MERTK in the rat trigeminal system: a potential novel target for cluster headache?

The trigeminal system is key to the pathophysiology of migraine and cluster headache, two primary headache disorders that share many features. Recently, MER proto-oncogene tyrosine kinase (MERTK), a cell surface receptor, was strongly associated with cluster headache through genetic studies. Further, the MERTK ligand galectin-3 has been found to be elevated in serum of migraine patients. In this study, MERTK and MERTK ligands were investigated in key tissue to better understand their potential implication in the pathophysiology of primary headache disorders. Immunohistochemistry was used to map MERTK and galectin-3 expression in rat trigeminal ganglia. RT-qPCR was used to assess MERTK gene expression in blood, and ELISA immunoassays were used for MERTK ligand quantification in serum from study participants with and without cluster headache. MERTK gene expression was elevated in blood samples from study participants with cluster headache compared to controls. In addition, MERTK ligand galectin-3 was found at increased concentration in the serum of study participants with cluster headache, whereas the levels of MERTK ligands growth arrest specific 6 and protein S unaffected. MERTK and galectin-3 were both expressed in rat trigeminal ganglia. Galectin-3 was primarily localized in smaller neurons and to a lesser extent in C-fibres, while MERTK was found in satellite glia cells and in the outer membrane of Schwann cells. Interestingly, a strong MERTK signal was found specifically in the region proximal to the nodes of Ranvier. The overexpression of MERTK and galectin-3 in tissue from study participants with cluster headache, as well as the presence of MERTK in rat peripheral satellite glia cells and Schwann cells in the trigeminal ganglia, further highlights MERTK signalling as an interesting potential future therapeutic target in primary headache.

Genetic and Phenotypic Profiling of Triptan Users in a Swedish Cluster Headache Cohort.

Up to 25% of individuals who live with cluster headache (CH), an extremely painful primary headache disorder, do not adequately respond to the first-line treatment, triptans. Studies have indicated that genetic variants can play a role in treatment response. Likewise, differences in clinical characteristics can give clues to mechanisms underlying triptan non-response. Our aim was to investigate five genetic variants previously implicated in triptan response and their relation to triptan usage in our Swedish CH cohort and to investigate potential distinctions in clinical characteristics. 545 CH patients were screened for the genetic variants rs1024905, rs6724624, rs4795541, rs5443, and rs2651899 with a case control design based on triptan usage. Analysis of clinical characteristics was based on self-reported questionnaire data from 893 patients. One genetic variant, rs1024905, was significantly associated with triptan non-usage in CH (Pc = 0.010). In addition, multi-allele effector analysis showed that individuals with a higher number of effector variants were less likely to use triptans (P = 0.007). Analysis of clinical characteristics showed that triptan users were more likely to have alcohol as a trigger (57.4% vs 43.4%, P = 0.002), have autonomic symptoms (95.1% vs 88.1%, P = 0.002), and be current smokers (27.0% vs 21.9%, P = 0.033) compared to non-users. These results support the hypothesis that genetic variants can play a role in triptan usage in CH and that patients with a typical CH phenotype are more likely to use triptans.

Use of Prescribed and Non-Prescribed Treatments for Cluster Headache in a Swedish Cohort.

BACKGROUND: Cluster headache (CH) is a debilitating condition, but current therapies leave CH patients in pain. The extent of this problem in Sweden is unknown. METHODS: An anonymized questionnaire was sent to 479 Swedish CH patients to investigate patterns and perceived effects of treatments. RESULTS: Three hundred fourteen answers were analyzed. The population was representative regarding age of onset and sex. Less than half (46%) were satisfied with their abortive treatments, 19% terminated functioning abortive treatments due to side effects. Additionally, 17% of chronic CH patients had not tried the first-line preventive drug verapamil. A small subset had tried illicit substances to treat their CH (0-8% depending on substance). Notably, psilocybin was reported effective as an abortive treatment by 100% (n = 8), and with some level of effect as a preventive treatment by 92% (n = 12). For verapamil, some level of preventive effect was reported among 68% (n = 85). CONCLUSIONS: Our descriptive data illustrate that many Swedish CH patients are undertreated, lack functional therapies, and experience side effects. Further studies are warranted to search for new treatment strategies as well as a revision of current treatment guidelines with the aim of reducing patient disease burden to the greatest extent possible.

