Genome-Wide Association Study of Alcohol Dependence Implicates KIAA0040 on Chromosome 1q

Abstract

Previous studies using SAGE (the Study of Addiction: Genetics and Environment) and COGA (the Collaborative Study on the Genetics of Alcoholism) genome-wide association study (GWAS) data sets reported several risk loci for alcohol dependence (AD), which have not yet been well replicated independently or confirmed by functional studies. We combined these two data sets, now publicly available, to increase the study power, in order to identify replicable, functional, and significant risk regions for AD. A total of 4116 subjects (1409 European-American (EA) cases with AD, 1518 EA controls, 681 African-American (AA) cases, and 508 AA controls) underwent association analysis. An additional 443 subjects underwent expression quantitative trait locus (eQTL) analysis. Genome-wide association analysis was performed in EAs to identify significant risk genes. All available markers in the genome-wide significant risk genes were tested in AAs for associations with AD, and in six HapMap populations and two European samples for associations with gene expression levels. We identified a unique genome-wide significant gene—KIAA0040—that was enriched with many replicable risk SNPs for AD, all of which had significant cis-acting regulatory effects. The distributions of −log(p) values for SNP-disease and SNP-expression associations for all markers in the TNN–KIAA0040 region were consistent across EAs, AAs, and five HapMap populations (0.369⩽r⩽0.824; 2.8 × 10⁻⁹⩽p⩽0.032). The most significant SNPs in these populations were in high LD, concentrating in KIAA0040. Finally, expression of KIAA0040 was significantly (1.2 × 10⁻¹¹⩽p⩽1.5 × 10⁻⁶) associated with the expression of numerous genes in the neurotransmitter systems or metabolic pathways previously associated with AD. We concluded that KIAA0040 might harbor a causal variant for AD and thus might directly contribute to risk for this disorder. KIAA0040 might also contribute to the risk of AD via neurotransmitter systems or metabolic pathways that have previously been implicated in the pathophysiology of AD. Alternatively, KIAA0040 might regulate the risk via some interactions with flanking genes TNN and TNR. TNN is involved in neurite outgrowth and cell migration in hippocampal explants, and TNR is an extracellular matrix protein expressed primarily in the central nervous system.

INTRODUCTION

Alcohol dependence (AD) is a complex disorder characterized by psychological and physiological dependence on ethanol. The 12-month prevalence of AD in the United States is 3.81% (Grant et al, 2004). Family, twin, and adoption studies have demonstrated that genetic factors constitute a significant component of the risk for AD. Candidate gene studies have shown that a large number of risk loci exist for AD in the dopaminergic, serotonergic, GABAergic, cholinergic, opioidergic, and endocannabinoidergic systems, as well as in the ethanol metabolic pathway. Several genome-wide association studies (GWASs) have reported additional risk loci for alcoholism (Treutlein et al, 2009; Bierut et al, 2010; Edenberg et al, 2010; Heath et al, 2011). The first GWAS in German males reported that 15 top-ranked SNPs (in PECR, ADH1C, CAST, ERAP1, PPP2R2B, ESR1, GATA4, CCDC41, and CDH13) (5.6 × 10⁻⁶⩽p⩽2.2 × 10⁻³) in a discovery sample were replicated in a follow-up data set; two in PECR (2q35) reached genome-wide significance in meta-analysis (α=5 × 10⁻⁸) (Treutlein et al, 2009). But the top-ranked SNPs (p<10⁻⁴) in this German discovery sample were not replicated by a second GWAS for AD (Bierut et al, 2010), which reported a different set of 15 top-ranked SNPs (in PKNOX2, CC2D2B, NOMO2, COL8A1, NXPH2, E2F8, FAM44B, SH3BP5, GRM5, ZNF285A, and TPK1) in a combined European-American (EA) and African-American (AA) sample from SAGE (the Study of Addiction: Genetics and Environment), all of which were nominally associated with alcoholism (1.9 × 10⁻⁷⩽p⩽9.8 × 10⁻⁶). However, these SNPs were neither genome-wide significant nor were they replicated in a family sample from COGA (the Collaborative Study on the Genetics of Alcoholism) or the German case–control sample. Furthermore, the top-ranked SNPs (p<10⁻⁶) in EAs (or AAs) were not replicated in AAs (or EAs) (Bierut et al, 2010). The third GWAS found no SNP with genome-wide significance in an EA COGA discovery sample. However, 6 SNPs in TMEM132C, EPHA7, OPA3, KCNMA1, DMRTA2, and SPTA1 for AD and 41 SNPs in SELL, SELE, LOC91431, PPARG, CTNN2, LEPR, and PDE4B for early-onset AD were replicated in an AA replication sample. Ten SNPs in CARS, OSBPL5, NAP1L4, BBX, SLC9A8, OPA3, TOX2, and CD53 for AD and 16 SNPs in SLC37A3, KCNMA1, CDH8, ZNF608, API5, CAT, and GRIN2C for early-onset AD were replicable between the EA case–control discovery sample and an EA family replication sample (Edenberg et al, 2010). Most recently, a GWAS in an Australian twin-family sample identified TMEM108 and ANKS1A as possible risk genes for alcohol consumption (Heath et al, 2011). Another GWAS meta-analysis in European populations identified AUTS2 as the risk gene for alcohol consumption (Schumann et al, 2011). However, these findings have not yet been replicated in independent samples or confirmed by functional studies, and hence the possibility of a false-positive result cannot yet be excluded.

