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Abstract

Cluster headache (CH) is a primary headache disorder where the aetiological and pathophysiological mechanisms still are largely unknown. An increased risk of CH in first- and second-degree relatives suggests the importance of genetic factors. Mutations of the P/Q type calcium channel alpha 1 subunit (CACNA1A) gene on chromosome 19p13 have been shown to cause several neurological disorders with a wide clinical spectrum, mainly episodic diseases. Missence mutations of the gene cause familial hemiplegic migraine (FHM) and it is also likely to be involved in the more common forms of migraine. The CACNA1A gene is thus a promising candidate gene for CH. In this study we performed an association analysis of an intragenic polymorphic (CA)n-repeat with marker D19S1150 and a (CAG)n-repeat in the 3′UTR region, in 75 patients with CH according to IHS criteria and 108 matched controls. Genotypes and allele frequencies were similarly distributed in patients and controls. Linkage disequilibrium between the two markers was similar in patients and controls. We conclude that an importance of the CACNA1A gene in sporadic CH is unlikely.

Keywords

CACNA1A cluster headache genetics

Introduction

Cluster headache (CH) is characterized by recurrent, unilateral attacks of severe headache. The attacks most commonly appear in clusters, so called cluster periods. CH is considered a neurovascular headache disorder with the trigeminovascular system and the cranial parasympathetic nervous system being involved (1–3), but the pathophysiology is still largely unknown. The genetic background to migraine and other headaches has attracted an increased interest in recent years (4, 5). Heredity has previously been regarded as being of minor importance for the aetiology of CH, but in recent years its importance has been increasingly recognized. Several studies have reported a positive family history of CH (6–14). Russell showed in a Danish genetic epidemiological survey a 14.1-fold significant increased risk of CH in first-degree relatives and a 2.3-fold increased risk in second-degree relatives (10, 12). Kudrow and Kudrow (7) showed in an American survey an 45.6-fold increased risk in first-degree relatives, but the familial occurrence in this study might have been overestimated. However, a recently published study has shown a 39-fold significantly increased risk for CH in first-degree relatives and an 8-fold increased risk in second-degree relatives in an Italian population (14). These studies strongly suggest a genetic influence of the disease. CH has been reported in five pairs of monozygotic twins that all were concordant for CH, also supporting the importance of genetic factors (15–18). A segregation analysis indicates that an autosomal dominant gene has a role in 3–4% of male patients and 7–10% of female CH patients (11). Thus most patients with CH are sporadic cases.

The brain-specific P/Q type calcium channel alpha 1A subunit gene CACNA1A on chromosome 19p13 has been shown to be involved in a few mainly episodic neurological disorders. Different types of mutations in this gene are described in three disorders: familial hemiplegic migraine (FHM), episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6). Four different missence mutations have been described in five unrelated FHM families (19–24). Two mutations disrupting the reading frame cause EA2 (20, 23, 25). In SCA6 there is an expansion of a polymorphic CAG-repeat within the open reading frame (26). Affected sib-pair analysis with highly informative microsatellite markers in the FHM locus on 19p13 in migraine with and without aura has shown significantly increased sharing of alleles, indicating involvement of the CACNA1A gene even in the more common forms of migraine (27, 28, 30). Another study has shown linkage to chromosome 19p13 in one of four families with familial typical migraine (29).

Calcium acts as an intracellular second messenger by regulating several events in the cell; both biochemical and neuronal events such as neurotransmitter release (31–33). P-type neuronal calcium channels mediate serotonin (5-HT) release (34, 35). Most models of migraine and CH suggest that serotonin has a central role in the pathophysiology. Verapamil, an L-calcium channel blocker, is effective as prophylactic treatment in CH although high doses are often needed. The mechanism of action is not yet known but it is likely that verapamil may act by non-L-channel mechanisms, perhaps by interacting with one or more of the other voltage-dependent calcium channels (36).