Sex Differences in the Allele Distribution of PGLYRP2 Variant rs892145 in Parkinson's Disease.

Introduction: Parkinson's disease (PD) is a complex multifactorial disease, involving genetic susceptibility, environmental risk factors, and gene-environmental interactions. The microbiota-gut-brain axis is hypothesized to play a role in the pathophysiology of PD, and peptidoglycan recognition proteins (PGLYRPs), which modulate the gut microbiota, are, therefore, relevant candidate genes for PD. Methods: Using quantitative real-time PCR, we genotyped three PGLYRP variants (rs892145, rs959117, and rs10888557) and performed an association analysis in 508 PD patients and 585 control individuals. We further conducted a meta-analysis of rs892145 and analyzed PGLYRP2 gene expression in lymphocytes from patients with PD and controls. Results: Although initial analysis of the three variants rs892145, rs959117, and rs10888557 and a meta-analysis of rs892145 did not reveal any association between the selected variants and PD, we found an interaction between sex and genotype for rs892145, with a marked difference in the allele distribution of rs892145 between male and female patients. As compared to controls, the T allele was less common in female patients (odds ratio = 0.76, P = 0.04) and more common in male patients (odds ratio = 1.29, P = 0.04). No difference was found in PGLYRP2 gene expression between PD patients and controls (P = 0.38), nor between sexes (P = 0.07). Discussion. Overall, this genetic screening in Swedish PD patients does not support previous results demonstrating associations of PGLYRP variants with the risk of PD. Meta-analysis of rs892145 revealed pronounced heterogeneity between previously published studies which is likely to have influenced the results. Taken together, the genetic and gene expression analyses suggest a possible link between genetic variants in PGLYRP2 and sex differences in PD. Because of the limited sample size in our study, these results need to be verified in independent cohorts before concluding.

Patients with cluster headache show signs of insomnia and sleep related stress: results from an actigraphy and self-assessed sleep study.

BACKGROUND: Cluster headache (CH) is a primary headache disorder which is characterized by circadian timing of headache attacks, usually at nighttime, in around two thirds of patients. Patients with CH often report sleep difficulties, though it is unknown whether this is a cause or a consequence of nightly headache attacks. OBJECTIVE: In this case-control study we have assessed sleep quality in study participants with CH in cluster bout respectively in remission, compared to a control group of neurologically healthy individuals to investigate the potential connection between sleep and CH. METHODS: Fifty study participants with CH and 42 controls were recruited for sleep assessment. Sleep was recorded using MotionWatch 8 actigraphs (CamNTech) for a period of two weeks. Study participants were instructed to wear the unit during rest and sleep and to fill out a sleep diary daily through the two-weeks period. RESULTS: Results from actigraphy recordings and sleep diaries suggested that patients with CH spend longer time in bed than controls (CH 8.1 hours vs. Controls 7.7 hours, p=0.03), but do not sleep more than controls (CH 6.7 hours vs. controls 6.5 hours, p=0.3). In addition, CH patients reported increased sleep latency (p=0.003), particularly during, but not restricted to, cluster bouts. Study participants with CH further reported higher levels of stress at bedtime (p=0.01), and they felt less well rested than controls (p=0.001). CONCLUSION: Our analysis suggests that sleep is negatively affected in CH both in cluster bout and in remission, manifesting in symptoms consistent with insomnia such as prolonged sleep latency and increased time in bed.

Cluster Headache Genomewide Association Study and Meta-Analysis Identifies Eight Loci and Implicates Smoking as Causal Risk Factor.

OBJECTIVE: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights. METHODS: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses. RESULTS: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine. INTERPRETATION: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor. ANN NEUROL 2023;94:713-726.

Investigating Vitamin D Receptor Genetic Markers in a Cluster Headache Meta-Analysis.

