On the Significance of Linkage Studies of Complex Traits

To the Editor:We read with interest the recent article by Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) reporting linkage of premature myocardial infarction (MI) to a locus on 1p34-36. The authors have recruited a large sample of families with premature coronary artery disease (CAD), as detected by catheterization, revascularization, or MI. Such large-scale approaches will be crucial to the identification of genetic variation underlying complex traits, including atherosclerotic CAD and MI, the leading killer of men and women in the Western world. We commend the authors for undertaking such an important study.Although their article reports the nominal LOD score of 11.68 for linkage of premature MI to 1p34-36 and a corrected pointwise P value of .00011, we note that the genomewide significance of the linkage statistic is not clear. We have some methodological concerns regarding the initial emphasis on results presented in their table 2 and figure 1: 1.The finding of 11 independent −log10P values >3.13 in the multipoint linkage approach (regions identified in single-point and multipoint W2 analyses overlap significantly) raises questions regarding the assertion that such a threshold corresponds to a LOD score >2.2, as proposed by Lander and Kruglyak (1995xGenetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Lander, E and Kruglyak, L. Nat Genet. 1995; 11: 241–247Crossref | PubMed | Scopus (4037)See all References1995). A LOD score >2.2 should occur once in a maximally informative genomewide scan, under the null hypothesis that no genetic linkage is present. It seems unlikely that 11 true genetic loci influencing a complex phenotype would be detected in a single study. It is more likely that the asymptotic P value statistic generated by the authors’ modified Haseman-Elston regression model is inflated.2.The marked attenuation of the multipoint P value of <10−12 to 10−4 on pointwise permutation testing at the 1p34-36 locus suggests that the nominal asymptotic P values are inflated. It is possible that the method of linkage analysis may have inflated the P value estimates. In particular, the treatment of the dichotomous MI phenotype as a continuous variable may not be appropriate. The assumption of equal variances required for a quantitative trait may not be valid for different numbers of affected and unaffected individuals in each family.3.The authors’ attempt to correct the pointwise empirical P value estimates for the number of markers tested is quite important for establishing the significance of their findings. However, the attempt may be inadequate to account for the testing of multiple markers. The authors refer to the simulation analyses by Wiltshire et al. (2002xEvaluating the results of genomewide linkage scans of complex traits by locus counting. Wiltshire, S, Cardon, LR, and McCarthy, MI. Am J Hum Genet. 2002; 71: 1175–1182Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References2002), which explored the influence of experimental deviations from the Lander-Kruglyak assumptions of completely informative linkage analyses. We are uncertain whether the empirical genomewide P value estimates derived from the Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) data correspond to the same nominal LOD-score thresholds identified in the Wiltshire et al. (2002xEvaluating the results of genomewide linkage scans of complex traits by locus counting. Wiltshire, S, Cardon, LR, and McCarthy, MI. Am J Hum Genet. 2002; 71: 1175–1182Abstract | Full Text | Full Text PDF | PubMed | Scopus (65)See all References2002) study using simulated data. Permutation testing of the Wang et al. (2004xPremature myocardial infarction novel susceptibility locus on chromosome 1P34-36 identified by genomewide linkage analysis. Wang, Q, Rao, S, Shen, G-Q, Li, L, Moliterno, DJ, Newby, LK, Rogers, WJ, Cannata, R, Zirzow, E, Elston, RC, and Topol, EJ. Am J Hum Genet. 