ORIGINAL ARTICLE

Molecular Analysis of Cluster Headache Federica Zarrilli, ScD, PhD,* Rossella Tomaiuolo, MD, PhD,wz Carlo Ceglia, ScD, PhD,wz Barbara Lombardo, ScD, PhD,wz Barbara Izzo, ScD, PhD,zy Giuseppe Castaldo, MD, PhD,wz Lucio Pastore, MD, PhD,wz and Roberto De Simone, MD8

Objectives: Cluster headache (CH) is characterized by severe, recurrent, unilateral attacks of extreme intensity and brief duration. Variants in a myriad of genes were studied in sporadic CH patients, often with conflicting results. Methods: We studied gene mutations in some candidate genes, hypocretin receptor 2, Clock, and alcohol dehydrogenase 4 (ADH4), in 54 unrelated sporadic CH patients and in 200 controls in 8 kindreds/families that included more affected and nonaffected cases. Furthermore, we performed the whole-genome scanning by comparative genomic hybridization, searching for rearrangements associated with DNA gain or loss in a subset of sporadic and familial CH and control participants. Results: The analysis of candidate genes revealed that only allele and genotype frequency of the 2 ADH4 mutations resulted significantly between sporadic CH and controls; the same mutations were homozygous in CH patients from 2 families. The comparative genomic hybridization analysis revealed 2 novel rearrangements that involved the intron regions of thyrotropin-releasing hormone-degrading enzyme and neurexin 3 (NRXN3) genes, respectively. The first arrangement was present either in CH or in controls, whereas the second one was specifically found in some sporadic and familial CH cases. Conclusions: Our data (although obtained on a small number of cases) confirm the genetic heterogeneity of CH, suggesting that mutations in the ADH4 gene and a novel rearrangement involving NRXN3 gene might be related to CH in a subset of cases. Key Words: CH, cluster headache, genetics, gene mutations, CGH

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luster headache (CH) is a primary headache that appears with severe, strictly unilateral periorbital pain attacks that typically last for 15 to 180 minutes and show

circadian and circannual cluster periods. About 10% to 15% of CH is chronic. Among CH patients, there is a large variability in the duration of attacks and cluster periods, number of attacks per day, accompanying symptoms, and response to therapy as reported by the ICHD-3b.1 Familiarity was observed in several CH patients and different models of inheritance were suggested. Mitochondrial DNA deletions were described in a single CH family2 and another study reported an association between CH and mutations of mitochondrial DNA.3 We described a large kindred with 8 affected members in which an autosomal recessive model was involved.4 About 5% of CH is transmitted as autosomal-dominant trait.5 In the last decade, mutations in a myriad of genes were studied in CH patients. Genes encoding ionic channels—as demonstrated for familial hemiplegic migraine—receptors, and carriers for neural amines6 and G proteins7 were the first candidates. More recently, the association between CH and mutations in genes such as hypocretin receptor 2 (HR2),8 alcohol dehydrogenase 4 (ADH4),9 and Clock10 was studied with conflicting results. All these studies compared the allele or genotype frequency of mutations in CH patients versus control populations. In the present study, we tested mutations in a series of candidate genes for CH in: (1) unrelated sporadic CH in comparison with participants from the general population; (2) kindreds/families with more patients with CH. In addition, we first analyzed the whole genome of a subset of CH patients and controls using the comparative genomic hybridization (CGH) looking for rearrangements that cause gain or loss of DNA.

