Association of TM6SF2 rs58542926 with Hepatic and Extrahepatic Alterations in Chronic Hepatitis C Patients


 Human genetic variants play major roles in predicting and prognosis of several diseases. The effect of rs58542926 variant in transmembrane 6 superfamily member 2 (TM6SF2) gene on liver fibrosis among patients with chronic hepatitis C (CHC) is still debatable. The aim of this study is to investigate the possible effects of this variant in CHC patients. The study comprised 351 subjects: 250 CHC patients with different fibrosis stages (F0-F4) and 101 healthy volunteers. TM6SF2 (rs58542926) genotype was determined for all subjects. Blood samples were collected for complete blood count and biochemical analysis and cohort subjects were genotyped for the variant TM6SF2 rs58542926. Fibrosis staging was performed using Fibrotest and Fibroscan standard tests. The presence of the minor allele was significantly associated with severe liver fibrosis, as well as thrombocytopenia as an extrahepatic alteration. In addition, there was a significant association between the minor allele and lower thrombopoietin levels. The association of TM6SF2 genotype with thrombocytopenia was explored by measuring plasma thrombopoietin (TPO) levels for CHC patients. The results showed an association with extrahepatic alteration (thrombocytopenia) through its effect on plasma TPO level, and consequently on platelets production, which raises questions about the role of this variant in HCV treatment outcome. In conclusion, the occurrence of the minor allele of the variant rs58542926 can be linked to severe fibrosis stages as well as thrombocytopenia, enabling this variant to be used as a diagnostic pharmacogenetic marker for predicting the risk of fibrosis onset in CHC patients.


Introduction
Hepatitis C virus is a pervasive virus affecting millions around the world, where Egypt shows one of the highest prevalence ratios of about 14% and about 40,000 deaths per year. Besides being a worldwide health hazard, the burden of the disease is still far from being controlled. A large percentage of the patients (70-80%) fails to get rid of the virus spontaneously during the acute phase and develop chronic infections 1

. Chronic Hepatitis C (CHC) viral infection is accompanied by both hepatic-and extrahepatic
complications so that mortality rates doubles in HCV carriers, as compared to healthy individuals 2 . Liver cirrhosis and hepatocellular carcinoma are the worst-expected hepatic complications of CHC infections.
However, the highest mortality rates from HCV infections has been correlated to progressive hepatic brosis 3 . Fibrosis is a structural damage to the liver tissue that ranges from mild changes limited to the portal and peripheral areas, to more severe changes that end up with cirrhosis in a time frame of approximately 20 years. Fibrosis is believed to be a result of many etiologies other than CHC, including obesity, alcohol intake and chronic viral infections. These multiple etiological factors suggest that genetic factors may also plays a pivotal role in the pathogenesis of chronic liver disease 4 .
Even with recent advances in the eradication of HCV using direct-acting antivirals (DAAs), a limited population of patients with genetic variances seems to be resistant to the treatment, or more susceptible to liver brosis secondary to CHC infection 5,6 . The transmembrane 6 superfamily 2 (TM6SF2) variant is located to locus 19p13.3-p12, and was identi ed by an exome-wide association study 7 . The TM6SF2 rs58542926 non-synonymous polymorphism was associated with non-alcoholic fatty liver disease (NAFLD) 7,8 , higher prevalence of brosis and alcohol-related cirrhosis 9 , triglyceride secretion 10 and myocardial infarction 11 . This variant was also shown to result in fat accumulation in the liver and reduce the very low density lipoprotein (vLDL) secretion in vitro 7 . Therefore, the association between the singlenucleotide polymorphism (SNP) rs58542926 in the TM6SF2 gene was hypothesized to play a role in liver brosis in CHC patients. At the beginning, the role of rs58542926 variant was debatable in case of CHC, until an Australian large scale functional study showed a signi cant correlation of this variant with liver brosis in CHC 12 . This study was followed by a meta-analysis which supported the role of this variant in histological changes in CHC infection, where it showed a robust association between the TM6SF2 variant and brosis development 13 . This study, therefore, aims at investigating the role of rs58542926 variant and its association with both hepatic and extrahepatic alteration in Egyptians CHC patients.

