XML Template (2015) [4.2.2015–3:57pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150002/APPFile/SG-CEPJ150002.3d


[1–9] [PREPRINTER stage]

Original Article

The association between candidate migraine susceptibility loci and severe migraine phenotype in a clinical sample

Cephalalgia 0(0) 1–9 ! International Headache Society 2015 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0333102415570492 cep.sagepub.com

Ann-Louise Esserlind1, Anne Francke Christensen1, Stacy Steinberg2, Niels Grarup3, Oluf Pedersen3, Torben Hansen3,4, Thomas Werge5, Thomas Folkmann Hansen5, Lise Lotte N Husemoen6, Allan Linneberg6,7,8, Esben Budtz-Jorgensen9, Maria Lurenda Westergaard1, Hreinn Stefansson2 and Jes Olesen1 Abstract Introduction: The objective of the study was to follow up and to test whether 12 previously identified migraine-associated single nucleotide polymorphisms were associated as risk factors and/or modifying factors for severe migraine traits in a Danish clinic-based population. Methods: Semi-structured migraine interviews, blood sampling and genotyping were performed on 1806 unrelated migraineurs recruited from the Danish Headache Center. Genotyping was also performed on a control group of 6415 people with no history of migraine. Association analyses were carried out using logistic regression and odds ratios were calculated assuming an additive model for risk. The proxies for severe migraine traits (early onset of migraine; many lifetime attacks, prolonged migraine and tendency to chronification of migraine) were tested against the 12 single nucleotide polymorphisms and a combined genetic score in both a case-control and case-only logistic regression model. Results: We successfully replicated five out of the 12 previously reported loci and confirmed the same direction of effects for all the 12 single nucleotide polymorphisms. In line with the recently published genome-wide association meta-analysis, the associations were significant for all migraine and migraine without aura but not for migraine with typical aura. Two single nucleotide polymorphisms (rs2274316 and rs11172113) conferred risk of many lifetime attacks in the case-control analysis. In the case-only analysis, only three single nucleotide polymorphisms showed nominal association with many lifetime attacks and prolonged migraine attacks. Conclusion: Our study supports previously reported findings on the association of several single nucleotide polymorphisms with migraine. It also suggests that the migraine susceptibility loci may be risk factors for severe migraine traits. Keywords Migraine, clinic-based sample, replication, genetics, susceptibility loci, genome-wide association studies Date received: 12 August 2014; revised: 21 October 2014; 11 December 2014; accepted: 27 December 2014


The Danish Headache Center, Department of Neurology, Glostrup Hospital, University of Copenhagen, Denmark 2 deCODE Genetics, Iceland 3 The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 4 Faculty of Health Sciences, University of Southern Denmark, Denmark 5 Institute of Biological Psychiatry, MHC Sct. Hans, Copenhagen Mental Health Services; Department of Clinical Medicine, University of Copenhagen; The Lundbeck Foundation Initiative for Integrative Psychiatric Research, iPSYCH, Denmark

6 Research Centre for Prevention and Health, the Capital Region of Denmark, Denmark 7 Department of Clinical Experimental Research, Glostrup University Hospital, Denmark 8 Faculty of Health and Medical Sciences, University of Copenhagen, Denmark 9 Department of Public Health, University of Copenhagen, Denmark

Corresponding author: Jes Olesen, Danish Headache Center, Department of Neurology, Glostrup Hospital DK-2600 Glostrup, Denmark. Email: [email protected]

XML Template (2015) [4.2.2015–3:57pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150002/APPFile/SG-CEPJ150002.3d


[1–9] [PREPRINTER stage]

2 Introduction Migraine with typical aura (MTA) and migraine without aura (MO) are prevalent and burdensome neurological disorders with high socioeconomic cost (1,2). The hereditary component of migraine has previously been estimated to be as high as 57% and is likely to be caused by multiple genetic factors, thus characterizing migraine as a common complex disorder (3–7). The prevailing method of genetically investigating complex disorders is the genome-wide association study (GWAS), in which the frequency of common variants is compared between the disease group and healthy controls. Several studies have attempted to elucidate genetic factors for migraine and the past years have witnessed promising advances. So far, three GWAS (8–10) and a genome-wide meta-analysis spearheaded by the International Headache Genetics Consortium have identified 12 genetic loci as conferring risk of migraine (11). This recent effort, a large-scale meta-analysis comprising 23,285 migraineurs and 95,425 controls, pooled mostly new results from 29 different GWAS and resulted in the identification of five new migraine-loci as well as confirmation of previously reported loci (11). Furthermore, the study demonstrated that the subgroup composed of clinic-based samples had larger effect sizes for all 12 genome-wide significant loci compared to migraine cases identified in population-based sampling; suggesting that the clinicbased cases may be genetically enriched compared to cases sampled from the general population (11). We aimed to evaluate if the 12 candidate single nucleotide polymorphisms (SNPs) could be replicated in an independent clinic-based migraine cohort and to test whether these migraine susceptibility loci were specifically associated with severe migraine characteristics inherent in clinically ascertained migraineurs recruited from a tertiary referral centre.