MATERIALS AND METHODS Received for publication November 15, 2013; revised February 13, 2014; accepted January 15, 2014. From the *Dipartimento di Bioscienze e Territorio, Universita` del Molise, Isernia, Italy; wDipartimento di Medicina Molecolare e Biotecnologie Mediche, Universita` di Napoli Federico II, Naples, Italy; yDipartimento di Medicina Clinica e Chirurgia, Universita` di Napoli Federico II, Naples, Italy; 8Dipartimento di Neuroscienze e Scienze Riproduttive e Odontostomatologiche, Universita` di Napoli Federico II, Naples, Italy; and zCEINGE-Biotecnologie Avanzate, Naples, Italy. Supported by a grant from Regione Campania (DGRC 1901/2009) and by POR Campania FSE 2007-13, Project CREME. It was a contribution of Campania region (public administration) aimed to fund research and diagnostics in the field of genes related to human disorders. The authors declare no conflict of interest. Reprints: Giuseppe Castaldo, MD, PhD, CEINGE-Biotecnologie Avanzate, Via Gaetano Salvatore 486, I-80145, Naples, Italy (e-mail: [email protected]). Supplemental Digital Content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website, www.clinicalpain.com. Copyright r 2014 by Lippincott Williams & Wilkins DOI: 10.1097/AJP.0000000000000075

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Patients The study was conducted according to guidelines of the Ethical Committee of our Medical School and was in accordance with the principles of the Helsinki II Declaration. The written informed consent was obtained from all participants. We studied 54 unrelated participants (42 male) affected by CH in which the anamnesis excluded other affected members in the family up to second-degree relatives (Supplementary Table S1, Supplemental Digital Content 1, http://links.lww.com/CJP/A84). In 4/54 cases (7.4%), CH was chronic; in the other 50 cases it was episodic. Supplementary Table S1 (Supplemental Digital Content 1, http://links.lww.com/CJP/A84) reports the main data of CH patients, that is, sex, duration of attacks (day/year and minutes/day), the number of attacks per day, and the age at onset. As side expression, in 3/54 cases (5.6%) CH was bilateral; in 3/54 cases (5.4%) the side of the attack was alternant; in the remaining 48 cases (88.9%) CH was unilateral. Furthermore, we studied Clin J Pain



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METHODS

8 kindred/families (Fig. 1) that included more members affected by CH and several nonaffected cases, all evaluated by a neurologist experienced in CH. In all sporadic and familial cases, CH was diagnosed according to ICHD-3b criteria.1 In addition, we studied 200 unrelated controls from the general population not affected by CH (all personally interviewed). From each participant included in the study, we collected a blood sample in EDTA (mostly at the time of sampling for diagnostic purposes).

Single Gene Analysis DNA was extracted from an EDTA blood sample using a commercial kit (Nucleon BACC; Amersham Biosciences, UK). The G1246A mutation of the HR2 gene,11 the T3092C mutation of the Clock gene,12 and ADH4 mutations rs1800759 and rs1126671 were analyzed by PCR followed by digestion with restriction enzymes.9

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FIGURE 1. Families’ pedigrees. Pedigree of 8 families/kindreds (A–H) with more affected cluster headache (CH) patients (in black). For each participant the genotypes for the following mutations are reported: ADH4 rs1800759 C > A; ADH4 rs1126671 G > A; HR2 G1246A; Clock T3092C; TRHDE gene deletion; NRXN3 gene deletion. N indicates not tested. r

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CGH Array Analysis DNA samples were analyzed with the Human Genome CGH Microarray kit 4X180K (Agilent Technologies, CA). The reference DNA (Promega, WI) consists of a pool of genomic DNA from 7 control samples. The CGH array contains 170,334 60-mer oligonucleotides probes covering

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the whole genome with an average spatial resolution of 13 kb. The procedures for DNA digestion, labeling, and hybridization were performed according to protocols provided by the manufacturer. Microarrays were scanned on an Agilent G2565CA and image files were quantified using Agilent’s Feature Extraction software (V10.10.1.1); r

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visualization of data was performed with the Agilent’s Genomic Work Bench Standard Edition (V6.5.0.58). Common aberrations were detected by the context-corrected common aberrations algorithm ADM-2 with a threshold = 6.0, minProbes = 1, and minAvgAbsLog Ratio = 0.2. The PCR cycling conditions were carried out with an initial denaturation at 951C for 15 minutes, followed by 6 cycles of 951C for 15 seconds, 611C for 30 seconds, and 721C for 30 seconds (6 cycles of touch down from 611C to 581C allowed to reduce the temperature by 0.51C every successive cycle), and 28 cycles at 951C for 15 seconds, 581C for 30 seconds, and 721C for 30 seconds with a final extension of 721C for 7 minutes.