Patient cohort
For gaining representative results, the study included 351 subjects (250 with CHC, and 101 healthy control subjects) from Assiut university hospital, Assiut, Egypt. The inclusion criteria taken in consideration for selecting CHC subjects included CHC patients who had a broscan and brotest ® with scoring for brosis stage before initiating the antiviral treatment. Patients who had evidence of other liver diseases by standard tests were excluded. An informed consent was obtained from all patients who agrees to be included in this study. The healthy control group included individuals with no history of chronic liver diseases. All experiments were performed in compliance with relevant laws and institutional guidelines and in accordance with the ethical standards of the Declaration of Helsinki. Ethical approval was obtained from the research ethics committee in the Faculty of Pharmacy, Minia University (No. HV01/2020) before the commencement of this work. Each subject included in the study signed a written informed consent, and was informed regarding the nature of the disease and the diagnostic procedures involved Clinical and laboratory assessment Samples and data were collected at the time of liver brosis staging assessment. viral load, serum bilirubin, serum albumin, Aspartate transaminase (AST), alanine transaminase (ALT), international normalized ratio (INR) and platelets count were evaluated using commercially available kits.

Staging of liver brosis
Both FibroScan ® , a non-invasive test for liver brosis assessment, and FibroTest ® , a highly sensitive serum biomarker test for evaluating liver brosis, were used for staging brosis to avoid the use of invasive techniques like taking biopsies 11 . Fibrosis was staged according to Metavir scoring system. Fibrosis was scored on a 5-point scale: stage zero (F0): no brosis; stage one (F1): portal brosis alone; stage two (F2): portal brosis with rare septa; stage three (F3): portal brosis with many septa; and stage four (F4): cirrhosis. A total of 73 patients out of 250 patients were categorized as F0-F1, 50 patients were categorized as F2, 26 patients as F3, while the remaining 91 patients were categorized as F4.

Genotyping
Genotyping for TM6SF2 rs58542926 (n=351) was performed using the TaqMan SNP genotyping allelic discrimination method according to manufacturer's instructions (Applied Biosystems, Foster City, CA). All genotyping was blinded to clinical variables.

Determination of TPO level
Thrombopoietin level was measured by a solid phase sandwich enzyme-linked immunosorbent assays (ELISA) (Invitrogen, USA) in plasma of CHC patients with known TM6SF2 rs58542926 genotype.

Statistical analysis
Statistical analyses of the data were carried out using GraphPad Prism version 8.0 (Graph pad software San Diego, USA). Difference in the median values were measured using the two-tailed Mann-Whitney ttest. P values less than 0.05 were considered statistically signi cant.

Results
Association between the rs58542926 genotype and clinical, viral, and metabolic characteristics in CHC.
Comparing the frequency of the minor allele frequency (MAF) of TM6SF2 rs58542926 in healthy group and CHC subjects showed almost similar values in both groups, where the minor allele represented about 10% in healthy group and 9% in CHC group (MAF = 0.06) (P = 0.7 for trend) ( Table 1). When the distribution of the major and minor alleles in males and females of each group was compared, it was found that the percent of females carrying the minor alleles was higher, however, the difference was statistically non-signi cant (p = 0.893). Patients with the TM6SF2 rs58542926 (TT/CT) genotype had no signi cant difference in HCV viral load compared to subjects with CC genotype, despite the lower mean value for viral load in the former group. When comparing CT/TT versus CC regarding the metabolic pro le, subjects with minor allele had signi cantly higher serum bilirubin, higher INR ratios and lower platelet count. It is to be noted that no signi cant differences were observed between the minor allele group (CT/TT) and the major allele one regarding the mean values of ALT, AST, serum albumin and BMI, as seen in table 2.

Association between TM6SF2 variant and plasma thrombopoietin level in CHC patients
After observing the signi cant association of the minor allele with thrombocytopenia, it was interesting to explore whether this low platelet count can be regarded to a decrease in thrombopoietin levels, the primary regulator of platelet production. Measuring plasma thrombopoietin level for 31 CHC patients showed a signi cant association between rs58542926 minor allele and low plasma thrombopoietin level as shown in Fig. 1 (p < 0.05).