Mutations of the CACNA1A gene result in a wide clinical spectrum varying from mild and fully reversible symptoms to severely progressive symptoms. The CACNA1A gene is thus an interesting candidate for conferring susceptibility to CH.

Materials and methods

Patients and control samples

Blood samples from 75 Caucasian outpatients with sporadic CH were collected, 56 (74.7%) males and 19 (25.3%) females. The mean age was 49.4 years (range 24–74 years). Six patients had chronic and 69 patients had episodic cluster headache. They had all been seen by us and clearly fulfilled the International Headache Society (IHS) criteria for CH (37). The control group consisted of 108 Caucasian persons, 74 (68.5%) males and 34 (31.5%) females. The mean age of the controls was 48.9 years (range 25–72 years). The control group, drawn from a larger group of controls to match for age and gender, consisted of blood donors, healthy volunteers and spouses of patients.

The study was approved by the local ethics committee at Huddinge University Hospital.

DNA isolation and genotyping

DNA was extracted from peripheral blood using a modified salting out method (38). For this association study we chose two highly polymorphic intragenic markers, the (CA)n-repeat marker D19S1150 (forward 5′-GGAGAAGCATAGAAAAGCCA-3′ and reverse 5′-CCTGTTGAAAACTCCTGACC-3′) and the (CAG)n triplet repeat sequence in the 3-prime-UTR (exon 47) with forward primer sequence S-5-F1 (5′-CACGTGTCCTATTCCCCTGTGATCC-3′) and reversed primer sequence S-5-R1(5′-TGGGTACCTCCGAGGGCCGCTGGTG-3′) as described by Zhuchenko et al. (26). For marker D19S1150, PCR was performed in a final volume of 7 μl consisting of 50 mm KCl, 10 mm Tris-HCl pH 8.3, 0.2 mMdNTP, 20 ng genomic DNA, 0.2 U Taq polymerase and 0.2 mm of each primer. PCR conditions involved initial denaturation at 94 °C for 5 min followed by 28 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for 30 s, annealing for 28 cycles. A final extension step at 72 °C for 7 min concluded the PCR. For the CAG-repeat, PCR was performed in a final volume of 8 μl consisting of 50 mm KCl, 10 mm Tris HCl pH 8.3, 2.5 mm MgCl₂, 5% v/v dimethylsulphoxide, 0.2 mm dNTP, 20 ng genomic DNA, 0.24 U Taq polymerase and 0.2 mm of each primer. PCR conditions involved initial denaturation at 94 °C for 2 min followed by 32 cycles of 94 °C for 30 s, 68 °C for 30 s and 72 °C for 30 s, annealing for 32 cycles. A final extension step at 72 °C for 5 min concluded the PCR.

Electrophoresis of the amplified DNA fragments was performed on polyacrylamide gels on an ABI 377 DNA sequencer. Fragment sizes were determined by comparison with internal lane size standards. The results were analysed using the GENESCAN/GENOTYPER software (version 1.1; PE Biosystems).

Statistical analysis

Allele and phenotype frequencies for patients and controls were compared using Fisher's exact test. The level of significance was chosen as P < 0.05. The large number of comparisons made in the study should be considered in evaluating deviations observed. We also performed a Monte Carlo simulation test to assess the likelihood of our data as described by Sham & Curtis (39). Linkage disequilibrium (LD) was assessed by Fisher's exact test and by chi-square analysis between allele pairs of the two markers. The sample size allows 80% power (with a two-sided CI of 95%) to detect a RR of 2.5 of an allele with a frequency of 13% in controls.

Results

In our dataset there were 11 alleles of the dinucleotide repeat polymorphisms (D19S1150), ranging in size from 144 to 168 bp. Phenotype and allele frequencies (defined as carriership of the different alleles) of this marker are shown in Tables 1 and 2. The 158 bp allele was somewhat more frequent in CH patients and the 156 bp allele was somewhat more frequent in controls, but none of the comparisons were statistically significant.