In the present study, we combined and reanalyzed the SAGE and COGA data sets, and used a new analytic strategy to identify additional risk loci for AD. First, we combined both data sets, hoping to increase the sample sizes and, in turn, the study's statistical power (site effects and sample overlapping were taken into account), thereby enhancing our ability to detect novel risk loci that were missed in previous studies. Second, we differentiated more fully the EAs and AAs in the analysis to increase population homogeneity, and controlled for admixture effects in the association tests. Third, we used the EAs as a discovery sample and the AAs as a replication sample, and different samples with distinct ethnicity to detect expression quantitative trait locus (eQTL) signals, as a confirmation of the variants' functional effects. Although using distinct samples in one study might increase the false-negative rates due to sample heterogeneity, replication in distinct samples makes the false-positive findings less likely. Replicable findings in distinct populations might be more generalizable to other populations, and would be more likely to be causal in nature. Fourth, allele frequencies could be different in distinct populations, or even exist in opposite phases; that is, a common allele in one population may be a rare allele in another population. Thus, distinct populations do not necessarily have the same risk markers associated with disease; alternatively, they could have the same risk markers, but have different phases of alleles in these markers associated with disease. That is, the effect sizes and effect directions of marker–disease associations may be not consistent, or could even be opposite across populations for each individual risk marker, such that meta-analysis may show weaker effects. Such markers are usually treated as nonreplicable and thus are discarded. However, to our hypothesis, when two distinct populations have common causal variants, there could be a risk region in LD with this putative causal variant in both populations, even though there are no individual risk alleles replicable between them. This is because in one population, a set of risk markers are in LD with the causal variant; but in another population, a different set of risk markers adjacent to the first set could be in LD with the causal variant. The risk marker sets in a causal region are different between populations, because they are not causal variants per se. Such a risk region may have a significant correlation between the distributions of −log(p) values of all markers across the entire region in different populations. Such regions have usually been missed in previous studies. Additionally, in the present study, the data sets for association studies and for eQTL analysis were different in many absolute statistics of genetic marker numbers, sample sizes, and study power. To study the consistency between them, we can only compare their relative statistics, that is, the distributions of relative significance strengths across whole regions, not individual markers. In a word, the present study aimed to identify replicable, functional, and significant risk regions for AD by increasing the study power and the sample homogeneity.

MATERIALS AND METHODS

Subjects

A total of 4316 SAGE (dbGaP study accession phs000092. v1.p1) subjects and 1957 COGA (dbGaP study accession phs000125.v1.p1) subjects were merged into a single data set; 1477 subjects in COGA who overlapped with SAGE were excluded. The demographic data of SAGE and COGA subjects have been presented previously (Bierut et al, 2010; Edenberg et al, 2010). After data cleaning (see below), 1409 EA cases (37.3% females; 38.3±10.2 years), 1518 EA controls (70.7% females; 39.4±10.4 years), 681 AA cases (37.2% females; 40.3±7.8 years), and 508 AA controls (66.7% females; 39.6±8.6 years) underwent analysis. Affected subjects met lifetime DSM-IV criteria (American Psychiatric Association, 1994) for AD. Controls were defined as individuals who had been exposed to alcohol (and possibly to other drugs) in sufficient amounts for a sufficient time, but had never become addicted to alcohol or other illicit substances (lifetime diagnoses). Additionally, controls were also screened to exclude individuals with major axis I disorders, including schizophrenia, mood disorders, and anxiety disorders. More demographic data are provided in Supplementary Materials and Methods. All subjects were de-identified in this study. All subjects were genotyped on the Illumina Human 1M beadchip.

Data Cleaning

Subjects with poor genotypic data and questionable diagnostic information, subjects with allele discordance, duplicated IDs, potential sample misidentification, sample relatedness, sample misspecification, gender anomalies, chromosome anomalies (such as aneuploidy and mosaic cell populations), missing race, non-EA and non-AA ethnicity, population group outliers, subjects with a mismatch between self-identified and genetically inferred ethnicity, and subjects with a missing genotype call rate ⩾2% across all SNPs were excluded step by step (Supplementary Table S1). Furthermore, SNPs with allele frequency difference in controls >2% between SAGE and COGA, SNPs with missing rate difference >2% between SAGE and COGA, and SNPs with allele discordance, chromosomal anomalies, or batch effect were excluded. We then filtered out the SNPs on all chromosomes with an overall missing genotype call rate ⩾2%, the monomorphic SNPs, and the SNPs with minor allele frequencies (MAFs) <0.01 in either EAs or AAs. The SNPs that deviated from Hardy–Weinberg equilibrium (HWE; p<10⁻⁴) within EA or AA controls were also excluded. This selection process yielded 805 814 SNPs in EAs and 895 714 SNPs in AAs. Details are provided in Supplementary Materials and Methods.

Association Analysis

1. Genome-wide association tests in the EA discovery sample: The allele frequencies were compared between cases and controls in EAs using genome-wide logistic regression analysis implemented in the program PLINK (Purcell et al, 2007). Diagnosis served as the dependent variable, alleles served as the independent variables, and ancestry proportions, sex, and age served as covariates. The p-values derived from these analyses are illustrated in Supplementary Figure S1 and the top-ranked risk SNPs (p<10⁻⁵) are listed in Table 1.

2. Association tests in the AA replication sample: Associations between the top-ranked risk SNPs in EAs were tested using logistic regression analysis in AAs. Additionally, associations for all available SNPs in the genome-wide significant risk genes identified in EAs were also tested in AAs (Figure 1). Meta-analysis was performed to derive the combined p-values for EAs and AAs.

