Of the 5269 SNPs passing Perlegen's quality control (QC) criteria analyzed in the Finnish case–control cohort, PLINK identified 12 SNPs with MAF < 0.01 and subsequently excluded them from further analysis. Importantly, the PLINK identity-by-state (IBS)-based cluster analysis determined that one cluster best fit the data, thus providing no evidence for population stratification within the Finnish cohort. Absence of stratification was further supported with PLINK reporting a genomic inflation factor (based on median chi-squared) of 1 (values >1 indicate population structure) and mean chi-squared statistic of 0.945 (a mean of 1 is expected under the null hypothesis of no stratification).
No evidence for association with MA was observed for SNPs within the footprints of the three FHM genes. Briefly, none of the 25 ATP1A2 and 26 SCN1A SNPs produced point-wise P-values <0.05. Although five of the 87 CACNA1A SNPs (rs10416717, rs2419233, rs7249323, rs11882861, rs2074880) did produce nominal point-wise P-values ≤0.05 (0.0131, 0.01822, 0.04142, 0.04257, 0.04915, respectively), they were not significant after adjustment for testing multiple SNPs.
Allelic association results −log10
) for all 5257 SNPs are presented in Figure (also see Supplementary Material, Table S1
). The smallest observed point-wise P
-value from allelic association analysis of 5257 SNPs in the Finnish 841 MA cases and 884 controls was obtained for SNP rs13276133 (χ12
= 12.49; point-wise P
= 0.00041) residing in the potassium voltage-gated channel, Shab-related, member 2 (KCNB2
) gene on chromosome 8q13.3. The next smallest P
-value obtained for SNPs within KCNQ3
on 8q24.22 (rs13267466; P
= 0.00064) and CLIC5
on 6p12.3 (rs2095771; P
= 0.00074) also showed encouraging evidence for association. An additional nine genes (ATP2C2, CACNA1E
, KCNE2, KCNK12
) produced promising association signals (P
< 0.005) (Table ). However, after multiple test correction for the analysis of 5257 SNPs via permutation, our smallest point-wise P
-value (rs13276133; P
= 0.00041) was not globally significant with a global EMP2 P
-value of 0.7713. Hence, none of the tested SNPs reached global statistical significance (EMP2 P
≤ 0.05) (Table ).
Allelic association results for Finnish MA cases versus controls. Horizontal red line indicates the required global significance threshold [−log10(P) = 4.7286] to ensure an overall type I error rate of 5%.
Single SNP association results for single nucleotide polymorphisms selected for replication from Finnish case–control cohort
Exploratory analysis of haplotypes spanning two to six contiguous SNPs also did not produce significant evidence for association (data not shown), with the smallest observed point-wise P-value (χ12 = 13.82; point-wise P = 0.00020) obtained for the specific 3-allele G-T-T haplotype of SNPs rs1998822–rs7076100–rs11598027 within the CACNB2 gene on chromosome 10p12.33. The omnibus 3-allele haplotype test for this three SNP window (correcting for haplotype tests for all observed 3-allele combinations) only produced a χ12 of 18.12 with point-wise P = 0.0012, compared with the single SNP point-wise P = 0.00198 for rs11598027. Given that these results are not corrected for testing, all other two to six SNP haplotypes, analysis of haplotypes in these data did not produce stronger evidence for association compared with the single SNP analyses.
In addition to the primary MA phenotype, we also performed exploratory association analyses with strict International Headache Society (IHS) migraine criteria (i.e. excluding the 114 MA individuals with aura symptoms not strictly fulfilling the IHS aura criteria, but diagnosed as MA by our clinical neurologist; Table ) and the key trait components of pulsation, photophobia and phonophobia (Table , Supplementary Material, Table S1
). Although these exploratory analyses did not provide increased evidence for association between MA and SNPs within the three FHM genes, they did produce the smallest observed P
= 16.00; point-wise P
= 0.000063), obtained for SNP rs12996816 residing in the KCNS3
gene on chromosome 2p24.2 with the pulsation phenotype; however, this result does not surpass the primary analysis global significance threshold of point-wise P
≤ 0.000019. Furthermore, given an additional global multiple test correction is required for examining four additional traits; these exploratory analyses do not alter the overall (negative) interpretation of our individual SNP association results.
Demographic factors of the Finnish case and control groups
Although no single SNP or haplotype reached statistical significance, multiple small effect association signals in multiple genes may nonetheless be present. To examine this possibility, we plotted the observed distribution of the 5257 rank-ordered chi-square (χ12) values obtained from the single SNP allelic association analysis using MA as trait, against the distribution of χ12 values expected under the null hypothesis of no association. The resulting QQ-plot (Fig. ) shows a definite, although non-significant preponderance of χ12 values ranging from around 9.573 (point-wise P = 0.00198) to 10.19 (point-wise P = 0.00141) [i.e. the top 11–15 χ12 values].
Figure 2. QQ-plot of Finnish case–control allelic association results for clinical migraine with aura end-diagnosis (closed black circles). The solid red line represents the expected (reference) distribution under the null. The broken red lines indicate (more ...)