TABLE 1

Phenotype frequencies of CACNA1A dinucleotide repeat (marker D19S1150) alleles in cluster headache patients and controls

Allele	Size in bp	Patients (n = 75)	Controls (n = 108)	P	OR (95% CI)
1	144	0	1	1.00	0.47
2	150	4	2	0.23	2.99
3	152	7	15	0.49	0.64
4	154	43	60	0.88	1.08
5	156	3	11	0.16	0.37 (0.914– 6.729)
6	158	11	7	0.08	2.48
7	160	21	37	0.42	0.75
8	162	27	40	1.00	0.96
9	164	2	5	0.70	0.57
10	166	10	9	0.33	1.69
11	168	1	1	1.00	1.45

TABLE 2

Allele frequencies of CACNA1A dinucleotide repeat (marker D19S1150) in cluster headache patients and controls

Allele	Size in bp	Patients (n = 150) ∗	%	Controls (n = 216) ∗	%	P	OR (95% CI)
1	144	0	0	1	0.5	0.23	2.93
2	150	4	2.7	2	0.9	0.52	0.70
3	152	8	5.3	16	7.4	1.00	0.48
4	154	54	36.0	73	33.8	0.74	1.10
5	156	3	2.0	13	6.0	0.073	0.319 (0.089–1.139)
6	158	13	8.7	8	3.7	0.065	2.47 (0.9961–6.111)
7	160	23	15.3	40	18.5	0.48	0.80
8	162	32	21.3	47	21.8	1.00	0.98
9	164	2	1.3	6	2.8	0.48	0.47
10	166	10	6.7	9	4.2	0.34	1.64
11	168	1	0.7	1	0.5	1.00	1.44

∗

Refers to numbers of alleles.

For the CAG-repeat in the 3′ coding region we detected alleles ranging in size from 112 to 142, with estimated numbers of repeats ranging from 4 to 14. The reported repeat lengths in control populations in other studies vary from 4 to 20 repeats (20, 26, 40, 41). We observed amplified fragments varying from 112 to 142 base pairs, corresponding to 4–14 repeats (Genebank accession number Z 80155). Phenotype and allele frequencies are shown in Tables 3 and 4. The 14 repeat allele was somewhat more frequent in the control group but differences were not significant.

TABLE 3

Phenotype frequencies of CACNA1A (SCA6) CAG-repeat alleles in exon 47 in cluster headache patients and controls

Allele size in bp	Repeats	Patients (n = 75)	Controls (n = 108)	P	OR (95% CI)
112	4	0	3	0.27	0.20
115	5	0	0
118	6	0	0
121	7	13	15	0.54	1.30
124	8	1	2	1.00	0.72
127	9	0	0
130	10	0	0
133	11	37	51	0.88	1.09
136	12	35	38	0.13	1.61
139	13	40	72	0.09	0.57 (0.3120–1.046)
142	14	3	9	0.36	0.46

TABLE 4

Allele frequencies of CACNA1A (SCA6) CAG-repeat in exon 47 in cluster headache patients and controls

Repeats	Patients (n = 150) ∗	%	Controls (n = 216) ∗	%	P	OR (95% CI)
4	0	0	3	1.4	0.27	0.20
7	13	8.7	16	7.4	0.70	1.19
8	1	0.7	2	0.9	1.00	0.72
11	46	30.7	60	27.8	0.56	1.15
12	38	25.3	41	19.0	0.16	1.45
13	49	32.7	85	39.4	0.23	0.75
14	3	2.0	9	4.2	0.37	0.47

∗

Refers to numbers of alleles.

The genotypes for the two polymorphisms in the chronic CH cases did not differ remarkably from the rest of the CH patients.

Chi-square comparisons of phenotype frequencies with Monte Carlo simulation confirmed the lack of significance (P = 0.398 for D19S1150 marker and P = 0.365 for the CAG-repeat).