3. Controlling for admixture effects: The ancestry proportions for each individual were estimated by integrating the ancestry information content of 3172 completely independent ancestry-informative markers (AIMs) using STRUCTURE (Pritchard et al, 2000). These AIMs were extracted from 1 million of markers by LD pruning (Purcell et al, 2007) (see details in Supplementary Materials and Methods). To control for the admixture effects on association analysis, ancestry proportions were included as covariates in the association analysis.

Table 1. Top-Ranked SNPs in EAs with P<10⁻⁵ for Associations.

Band	SNP	Genes	P-value	FDR	OR
1q24–q25	rs1057239a	KIAA0040	2.8 × 10−7	0.038	1.32
1q24–q25	rs1894709a	KIAA0040	3.9 × 10−7	0.039	1.31
1q23–q24	rs6701037a	KIAA0040	1.3 × 10−6	0.043	1.30
1q24–q25	rs6425323a	KIAA0040	1.9 × 10−6	0.043	1.29
1q24–q25	rs1057302a	KIAA0040	2.5 × 10−6	0.044	1.29
1p35.1	rs4949400a	SERINC2	2.3 × 10−7	0.043	1.32
1p35.1	rs4949402a	SERINC2	2.6 × 10−7	0.043	1.32
1p35.1	rs1039630a	SERINC2	2.6 × 10−7	0.043	1.32
1p35.1	rs2275435a	SERINC2	3.0 × 10−7	0.044	1.31
1p35.1	rs4478858a	SERINC2	4.4 × 10−7	0.044	1.31
1p35.1	rs10914386	SERINC2	9.0 × 10−6	>0.05	1.30
5q12.1	rs7445832a	HTR1A	2.8 × 10−7	0.041	1.38
1p34.3	rs11583322	STK40	4.0 × 10−7	>0.05	0.76
5q23.3	rs257906	SLC27A6	5.0 × 10−7	>0.05	1.43
5q23.3	rs10478829	SLC27A6	5.3 × 10−7	>0.05	1.43
10q25.3	rs4751971	PNLIPRP3	2.4 × 10−6	>0.05	3.60
13q11	rs1867248	TUBA3C	2.6 × 10−6	>0.05	1.33
12q21.3	rs882968	ATP2B1	3.6 × 10−6	>0.05	1.29
5q11.2–q13	rs4700575	HTR1A	3.8 × 10−6	>0.05	1.35
5q11.2–q13	rs2169520	HTR1A	4.0 × 10−6	>0.05	1.35
13q12	rs7983722	ATP8A2	4.1 × 10−6	>0.05	6.48
12q24.1	rs4964684	CMKLR1	5.1 × 10−6	>0.05	4.18
12q23.3	rs1896086	KIAA0789	5.4 × 10−6	>0.05	4.37
12q23.3	rs7971309	KIAA0789	5.4 × 10−6	>0.05	4.37
3p13	rs6802792	RYBP	5.5 × 10−6	>0.05	9.80
7q31	rs17142876	KCND2	5.6 × 10−6	>0.05	1.51
7q31	rs728115	KCND2	5.6 × 10−6	>0.05	1.49
8q11–q12	rs9298318	SNTG1	6.9 × 10−6	>0.05	6.27
8q13.3	rs12549296	EYA1	8.8 × 10−6	>0.05	1.81
11q24.2	rs750338	PKNOX2	9.5 × 10−6	>0.05	1.32
2q22.1	rs1869324	THSD7B	9.8 × 10−6	>0.05	1.44
5q14	rs6888626	COX7C	9.9 × 10−6	>0.05	6.66
12q24.33	rs2218917	TMEM132D	1.0 × 10−5	>0.05	9.04

Abbreviations: FDR, genome-wide false discovery rate; OR, odds ratio.

^aIndicates FDR<0.05.

Figure 1.

Regional association and eQTL plots around TNN–KIAA0040 region. Left y-axis corresponds to −log(p) value; right y-axis corresponds to recombination rates; quantitative color gradient corresponds to r²; red squares represent peak SNPs. (a) Regional association plot in EAs for a 10 Mb region around the peak association SNP (rs1057239); (b, c) regional association plots in EAs and AAs for a 1 Mb region around the peak association SNPs; (d–k) regional eQTL plots in HapMap and European populations for a 1 MB region around the peak functional SNPs; and (l) LD map for TNN–KIAA0040 region.

Functional Analysis (Cis-Acting Genetic Regulation of Expression Analysis)

1. Cis-acting expression of QTL (Cis-eQTL) analysis on the risk SNPs in lymphoblastoid cell lines: To examine relationships between all risk SNPs (Table 2) and local mRNA expression levels, we used expression data of 14 925 transcripts (14 072 genes) in 270 unrelated HapMap individuals from six populations (CEU-Children, CEU-Parent, CHB, JPT, YRI-Children, and YRI-Parent) (Stranger et al, 2005). Differences in the distribution of mRNA expression levels between SNP genotypes were compared using a Wilcoxon-type trend test. The p-values of <0.05 are listed in Table 2. Effects of SNPs 1 Mb around the association peak SNP (rs1057239) are illustrated in Figure 1d–i.

2. Cis-eQTL analysis on the risk SNPs in other two primary human cells: To examine whether the risk SNPs influence the local exon- or transcript-level expression changes, we also tested the associations between the genotypes of these risk SNPs and the expression levels of exons and transcripts of local genes in the other two European samples (Table 2 and Figure 1j and k). Expression data in 93 autopsy-collected frontal cortical brain tissue samples with no defined neuropsychiatric condition and 80 peripheral blood mononuclear cell (PBMC) samples collected from living healthy donors obtained from a study (Heinzen et al, 2008) at Duke University (Duke samples) were evaluated. Each of these associations was analyzed using a linear regression model by correcting for age, sex, source of tissues, and principle component scores.