Applying the false discovery rate (FDR) approach to the 5257 rank-ordered allelic association P-values produced q-values (FDRs) of 0.673 and 0.692 for the 1st–11th and 12th–15th smallest P-values, respectively. Assigned q-values quickly and substantially increased in magnitude for the 16th (q-value = 0.773), 17th–23rd (q-value = 0.950) and remaining 24th–5257th ranked P-values (q-value = 0.9997). These results indicate that there are a maximum of four to five likely true-positives contained within the top 15 most significant allelic associations [i.e. true positive probability (1 − q-value) multiplied by the total number in the set with equivalent or smaller q-values: (1– 0.6922) × 15 = 4.62]. Also, our stage-1 nominal significance threshold for allelic association with MA in the Finnish cohort demarcates q-values of 0.950 and 0.9997, thus supporting P < 0.005 as a reasonable compromise between following-up true risk loci and the total number of loci to follow-up (i.e. minimizing the multiple-test burden while maintaining high replication power).
A total of 66 SNPs within 12 genes (Table ) were selected for follow-up in the replication cohorts (see Supplementary Material, Tables S2a and S2b
for estimates of power to replicate the Finnish association results). The 66 SNPs were primarily selected upon the basis of having the smallest allelic association results among all the Finnish MA cases and controls (P
< 0.005). The SNPs within these 12 genes were also selected if they produced nominal evidence (point-wise P
< 0.05) for MA and/or produced more significant P
-values, compared with the primary MA phenotype, for association with the traits examined in the exploratory association analyses (Table , Supplementary Material, Table S1
). In addition to the investigation of multiple effects within each gene, we selected multiple associated SNPs within the 12 most promising genes to help guard against potential assay failure in the follow-up replication studies. Counts of case–control individuals successfully genotyped are provided in Table .
Replication cohort sizes for individuals successfully genotyped
All 66 SNPs, except those which failed, were tested first in the Leiden sample with MA and MO (one SNP failed). Following the Leiden results, 60 SNPs were successfully tested in the population-based Brisbane sample with MA and MO. Finally, 44 SNPs were successfully tested in the Cologne and Munich clinic-based German MA samples. We note that due to the Munich cohort consisting of only migraine cases, two German-specific analyses were performed; (i) Cologne cases versus Cologne controls and (ii) combined Cologne cases and Munich cases versus Cologne controls. Post hoc analyses (not presented) in the replication cohorts containing a mixture of migraine sub-groups did not identify significant allele frequency differences between MO, MA and the latent class analysis (LCA)-based migraine groups, thus supporting the combination of these sub-groups. After QC checks, some genotype assays were failed in each cohort.
The results from association analysis of the 66 replication SNPs are presented in Table . Although a small number of SNPs showed significant evidence for replication (EMP2 P
≤ 0.05) in the Brisbane [CLIC5
SNP rs3729618 (EMP2 P
= 0.0459)] and Cologne cohort [KCNE2
SNPs rs1467847 (EMP2 P
= 0.0139) and rs1013063 (EMP2 P
= 0.0193)], none of the SNPs produced consistent evidence for replication across the cohorts. Furthermore, none of the SNPs were significant in the combined replication cohort Cochran-Mantel-Haenszel (CMH) test after correction for testing 66 SNPs. Analysis of the Leiden, Brisbane and/or combined replication MA subset did not alter the overall results. Due to the associated CLIC5
alleles in the Finnish and Brisbane cohorts being in opposite directions, the KCNE2
associations remain as the sole result which could justify further investigation. See Supplementary Material, Table S3
for point-wise replication association analysis results for all 66 replication SNPs in the four individual replication cohorts and combined replication sample.
Replication association analysis results
Interestingly, a SNP × SNP epistasis analysis of the 66 replication SNPs in the Finnish case–control sample detected an interaction between KCNB2
SNP rs1431656 on chromosome 8q13.3 and CACNB2
SNP rs7076100 on chromosome 10p12.33 (point-wise P
= 0.0000199), which remained significant after conservatively correcting for all 2145 possible interaction tests [i.e. treating the 66 replication SNPs as independent] (global P
= 0.042). Accounting for non-independence between SNPs due to intermarker linkage disequilibrium (LD) (22
), the global P
-value for the rs1431656 × rs7076100 interaction is P
= 0.022. This interaction was driven by the rs1431656–rs7076100 G-A haplotype providing increased protection against migraine occurrence (Table ). An interaction between these two SNPs was not observed in any of the replication samples (Table for comparison of population sample allele frequencies). However, other SNPs within these same genes, KCNB2
, provided suggestive evidence for interaction in the Brisbane cohort and marginally significant evidence for interaction in the combined Cologne and Munich cohort (i.e. between CACNB2
SNP rs8181477 and KCNB2
SNPs rs7006287 and rs11782118, point-wise P
= 0.004017 and P
= 0.004454, respectively). A more detailed description of the interaction analysis is provided in the Supplementary Material
Allele frequency data for rs1431656 and rs7076100 interaction