Patients diagnosed with the primary headache disorder known as cluster headache (CH) commonly report that their headache attacks occur in patterns of both circadian and seasonal rhythmicity. Vitamin D is essential for a variety of bodily functions and vitamin D levels are largely regulated by daylight exposure in connection with seasonal variation. For this Sweden-based study, the association between CH and three single-nucleotide polymorphisms in the vitamin D receptor gene, rs2228570, rs1544410, and rs731236, were investigated, as well as CH bouts and trigger factors in relation to seasonal and weather changes. Over 600 study participants with CH and 600 controls were genotyped for rs2228570, and genotyping results for rs1544410 and rs731236 were obtained from a previous genome-wide association study. The genotyping results were combined in a meta-analysis, with data from a Greek study. No significant association was found between rs2228570 and CH or the CH subtype in Sweden, nor did the meta-analysis show significant results for any of the three markers. The most common period of the year to experience CH bouts in Sweden was autumn, and conditions linked to weather or weather changes were also identified as potential triggers for CH bouts for a quarter of the responders who reported bout triggers. Though we cannot rule out vitamin D involvement in CH, this study does not indicate any connection between CH and the three vitamin D receptor gene markers.

Embracing Monogenic Parkinson's Disease: The MJFF Global Genetic PD Cohort.

BACKGROUND: As gene-targeted therapies are increasingly being developed for Parkinson's disease (PD), identifying and characterizing carriers of specific genetic pathogenic variants is imperative. Only a small fraction of the estimated number of subjects with monogenic PD worldwide are currently represented in the literature and availability of clinical data and clinical trial-ready cohorts is limited. OBJECTIVE: The objectives are to (1) establish an international cohort of affected and unaffected individuals with PD-linked variants; (2) provide harmonized and quality-controlled clinical characterization data for each included individual; and (3) further promote collaboration of researchers in the field of monogenic PD. METHODS: We conducted a worldwide, systematic online survey to collect individual-level data on individuals with PD-linked variants in SNCA, LRRK2, VPS35, PRKN, PINK1, DJ-1, as well as selected pathogenic and risk variants in GBA and corresponding demographic, clinical, and genetic data. All registered cases underwent thorough quality checks, and pathogenicity scoring of the variants and genotype-phenotype relationships were analyzed. RESULTS: We collected 3888 variant carriers for our analyses, reported by 92 centers (42 countries) worldwide. Of the included individuals, 3185 had a diagnosis of PD (ie, 1306 LRRK2, 115 SNCA, 23 VPS35, 429 PRKN, 75 PINK1, 13 DJ-1, and 1224 GBA) and 703 were unaffected (ie, 328 LRRK2, 32 SNCA, 3 VPS35, 1 PRKN, 1 PINK1, and 338 GBA). In total, we identified 269 different pathogenic variants; 1322 individuals in our cohort (34%) were indicated as not previously published. CONCLUSIONS: Within the MJFF Global Genetic PD Study Group, we (1) established the largest international cohort of affected and unaffected individuals carrying PD-linked variants; (2) provide harmonized and quality-controlled clinical and genetic data for each included individual; (3) promote collaboration in the field of genetic PD with a view toward clinical and genetic stratification of patients for gene-targeted clinical trials. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Sex Differences in Clinical Features, Treatment, and Lifestyle Factors in Patients With Cluster Headache.

BACKGROUND AND OBJECTIVES: Cluster headache is considered a male-dominated disorder, but we have previously suggested that female patients may display a more severe phenotype. Studies on sex differences in cluster headache have been conflicting; therefore, this study, with the largest validated cluster headache material at present, gives more insights into sex-specific characteristics of the disease. The objective of this study was to describe sex differences in patient demographics, clinical phenotype, chronobiology, triggers, treatment, and lifestyle in a Swedish cluster headache population. METHODS: Study participants were identified by screening medical records from 2014 to 2020, requested from hospitals and neurology clinics in Sweden for the ICD-10 code G44.0 for cluster headache. Each study participant answered a detailed questionnaire on clinical information and lifestyle, and all variables were compared with regard to sex. RESULTS: A total of 874 study participants with a verified cluster headache diagnosis were included. Of the participants, 575 (66%) were male and 299 (34%) were female, and biological sex matched self-reported sex for all. Female participants were to a greater extent diagnosed with the chronic cluster headache subtype compared with male participants (18% vs 9%, p = 0.0002). In line with this observation, female participants report longer bouts than male participants (p = 0.003) and used prophylactic treatment more often (60% vs 48%, p = 0.0005). Regarding associated symptoms, female participants experienced ptosis (61% vs 47%, p = 0.0002) and restlessness (54% vs 46%, p = 0.02) more frequently compared with male participants. More female than male study participants had a positive family history of cluster headache (15% vs 7%, p = 0.0002). In addition, female participants reported diurnal rhythmicity of their attacks more often than male participants (74% vs 63%, p = 0.002). Alcohol as a trigger occurred more frequently in male participants (54% vs 48%, p = 0.01), whereas lack of sleep triggering an attack was more common in female participants (31% vs 20%, p = 0.001). DISCUSSION: With this in-depth analysis of a well-characterized cluster headache population, we could demonstrate that there are significant differences between male and female participants with cluster headache, which should be regarded at the time of diagnosis and when choosing treatment options. The data suggest that female patients generally may be more gravely affected by cluster headache than male patients.

