Molecular Psychiatry (2011) 16, 516–532 & 2011 Macmillan Publishers Limited All rights reserved 1359-4184/11 www.nature.com/mp ORIGINAL ARTICLE Poor replication of candidate genes for major depressive disorder using genome-wide association data FJ Bosker1,8, CA Hartman1,8, IM Nolte2,8, BP Prins2, P Terpstra2, D Posthuma3,4, T van Veen5, G Willemsen3, RH DeRijk5, EJ de Geus3, WJ Hoogendijk6, PF Sullivan7, BW Penninx1,5,6, DI Boomsma3, H Snieder2,8 and WA Nolen1,8 1Department of Psychiatry, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 2Unit of Genetic Epidemiology and Bioinformatics, Department of Epidemiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands; 3Department of Biological Psychology, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands; 4Department of Medical and Functional Genomics, VU University Medical Center, Amsterdam, The Netherlands; 5Department of Psychiatry, Leiden University Medical Center, Leiden, The Netherlands; 6Department of Psychiatry, VU University Medical Center, Amsterdam, The Netherlands and 7Department of Genetics, University of North Carolina, Chapel Hill, NC, USA Data from the Genetic Association Information Network (GAIN) genome-wide association study (GWAS) in major depressive disorder (MDD) were used to explore previously reported candidate gene and single-nucleotide polymorphism (SNP) associations in MDD. A systematic literature search of candidate genes associated with MDD in case–control studies was performed before the results of the GAIN MDD study became available. Measured and imputed candidate SNPs and genes were tested in the GAIN MDD study encompassing 1738 cases and 1802 controls. Imputation was used to increase the number of SNPs from the GWAS and to improve coverage of SNPs in the candidate genes selected. Tests were carried out for individual SNPs and the entire gene using different statistical approaches, with permutation analysis as the final arbiter. In all, 78 papers reporting on 57 genes were identified, from which 92 SNPs could be mapped. In the GAIN MDD study, two SNPs were associated with MDD: C5orf20 (rs12520799; P = 0.038; odds ratio (OR) AT = 1.10, 95% CI 0.95–1.29; OR TT = 1.21, 95% confidence interval (CI) 1.01–1.47) and NPY (rs16139; P = 0.034; OR C allele = 0.73, 95% CI 0.55– 0.97), constituting a direct replication of previously identified SNPs. At the gene level, TNF (rs76917; OR T = 1.35, 95% CI 1.13–1.63; P = 0.0034) was identified as the only gene for which the association with MDD remained significant after correction for multiple testing. For SLC6A2 (norepinephrine transporter (NET)) significantly more SNPs (19 out of 100; P = 0.039) than expected were associated while accounting for the linkage disequilibrium (LD) structure. Thus, we found support for involvement in MDD for only four genes. However, given the number of candidate SNPs and genes that were tested, even these significant may well be false positives. The poor replication may point to publication bias and false-positive findings in previous candidate gene studies, and may also be related to heterogeneity of the MDD phenotype as well as contextual genetic or environmental factors. Molecular Psychiatry (2011) 16, 516–532; doi:10.1038/mp.2010.38; published online 30 March 2010 Keywords: candidate genes; genome-wide association study; major depressive disorder; replication; single-nucleotide polymorphisms Introduction nesda.nl) and the Netherlands Twin Registry (NTR; www.tweelingenregister.org) took part in the Genetic Major depressive disorder (MDD) is a multi-factorial Association Information Network (GAIN; http:// disease, with both genetic and environmental factors www.fnih.org/GAIN) project to enable a genome-wide likely to have a role in its etiology. The Netherlands association study (GWAS) using a 600K Perlegen chip Study of Depression and Anxiety (NESDA; www. (Perlegen Sciences, Mountain View, CA, USA).1 Within the GAIN MDD study,2 1862 participants with Correspondence: Dr FJ Bosker, Department of Psychiatry, a diagnosis of MDD and 1860 controls at low liability University Medical Center Groningen, University of Groningen, for MDD were selected for genome-wide genotyping. PO Box 30.001, 9700 RB Groningen, The Netherlands. A GWAS approach allows a hypothesis-free search E-mail: [email protected] for potential new susceptibility genes. The downside 8These authors contributed equally to this work. Received 3 August 2009; revised 18 February 2010; accepted 22 of a GWAS is that a strict statistical adjustment for February 2010; published online 30 March 2010 the large number of single-nucleotide polymorphisms Poor replication of candidate genes for MDD using GWAS FJ Bosker et al 517 (SNPs; in the GAIN MDD study 435 291 SNPs) is