Statistical Analysis The comparison of allele and genotype frequency of the different mutations between CH patients and controls was made using the w2 test (using Yates’ correction test in 2 2 table comparisons). For genes that showed multiple mutations, we conducted a multiple comparison test. Furthermore, for each mutation, we verified the correspondence to the Hardy-Weinberg distribution (using a P-level of 0.01).

RESULTS Analysis of Candidate Genes in Sporadic CH Patients The distribution of all allele and genotype mutations was in Hardy-Weinberg equilibrium, with the exception of HR2 gene G1246A mutation in the control population. We analyzed a series of mutations in genes previously related to CH in 54 sporadic CH patients and in 200 control participants from the general population. Tables 1 and 2 summarize the results. In detail, the allele frequency of the A mutant allele of either the rs1800759 and of the rs1126671 of the ADH4 gene was significantly higher in CH patients as compared with controls (P = 0.03 in both the cases, Table 1). The genotype frequency of either mutations was not significantly different between CH and controls (Table 1). However, the frequency of the AA genotype of both the mutations was higher (although not significantly) in CH patients and for the rs1800759, comparing the frequency of the AA variant genotype versus the sum of the CA and the CC ones, the difference was significant between CH and controls (P = 0.03, data not shown). The genotype and the allele frequency of the HR2 G1246A and of the Clock gene T3092C mutations (Table 2) were not significantly different between CH patients and participants from the general population.

Analysis of Candidate Genes in CH Families Thus, we analyzed the mutations in genes previously related to CH in 8 families/kindreds with more affected CH patients (Figs. 1A–H). Family A includes 2 CH patients (ie, case no. 11 and the father, case no. 8). We analyzed the DNA from the case no. 11 and from the mother (case no. 7, nonaffected). Both the participants had the same genotype for all the genes, and all such genotypes were those not previously related to CH. Family B includes 3 CH patients (ie, 1 died and 2 living affected siblings). In this family, we analyzed DNA from both living CH patients and from several nonaffected participants along 3 generations. All genotypes obtained in all participants were those not previously related to CH, with the exception of both the mutations of ADH4 gene, that showed the homozygous r

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TABLE 1. Genotype (Panel A) and Allele Frequency (Panel B), Number and (%) of ADH4 Mutations rs1800759 and rs1126671 in Sporadic CH Patients as Compared With the GP

v2 Test rs1800759 Panel A CC CA AA Panel B C A rs1126671 Panel A GG GA AA Panel B G A

CH (n = 54) 19 (35.2) 28 (51.8) 7 (13.0) CH (n = 108) 66 (61.1) 42 (38.9)

GP (n = 200) 88 (44.0) 100 (50.0) 12 (6.0) GP (n = 400) 276 (69.0) 124 (31.0)

CH (n = 54) 18 (33.3) 25 (46.3) 11 (20.4) CH (n = 108) 61 (56.5) 47 (43.5)

GP (n = 200) 78 (39.0) 104 (52.0) 18 (9.0) GP (n = 400) 276 (69.0) 124 (31.0)

NS

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CH indicates cluster headache; GP, general population; NS not significant; italicized numerals are P-values.

variant allele in the 2 CH patients (ie, AA), whereas in all nonaffected participants from the family showed the homozygous wild-type (ie, CC and GG for the 2 variants, respectively) or the heterozygous (ie, CA and GA for the 2 variants, respectively) genotype. Again in family C, there were 2 CH siblings and some nonaffected participants, and we analyzed some DNA samples along 2 generations. For the G1246A mutation of the HR2 and both the mutations of the ADH4 gene, the 2 CH siblings had discordant genotypes. For Clock, the genotypes were concordant in the 2 CH siblings and were always those commonly observed in the general population. In family D, there were 3 affected siblings, 2 of which were alive; these latter were analyzed together with a nonaffected sibling. In this family, both the affected siblings were homozygous variant (ie, AA) for both the ADH4 mutations, whereas the nonaffected sibling was homozygous for the wild-type allele for both of them. All TABLE 2. Genotype (Panel A) and Allele Frequency (Panel B), Number and (%) of HR2 G1246A and Clock T3092C Mutations in Sporadic CH Patients as Compared with the GP

v2 Test HR2 G1246A Panel A GG GA AA Panel B G A Clock T3092C Panel A TT TC CC Panel B T C

