Investigating the association of toll-like receptor 9 rs5743836, rs352140, and rs187084 gene polymorphisms and their mRNA levels with different hepatic fibrosis stages of non-alcoholic fatty liver patients compared to healthy controls

Document Type : Original article

Authors

1 Gastroenterology and Liver Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

2 Department of Genetics, Damghan Branch, Islamic Azad University, Damghan, Iran.

3 School of Biology, Damghan University, Damghan, Iran

Abstract

Recent studies have shown that the level of hepatocyte-derived mitochondrial DNA is elevated in plasma samples obtained from mice and NASH patients, and it has the ability to toll-like receptor 9 (TLR9) activation resulting in steatosis, hepatocyte injury, and fibrosis. In this study, we explored the association between TLR9 rs5743836, rs352140, and rs187084 polymorphism and its plasma mRNA level in non-alcoholic fatty liver (NAFL) patients with different liver fibrosis scores compared to healthy controls. Seventy Iranian patients diagnosed with NAFL, based on fibroscan testing results, were divided into F0-F1 (N=33), F2-F3 (N=19), and F4 (N=18) hepatic fibrosis groups and compared to 22 healthy controls. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and the mRNA expression level of TLR9 was determined using Real-Time PCR analysis. Results showed no significant association between allelic and genotypic distribution frequency of TLR9 rs5743836, rs352140, and rs187084 polymorphisms in NAFL patients with hepatic fibrosis compared to healthy controls (P>0.05). However, the mRNA level of TLR9 was significantly elevated in correlation with hepatic fibrosis progression in NAFL patients compared to healthy controls (P<0.05). As a preliminary study, our data showed a correlative overexpression of TLR9 mRNA with hepatic fibrosis progression in NAFL patients without the effectiveness of TLR9 gene polymorphisms.

Keywords


  1. Chalasani N, Younossi Z, Lavine JE, Charlton M, Cusi K, Rinella M, Harrison SA, Brunt EM, Sanyal AJ. The diagnosis and management of nonalcoholic fatty liver disease: Practice guidance from the American Association for the Study of Liver Diseases. Hepatology 2018;67:328-357.
  2. Moghaddasifar I, Lankarani KB, Moosazadeh M, Afshari M, Ghaemi A, Aliramezany M, Afsar Gharebagh R, Malary M. Prevalence of non-alcoholic fatty liver disease and its related factors in Iran. Int J Organ Transplant Med 2016;7:149-160.
  3. Hardy T, Oakley F, Anstee QM, Day CP. Nonalcoholic fatty liver disease: Pathogenesis and disease spectrum. Annu Rev Pathol 2016;11:451-496.
  4. Nasiri M, Karimi MH, Azarpira N, Saadat I. Gene expression profile of toll-like receptor/adaptor/interferon regulatory factor/cytokine axis during liver regeneration after partial ischemia-reperfusion injury. Exp Clin Transplant 2020;18:215-223.
  5. Nasiri M, Saadat M, Karimi MH, Azarpira N, Saadat I. Evaluating mRNA expression levels of the TLR4/IRF5 signaling axis during hepatic ischemia-reperfusion injuries. Exp Clin Transplant 2019;17:648-652.
  6. Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, Olefsky JM, Brenner DA, Seki E. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology 2010;139:323-334.
  7. Beutler BA. TLRs and innate immunity. Blood 2009;113:1399-1407.
  8. Lazarus R, Klimecki WT, Raby BA, Vercelli D, Palmer LJ, Kwiatkowski DJ, Silverman EK, Martinez F, Weiss ST. Single-nucleotide polymorphisms in the Toll-like receptor 9 gene (TLR9): frequencies, pairwise linkage disequilibrium, and haplotypes in three U.S. ethnic groups and exploratory case-control disease association studies. Genomics 2003;81:85-91.
  9. Zayed RA, Omran D, Mokhtar DA, Zakaria Z, Ezzat S, Soliman MA, Mobarak L, El-Sweesy H, Emam G. Association of toll-like receptor 3 and toll-like receptor 9 single nucleotide polymorphisms with hepatitis C virus infection and hepatic fibrosis in Egyptian patients. Am J Trop Med Hyg 2017;96:720-726.
  10. Shastry BS. SNPs: impact on gene function and phenotype. Methods Mol Biol 2009;578:3-22.
  11. Castera L, Vergniol J, Foucher J, Le Bail B, Chanteloup E, Haaser M, Darriet M, Couzigou P, De Ledinghen V. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128:343-350.
  12. Junjie X, Songyao J, Minmin S, Yanyan S, Baiyong S, Xiaxing D, Jiabin J, Xi Z, Hao C. The association between toll-like receptor 2 single-nucleotide polymorphisms and hepatocellular carcinoma susceptibility. BMC Cancer 2012;12:57-67.
  13. Kikuchi K, Lian ZX, Kimura Y, Selmi C, Yang GX, Gordon SC, Invernizzi P, Podda M, Coppel RL, Ansari AA, Ikehara S, Miyakawa H, Gershwin ME. Genetic polymorphisms of toll-like receptor 9 influence the immune response to CpG and contribute to hyper-IgM in primary biliary cirrhosis. J Autoimmun 2005;24:347-352.
  14. Youssef SS, Hamdy NM. SOCS1 and pattern recognition receptors: TLR9 and RIG-I; novel haplotype associations in Egyptian fibrotic/cirrhotic patients with HCV genotype 4. Arch Virol 2017;162:3347-3354.

  15. Fischer J, Weber ANR, Böhm S, Dickhöfer S, El Maadidi S, Deichsel D, Knop V, Klinker H, Möller B, Rasenack J, Wang L, Sharma M, Hinrichsen H, Spengler U, Buggisch P, Sarrazin C, Pawlita M, Waterboer T, Wiese M, Probst-Müller E, Malinverni R, Bochud PY, Gardiner C, O'Farrelly C, Berg T. Sex-specific effects of TLR9 promoter variants on spontaneous clearance of HCV infection. Gut 2017;66:1829-1837.
  16. Watanabe A, Hashmi A, Gomes DA, Town T, Badou A, Flavell RA, Mehal WZ. Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9. Hepatology 2007;46:1509-1518.
  17. Garcia-Martinez I, Santoro N, Chen Y, Hoque R, Ouyang X, Caprio S, Shlomchik MJ, Coffman RL, Candia A, Mehal WZ. Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9. J Clin Invest 2016;126:859-864.
  18. Caldwell SH, Lee VD, Kleiner DE, Al-Osaimi AM, Argo CK, Northup PG, Berg CL. NASH and cryptogenic cirrhosis: a histological analysis. Ann Hepatol 2009;8:346-352.
  19. Takahashi Y, Fukusato T. Histopathology of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol 2014;20:15539-15548.