Multimorbidity and Sickness Absence/Disability Pension in Patients With Cluster Headache and Matched References: A Swedish Register-Based Study.

BACKGROUND AND OBJECTIVES: Multimorbidity among patients with cluster headache (CH) is considered to be high, but large studies are lacking. The aims were to explore the occurrence of diagnosis-specific multimorbidity among patients with CH and matched references and possible associations of this with their sickness absence and disability pension. METHODS: We performed a register-based study of patients with CH and matched references, regarding their multimorbidity, sickness absence, and disability pension. Data were obtained from 2 nationwide registers: Statistics Sweden's Longitudinal Integration Database for Health Insurance and Labor Market Studies (LISA) (for sociodemographics in 2009, sickness absence, and disability pension in 2010) and The National Board of Health and Welfare's specialized outpatient and inpatient registers for diagnosis-specific health care in 2001-2010 (for identifying patients with CH and multimorbidity, defined by ICD-10 codes). The prevalence and number of net days of sickness absence and/or disability pension in 2010 were calculated, in general and by multimorbidity. Odds ratios (OR) with 95% confidence intervals (CIs) were calculated for comparison of each diagnostic group with references without the chosen morbidity. RESULTS: We analyzed 3,240 patients with CH, aged 16-64 years, and living in Sweden in 2010 and 16,200 matched references. A higher proportion of patients with CH had multimorbidity (91.9%) than of references (77.6%), OR 3.263 (95% CI 2.861-3.721), both in general and regarding all analyzed diagnostic groups. Differences were particularly high for diagnoses relating to the nervous (CH 51.8% vs references 15.4%), OR 5.922 (95% CI 5.461-6.422), and musculoskeletal (CH 39.0% vs references 23.7%), OR 2.057 (95% CI 1.900-2.227), systems. Multimorbidity rates were overall higher among women in patients with CH (96.4% vs men 89.6%). Patients with CH had a higher mean number of days of sickness absence and disability pension compared with references, 63.15 vs 34.08 days. Moreover, multimorbidity was associated with a higher mean number of such days in patients with CH, 67.25, as compared with references, 40.69 days. DISCUSSION: The proportions of multimorbidity were high in both patients with CH and references, however, higher in the patients with CH, who also had higher sickness absence and disability pension levels. In particular, CH patients with multimorbidity and of female sex had high sickness absence and disability pension levels.

Glucocerebrosidase variant T369M is not a risk factor for Parkinson's disease in Sweden.

INTRODUCTION: Genetic variants in the Beta-glucocerebrosidase gene (GBA1) is a known risk factor for Parkinson's disease. The GBA1 mutations L444P, N370S and many other have been shown to associate with the disease in populations with diverse background. Some GBA1 polymorphisms have a less pronounced effect, and their pathogenicity has been debated. We have previously found associations with L444P, N370S and E326K and Parkinson's disease in Sweden. METHOD: In this study we used pyrosequencing to genotype the T369M variant in a large Swedish cohort consisting of 1,131 patients with idiopathic Parkinson's disease, and 1,594 control subjects to evaluate the possibility of this variant conferring an increased risk for Parkinson's disease. RESULTS: The minor allele frequency was 2.15% in patients and 1.76% in controls. Statistical analysis showed that there was no significant difference in allele frequency between patients and control subjects, p-value 0.37, Odds Ratio 1.23 with a 95% confidence interval of 0.82-1.83. CONCLUSION: Our results suggest that T369M is not a risk factor for Parkinson's disease in the Swedish population.

Genome-wide analysis of 102,084 migraine cases identifies 123 risk loci and subtype-specific risk alleles.