Table 2. P-Values for Associations and Cis-Acting Regulatory Effects of the SNPs in TNN–KIAA0040 Region.

SNP	A1	EA		AA		P-values for eQTL in six HapMap populations						P-values for eQTL in Europeansa
		OR	P-value	OR	P-value	CEU_children	CEU_parent	CHB	JPT	YRI_children	YRI_parent	Transcript (B)	Exon 1 (P)	Exon 4 (P)
rs12094153	A	1.210	3.9 × 10−4	—	—	—	—	—	—	—	—	—	2.8 × 10−3	—
rs4651322	T	0.889	0.034	—	—	—	—	—	6.0 × 10−3	—	—	—	—	—
rs2157588	A	1.219	1.9 × 10−4	—	—	—	—	—	—	—	—	—	—	—
rs12563833	A	0.893	0.047	—	—	—	—	—	0.023	—	—	—	—	—
rs1018829	T	0.839	6.8 × 10−3	—	—	—	—	—	0.035	—	—	—	—	—
rs10489328	A	0.848	0.017	—	—	—	—	—	—	—	—	0.023	—	—
rs2018318	T	0.836	9.9 × 10−3	—	—	—	—	—	—	—	—	—	—	—
rs6701037	C	1.295	1.3 × 10−6	—	—	—	—	—	0.025	0.037	6.0 × 10−4	—	9.4 × 10−4	—
rs10912899	A	0.871	9.4 × 10−3	—	—	—	—	—	—	—	0.014	—	—	3.2 × 10−4
rs4650707	A	0.741	0.024	—	—	—	—	—	—	—	—	—	—	—
rs6425323	T	1.291	1.9 × 10−6	—	—	0.010	8.9 × 10−3	—	0.025	0.038	5.0 × 10−4	—	—	—
rs1057302	C	1.286	2.5 × 10−6	—	—	0.013	4.8 × 10−3	—	0.025	—	2.0 × 10−4	—	—	—
rs3737933	C	0.850	0.023	1.757	0.040	4.0 × 10−3	4.0 × 10−3	0.024	0.035	—	—	—	—	—
rs1057285	G	—	—	1.412	5.1 × 10−4	1.4 × 10−3	0.014	—	—	—	—	—	—	—
rs4650716	C	1.249	2.2 × 10−5	—	—	—	—	—	—	0.025	—	—	—	—
rs2269650	A	0.842	1.4 × 10−3	—	—	—	—	—	—	—	—	—	—	8.6 × 10−5
rs2072035	A	0.841	1.3 × 10−3	—	—	—	—	—	—	—	—	—	—	—
rs1057239	T	1.318	2.8 × 10−7	—	—	—	0.028	—	—	0.011	—	—	—	—
rs1894709	A	1.313	3.9 × 10−7	—	—	—	8.0 × 10−3	—	—	0.016	—	—	—	—
rs10489326	T	1.242	3.4 × 10−5	—	—	—	—	—	—	0.021	—	—	—	—
rs2269655	T	—	—	1.875	0.026	0.020	0.039	0.031	—	—	—	6.9 × 10−3	—	—
rs2861158	A	0.863	0.011	—	—	—	—	—	—	—	—	—	—	—
rs16847872	G	—	—	1.872	0.026	0.020	0.039	0.031	—	—	—	—	—	—
rs12136973	C	0.865	0.012	—	—	—	—	—	0.040	—	—	—	—	2.5 × 10−3
rs1008459	G	0.881	0.029	—	—	—	—	0.018	0.028	—	—	—	—	1.1 × 10−3
rs2272785	A	0.879	0.025	—	—	—	—	0.018	0.044	—	—	—	—	—
rs2272784	C	0.823	2.8 × 10−4	—	—	—	—	—	—	—	—	—	—	7.7 × 10−6
rs3753555	C	0.823	2.8 × 10−4	—	—	—	—	—	—	—	—	—	—	7.7 × 10−6

Abbreviations: eQTL, expression quantitative trait locus analysis.

^aExon-level expression changes in brain (B) and PBMC (P) tissues were corrected for 4 exons and 2 tissues (α=0.0063).

‘—', p>0.05 or not available.

All markers are in HWE.

