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Original articles • Clinics 77 • 2022 • https://doi.org/10.1016/j.clinsp.2022.100094   copy

Effect of MTTP -493G/T, I128T, Q95H and Q244E polymorphisms on hepatic steatosis in patients with chronic hepatitis

Authorship SCIMAGO INSTITUTIONS RANKINGS

Abstract

Background:

Chronic hepatitis C is characterized by a progressive deterioration of liver function and is involved in metabolic complications, such as hepatic steatosis.

Objective:

The aim of this study was to investigate the role of host and viral characteristics associated with -493G/T (rs1800591), I128T (rs3816873), Q95H (rs61733139), and Q244E (rs17599091) Single Nucleotide Polymorphisms (SNPs) in the Microsomal Triglyceride Transfer Protein (MTTP) gene on hepatic steatosis in chronic hepatitis C.

Methods:

SNPs were genotyped by PCR-RFLP and analyzed in combination with host and viral characteristics by multiple logistic regression in different genetic models of inheritance.

Results:

The authors analyzed 236 patients with chronic hepatitis C, and 53% had hepatic steatosis. The mutated allele frequencies were > 5%, and the genotypes were in Hardy-Weinberg equilibrium (p ≥ 0.05). It was observed that patients with HCV genotype 3 infection (OR = 2.74, 95% CI 1.24‒6.06, p = 0.013), female sex (OR = 2.28, 95% CI 1.21‒4.28, p = 0.011) and moderate- and high-intensity liver inflammatory activity (A2-A3) (OR = 3.61, 95% CI 1.86‒7.01, p < 0.001) alone exhibited a higher risk of steatosis. The results of multiple logistic regression analysis for interaction showed that for the -493G/T SNP, when the GT/TT genotype (dominant model) and the GT genotype (codominant model) were each combined with HCV genotype 3 infection, an 11.51-fold (95% CI 2.08‒63.59, p = 0.005) and a 15.69-fold (95% CI 2.46‒99.85, p = 0.004) increased risk of steatosis, respectively, was observed. For the I128T SNP, when both the IT/TT genotype (dominant model) and the IT genotype (codominant model) were combined with HCV genotype 3 infection, an 8.51-fold (95% CI 1.59‒45.54, p = 0.012) and an 8.40 fold (95% CI 1.51‒46.91, p = 0.015) increased risk of steatosis, respectively, was observed.

Conclusion:

The present study showed that the viral genotype combined with the -493G/T and I128T SNPs in the MTTP gene influences hepatic steatosis.

Keywords:
Chronic hepatitisC; Genetic models of inheritance; Hepatic steatosis; Microsomal Triglyceride Transfer Protein (MTTP); Single Nucleotide Polymorphisms (SNPs)

Highlights

  • Important etiologies of chronic liver disease are viral hepatitis.

  • Viral hepatitis B and C causes 1.1 million deaths per year.

  • Hepatic steatosis (liver fat accumulation) is a metabolic complication of hepatitis C.

  • The underlying mechanisms involving steatosis include genetic polymorphisms.

  • -493G/T and I128T polymorphisms in the MTTP gene are relevant in hepatic steatosis.

Highlights

  • Important etiologies of chronic liver disease are viral hepatitis.

  • Viral hepatitis B and C causes 1.1 million deaths per year.

  • Hepatic steatosis (liver fat accumulation) is a metabolic complication of hepatitis C.

  • The underlying mechanisms involving steatosis include genetic polymorphisms.

  • -493G/T and I128T polymorphisms in the MTTP gene are relevant in hepatic steatosis.

Introduction

Chronic liver disease is characterized by a progressive deterioration of liver function for more than six months, and this process is related to the persistent inflammation, destruction, and regeneration of the liver parenchyma. The most common etiologies are the presence of alcoholic liver disease, autoimmune or genetic causes, drugs, Nonalcoholic Fatty Liver Disease (NAFDL)/Nonalcoholic Steatohepatitis (NASH), and chronic viral hepatitis.11 Sharma A, Nagalli S. Chronic liver disease. StatPearls. Treasure IslandFL: StatPearls Publishing; 2022 [Internet]. Regarding viral hepatitis, hepatitis C causes inflammation in the liver that is caused by Hepatitis C Virus (HCV). According to the World Health Organization (WHO), an estimated 58 million people are chronically infected with HCV worldwide.22 World Health Organization. Hepatitis C. https://www.who.int/news-room/fact-sheets/detail/hepatitis-c (15 March 2022, date last accessed).
https://www.who.int/news-room/fact-sheet... Viral hepatitis (hepatitis B and C) causes 1.1 million deaths per year and 3.0 million new infections. Among hepatitis C patients, approximately 62% of those diagnosed receive specific treatment.33 World Health Organization. Global Progress Report on HIV, Viral Hepatitis, and Sexually Transmitted Infections, 2021. Accountability for the Global Health Sector Strategies 2016-2021: Actions for impact. 2021. In addition, hepatitis C is involved in several metabolic complications, such as insulin resistance, hepatic steatosis, hyperlipidemia, and metabolic syndrome.44 Chaudhari R, Fouda S, Sainu A, Pappachan JM. Metabolic complications of hepatitis C virus infection. World J Gastroenterol 2021;27(13):1267–82.