We analysed the extent of LD between specific alleles of the two markers. The distribution of genotypes was similar in patients and controls, with a minor increase of the combined alleles, 162/139, in patients (23% vs. 13% in controls, OR = 1.8, P = 0.16, data not shown). The 154 allele occurred more often together with the 136 allele in both patients and controls (OR = 2.39, P < 0.01). This indicates a slight LD between the two markers that appeared to be similar in patients and controls.

Discussion

This study, to our knowledge the first of its kind in CH, offers no support for an association between CH and two polymorphic markers of the CACNA1A gene, the (CA)n repeat marker D19S1150 and the coding (CAG)n poly glu SCA6 expansion polymorphism. The observed slight differences in phenotype and allele frequencies fall well inside what would be expected by chance. Based on the power estimations performed, we conclude that it is unlikely that genetic variation within this gene contributes greatly to the susceptibility to CH.

CACNA1A gene mutations are described in a few neurological disorders, calcium acts as an important messenger in the regulation of many neuronal events and verapamil, an L-channel-blocker, is an effective prophylactic treatment in CH as mentioned in the introduction. Given these findings, the CACNA1A gene becomes a relevant candidate in conferring CH susceptibility, especially as it is preferentially expressed within the central nervous system. Therefore, even if no evidence for an association was found with CH, other subunits of the same or other calcium channels may be selected in subsequent studies.

It is likely that a number of individuals in the control group would suffer from common or classical migraine. As the CACNA1A gene could just as well be considered a candidate gene for migraine as for CH, the hypothetical presence of an association with this gene in migraine would somewhat diminish the statistical power of the present study. However, this negative effect is not likely to be large, and there is not yet enough data available to evaluate the importance of the CACNA1A gene in the more common forms of migraine.

We chose to study two highly polymorphic multi-allelic markers of the CACNA1A gene, assuming that either they should be of functional importance for CH themselves or that they should be in linkage disequilibrium (LD) with other hypothetical disease-causing mutations. Indeed, the significant LD observed between pairs of the more common alleles of the two markers suggests that LD is quite extensive within this gene, at least between relatively recent polymorphisms. On the other hand, functionally important single nucleotide polymorphisms could be of an older date, which would have allowed LD to weaken. In that situation, only an analysis of the polymorphisms themselves would give the final answer to whether an association exists. So far, no such polymorphisms have been reported within this gene and we did not consider the effort of sequencing long sequences of this gene in large numbers of individuals as relevant at this stage. Thus, lacking other direct indications of an importance of this gene in CH, we consider the present data sufficient for the time being. Clearly a minor contribution to sporadic CH or a major contribution in a subgroup, for example familial cases of CH, cannot be ruled out. The 19p13 chromosomal region remains of great interest in the genetics of neurological disorders.

Footnotes

Acknowledgements

This study was supported by grants from the Karolinska Institute, Astra Zeneca and Glaxo Wellcome.

References

1 Fanciullacci

Allesandro

Figini

Gepetti

Michelacci

. Increase in plasma calcitonin gene-related peptide from extracerebral circulation during nitroglycerin-induced cluster headache attacks. Pain 1995; 60:119–23.

2 Goadsby

Edvinsson

. Human in vivo evidence for trigeminovascular activation in cluster headache. Neuropeptide changes and effects of acute attacks therapies. Brain 1994; 117(3):427–34.

3 May

Bahra

Buchel

Frackowiak

Goadsby

. Hypothalamic activation in cluster headache attacks. Lancet 1998; 352(9124):275–8.

4 Russell

Olesen

. Genetic epidemiologic studies in migraine, cluster headache and other disorders. In: Olesen

Bousser

M-G

, eds. Genetics of Headache Disorders. Philadelphia: Lippincott, Williams & Wilkins, 2000:13–8.