2004; 74: 262–271Abstract | Full Text | Full Text PDF | PubMed | Scopus (142)See all References2004) data by use of the Wiltshire approach might provide greater confidence regarding the genomewide significance of the study findings, but this approach admittedly represents a significant computational burden.4.Genomewide linkage analyses at 10 cM may not extract maximally the identity-by-descent information for the sample under study. A fine-mapping study at higher density across a region of interest may show a change in the maximum-LOD-score estimate. An increase in the LOD-score estimate with better information extraction might be reassuring, but a fall in the LOD score may signal a false-positive finding. We would encourage the authors to perform and publish the results of a higher-density map.5.The study cohort was recruited on the basis of a composite definition of premature CAD. Was the broader CAD phenotype (including MI) the primary phenotype prespecified in the linkage analysis? The reported MI linkage analysis represents a subgroup of the subjects enrolled; the “less-restrictive” CAD phenotype was also tested and revealed no suggestive or significant linkage evidence. Could the authors clarify their original primary analysis and whether additional subgroups were analyzed? A true empirical P value would also account for the multiple phenotypes tested.On review of the study, the declaration of a finding of genomewide significance may not be as strongly supported as suggested by the authors. The results of this linkage analysis do not contain much overlap with those of similar analyses, and this certainly could result from differences in phenotype definition, environmental exposures, or study design (Pajukanta et al. 2000xTwo loci on chromosomes 2 and X for premature coronary heart disease identified in early- and late-settlement populations of Finland. Pajukanta, P, Cargill, M, Viitanen, L, Nuotio, I, Kareinen, A, Perola, M, Terwilliger, JD, Kempas, E, Daly, M, Lilja, H, Rioux, JD, Brettin, T, Viikari, JS, Ronnemaa, T, Laakso, M, Lander, ES, and Peltonen, L. Am J Hum Genet. 2000; 67: 1481–1493Abstract | Full Text | Full Text PDF | PubMed | Scopus (126)See all References2000; Francke et al. 2001xA genome-wide scan for coronary heart disease suggests in Indo-Mauritians a susceptibility locus on chromosome 16p13 and replicates linkage with the metabolic syndrome on 3q27. Francke, S, Manraj, M, Lacquemant, C, Lecoeur, C, Lepretre, F, Passa, P, Hebe, A, Corset, L, Yan, SL, Lahmidi, S, Jankee, S, Gunness, TK, Ramjuttun, US, Balgobin, V, Dina, C, and Froguel, P. Hum Mol Genet. 2001; 10: 2751–2765Crossref | PubMedSee all References2001; Broeckel et al. 2002xA comprehensive linkage analysis for myocardial infarction and its related risk factors. Broeckel, U, Hengstenberg, C, Mayer, B, Holmer, S, Martin, LJ, Comuzzie, AG, Blangero, J, Nurnberg, P, Reis, A, Riegger, GA, Jacob, HJ, and Schunkert, H. Nat Genet. 2002; 30: 210–214Crossref | PubMed | Scopus (252)See all References2002; Harrap et al. 2002xGenome-wide linkage analysis of the acute coronary syndrome suggests a locus on chromosome 2. Harrap, SB, Zammit, KS, Wong, ZY, Williams, FM, Bahlo, M, Tonkin, AM, and Anderson, ST. Arterioscler Thromb Vasc Biol. 2002; 22: 874–878Crossref | PubMed | Scopus (101)See all References2002; Chiodini and Lewis 2003xMeta-analysis of 4 coronary heart disease genome-wide linkage studies confirms a susceptibility locus on chromosome 3q. Chiodini, BD and Lewis, CM. Arterioscler Thromb Vasc Biol. 2003; 23: 1863–1868Crossref | PubMed | Scopus (63)See all References2003). Replication of linkage analyses for complex cardiovascular traits has often proven challenging, and the difficulty in achieving replication for MI underscores the many difficulties in the conduct and interpretation of such linkage analyses (Altmuller et al. 2001xGenomewide scans of complex human diseases: true linkage is hard to find. Altmuller, J, Palmer, LJ, Fischer, G, Scherb, H, and Wjst, M. Am J Hum Genet. 2001; 69: 936–950Abstract | Full Text | Full Text PDF | PubMed | Scopus (378)See all References2001).Identifying genetic factors underlying linkage peaks in this and related studies of MI will require considerable expenditure of resources and should proceed on the basis of the strongest possible evidence. We encourage the systematic comparison of available and accruing linkage data across studies in various CAD phenotypes, including continued assessment of the most appropriate linkage methods.