Association between TM6SF2 variant and brosis stage in CHC patients
After evaluation of the brosis stage with Fibrotest and Fibroscan, we next analyzed the association between TM6SF2 rs58542926 genotype and liver brosis stage. Interestingly, The rs58542926 minor allele showed a signi cant association with severe brosis stages (70% of subjects had staging of F3 or more) compared to the major allele where 65% of subjects showed brosis stages of F0-F2 at most. (P = 0.005) as shown in Fig. 2.

Discussion
The current study explored the effect of the TM6SF2 E167K variant on liver brosis severity in Egyptian patients with CHC. Based on our ndings, it is evident that the TM6SF2 E167K variant favors the development of both hepatic and extrahepatic alterations. The TM6SF2 E167K minor allele was associated with severe liver brosis stages as reported before 13 , but we also found an association with thrombocytopenia in an Egyptian cohort of 250 patients with CHC viral infection. Previous studies, however, analyzed the contribution of TM6SF2 E167K variant to the pathogenicity of nonalcoholic steatohepatitis [14][15][16] .
The most important hepatic alterations which occur due to CHC infection are liver cirrhosis, hepatocellular carcinoma (HCC) and end-stage liver disease, where the progression from brosis to cirrhosis can take ten to thirty years 17 .
In addition, CHC patients with chronic HCV infection also suffer from several extra-hepatic pathologies that range from stroke and myocardial infarction to diabetes and autoimmune responses like rheumatoid arthritis. Thrombocytopenia is a major concern in CHC patients, affection about 45 % of them. This decrease in platelet count can induce bleeding manifestations, which strongly can in uence the initiation and continuation of antiviral therapy in the corresponding cases. Thrombocytopenia can be observed in the majority of HCV patients with advanced brosis and/or cirrhosis, compared with the non-cirrhotic patients 18, 19 . Thrombocytopenia can limit not only diagnostic but also therapeutic procedures and treatments, and increases risk of complications, especially excessive bleeding 20 .
Several etiologies have been proposed for the pathogenesis of thrombocytopenia in CHC patients including excessive destruction of platelets either via autoimmune reactions or platelet sequestration as a result of splenomegaly [21][22][23] . In addition, two further mechanisms are also possible as a cause for CHCassociated thrombocytopenia, including virus-induced bone marrow suppression and decreased TPO production 20 . There are also data supporting the coexistence of several mechanisms causing thrombocytopenia in response to chronic HCV and cirrhosis 21 . Therefore, more research is needed to clarify the underlying factors behind the pathogenesis of chronic liver diseases-associated thrombocytopenia.
Human genetic variants are gaining much interest in the last decade for their role in chronic liver diseaseseverity and its-associated thrombocytopenia. Interestingly, a previous study showed a signi cant association between the minor allele of rs11697186 (located in DDRGK1 gene) and the decrease in platelet count that was observed during the treatment of HCV infection with peg-interferon 24 .
In the current study, we found a signi cant association between the minor allele (T allele) and the occurrence of thrombocytopenia which agrees with previous research reporting this extrahepatic manifestation in CHC patients 4,18,21,25 . It is worth to note that the observed thrombocytopenia in the current study was linked to lower TPO plasma levels these ndings are in agreement with data from previous studies which have detected a negative correlation between the production of TPO and the degree of liver brosis, one possible explanation for the association between the TM6SF2 minor allele and low plasma TPO level is the association of this allele with advanced-stage liver brosis. As brosis advances to cirrhosis, liver shrinks and loses its ability to synthesize TPO, resulting in inappropriately low levels of TPO 26 .

Conclusion
In conclusion, our study shed the light on the association of the minor allele of rs58542926 and serum TPO levels with the brosis stage in Egyptian CHC patients, and links the minor allele to the severe brosis stages in such cohort. This introduces the minor variant of TM6SF2 rs58542926 as a pharmacogenetic diagnostic tool for predicting brosis progression in CHC patients.