5 Russell

. Genetics of cluster headache. In: Olesen

Tfelt-Hansen

Welch

KMA

, eds. The Headaches 2nd edn. Philadelphia: Lippincott, Williams & Wilkins, 2000:679–82.

6 D'Amico

Leone

Moschiano

Bussone

. Familial cluster headache: report of three families. Headache 1996; 36(1):41–3.

7 Kudrow

Kudrow

. Inheritance of cluster headache and its possible link to migraine. Headache 1994; 34(7):400–7.

8 Montagna

Mochi

Prologo

Sangiorgi

Pierangeli

Cevoli

et al.Heritability of cluster headache. European J Neurology 1998; 5:343–5.

9 Russell

Andersson

Iselius

. Cluster headache is an inherited disorder in some families. Headache 1996; 36(10):608–12.

10.

10 Russell

Andersson

Thomsen

. Familial occurrence of cluster headache. J Neurol Neurosurg Psychiatry 1995; 58(3):341–3.

11.

11 Russell

Andersson

Thomsen

Iselius

. Cluster headache is an autosomal dominantly inherited disorder in some families: a complex segregation analysis. J Med Genet 1995; 32(12):954–6.

12.

12 Russell

. Genetic epidemiology of migraine and cluster headache. Cephalalgia 1997; 17(6):683–701.

13.

13 Spierings

Vincent

. Familial cluster headache: occurrence in three generations. Neurology 1992; 42(7):1399–400.

14.

14 Leone

Russell

Rigamonti

Attanasio

Grazzi

D'Amico

Usai

Bussone

. Increased familial risk of cluster headache. Neurology 2001; 56(9):1233–6.

15.

15 Eadie

Sutherland

. Migrainous neuralgia. Med J Australia 1996; 1:1053–7.

16.

16 Couturier

Hering

Steiner

. The first report of cluster headache in identical twins [see comments]. Neurology 1991; 41(5):761.

17.

17 Roberge

Bouchard

Simard

Gagne

. Cluster headache in twins [letter; comment]. Neurology 1992; 42(6):1255–6.

18.

18 Sjaastad

Shen

Stovner

Elsas

. Cluster headache in identical twins. Headache 1993; 33(4):214–7.

19.

19 Ophoff

Terwindt

Vergouwe

Frants

Ferrari

. Familial hemiplegic migraine: involvement of a calcium neuronal channel. Neurologia 1997; 12 (Suppl. 5):31–7.

20.

20 Ophoff

Terwindt

Vergouwe

van Eijk

Oefner

Hoffman

et al.Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+ channel gene CACNL1A4. Cell 1996; 87(3):543–52.

21.

21 Ophoff

Terwindt

Vergouwe

Frants

Ferrari

. Wolff Award 1997. Involvement of a Ca2+ channel gene in familial hemiplegic migraine and migraine with and without aura. Dutch Migraine Genetics Research Group. Headache 1997; 37(8):479–85.

22.

22 Ophoff

Terwindt

Vergouwe

. Involvement of a Ca2+ channel gene in familial hemiplegic migraine with and without aura. Headache 1997; 37:479–85.

23.

23 Terwindt

Ophoff

Haan

Sandkuijl

Frants

Ferrari

. Migraine, ataxia and epilepsy: a challenging spectrum of genetically determined calcium channelopathies. Dutch Migraine Genetics Research Group. Eur J Hum Genet 1998; 6(4):297–307.

24.

24 Terwindt

Ophoff

Haan

Vergouwe

van Eijk

Frants

et al.Variable clinical expression of mutations in the P/Q-type calcium channel gene in familial hemiplegic migraine. Dutch Migraine Genetics Research Group. Neurology 1998; 50(4):1105–10.

25.

25 Jodice

Mantuano

Veneziano

Trettel

Sabbadini

Calandriello

et al.Episodic ataxia type 2 (EA2) and spinocerebellar ataxia type 6 (SCA6) due to CAG repeat expansion in the CACNA1A gene on chromosome 19p. Hum Mol Genet 1997; 6(11):1973–8.