Migraine affects over a billion individuals worldwide but its genetic underpinning remains largely unknown. Here, we performed a genome-wide association study of 102,084 migraine cases and 771,257 controls and identified 123 loci, of which 86 are previously unknown. These loci provide an opportunity to evaluate shared and distinct genetic components in the two main migraine subtypes: migraine with aura and migraine without aura. Stratification of the risk loci using 29,679 cases with subtype information indicated three risk variants that seem specific for migraine with aura (in HMOX2, CACNA1A and MPPED2), two that seem specific for migraine without aura (near SPINK2 and near FECH) and nine that increase susceptibility for migraine regardless of subtype. The new risk loci include genes encoding recent migraine-specific drug targets, namely calcitonin gene-related peptide (CALCA/CALCB) and serotonin 1F receptor (HTR1F). Overall, genomic annotations among migraine-associated variants were enriched in both vascular and central nervous system tissue/cell types, supporting unequivocally that neurovascular mechanisms underlie migraine pathophysiology.

PER Gene Family Polymorphisms in Relation to Cluster Headache and Circadian Rhythm in Sweden.

The trigeminal autonomic cephalalgia, cluster headache (CH), is one of the most painful disorders known to man. One of the disorder's most striking features is the reported diurnal rhythmicity of the attacks. For a majority of patients, the headache attacks occur at approximately the same time every day. Genetic variants of genes involved in the circadian rhythm such as Period Circadian Regulator 1, 2, and 3 (PER1, 2 and 3) are hypothesized to have an effect on the rhythmicity of the attacks. Six PER1, 2 and 3 genetic markers; the indel rs57875989 and five single nucleotide polymorphisms (SNPs), rs2735611, rs2304672, rs934945, rs10462020, and rs228697, were genotyped, using TaqMan® or regular polymerase chain reaction (PCR), in a Swedish CH case control material. Logistic regression showed no association between CH and any of the six genetic variants; rs57875989, p = 0.523; rs2735611, p = 0.416; rs2304672, p = 0.732; rs934945, p = 0.907; rs10462020, p = 0.726; and rs228697, p = 0.717. Furthermore, no difference in allele frequency was found for patients reporting diurnal rhythmicity of attacks, nor were any of the variants linked to diurnal preference. The results of this study indicate no involvement of these PER genetic variants in CH or diurnal phenotype in Sweden.

The molecular clock gene cryptochrome 1 (CRY1) and its role in cluster headache.

BACKGROUND: Cluster headache is a severe primary headache disorder commonly featuring a strikingly distinct circadian attack pattern. Therefore, the circadian system has been suggested to play a crucial role in the pathophysiology of cluster headache. Cryptochromes are key components of the molecular clock generating circadian rhythms and have previously been shown to be associated with several psychiatric disorders, including seasonal affective disorder, bipolar disorder, and depression. METHODS: In this case-control study, we investigated the role of cryptochrome (CRY) genes in cluster headache by screening 628 cluster headache patients and 681 controls from Sweden for four known genetic variants in the CRY1 (rs2287161 and rs8192440) and CRY2 (rs10838524 and rs1554338) genes. In addition, we analyzed CRY1 gene expression in primary fibroblast cell lines from eleven patients and ten controls. RESULTS: The exonic CRY1 variant rs8192440 was associated with cluster headache on allelic level (p=0.02) and this association was even more pronounced in a subgroup of patients with reported diurnal rhythmicity of attacks (p=0.002). We found a small significant difference in CRY1 gene expression between cluster headache patients and control individuals (p=0.04), but we could not identify an effect of the associated variant rs8192440 on CRY1 expression. CONCLUSIONS: We discovered a disease-associated variant in the CRY1 gene and slightly increased CRY1 gene expression in tissue from cluster headache patients, strengthening the hypothesis of circadian dysregulation in cluster headache. How this gene variant may contribute to the pathophysiology of the disease remains subject to further studies.

Genome-Wide Association Study Identifies Risk Loci for Cluster Headache.