Materials and methods Study population The migraine sample comprised 2463 participants from both a recent and a previously collected migraine cohort from the Danish Headache Center (DHC), Glostrup Hospital. DHC is a national tertiary referral centre that receives headache patients from general practitioners, neurology departments and neurology practices. The study protocol and detailed information about the migraine cohort have been described previously (12,13). A semi-structured interview was conducted using a previously validated questionnaire. The interviews were performed one-to-one at the

Cephalalgia 0(0) DHC or over the telephone by trained physicians or senior medical students. The survey included all the pertinent clinical data for correct migraine diagnosing according to the ICHD-2 criteria (14). Standard blood sampling was performed for the purpose of DNAextraction and genotyping. In an effort to minimize both a clinical and a potential genetic heterogeneity, cases diagnosed with ‘probable’ migraine with regard to headache characteristics and accompanying symptoms were excluded. We did, however, choose to include the cases with tendency of prolonged migraine attacks since this was a feature of migraine severity which we intended to study. In addition, we also filtered for relatedness retaining only the proband. Cases with missing genotypes were excluded. The remaining sample comprised 1806 individuals, out of which 796 had MTA and 1010 had MO. The skewed proportion of MO and MTA in our cohort compared to the population prevalence was deliberate as we meant to recruit an equal number of cases with MTA and MO for our genetic studies. The mean age was 44.7 (12.4) years and the male to female ratio was 1:4.6 (322:1484). The control samples included 6415 healthy and unrelated individuals of Danish ethnicity enrolled as controls for epidemiological and genetic studies in Denmark. These controls were pooled from three sources: 458 were migraine-free controls from the DHC, recruited using the same semi-structured questionnaire mentioned above; 1326 blood donors, who reported the absence of any pre-existing medical condition or taking any medication were recruited under the auspices of the Danish Blood Donor Study (M-20090237); 4631 persons with no self-reported history of severe headaches from the population-based Inter99 study sampled at the Research Centre for Prevention and Health, Glostrup Hospital, Denmark. The Inter99 study (ClinicalTrials.gov: NCT00289237) is a randomized, non-pharmacological intervention study for the prevention of ischaemic heart disease (15). The present study protocol was in accordance with the Helsinki Declaration and approved by the Danish Ethical Standards Committee and Danish Data Protection Agency (Protocol number: H-2– 2010–122). Written informed consent was obtained from all participants.

SNP selection and genotyping For genotyping we selected migraine-associated SNPs that exceeded the threshold for genome-wide significant association (P < 5  108) in the abovementioned migraine meta-analysis (11). The 12 markers that were available at the time of this study were included (rs2651899/

XML Template (2015) [4.2.2015–3:57pm] //blrnas3.glyph.com/cenpro/ApplicationFiles/Journals/SAGE/3B2/CEPJ/Vol00000/150002/APPFile/SG-CEPJ150002.3d


[1–9] [PREPRINTER stage]


Esserlind et al. PRDM16; rs10915437/near AJAP1; rs12134493/near TSPAN2; rs2274316/MEF2D; rs7577262 -surrogate marker (r2 ¼ 1.00) for rs6741751/TRPM8; rs6790925/ near TGFBR2; rs9349379/PHACTR1; rs13208321 -surrogate marker (r2 ¼ 0.91) for rs11759769/FHL5; rs4379368/c7orf10; rs10504861/near MMP16; rs6478241/ASTN2; rs11172113/LRP1). All samples were typed using Centaurus assays at deCODE genetics, Reykjavik, Iceland. For each SNP, genotype call rate was at least 95% in both cases and controls and a test for deviation from the Hardy– Weinberg equilibrium in controls was non-significant. For rs6790925, plates containing 347 samples could not be included due to poor genotyping quality.