Correction for Multiple Testing on Association and Cis-eQTL Analysis

To mitigate false-positive rates, genome-wide associations in the discovery stage need to be corrected for multiple testing. Apparently, Bonferroni correction (α=5 × 10⁻⁸) was overly conservative because it treated all 1 million markers in the genome as independent ones (which is impossible). Alternatively, a WTCCC-defined α (=5 × 10⁻⁷) might be more appropriate to the present study (The Wellcome Trust Case Control Consortium, 2007). As complements to this correction, we also corrected the discovery findings by genome-wide false discovery rate (FDR; Benjamini and Hochberg, 1995) and replicated and confirmed the discovery findings by replication and confirmation designs. Only when an association survived WTCCC-defined genome-wide correction (p<5 × 10⁻⁷) together with FDR<0.05, and was replicated in an independent sample and confirmed by functional studies, should it be taken as ‘significant'. In the present study, we used multiple samples to replicate and confirm the discovery findings, which significantly reduced the chance of false-positive findings (ie, FDR). First, we used AAs, the population most genetically distinct from EAs in the United States, to replicate the association analysis and make the replicable findings more generalizable to other populations. Second, we aimed to detect replicable regions, not individual markers, to avoid missing risk regions because of the population specificity of allele frequencies introduced above. Many risk markers, rather than a single marker, were detected in the risk regions, which reduced the chance of false-positive association findings. Third, functional analysis as confirmation of association analysis further reduced the chance of false-positive findings. Additionally, functional analysis in multiple different populations, which differed from the populations for association analysis, made the findings more generalizable too. Fourth, −log(P) value distributions across the discovery sample and the replication and confirmation samples were compared for the similarity using Pearson correlation analysis. The consistency between them would significantly reduce the chance of false-positive findings. Therefore, α could be set at 0.05 for the findings in replication and confirmation samples if they replicated or confirmed the discovery findings (except for exon-level cis-eQTL findings that were corrected for the number of exons and the types of tissues; ie, α was set at 0.0063 for KIAA0040; see Table 2).

Functional Analysis (Trans-Acting Genetic Regulation of Expression Analysis)

1. Transcriptome-wide trans-eQTL analysis on the risk SNPs: To examine whether the risk SNPs regulated other transcript expression, we tested the associations between the genotypes of these risk SNPs and the transcript expression levels across the transcriptome. The transcriptome-wide expression levels in two human primary cells (brain and PBMC) in the Duke samples (Heinzen et al, 2008) were assessed using Affymetrix Human ST 1.0 exon arrays. Associations between genotypes and transcriptome-wide expression levels were analyzed using linear regression implemented in PLINK (Purcell et al, 2007) by incorporating all covariates. A total of 2 047 023 transcript expression data records in the brain set, 1 760 880 transcript expression data records in the PBMC set, and 28 risk SNPs were tested, and hence α was set at 8.7 × 10⁻¹⁰ for the brain set and 1.0 × 10⁻⁹ for the PBMC set, respectively.

2. Genome-wide trans-eQTL analysis of KIAA0040 transcript expression: To examine what polymorphisms across genome regulated the transcript expression of KIAA0040, we scanned the whole genome and tested the associations between the transcript expression level of KIAA0040 and the genotypes across whole genome. The same Duke samples including both tissues as described above were tested (Heinzen et al, 2008). Genome-wide genotyping was performed using Illumina Human Hap550K chip. Strict data cleaning was performed before association analysis using previously published methods (Fellay et al, 2007). Associations between KIAA0040 transcript expression level and genotypes across genome were analyzed using linear regression implemented in PLINK, by incorporating all covariates. A total of 571 738 SNPs and 2 tissues were tested, and hence α was set at 4.4 × 10⁻⁸.

3. Transcriptome-wide expression correlation analysis: The expression of 14 925 transcripts was examined in Duke samples (Heinzen et al, 2008). Correlations between expression of KIAA0040 transcript and expression of other genes across transcriptome were tested in the brain and PBMC, respectively. The α was set at 1.7 × 10⁻⁶ (=0.05/(14 925 × 2) in which ‘2' is the types of tissues tested).

Functional Analysis (RNA Secondary Structure Analysis)

Each unique DNA sequence across a gene, whether common, rare, or with a unique mutation, could have a different consequent RNA secondary structure. Alteration of RNA secondary structure could influence the efficiency of splicing, translation, and/or binding of regulatory factors. These influences could affect disease susceptibility. Because a test of this hypothesis is beyond the scope of the present report, we used the program MFOLD (Zuker, 2003) to predict an alteration in RNA secondary structures. The upstream and downstream sequences (800 bp) around a variant were retrieved from NCBI dbSNP based on the SNP accession number. The RNA secondary structures of the retrieved sequences with either the common or variant allele were constructed by MFOLD. All parameters were set in default for the most stable secondary structure folding prediction. A ΔG value for each structure corresponding to each allele was derived (Supplementary Figure S2). The ΔG is a metric of stickiness when constructing the RNA secondary structure, where stickiness represents how thermodynamically stable a structure may be. The larger the absolute value of a negative ΔG is, the more stable a structure may be; conversely, the larger the absolute value of a positive ΔG is, the less stable a structure may be. Alterations in the most stable secondary structures of the sequences were visualized by comparing these structures with either common or rare alleles in parallel (Supplementary Figure S2).

In view of the fact that the length (800 bp) of sequence that the program can accept is shorter than the mature mRNA, the program does not account for the multiple mRNA-binding proteins that influence the conformation of the mature mRNA. Thus, this functional analysis is exploratory.

RESULTS

Population stratification effects on associations were controlled after separating EAs and AAs in the analysis. The admixture degrees of our cleaned EA and AA samples were very low. They were 1.4% in EAs and 6.2% in AAs, respectively, and did not affect our association findings significantly (data not shown). As a result, in the present study, we only showed the p-values after these effects had been controlled.

The p-values (<10⁻⁵) for the associations between the top-ranked SNPs and AD in the EA discovery sample are listed in Table 1, which includes 33 SNPs in 21 genes. All these SNPs in controls were in HWE in both EAs and AAs. Associations for five SNPs in KIAA0040, five SNPs in SERINC2, and one SNP in HTR1A in EAs had a genome-wide FDR<0.05; eight of which were genome-wide significant (p<5 × 10⁻⁷) (Table 1). No associations for these SNPs (but other markers) were significant in AAs. The region surrounding KIAA0040 in AAs overlapped extensively with that in EAs (Figure 1c). This region was enriched with risk or functional signals across EAs, AAs, six HapMap populations, and two primary tissues in Europeans.