retrieved the abstracts, and if considered relevant, the required before associations can be considered sig- full paper. nificant on a genome-wide level,3 and replication of In the next step, we selected all papers fulfilling the such findings in independent cohorts is mandatory to following five inclusion criteria: (1) The study had to exclude false-positive findings.4 be a candidate gene case–control association study. Another potential use of a GWAS is to use the Linkage and fine-mapping studies were excluded. (2) results for a large-scale replication study of previous The primary diagnosis of the patients in the candidate candidate gene studies. Application of such previous gene study had to be MDD to enable comparison with knowledge within the context of a GWAS allows less the GAIN MDD study. Therefore, we excluded studies stringent significance thresholds than those for the involving patients: (i) with a depressive episode in hypothesis-free GWAS analysis.5,6 So far, the role of the course of bipolar disorder, (ii) with a primary candidate genes in MDD has been the subject of many psychotic disorder such as schizophrenia and a association studies. Unfortunately, there is little secondary depression, (iii) with a seasonal affective consistency between candidate gene studies for disorder not being MDD with a seasonal pattern, (iv) multifactorial diseases such as MDD (see, for exam- with a primary anxiety disorder (that is, panic ple, Hirschhorn et al.7 and Munafo8). disorder, agoraphobia or social phobia) or obsessive In the current study we attempted to replicate compulsive disorder and a secondary depression and significant findings from previous candidate gene (v) with MDD plus an additional specific comorbid studies in MDD. To this end we conducted a disorder or condition, for example, MDD plus systematic review of the literature and selected those alcoholism. However, we allowed subgroups within genes that were reported to be significantly associated MDD, for example, MDD in women or in men, with MDD at least once. The GWAS data from recurrent MDD or early-onset MDD. (3) The sample the GAIN MDD study were used to screen all the of a specific study consisted of at least 30 patients identified candidate genes in two ways: (1) for with MDD and 30 healthy controls. (4) The findings association with the specific SNPs reported in the on the association with MDD of any variant within the literature; and (2) for association with any of the SNPs candidate gene (either a SNP, a microsatellite marker (genotyped or imputed) from the Perlegen chip within or a haplotype) had reached a statistical significance the identified genes. at the level of P < 0.05. (5) Finally, the genetic association had to be with the diagnosis of MDD and not with other aspects such as associated Materials and methods personality features (for example, neuroticism) or Selection of studies reporting on candidate genes factors related to treatment response. This resulted in To prevent any bias from the results of the GAIN MDD 78 papers. study, we identified candidate genes for MDD before In order not to miss potential true-positive findings, the results from the GAIN MDD study became we did not exclude candidate genes with associated available. Therefore, a so-called ‘enhanced search’ markers that had low allele frequencies or that was performed in Medline through PubMed on 1 deviated from Hardy–Weinberg equilibrium. Two September 2007 using the following search terms: investigators (FB and CH) independently made a ((‘genes’ (TIAB) NOT Medline(SB])) OR ‘genes’ selection from the initial list of papers using the (MeSH terms) OR gene (text word)) OR (‘genes’(MeSH above-mentioned criteria. When both reached con- terms) OR genes (text word)) OR snp (all fields) OR sensus, the paper was included or excluded; in case (‘single nucleotide polymorphism’ (text word of disagreement, consensus was obtained with assis- OR ‘polymorphism, single nucleotide’ (MeSH terms) tance of two other authors (WN and HS). As a final OR snps (text word)) OR (‘genetic polymorphism’ step, one author (IN) double checked whether all (text word) OR ‘polymorphism, genetic’ (MeSH terms) selected papers fulfilled the aforementioned selection OR polymorphism (text word)) OR polymorphisms criteria 1–5. Figure 1 shows a flowchart of how we (all fields) AND (‘major depressive disorder’ (text retrieved the 78 papers for the present study. word) OR ‘depressive disorder, major’ (MeSH Terms) OR major depression (text word)) AND ‘humans’ Bioinformatic tools (MeSH terms). This resulted in 641 hits. We addi- For many SNPs no reference SNP identification tionally scrutinized the reference list of the meta- number (rs-id) was given in the original