Statistical analysis Follow-up case-control analysis of the 12 SNPs. The followup association analysis for the 12 SNPs mentioned above was carried out using logistic regression with 1806 migraineurs and 6415 healthy controls. The analysis was performed for MTA and MO both separately and combined. Odds ratios (ORs) were calculated assuming an additive model for risk. The level of significance was set at P < 0.004 (0.05/12). Association analysis of the 12 SNPs and severe migraine characteristics in a case-control and a case-only logistic regression model. Migraine is sub-classified as MTA and MO (14), but as the primary aim of this analysis was to investigate clinical migraine as a subgroup, with a focus on severe migraine characteristics, we have chosen not to discriminate between the two subtypes. In a logistic regression analysis assuming an additive genetic effect, we investigated the single locus association and the association of a cumulative risk score, with severe migraine traits. This was done, first, by comparing severe migraine cases with non-migraineurs, i.e. healthy controls (case-control analysis) and, second, the same analysis was carried out again comparing cases with severe migraine traits with migraine cases without these severe sub-phenotypes (case-only analysis). The rationale for using these two distinct logistic regression models is that they represent two fundamentally different assumptions. The first model represents a conventional way to re-assess the migraine risk alleles that were identified in a case-control association study and investigates if the SNPs confer risk of severe migraine. In the latter model we perform a conditional analysis to test the influence of the variants on specific traits within a severely affected migraine population. Thus, in the case-only analysis we aim to test whether the risk alleles have a modifying effect on specific severe migraine traits.

To our best knowledge, no previous studies have described severe migraine characteristics or traits: hence, these traits were defined by consensus among headache specialists at the DHC as traits that are frequently reported by migraineurs at a tertiary referral centre for headache (Olesen, internal communication). Severe migraine characteristics were defined as the following: early onset of migraine (migraine onset ¼/100 migraine attacks during the entire lifetime); tendency to have prolonged migraine attacks (migraine attacks lasting >72 hours); chronification of migraine (headache ¼/>15 days per month and having at least eight migraine attacks monthly over a period of 3 months. This characteristic was not adjusted for prior medication overuse). The effect allele was set as the unfavourable allele associated with migraine as determined by the previous report (11). In an additive model based on an expected allelic dosage model for SNP markers, we calculated a multi-locus cumulative genetic score for each individual by adding up the number of risk alleles from the original study (11). We also calculated a cumulative genetic risk score using only the seven SNPs that were significant for ‘all migraine’ in the original study (11), since these variants were significantly associated with migraine in a larger sample size. The level of significance was set at P < 0.001 (0.05/ 52). Data on the binary end-points were extracted from the semi-structured interview. Results were presented as ORs, 95% confidence intervals (CI) and P-values. All of the analyses were adjusted for sex and age. Statistical analysis was performed using statistical software SAS version 9.3 (Copyright (c) 2002–2008 by SAS Institute Inc., Cary, NC, USA) for Microsoft Windows 10.

Results All the 12 candidate migraine SNPs showed the same direction of effects as in the original study (11) and seven out of the 12 SNPs were replicated in our clinical migraine cohort (Table 1). However, the two SNPs rs6478241 (ASTN2) and rs6790925 (near TGFBR2) only reached nominal significance resulting in five out the 12 SNPs succeeding the significance threshold for association with migraine after correction for multiple testing (Table 1; P < 0.004). A two-tailed binomial test showed that the observed consistency of effects with the previous study (11) is significantly different than what would be expected by chance for the 12 SNPs (P ¼ 2  104). In accordance with the GWAS metaanalysis data (11), our data show that the SNPs show

1:3073572 1:4082866 1:115479469 1:154712866 2:234483608 3:30455089 6:13011943 6:96967075 7:40432725 8:89617948 9:118292450 12:55813550

rs2651899 rs10915437 rs12134493 rs2274316 rs6741751c rs6790925 rs9349379 rs11759769d rs4379368 rs10504861 rs6478241 rs11172113


Gene/Nearby gene C G A C A T G A T T A C

Alleleb 0.95 0.94 1.08 1.16 0.87 1.08 0.87 1.11 1.05 0.92 1.09 0.98

(0.86–1.06) (0.84–1.05) (0.92–1.27) (1.04–1.29) (0.72–1.04) (0.98–1.21) (0.78–0.97) (0.99–1.26) (0.89–1.24) (0.80–1.06) (0.98–1.22) (0.88–1.09)

MTA OR (95% CI) 1.04 1.02 1.25 1.27 0.94 1.09 0.85 1.18 1.09 0.99 1.14 0.79

3.7  101 2.5  101 3.4  101 8.1  103 1.1  101 1.3  101 1.1  102 7.3  102 5.4  101 2.4  101 1.3  101 7.1  101 (0.95–1.15) (0.93–1.13) (1.09–1.44) (1.16–1.40) (0.81–1.10) (0.99–1.19) (0.77–0.94) (1.06–1.32) (0.94–1.26) (0.89–1.14) (1.03–1.25) (0.72–0.87)

MO OR (95% CI)

MTA P-value* 4.0  101 6.5  101 1.5  103

The association between candidate migraine susceptibility loci and severe migraine phenotype in a clinical sample.

The objective of the study was to follow up and to test whether 12 previously identified migraine-associated single nucleotide polymorphisms were asso...
167KB Sizes 0 Downloads 8 Views