This risk region spanned three LD blocks, including the first block from rs12094153 to rs1018829 in TNN, the second block from rs10489328 in TNN to rs1008459 in KIAA0040 (38 kb), and the third block from rs2272785 to rs3753555 in KIAA0040 (Figure 1l). The broader region (within 1 Mb; Figure 1b) outside this TNN–KIAA0040 region yielded no association signal with p<0.01 in EAs. In all, 25 and 4 SNPs in this region were nominally associated with AD in EAs (2.8 × 10⁻⁷⩽p⩽0.047) and AAs (5.1 × 10⁻⁴⩽p⩽0.040), respectively. All association signals in this risk region with p<10⁻⁵ in EAs were located in the second LD block in KIAA0040 (Figure 1l). All risk SNPs were in HWE (p>0.05) in both cases and controls in both EAs and AAs (Table 2). Meta-analysis of EAs and AAs did not change the p-values for these risk SNPs significantly (data not shown). The allele frequencies of all SNPs in EA and AA controls were similar to those in CEU and YRI, respectively, in the HapMap database (see Supplementary Table S2).

eQTL analysis showed that all risk SNPs in this region had cis-acting regulatory effects on KIAA0040 mRNA expression in at least one population; all of those with p<10⁻⁵ in EAs had cis-acting effects in at least two populations; and five risk SNPs had significant effects in at least four populations (Table 2).

In EAs, throughout the Chromosome 1q, this region was the only one that had association signals at p<10⁻⁵. Within 17.7 Mb around TNN–KIAA0040, this region was also the only one with association signals at p<0.001. Similarly, in AAs, within 10 Mb around TNN–KIAA0040, this region was the only one that had association signals at p<0.001. Furthermore, within 775 Kb around TNN–KIAA0040, this region was the only one with association signals at p<0.01. Additionally, within the 1 Mb range, the most significant functional SNPs in the HapMap CEU-Child (rs1057285; p=0.0014; Figure 1d), CEU-Parent (rs3737933; p=0.004; Figure 1e), YRI-Parent (rs1057302; p=0.0002; Figure 1f), and Europeans (brain tissue) (rs2269655; p=0.0069; Figure 1j) were all located in the second LD block, that is, KIAA0040, in this risk region (Figure 1l). The only exception was JPT, in which the most significant functional SNP in this risk region (rs4651322; p=0.006; Figure 1i) was located in the first LD block, that is, TNN; and this peak SNP was the second most significant SNP within the 1 Mb range.

The −log(p) values for all available SNPs (n=40) within TNN–KIAA0040 region are plotted in Figure 1. The distributions of −log(p) values were highly consistent across EAs, AAs, CEU-Child, CEU-Parent, YRI-Child, YRI-Parent, and CHB (0.369⩽r⩽0.824; 2.8 × 10⁻⁹⩽p⩽0.032; Table 3). The peak SNPs among each of these populations were in high LD. This was especially the case for the SNP showing the most significant expression differences in the CEU-Child population (rs1057285, p=0.0014), which was also the peak SNP associated with AD in AAs (rs1057285; p=5.1 × 10⁻⁴) (Figure 1c vs d). And the SNP showing the most significant expression differences in the YRI-Child population (rs1057239, p=0.0108) was the peak SNP associated with AD in EAs (rs1057239; p=2.8 × 10⁻⁷) (Figure 1b vs g). The peak SNPs in AAs (rs1057285), CEU-Child (rs1057285), CEU-Parent (rs3737933), and YRI-Parent (rs1057302) were closely located together (Figure 1l). The more closely the peak SNPs were located (Figure 1l), the more significant the correlations were between the −log(p) value distributions across the whole region (Table 2), which suggested that the peak SNP captured most of the information across that region. The more significant those correlations were, the more consistent (replicable) between two populations the risk regions were. Thus, the distance between peak SNPs reflected the strength of replicability of association or function signals between populations.

Table 3. Correlation of Distributions of –Log(P) Values for Associations and Cis-Acting Effects in TNN–KIAA0040 Region Between Different Populations.

Populations	Pearson correlation coefficients (r)
	EA	AA	CEU-child	CEU-parent	CHB	JPT	YRI-child	YRI-parent	Europeansa
P-values
EA		0.385	0.091	0.405	−0.245	−0.198	0.824	0.402	−0.141
AA	0.025		0.814	0.608	0.244	0.139	0.202	0.124	0.229
CEU-child	0.604	2.8 × 10−9		0.769	0.369	0.251	0.215	0.450	0.306
CEU-parent	0.014	8.3 × 10−5	7.0 × 10−8		0.105	0.158	0.422	0.510	0.148
CHB	0.162	0.164	0.032	0.556		0.215	−0.190	−0.036	0.090
JPT	0.270	0.440	0.158	0.380	0.229		−0.286	0.318	−0.162
YRI-child	1.2 × 10−8	0.268	0.244	0.018	0.306	0.125		0.207	0.119
YRI-parent	0.028	0.507	0.013	0.004	0.848	0.093	0.264		−0.094
Europeans	0.552	0.331	0.190	0.532	0.707	0.509	0.627	0.710

^aTranscript expression in brain tissue.

Transcriptome-wide trans-acting eQTL analysis showed that 10 SNPs in this region nominally regulated transcript expression of multiple genes across the transcriptome (Supplementary Table S3). Genome-wide trans-acting eQTL analysis showed that transcript expression of KIAA0040 was marginally regulated by multiple genes across the genome (Supplementary Table S4). However, after Bonferroni correction, none of them remained significant.

