Valutazione dell’espressione del gene TERT e della lunghezza dei telomeri dei leucociti in pazienti affetti da cefalea a grappolo

Introduction Cluster headache (CH) is a rare form of primary headache, characterized by severe unilateral orbital pain attacks lasting 15 - 180 minutes, associated with ipsilateral autonomic trigeminal symptoms. Although several hypotheses on the underlying neurobiological underpinnings of CH have been proposed, there is still uncertainty on its specific pathophysiology. Recently, a gene expression study suggested a potential role of the telomerase complex, an enzyme that counteracts the shortening of telomeres, proteins complexes capping the chromosome ends, which ensure genome stability and integrity and allow the complete replication of the double helix. Indeed, leukocyte telomeres length (LTL) may represent a molecular signature of the biological impact of stress and pain. Further, an accelerated LTL shortening of leukocyte telomeres has been observed in migraine, another form of primary headache. Lithium, a mood stabilizer, is one of the prophylactic therapies indicated for the treatment of CH. The differential effect of lithium on the modulation of telomerase reverse transcriptase (TERT) gene expression, which encodes the catalytic subunit of the telomerase enzyme, has been shown on human cell lines and on rats. In addition, lithium has been shown to counteract LTL shortening, both in animal models (rats) and in patients suffering from bipolar disorder. Objectives 1) To evaluate whether there are differences in TERT gene expression levels in lymphoblasts derived from CG patients compared to healthy controls; 2) Evaluate the effect of lithium on TERT gene expression levels in the same sample of CG patients and in a sample of healthy controls; 3) Evaluate, in a larger sample, whether there are differences in LTL between CG patients and healthy controls. Materials and methods Evaluation of TERT gene expression levels was performed on the dataset obtained from our previous transcriptomics study (Squassina et al., 2013; Costa et al., 2015). The sample included 8 patients with episodic CG and 10 healthy controls. Genome-wide expression levels were obtained by GeneChip Human Gene ST 1.0 microarray (Affymetrix, USA) on RNA extracted from lymphoblastoid cells. For each subject, the lymphoblastoid culture was divided into two aliquots, one of which was treated with 1 mM LiCl for 7 days, while the other was kept in culture for 7 days in non-LiCl. LTL was evaluated on an extended sample of 51 patients with CG and 100 healthy controls. The qualitative and quantitative evaluation of the extracted DNA was carried out by spectrophotometric analysis. The evaluation of LTL was performed on genomic DNA using the Real Time Quantitative PCR (QPCR) technique, developed by Cawthon (2002), whose protocol has been modified in our laboratory. Results The study showed a reduced expression of the TERT gene in patients with CH compared to healthy controls [fold change (FC) = 0.82, p = 0.006]. In addition, in vitro lithium treatment was found to increase TERT gene expression levels in patients with CH (FC = 1.12, p = 0.042) but not in controls (FC = 0.93, p = 0.11). The study did not show significant differences in LTL between the two groups in the extended sample. Conclusions The results of the TERT gene expression study support the hypothesis of a potential involvement of the telomerase complex in the pathogenesis of CH, and confirm the previous evidence of a differential effect of lithium on the modulation of the expression of this gene. However, the results of the LTL study suggest the absence of significant differences in LTL between CH patients and controls. Further studies are needed to better understand the role of alterations in telomere dynamics in the pathogenesis of CH and in the mechanism of action of lithium.