CH (n = 54) 43 (79.6) 9 (16.7) 2 (3.7) CH (n = 108) 95 (88.0) 13 (12.0)

GP (n = 200) 165 (82.5) 27 (13.5) 8 (4.0) GP (n = 400) 369 (92.2) 31 (7.8)

CH (n = 54) 28 (51.8) 23 (42.6) 3 (5.6) CH (n = 108) 79 (73.1) 29 (26.9)

GP (n = 200) 120 (60.0) 72 (36.0) 8 (4.0) GP (n = 400) 312 (78.0) 88 (22.0)

NS

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CH indicates cluster headache; GP, general population; NS not significant.

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the other genes were wild type or heterozygous either in the 2 affected or in the nonaffected sibling. Family E included 4 CH patients along 3 generations; we studied 3 CH and 2 nonaffected participants. None of the genes showed the homozygous variant haplotype in none of the participants. Family F included 3 CH patients in the same generation, 2 of which were analyzed together with 2 nonaffected participants. None of the examined genes showed the homozygous variant haplotype. In family G, we studied 2 CH (a patient and the father) and a nonaffected participant. For all genes, we invariably observed the wild-type or the heterozygous genotype. Finally, in the large kindred H, there were several CH patients analyzed together with some nonaffected ones. None of the 3 genes resulted in being mutated in any case.

CGH Array Analysis We performed the whole-genome scanning by CGH searching for large rearrangements causing DNA gain or loss in 10 sporadic CH patients and in 7 controls from the general population. Furthermore, the analysis was performed on DNA from some familial CH and nonaffected participants from the same families (ie, families C, D, F, G, and H). The analysis revealed 2 novel rearrangements. The first arrangement, that is, arr 12q21.1(72,739,818-72,739,877)x0 resulted in a 63 bp deletion involving intron 2 of thyrotropin-releasing hormonedegrading enzyme (TRHDE) gene. The deletion was confirmed in all participants by PCR analysis using oligonucleotides placed on either side of alteration (Supplementary Table S2, Supplemental Digital Content 2, http://links. lww.com/CJP/A85); the condition of the absence of deletion allowed the amplification of a product of 551 bp. The second rearrangement was arr 14q31.1(80,110,45180,110,510)x0 that resulted in a 200 bp deletion involving intron 13 of neurexin 3 (NRXN3) gene. Also such a deletion was confirmed using oligonucleotides shown in Supplementary Table S2 (Supplemental Digital Content 2, http:// links.lww.com/CJP/A85). The absence of the deletion allowed the amplification of a product of 500 bp. The first deletion (ie, that involving TRHDE gene) was found in all 7 control subjects from the general population (3 homozygous and 4 heterozygous cases) and in 6/10 sporadic CH patients (3 homozygous and 3 heterozygous). The deletion was also studied in some families (ie, families C, D, F, G, and H, Fig. 1). In family D, the deletion was absent in a CH patient and in a control sibling, and the same was true in family F. In family H, the deletion was absent or heterozygous in 6 CH patients and in 4 controls. In family C, the deletion was homozygous in 2 CH patients and absent in the control sibling, as it was in family G. The second deletion (ie, that involving the NRXN3 gene) was found heterozygous in 3/10 sporadic CH patients, whereas it was absent in all 7 control subjects from the general population. The deletion was also studied in some families (ie, families C, D, F, G, and H). In families C, D, F, and G, the deletion was absent in CH patients and in control siblings. Only in family H the deletion was identified (heterozygous in 1 patient and homozygous in another patient). However, in the same family, 4 CH patients and 2 CH-free participants were wild type.