Transcriptome-wide expression correlation analysis showed that the expression of KIAA0040 was significantly correlated with the expression of many genes (α=1.7 × 10⁻⁶; data not shown). These genes included some alcoholism-related genes (see Discussion), such as SOD2 (p=8.8 × 10⁻¹¹) and ADH1C (p=1.2 × 10⁻⁶) in brain, and FAM44B (p<2 × 10⁻¹⁶), IPO11 (p=1.9 × 10⁻¹⁴), ERAP1 (p=1.2 × 10⁻¹¹), GRIN2C (p=2.2 × 10⁻¹¹), PECR (p=5.9 × 10⁻¹¹), BBX (p=7.8 × 10⁻¹¹), NRD1 (p=9.7 × 10⁻¹⁰), API5 (p=1.1 × 10⁻⁹), DRD2 (p=1.9 × 10⁻⁹), LEPR (p=2.9 × 10⁻⁹), ADH5 (p=4.1 × 10⁻⁹), GRM5 (p=7.2 × 10⁻⁹), TH (p=1.2 × 10⁻⁸), MTHFR (p=1.6 × 10⁻⁸), CARS (p=4.1 × 10⁻⁸), TTC12 (p=4.5 × 10⁻⁸), NXPH2 (p=2.2 × 10⁻⁸), CAST (p=7.7 × 10⁻⁸), HNMT (p=1.2 × 10⁻⁷), HTR1B (p=1.7 × 10⁻⁷), OLFM3 (p=3.6 × 10⁻⁷), PPP1R1B (p=7.1 × 10⁻⁷), OPRD1 (p=7.2 × 10⁻⁷), DRD3 (p=7.7 × 10⁻⁷), CRHR1 (p=1.3 × 10⁻⁶), and PPP2R2B (p=1.5 × 10⁻⁶) in PBMC.

Several risk SNPs were predicted to significantly alter the RNA secondary structure, including rs2157588 in TNN, and rs4650707, rs3737933, rs4650716, rs1894709, rs2861158, and rs16847872 in KIAA0040 (Supplementary Figure S2 and Table S2). Eight KIAA0040 markers were located in a transcription factor binding site (TFBS); two KIAA0040 markers, that is, rs2861158 and rs2272784, were located in an exonic splicing silencer or enhancer (Supplementary Table S2).

DISCUSSION

In the present study, after merging 480 COGA subjects into the SAGE sample, the results were highly similar to those in a previous study that used the SAGE sample alone (Bierut et al, 2010). The top-ranked risk SNPs (p<10⁻⁵) in EAs, AAs, and combined EAs and AAs in that previous study (Bierut et al, 2010) were confirmed by our analysis (see Supplementary Table S5). In the present study, we found genome-wide significant association signals (p<5 × 10⁻⁷ together with FDR<0.05) for AD for three genes (KIAA0040, SERINC2 and HTR1A) in EAs. Two of these genes, that is, KIAA0040 and HTR1A, were also among the top-ranked genes in EAs in the prior study (Bierut et al, 2010); in addition, KIAA0040 as a risk gene for AD was confirmed by another GWAS meta-analysis for SAGE, COGA, and an Australian family sample (Wang et al, 2011). However, this was not previously replicated in AAs and not confirmed by functional studies.

In the present study, using a new analytic strategy and integrating evidence from the functional analysis, we were able to present additional important information that was not obtained previously. We found that, among the three significantly associated genes, only the region around KIAA0040 overlapped extensively between EAs and AAs, which would be expected mostly for functional regions that harbor the same causal variant in both populations. This was consistent with the eQTL findings; that is, all risk SNPs in this region had expression effects. We thus concluded that KIAA0040 might harbor a causal variant for AD.

Multiple pieces of evidence support our conclusion. First, KIAA0040 contained two genome-wide significant markers and several other marginally significant markers in EAs. Second, KIAA0040 was the only gene that had association signals with p<10⁻⁵ throughout Chromosome 1q in EAs. Similarly, in AAs, within 10 Mb around this region, KIAA0040 was the only gene with association signals at p<0.001. Furthermore, within the 1 Mb range, the most significant functional SNPs in four HapMap populations (Stranger et al, 2005) and one Duke sample (Heinzen et al, 2008) were all located in KIAA0040. It is thus likely that the putative causal variant for AD was located within KIAA0040. Third, eQTL analysis showed that all risk SNPs in this TNN–KIAA0040 region had cis-acting regulatory effects, and such effects in KIAA0040 appeared in two to five different populations, which is highly unlikely to have occurred by chance. These effects suggest that KIAA0040 per se might play a direct functional role in AD. Fourth, many KIAA0040 SNPs had significant potential to alter the RNA secondary structures; many KIAA0040 markers were located in a TFBS and two KIAA0040 markers were located in an exonic splicing silencer or enhancer. Fifth, the overall –log(P) value distributions for gene–disease associations and for gene expression were correlated across at least seven populations, suggesting that the majority of the functions of TNN–KIAA0040 might contribute to the risk for AD, and that the regulatory pathway through which these SNPs cause AD might be related to the TNN and KIAA0040 proteins per se.

In summary, (1) TNN–KIAA0040 was enriched with many risk SNPs, (2) association signal distributions were consistent between EAs and AAs, (3) functional signal distributions were consistent across multiple populations, (4) association and functional signal distributions were consistent, and, especially, (5) many peak association and functional SNPs were concentrated in one LD block, suggesting that chance alone was unlikely to account for the findings. Taken together, these findings strongly support the hypothesis that TNN–KIAA0040, especially KIAA0040, might harbor a causal variant for AD.