The development of hepatic steatosis influences disease progression. In addition, persistence and even an increase in the degree of steatosis are observed in patients who achieve a Sustained Virologic Response (SVR) after specific treatment with Direct-Acting Antiviral (DAA) drugs one year after treatment ends.55 Rout G, Nayak B, Patel AH, Gunjan D, Singh V, Kedia S, et al. Therapy with oral directly acting agents in hepatitis c infection is associated with reduction in fibrosis and increase in hepatic steatosis on transient elastography. J Clin Exp Hepatol 2019;9(2):207–14., 66 Kawagishi N, Suda G, Nakamura A, Kimura M, Maehara O, Suzuki K, et al. Liver steatosis and dyslipidemia after HCV eradication by direct acting antiviral agents are synergistic risks of atherosclerosis. PLoS One 2018;13(12):e0209615., 77 Soliman H, Ziada D, Hamisa M, Badawi R, Hawash N, Salama M, et al. The effect of HCV eradication after direct-acting antiviral agents on hepatic steatosis: a prospective observational study. Endocr Metab Immune Disord Drug Targets 2022;22(1):100–7. Interestingly, diabetes mellitus was reported to be a factor that independently affected liver stiffness after DAA treatment despite SVR88 Pontual DM, Nabuco LC, Luiz RR, Cardoso AC, Perez RM, Villela-Nogueira CA. Diabetes influences liver stiffness in chronic hepatitis C patients with and without virological cure: a longitudinal study. Clinics 2021;76:e3236. The underlying mechanisms involving the presence of steatosis, especially before treatment, can be alcohol consumption, being overweight, obesity, diabetes type 2 mellitus, HCV genotype, Human Immunodeficiency Virus (HIV) coinfection, and genetic polymorphisms in genes, such as Transmembrane six Superfamily member 2 (TM6SF2), Patatin-Like Phospholipase Domain Containing 3 (PNPLA3) and Microsomal Triglyceride Transfer Protein (MTTP).99 Valenti L, Rumi M, Galmozzi E, Aghemo A, Del Menico D, De Nicola S, et al. Patatin-like phospholipase domain-containing 3 I148M polymorphism, steatosis, and liver damage in chronic hepatitis C. Hepatology 2011;53(3):791–9., 1010 Adinolfi LE, Restivo L, Marrone A. The predictive value of steatosis in hepatitis C virus infection. Expert Rev Gastroenterol Hepatol 2013;7(3):205–13., 1111 Magri MC, Prata TVG, Manchiero C, Dantas BP, Mazza CC, Tengan FM. Genetic variation in the microsomal triglyceride transfer protein (-493G/T) is associated with hepatic steatosis in patients infected with hepatitis C virus. BMC Infect Dis 2017;17:235., 1212 Manchiero C, Nunes AKS, Magri MC, Dantas BP, Mazza CC, Barone AA, et al. The rs738409 polymorphism of the PNPLA3 gene is associated with hepatic steatosis and fibrosis in Brazilian patients with chronic hepatitis C. BMC Infect Dis 2017;17(1):780., 1313 Stevenson HL, Utay NS. Hepatic steatosis in HCV-infected persons in the direct-acting antiviral era. Trop Dis Travel Med Vaccines 2016;2:1–7. In this context, the role of host and viral characteristics associated with genetic polymorphisms in liver fat accumulation in chronic hepatitis C was investigated in the present study. The aim of the present study was to determine whether the -493G/T (rs1800591), I128T (rs3816873), Q95H (rs61733139), and Q244E (rs17599091) Single Nucleotide Polymorphisms (SNPs) in the MTTP gene are linked to the presence of hepatic steatosis in patients with chronic hepatitis C.