OBJECTIVE: This study was undertaken to identify susceptibility loci for cluster headache and obtain insights into relevant disease pathways. METHODS: We carried out a genome-wide association study, where 852 UK and 591 Swedish cluster headache cases were compared with 5,614 and 1,134 controls, respectively. Following quality control and imputation, single variant association testing was conducted using a logistic mixed model for each cohort. The 2 cohorts were subsequently combined in a merged analysis. Downstream analyses, such as gene-set enrichment, functional variant annotation, prediction and pathway analyses, were performed. RESULTS: Initial independent analysis identified 2 replicable cluster headache susceptibility loci on chromosome 2. A merged analysis identified an additional locus on chromosome 1 and confirmed a locus significant in the UK analysis on chromosome 6, which overlaps with a previously known migraine locus. The lead single nucleotide polymorphisms were rs113658130 (p = 1.92 × 10-17 , odds ratio [OR] = 1.51, 95% confidence interval [CI] = 1.37-1.66) and rs4519530 (p = 6.98 × 10-17 , OR = 1.47, 95% CI = 1.34-1.61) on chromosome 2, rs12121134 on chromosome 1 (p = 1.66 × 10-8 , OR = 1.36, 95% CI = 1.22-1.52), and rs11153082 (p = 1.85 × 10-8 , OR = 1.30, 95% CI = 1.19-1.42) on chromosome 6. Downstream analyses implicated immunological processes in the pathogenesis of cluster headache. INTERPRETATION: We identified and replicated several genome-wide significant associations supporting a genetic predisposition in cluster headache in a genome-wide association study involving 1,443 cases. Replication in larger independent cohorts combined with comprehensive phenotyping, in relation to, for example, treatment response and cluster headache subtypes, could provide unprecedented insights into genotype-phenotype correlations and the pathophysiological pathways underlying cluster headache. ANN NEUROL 2021;90:193-202.

Calcitonin Gene-Related Peptide (CGRP) and Cluster Headache.

Cluster headache (CH) is a severe primary headache with a prevalence of 1/1000 individuals, and a predominance in men. Calcitonin gene-related peptide (CGRP) is a potent vasodilator, originating in trigeminal neurons and has a central role in CH pathophysiology. CGRP and the CGRP receptor complex have recently taken center stage as therapeutic targets for primary headaches, such as migraine. Multiple CGRP and CGRP receptor monoclonal antibodies, as well as small molecule antagonists (gepants) are on their way constituting a new frontier of migraine and possibly CH medication. During a CH attack, there is an activation of the trigeminal-autonomic reflex with the release of CGRP, and inversely if CGRP is administered to a CH patient in an active disease phase, it triggers an attack. Increased levels of CGRP have been found in ipsilateral jugular vein blood during the active phase of CH. This process is hypothesized to have a key role in the intense pain perception and in the associated distinctive vasodilation. So far, clinical tests of CGRP antibodies have been inconclusive in CH patients. This review summarizes the current state of knowledge on the role of CGRP in CH pathology, and as a target for future treatments.

Analysis of NOS Gene Polymorphisms in Relation to Cluster Headache and Predisposing Factors in Sweden.

Cluster headache is characterized by activation of the autonomic-trigeminal reflex. Nitric oxide can trigger headaches in patients, and nitric oxide signaling is known to be affected in cluster headache. Based on the hypothesis of nitric oxide being involved in cluster headache pathophysiology we investigated nitric oxide synthases as potential candidate genes for cluster headache. We analyzed eight variants in the three forms of nitric oxide synthase (NOS) genes, inducible NOS (iNOS), endothelial NOS (eNOS) and neuronal NOS (nNOS), and tested for association with cluster headache. Swedish cluster headache patients (n = 542) and controls (n = 581) were genotyped using TaqMan® assays on an Applied Biosystems 7500 qPCR cycler. This is the largest performed genetic study on NOS involvement in cluster headache so far. We found an association between cluster headache and one iNOS haplotype consisting of the minor alleles of rs2297518 and rs2779249 (p = 0.022). In addition, one of the analyzed nNOS variants, rs2682826, was associated with reported triptan use (p = 0.039). Our data suggest that genetic variants in NOS genes do not have a strong influence on cluster headache pathophysiology, but that certain combinations of genetic variants in NOS genes may influence the risk of developing the disorder or triptan use.

Genetic Screening of Plasticity Regulating Nogo-Type Signaling Genes in Migraine.

