Gene of gamma-glutamylcyclotransferase, a key enzyme of glutathione catabolism, and predisposition to ischemic stroke: association analysis and functional annotation of gene polymorphisms

Genetic epidemiological studies have established that polymorphisms of the genes encoding antioxidant defense enzymes represent meaningful predictors for the risk and severity of ischemic stroke (IS). The aim of this study was to investigate associations of IS with three common single nucleotide polymorphisms (SNPs) such as rs38420, rs4270 and rs6462210 in gamma-glutamylcyclotransferase (GGCT) gene, a key enzyme of glutathione catabolism. DNA samples obtained from 1288 unrelated individuals of Slavic origin, including 600 patients with IS and 688 healthy volunteers were included in the study. Genotyping of the GGCT polymorphisms was done using an iPLEX-based technology by the MassARRAY-4 system. Functional annotation of SNPs was performed using numerous online bioinformatics tools and resources. We found an association between genotype T/T rs6462210 and a decreased risk of ischemic stroke (OR = 0.36 95% CI 0.15-0.85, p=0.01). The haplotype rs38420A-rs4270T-rs6462210C showed a clear tendency in association with decreased disease risk (p=0.057). Bioinformatics analysis showed that the phenotypic effects of the SNPs are characterized by a weak transcriptional activity of the GGCT gene mainly in blood cells and arteries as a result of chemical modifications of chromatin. For the rs4270 polymorphism, which is in close linkage disequilibrium with SNP rs6462210 (D’=0.966, p<0.01), we found a binding site for miRNA hsa-miR-1246 which is capable to block the GGCT expression, thereby reducing the formation of L-cysteine, a precursor of glutathione. The study showed for the first time that GGCT gene may contribute to the development of ischemic stroke. Experimental studies focusing on the regulatory potential of GGCT gene polymorphisms are required for the pathophysiological interpretation of the identified associations.

ischemic stroke, glutathione metabolism, gamma-glutamylcyclotransferase (GGCT), single nucleotide polymorphism (SNP), gene expression, histone modifications, SNP annotation

1. Feigin V.L., Lawes C.M., Bennett D.A., Anderson C.S. Stroke epidemiology: a review of population-based studies of incidence, prevalence, and case-fatality in the late 20th century. Lancet Neurol. 2003;2(1):43-53.

2. Hassan A., Markus H.S. Genetics and ischaemic stroke. Brain. 2000;123 (Pt 9):1784-1812.

3. Dichgans M. Genetics of ischaemic stroke. Lancet Neurol. 2007;6(2):149-161.

4. Allen C.L., Bayraktutan U. Oxidative stress and its role in the pathogenesis of ischaemic stroke. Int J Stroke. 2009;4(6):461-470.

5. Ciancarelli I., Di Massimo C., De Amicis D., Carolei A., Tozzi Ciancarelli M.G. Evidence of redox unbalance in post-acute ischemic stroke patients. Curr Neurovasc Res. 2012;9(2):85-90.

6. Chehaibi K., Trabelsi I., Mahdouani K., Slimane M.N. Correlation of Oxidative Stress Parameters and Inflammatory Markers in Ischemic Stroke Patients. J Stroke Cerebrovasc Dis. 2016;25(11):2585-2593.

7. Kölsch H., Linnebank M., Lütjohann D., Jessen F., Wüllner U., Harbrecht U., Thelen K.M., Kreis M., Hentschel F., Schulz A., von Bergmann K, Maier W., Heun R. Polymorphisms in glutathione S-transferase omega-1 and AD, vascular dementia, and stroke. Neurology. 2004;63(12):2255-2260.

8. Türkanoğlu A., Can Demirdöğen B., Demirkaya S., Bek S., Adali O. Association analysis of GSTT1, GSTM1 genotype polymorphisms and serum total GST activity with ischemic stroke risk. Neurol Sci. 2010;31(6):727-34.

9. Bilgin E., Can Demirdöğen B., Türkanoğlu Özçelik A., Demirkaya Ş, Adalı O. Association analysis of Glutathione S-transferase omega-1 and omega-2 genetic polymorphisms and ischemic stroke risk in a Turkish population. Neurol Res. 2019;41(2):118-124.

10. Voetsch B., Jin R.C., Bierl C., Benke K.S., Kenet G., Simioni P., Ottaviano F., Damasceno B.P., Annichino-Bizacchi J.M., Handy D.E., Loscalzo J. Promoter polymorphisms in the plasma glutathione peroxidase (GPx-3) gene: a novel risk factor for arterial ischemic stroke among young adults and children. Stroke. 2007;38(1):41-49.

11. Polonikov A., Vialykh E., Vasil’eva O., Bulgakova I., Bushueva O., Illig T., Solodilova M. Genetic variation in glutathione S-transferase genes and risk of nonfatal cerebral stroke in patients suffering from essential hypertension. J Mol Neurosci. 2012;47(3):511-513.

12. Lee B.J., Marchionni L., Andrews C.E., Norris A.L., Nucifora L.G., Wu Y.C., Wright R.A., Pevsner J., Ross C.A., Margolis R.L., Sawa A., Nucifora F.C. Jr. Analysis of differential gene expression mediated by clozapine in human postmortem brains. Schizophr Res. 2017;185:58-66.

13. Пономаренко И.В. Отбор полиморфных локусов для анализа ассоциаций при генетико-эпидемиологических исследованиях. Научный результат. Медицина и фармация. 2018;(4):40-54.

14. Solé X., Guinó E., Valls J., Iniesta R., Moreno V. SNPStats: a web tool for the analysis of association studies. Bioinformatics. 2006;22(15):1928-1929.

15. Callegari A., Liu Y., White C.C., Chait A., Gough P., Raines E.W., Cox D., Kavanagh T.J., Rosenfeld M.E. Gain and loss of function for glutathione synthesis: impact on advanced atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2011;31(11):2473-2482.

16. Shimizu H., Kiyohara Y., Kato I., Kitazono T., Tanizaki Y., Kubo M., Ueno H., Ibayashi S., Fujishima M., Iida M. Relationship between plasma glutathione levels and cardiovascular disease in a defined population: the Hisayama study. Stroke. 2004;35(9):2072-2077.

17. Zhou Y., Danbolt N.C. Glutamate as a neurotransmitter in the healthy brain. J Neural Transm (Vienna). 2014;121(8):799-817.

18. Li P., Teng F., Gao F., Zhang M., Wu J., Zhang C. Identification of circulating microRNAs as potential biomarkers for detecting acute ischemic stroke. Cell Mol Neurobiol. 2015;35(3):433-447.

19. Wu Y., Zhang R., Zhou C., Xu Y., Guan X., Hu J., Xu Y., Li S. Enhanced expression of vascular cell adhesion molecule-1 by corticotrophin-releasing hormone contributes to progression of atherosclerosis in LDL receptor-deficient mice. Atherosclerosis. 2009;203(2):360-370.