DISCUSSION We studied mutations in candidate genes in sporadic CH patients and in families that include more affected

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participants (to the best of our knowledge, this is the first study on gene mutations in CH families). Furthermore, given the complexity and variability of the CH phenotype, we analyzed, in a subset of cases, the whole genome by CGH13 looking for genomic alterations associated with gain or loss of DNA, never previously studied in CH patients. Going to variants in candidate genes, both the mutations in the ADH4 gene show a significantly higher frequency of the mutant allele in sporadic CH patients as compared with controls; this depends on the higher (although not significant) occurrence of the homozygous mutant genotype of both the mutations in CH patients (ie, 13.0% of CH patients for the rs1800759 and 20.4% for the rs1126671). Similarly, in 2 families (families B and D), the homozygous genotype for both the ADH4 mutations segregates with CH, whereas in the other 6 families neither patients nor controls showed such genotype. Our data are partially in agreement with another study9 that reported a higher occurrence of the mutant haplotype of the rs1126671 (and not of the rs1800759) of ADH4 in CH patients. Thus, we confirm that in a subset of CH patients the ADH4 gene, which encodes an enzyme subunit involved in the ethanol metabolism, may have a role. Interestingly, in the 2 CH patients from family B and in 1 member of the family D (all mutant homozygous for both the ADH4 variants), ethanol was referred as a trigger of the CH attack. On the contrary, mutations in the 2 other candidate genes do not correlate with CH neither in sporadic nor in familial CH patients. In fact, our data excluded differences for the allele and genotype frequency of HR2 G1246A mutation between CH patients and controls, unlike a previous study on 109 CH cases and 211 controls reported a 5-fold higher risk for CH for participants bearing the GG genotype.11 No differences were obtained for the allele and genotype frequency of the T3092C variant of the Clock gene (a well-known biological rhythm gene) between sporadic CH and controls, in agreement with 2 previous studies on Italian patients.10,12 Furthermore, none of our familial CH patients was homozygous for the variant haplotype. The CGH scanning, performed on a subset of participants, revealed 2 novel rearrangements. The first was a 63 bp deletion involving intron 2 of TRHDE gene.14 The gene encodes an ectoenzyme involved in the specific inactivation of the neuronal TRH that, in addition to the endocrine function, exerts a wide activity as neuromodulator and neurotransmitter. Some variants involving amino acids of the TRHDE protein reduce its activity,15 although it is difficult to predict the effect of the novel intron deletion identified in the present study because the enzyme is mainly expressed at the brain level. However, the deletion was found—heterozygous or homozygous—in most control and CH patients either sporadic or familial. Thus, we exclude a relevant pathogenic role of such novel mutation. The second rearrangement was a 200 bp deletion involving intron 13 of NRXN3 gene encoding the corresponding neurexin 3, a member of a complex family of isoproteins involved in a myriad of activities in the nervous system, particularly at the synapses level.16 Polymorphisms in the NRXN3 gene were correlated with behavior disorders,17 smoking behavior,18 and alcohol dependence19 and, more recently, rare deletions within the gene were correlated with the autism spectrum disorder.20 However, such deletions involve the coding regions of the gene, whereas the novel deletion identified in the present study involves only an intron region. In any case, given the very r