Migraine is the sixth most prevalent disease in the world and a substantial number of experiments have been conducted to analyze potential differences between the migraine brain and the healthy brain. Results from these investigations point to the possibility that development and aggravation of migraine may include grey matter plasticity. Nogo-type signaling is a potent plasticity regulating system in the CNS and consists of ligands, receptors, co-receptors and modulators with a dynamic age- and activity-related expression in cortical and subcortical regions. Here we investigated a potential link between migraine and five key Nogo-type signaling genes: RTN4, OMGP, MAG, RTN4R and LINGO1, by screening 15 single nucleotide polymorphisms (SNPs) within these genes. In a large Swedish migraine cohort (749 migraine patients and 4032 controls), using a logistic regression with sex as covariate, we found that there was no such association. In addition, a haplotype analysis was performed which revealed three haplotype blocks. These blocks had no significant association with migraine. However, to robustly conclude that Nogo-type genotypes signaling do not influence the prevalence of migraine, further studies are encouraged.

Anoctamin 3: A Possible Link between Cluster Headache and Ca2+ Signaling.

Cluster headache is a severe primary headache characterized by extremely painful attacks of unilateral headache. Verapamil is commonly used as a prophylactic treatment with good effect. In order to search for new pathways involved in the pathophysiology of cluster headache, we analyzed genetic variants that were previously linked to verapamil response in migraine in a Swedish cluster headache case-control sample. We used TaqMan qPCR for genetic screening and performed a gene expression analysis on associated genes in patient-derived fibroblasts, and further investigated which reference genes were suitable for analysis in fibroblasts from cluster headache patients. We discovered a significant association between anoctamin 3, a gene encoding a calcium-activated ion channel, and cluster headache. The association was not dependent on verapamil treatment since the associated variant, rs1531394, was also overrepresented in patients not using verapamil. No difference was found in the anoctamin 3 gene expression between controls and patients. Also, we determined that TBP, IPO8 and PDHB were suitable reference genes in cluster headache fibroblasts. This finding is the first report of an association between a variant in a gene encoding an ion-channel and cluster headache, and the first significant genetic evidence of calcium involvement in cluster headache pathophysiology.

Analysis of HCRTR2 Gene Variants and Cluster Headache in Sweden.

OBJECTIVE: The purpose of this study was to investigate the HCRTR2 gene variants rs3122156, rs2653342, and rs2653349 in a large homogenous Swedish case-control cohort in order to further evaluate the possible contribution of HCRTR2 to cluster headache. BACKGROUND: Cluster headache is a severe neurovascular disorder and the pathophysiology is not yet fully understood. Due to striking circadian and circannual patterns of this disease, the hypothalamus has been a research focus in cluster headache. Several studies with many different cohorts from Europe have investigated the hypocretin receptor 2 (HCRTR2) gene, which is expressed in the hypothalamus. In particular, one HCRTR2 single nucleotide polymorphism, rs2653349, has been subject to a number of genetic association studies on cluster headache, with conflicting results. Two other HCRTR2 gene variants, rs2653342 and rs2653349, have been reported to be linked to cluster headache in an Italian study. METHODS: We genotyped a total of 517 patients diagnosed with cluster headache and 581 controls, representing a general Swedish population, for rs3122156, rs2653342, and rs2653349 using quantitative real-time PCR. Statistical analyses of genotype, allele, and haplotype frequencies for the 3 gene variants were performed comparing patients and controls. RESULTS: For rs3122156, the minor allele frequency in patients was 25.9% compared to 29.9% in controls (P = .0421). However, this significance did not hold after correction for multiple testing. The minor allele frequencies for rs2653342 (14.7% vs 14.7%) and rs2653349 (19.5% vs 18.8%) were similar for patients and controls. Furthermore, we found one haplotype that was significantly less common in patients than controls (P = .0264). This haplotype included the minor allele for rs3122156 and the major alleles for rs2653342 and rs2653349. Significance did not hold after applying a permutation test. CONCLUSIONS: Our data show a trend for association between cluster headache and the HCRTR2 polymorphism rs3122156, where the minor allele seems to be a protective factor. However, the other 2 HCRTR2 gene variants, including the previously reported rs2653349, were not associated with cluster headache in our Swedish material. A comparison with previous studies points to variance in genotype and allele frequencies among the different populations, which most likely contributes to the opposing results regarding rs2653349. Although the results from this study do not strongly support an association, HCRTR2 remains an interesting candidate gene for involvement in the pathophysiology of cluster headache.