KIAA0040 encodes an HLA-DR11-restricted T-cell epitope. It is expressed in multiple tissues and organs including brain. It was worth noting that expression of KIAA0040 was significantly associated with the expression of many genes that have previously been associated with AD (although some of these associations were reported by a candidate gene approach and were not yet well replicated). These genes are in the dopaminergic (DRD2-TTC12, DRD3, TH, and PPP1R1B), serotonergic (HTR1B), glutamatergic (GRM5 and GRIN2C), histaminergic (HNMT), and opioidergic (OPRD1) systems, as well as in the ethanol metabolic pathway (ADH1C and ADH5) (Bierut et al, 2010; Edenberg et al, 2010; Connor et al, 2002; Yang et al, 2008; Dick and Foroud, 2003; Dahmen et al, 2005; Tabakoff et al, 2009; Sun et al, 2002; Oroszi et al, 2005; Zhang et al, 2008; Cichoz-Lach et al, 2007; Luo et al, 2006). These findings suggest that KIAA0040 might also be implicated in AD via these neurotransmitter systems or metabolic pathways.

The causal variant is more likely to be located in KIAA0040 than TNN, because (1) there were more risk SNPs in KIAA0040 than TNN; (2) all risk SNPs with p<10⁻⁵ in EAs were located in KIAA0040; (3) all functional markers that had significant cis-acting regulatory effects in at least two populations were located in KIAA0040; (4) all risk markers in AAs were located in KIAA0040; and (5) most peak association and functional SNPs were located in KIAA0040. However, most of these risk SNPs were common variants and were predicted to lack any phenotypic effect (by Polyphen-2; Adzhubei et al, 2010; Supplementary Table S2), so that the causal variant might not be any one of these risk markers per se. Future studies should aim to identify the causal variants by sequencing the entire TNN–KIAA0040 region, especially KIAA0040.

TNN and TNR flank KIAA0040. They are closely linked, 8.9 and 129.7 kb distant from KIAA0040, respectively. TNN, which encodes tenascin-N, is involved in neurite outgrowth and cell migration in hippocampal explants. TNR, which encodes tenascin-R, is an extracellular matrix protein expressed primarily in the central nervous system and has been related to multiple brain diseases. Recent GWASs reported that (1) two SNPs in KIAA0040 (rs1008459 and rs12136973) were significantly associated with amyotrophic lateral sclerosis (Schymick et al, 2007) and two other SNPs in KIAA0040 (rs760486 and rs3766685) were marginally associated with Alzheimer's disease (Li et al, 2008); (2) several SNPs in TNN (rs1009418, rs12065394, rs6672099, rs6681984, and rs16847787) were marginally associated with narcolepsy, a neurological sleep disorder (Miyagawa et al, 2008); and (3) one inter-KIAA0040–TNR SNP (rs875326) was marginally associated with treatment response of schizophrenia to an antipsychotic medication (iloperidone) (Lavedan et al, 2009). These findings support a role for the TNN–KIAA0040–TNR compound locus in the risk for medical disorders, particularly those involving the central nervous system. In addition to the two aforementioned interpretations for our association findings in the present study (ie, KIAA0040 might harbor a causal variant and directly contribute to the risk for AD, or it might be implicated in AD via neurotransmitter systems or metabolic pathways), these GWAS findings suggest to us an alternative interpretation that KIAA0040 might regulate the risk for AD via flanking genes TNN or TNR.

The present study has limitations. First, the correction for multiple testing remains controversial. If corrected by Bonferroni correction (α=5 × 10⁻⁸), which is conservative, our findings were only marginally significant. They warrant more validation independently in the future. Second, although λ=1.07 in EAs and λ=1.04 in AAs in QQ plots indicated that the inflation of p-values was not significant, we do not exclude the possibility that a small proportion of inflation might still exist, which might result from the heterogeneity of some unknown factors.

Dr Kranzler has been a paid consultant for Alkermes, GlaxoSmithKline, and Gilead. He serves as a member of an Advisory Board for Lundbeck. He also reports associations with Eli Lilly, Janssen, Schering Plough, Lundbeck, Alkermes, GlaxoSmithKline, Abbott, and Johnson & Johnson, as these companies provide support to the ACNP Alcohol Clinical Trials Initiative (ACTIVE) and Dr Kranzler receives support from ACTIVE.

Dr Krystal has been a paid consultant for Aisling Capital, LLC, AstraZeneca Pharmaceuticals, Brintnall & Nicolini, Easton Associates, Gilead Sciences, GlaxoSmithKline, Janssen Pharmaceuticals, Lundbeck Research USA, Medivation, Merz Pharmaceuticals, MK Medical Communications, F Hoffmann-La Roche, SK Holdings, Sunovion Pharmaceuticals, Takeda Industries and Teva Pharmaceutical Industries. He serves as a member of Scientific Advisory Boards for Abbott Laboratories, Bristol-Myers Squibb, Eisai, Eli Lilly, Forest Laboratories, Lohocla Research Corporation, Mnemosyne Pharmaceuticals, Naurex, Pfizer Pharmaceuticals, and Shire Pharmaceuticals. He is the Editor for Biological Psychiatry, a member of Board of Directors of Coalition for Translational Research in Alcohol and Substance Use Disorders, and the President Elect for American College of Neuropsychopharmacology. He also gets support from Tetragenex Pharmaceuticals. The other authors declare no conflict of interest.