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complex regulation of neurexin expression and the number of alternative splicing (both phenomena that involve noncoding regions), we cannot exclude a pathogenic role of the novel deletion that could impair the interaction of the gene with regulatory factors. In contrast, it was found only in CH patients (3/10 sporadic cases were heterozygous for the deletion) and in none of the controls; similarly, it was found only in 2 CH patients (one heterozygous and the other homozygous) of 1 of the 5 CH families in which it was studied. These data are preliminary, but they disclose a novel possible association between a genetic factor and CH. The analysis extended to other CH cases, in vitro expression studies to define the functional effect of the novel deletion, and a systematic search of the NRXN3 genotype in CH patients will elucidate the correlations between the NRXN3 gene and CH. To conclude, our data, although obtained on a preliminary population, confirm the genetic heterogeneity of CH reinforcing the opinion that more complex mechanisms, as epigenetics, have a role in the pathogenesis of CH.10 Mutations in the ADH4 gene and a novel rearrangement involving NRXN3 gene might be related to CH in a small number of cases. Larger studies are required to verify differences between chronic and episodic CH patients. REFERENCES 1. Headache Classification Committee of the International Headache Society (HIS). The International Classification of Headache Disorders, 3rd edition (beta version). Cephalalgia. 2013; 33:629–808. 2. Odawara M, Tamaoka A, Mizusawa H, et al. A case of cluster headache associated with mitochondrial DNA deletions. Muscle Nerve. 1997;20:394–395. 3. Shimomura T, Kitano A, Marukawa H, et al. Point mutation in platelet mitochondrial tRNA-Leu (URR) in patient with cluster headache. Lancet. 1994;344:625. 4. De Simone R, Fiorillo C, Bonuso S, et al. A Cluster Headache family with possible autosomal recessive inheritance. Neurology. 2003;61:578–579. 5. Montagna P. The primary headaches: genetics, epigenetics and a behavioural genetic model. J Headache Pain. 2008;9:57–69. 6. Montagna P. Molecular genetics of migraine headaches: a review. Cephalalgia. 2000;20:3–14.

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7. Galeotti N, Ghelardini C, Zoppi M, et al. Hypofunctionality of Gi proteins as aetiopathogenic mechanism for migraine and cluster headache. Cephalalgia. 2001;21:38–45. 8. Rainero I, Rubino E, Valfre` W, et al. Association between the G1246A polymorphism of the hypocretin receptor 2 gene and cluster headache: a meta-analysis. J Headache Pain. 2007;8: 152–156. 9. Rainero I, Rubino E, Gallone S, et al. Cluster headache is associated with the alcohol dehydrogenase 4 (ADH4) gene. Headache. 2010;50:92–98. 10. Cevoli S, Mochi M, Pierangeli G, et al. Investigation of the T3111C CLOCK gene polymorphism in cluster headache. J Neurol. 2008;255:299–300. 11. Rainero I, Gallone S, Valfre` W, et al. A polymorphism of the hypocretin receptor 2 gene is associated with cluster headache. Neurology. 2004;63:1286–1288. 12. Rainero I, Rivoiro C, Gallone S, et al. Lack of association between the 3092 T > C Clock gene polymorphism and cluster headache. Cephalalgia. 2005;25:1078–1081. 13. Castaldo G, Lembo F, Tomaiuolo R. Molecular diagnostics: between chips and customized medicine. Clin Chem Lab Med. 2010;48:973–982. 14. Schomburg L, Turwitt S, Prescher G, et al. Human TRHdegrading ectoenzyme cDNA cloning, functional expression, genomic structure and chromosomal assignment. Eur J Biochem. 1999;265:415–422. 15. Papadopoulos T, Kelly JA, Bauer K. Mutational analysis of the thyrotropin-releasing hormone degrading ectoenzyme. Similarities and differences with other members of the M1 family of aminopeptidases and thermolysin. Biochemistry. 2001;40:9347–9355. 16. Bottos A, Rissone A, Bussolino F, et al. Neurexins and neuroligins: synapses look out of the nervous system. Cell Mol Life Sci. 2011;68:2655–2666. 17. Stoltenberg SF, Lehmann MK, Christ CC, et al. Associations among types of impulsivity, substance use problems and Neurexin-3 polymorphisms. Drug Alcohol Depend. 2011;119: 31–38. 18. Docampo E, Ribase´s M, Grataco`s M, et al. Association of neurexin 3 polymorphisms with smoking behavior. Genes Brain Behav. 2012;11:704–711. 19. Hishimoto A, Liu QR, Drgon T, et al. Neurexin 3 polymorphisms are associated with alcohol dependence and altered expression of specific isoforms. Hum Mol Genet. 2007;16: 2880–2891. 20. Vaags AK, Lionel AC, Sato D, et al. Rare deletions at the neurexin 3 locus in autism spectrum disorder. Am J Hum Genet. 2012;90:133–141.

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Molecular analysis of cluster headache.

Cluster headache (CH) is characterized by severe, recurrent, unilateral attacks of extreme intensity and brief duration. Variants in a myriad of genes...
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