26.

26 Zhuchenko

Bailey

Bonnen

Ashizawa

Stockston

Amos

et al.Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the alpha 1A-voltage-dependent calcium channel. Nat Genet 1997; 15(1):62–9.

27.

27 May

Ophoff

Terwindt

Urban

van Eijk

Haan

et al.Familial hemiplegic migraine locus on 19p13 is involved in the common forms of migraine with and without aura. Hum Genet 1995; 96(5):604–8.

28.

28 Terwindt

Ophoff

Sandkuijl

. Involvement of the familial hemiplegic migraine gene on 19p13 in migraine with and without aura. Cephalalgia 1997; 17:232.

29.

29 Nyholt

Lea

Goadsby

Brimage

Griffiths

. Familial typical migraine: linkage to chromosome 19p13 and evidence for genetic heterogeneity. Neurology 1998; 50(5):1428–32.

30.

30 Terwindt

Ophoff

van Eijk

Vergouwe

Haan

Frants

et al.Involvement of the CACNA1A gene containing region on 19p13 in migraine with and without aura. Neurology 2001; (56):1028–32.

31.

31 Dunlap

Luebke

Turner

. Exocytotic Ca2+ channels in mammalian central neurons. Trends Neurosci 1995; 18(2):89–98.

32.

32 Ophoff

Terwindt

Ferrari

Frants

. Genetics and pathology of voltage-gated Ca2+ channels. Histol Histopathol 1998; 13(3):827–36.

33.

33 Stea

Soong

Snutch

. Voltage gated calcium channels. In: North

, ed. Handbook of Receptors and Channels. Ligand and Voltage Gated ion Channels. Boca Raton, FL: CRC Press, 1995:113–53.

34.

34 Codignola

Tarroni

Clementi

Pollo

Lovallo

, Carbone. Calcium channel subtypes controlling serotonine release from human small cell lung carcinoma cell lines. J Biol Chem 1993; 268(35):26240–7.

35.

35 Frittoli

Gobbi

Mennini

. Involvement of P-type Ca2+ channels in the K (+)-d-fenfluramine induced [3H]5-HT release from rat hippocampal synaptosome. Neuropharmacology 1994; 33(6):833–5.

36.

36 Olesen

. Future treatment for cluster headache. In: Olesen

Goadsby

, eds. Cluster Headache and Related Conditions. Oxford: Oxford Univerity Press, 1999:271–2.

37.

37 International Headache Society. Classification and diagnostic criteria for headache disorders, cranial neuralgias and facial pain. Cephalalgia 1988; 8 (Suppl. 7):1–96.

38.

38 Miller

Dykes

Polesky

. A simple salting out procedure for extracting DNA from human nucleated cells. Nucl Acids Res 1988; 16(3):1215.

39.

39 Sham

Curtis

. Monte Carlo tests for associations between disease and alleles at highly polymorphic loci. Ann Hum Genet 1995; 59(1):97–105.

40.

40 Matsuyama

Kawakami

Maruyama

Izumi

Komure

Udaka

et al.Molecular features of the CAG repeats of spinocerebellar ataxia 6 (SCA6). Hum Mol Genet 1997; 6(8):1283–7.

41.

41 Ishikawa

Tanaka

Saito

Ohkoshi

Fujita

Yoshizawa

et al.Japanese families with autosomal dominant pure cerebellar ataxia map to chromosome 19p13.1-p13.2 and are strongly associated with mild CAG expansions in the spinocerebellar ataxia type 6 gene in chromosome 19p13.1. Am J Hum Genet 1997; 61(2):336–46.

CACNA1A Gene Polymorphisms in Cluster Headache

Abstract

Keywords

Introduction

Materials and methods

Patients and control samples

DNA isolation and genotyping

Statistical analysis

Results

Discussion

Footnotes

Acknowledgements

References