, Thompson EE. Rethinking genetic models of asthma: the role of environmental modifiers. Curr Opin Immunol. 2005;17:1–9
  28. Weiss LA, Pan L, Abney M, Ober C. The sex-specific genetic architecture of quantitative traits in humans. Nat Genet. 2006;38:218–222
  29. Vercelli D. Learning from discrepancies: CD14 polymorphisms, atopy and the endotoxin switch. Clin Exp Allergy. 2003;33:153–155
  30. Martinez FD. Gene-environment interactions in asthma and allergies: a new paradigm to understand disease causation. Immunol Allergy Clin North Am. 2005;25:709–721
  31. Braun-Fahrlander C, Riedler J, Herz U, Eder W, Waser M, Grize L, et al. Environmental exposure to endotoxin and its relation to asthma in school-age children. N Engl J Med. 2002;347:869–877
  32. Schram D, Doekes G, Boeve M, Douwes J, Riedler J, Ublagger E, et al. Bacterial and fungal components in house dust of farm children, Rudolf Steiner school children and reference children—the PARSIFAL Study. Allergy. 2005;60:611–618
  33. Bottema RW, Reijmerink NE, Koppelman GH, Kerkhof M, Postma DS. Phenotype definition, age, and gender in the genetics of asthma and atopy. Immunol Allergy Clin North Am. 2005;25:621–639
  34. von Mutius E, Schmid S. The PASTURE project: EU support for the improvement of knowledge about risk factors and preventive factors for atopy in Europe. Allergy. 2006;61:407–413

 Disclosure of potential conflict of interest: D. Vercelli receives grant support from the NIH and is on the speakers' bureau for Merck. F. Martinez receives grant support from the NIH and is on the speakers' bureau for Merck.

PII: S0091-6749(06)00736-6

doi:10.1016/j.jaci.2006.03.030

The Journal of Allergy and Clinical Immunology
Volume 117, Issue 6 , Pages 1303-1305, June 2006