Investigation of the T3111C CLOCK gene polymorphism in cluster headache

Abstract

Sirs: Cluster headache (CH) is a primary headache characterized by severe unilateral pain, ipsilateral autonomic symptoms and, in many cases, restlessness. The etiology of CH is still unclear, although clinical, endocrine and neuroimaging studies implicate the hypothalamus. Hypothalamic involvement is also supported by the efficacy of stereotactic deep brain stimulation of the posterior hypothalamic area in chronic drug resistant CH [1]. The increased familial risk of CH suggests a genetic liability with inheritance likely to be autosomal dominant with low penetrance, although autosomal recessive or multifactorial inheritance have also been suggested in some families [2]. A polymorphism of the hypocretin receptor 2 gene has recently been associated with CH [3], a remarkable association since hypocretin-containing cells are located exclusively in the posterolateral hypothalamus. A striking feature of CH is its diurnal and seasonal periodicity, suggesting that circadian and infradian rhythms regulate CH attacks. Polymorphisms in the CLOCK gene, a highly conserved circadian gene, influence the circadian phase in humans [4] and circadian mood fluctuations in bipolar patients [5]. We performed a genetic association study to evaluate whether the T3111C CLOCK polymorphism (also known as T3092C) might be related to CH susceptibility under the assumption of autosomal dominant inheritance. A total of 101 consecutive, unrelated Italian patients (79 men and 22 women) with CH (85 episodic and 16 chronic CH) gave informed consent to the study. All patients were diagnosed after direct interview by a neurologist expert in headache disorders and according to ICHD-II criteria [6]. Of the 101 patients, 87 described a clear circadian rhythmicity of the attacks. 100 healthy individuals, in whom CH and migraine were excluded by direct interview, served as controls. The control sample came from the same geographical region as the CH patients. We analyzed a single nucleotide polymorphism, T3111C, in the 3’ flanking region of the CLOCK gene [4]. DNA was extracted from whole blood by standard methods [7]. PCR primers and methods were those reported by Katzenberg et al. [4]. The T3111C polymorphism was detected by enzymatic digestion with Sdu I (Fermentas) and electrophoresed on MetaPhor agarose 2 % gel. The allelic and genotypic frequencies were compared by means of the Fisher’s exact test. The Hardy-Weinberg equilibrium was verified for all tested populations, and alleles were in Hardy-Weinberg equilibrium in both controls and patients. We found no significant difference in allelic and genotypic frequencies between the total CH patient group and the control subjects (χ2 = 1.261, p = 0.261; χ2 = 5.791, p = 0.055) (Table 1). Nor was a significant difference found when considering the 87 CH patients with circadian rhythmicity of the attacks alone. Our study does not support the hypothesis that the T3111C CLOCK polymorphism is associated with CH. It therefore strengthens the similar negative results reported previously by Rianero et al. [8]. Our analysis moreover resulted in nonsignificant differences when the 87 CH patients reporting a clear periodicity of the attacks were considered separately. It thus appears that CLOCK gene T3111C polymorphism does not influence CH, but our result of course cannot exclude that polymorphic variations at other biological rhythm genes LETTER TO THE EDITORS