Materials and methods

Patient selection

Patients with chronic hepatitis C were selected from the Clinical Hospital of the School of Medicine at the University of Sao Paulo (HCFMUSP) in Brazil. The present study was approved by the Ethics Committee (Ethics Committee for Analysis of Research Projects) of HCFMUSP. The protocol followed the guidelines of the 1975 Declaration of Helsinki, and informed consent was obtained from all participants. The actual research did not conflict with any treatment or medical advice. The present study is part of a sequence of previously published studies.1111 Magri MC, Prata TVG, Manchiero C, Dantas BP, Mazza CC, Tengan FM. Genetic variation in the microsomal triglyceride transfer protein (-493G/T) is associated with hepatic steatosis in patients infected with hepatitis C virus. BMC Infect Dis 2017;17:235.,1414 Prata TVG, Silva DSRD, Manchiero C, Dantas BP, Mazza CC, Nunes AKS, et al. MTTP polymorphisms and hepatic steatosis in individuals chronically infected with hepatitis C virus. Arch Virol 2019;164(10):2559–63.

The inclusion criteria were patients who presented positive anti-HCV antibody and HCV-RNA results for more than six months had undergone histopathological analysis after liver biopsy, and were older than 18 years. The exclusion criteria were patients who were coinfected with HIV or Hepatitis B Virus (HBV), patients who received previous HCV treatment, and the presence of liver conditions of other etiologies, such as autoimmune liver disease, primary biliary cirrhosis, and Wilson's disease. The patients were included in the study and had biological samples collected before initiating any hepatitis C treatment.

A total of 236 patients with chronic hepatitis C infection under follow-up at the outpatient clinic of infectious diseases in the HCFMUSP who met the criteria described were included from 2010 to 2012. The calculation of the minimum sample size required was performed considering the following parameters: the alpha value of 5%; a beta value of 20% and, consequently, a power of 80% and standard error type 2. The recommended equation for this type of study was used and was as follows: n = π (1-π)/e2.1515 Kirkwood BR, Sterne JAC. Essential medical statistics. 2nd ed. Massachusetts: Black-well Science; 2006. This calculation was performed considering the frequency of 27.5% of the recessive allele (T) for the -493G/T SNP in the MTTP gene as described by Mirandola et al.1616 Mirandola S, Osterreicher CH, Marcolongo M, Datz C, Aigner E, Schlabrakowski A, et al. Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C. Liver Int 2009;29(4):557–65. The minimum sample size was calculated to be 225 patients. At the time of enrollment, all patients with chronic hepatitis C attending this outpatient clinic underwent liver biopsy. After histopathological analysis of the liver fragment, the patients were divided into two groups as follows: patients with hepatic steatosis (n = 125) and patients without hepatic steatosis (n = 111).

Data collection

Data regarding epidemiological and demographical factors and laboratory tests were collected from medical records. The reference values of biochemical tests were as follows: Alanine Aminotransferase (ALT) levels: ≥ 41 U/L, Aspartate Aminotransferase (AST) levels: ≥ 37 U/L, Gamma Glutamyl Transpeptidase (GGT) levels: > 61 U/L, insulin levels: ≥ 25 µU/mL, glucose levels: >99 mg/dL, triglyceride levels: ≥ 200 mg/dL, total cholesterol levels: ≥ 200 mg/dL, High-Density Lipoprotein (HDL) levels: ≤ 60 mg/dL, Low-Density Lipoprotein (LDL) levels: ≥ 130 mg/dL, and Very-Low-Density Lipoprotein (VLDL) levels: ≥ 40 mg/dL. HOMA-IR index (homeostasis model assessment of insulin resistance) was calculated as fasting insulin levels (μU/mL) × fasting glucose levels (mmoL/L)/22.5, and its reference value was ≥ 3,0. The reference value for HCV viral load was <850,000 IU/mL, and that for alcohol consumption was ≥20 g/day. Metabolic syndrome was defined by the appearance of three or more of the following alterations: high triglyceride levels (≥ 150 mg/dL), low HDL levels (≤ 40 mg/dL), diagnosis of diabetes or fasting blood glucose levels ≥ 100 mg/dL and the diagnosis of high blood pressure or systolic blood pressure ≥ 130 mmHg and/or diastolic blood pressure ≥ 85 mmHg.

Histopathological analysis after liver biopsy of all patients was performed according to Kleiner et al.1717 Kleiner DE, Brunt EM, Van Natta M, Behling C, Contos MJ, Cummings OW, et al. Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005;41 (6):1313–21. classification for the assessment of hepatic steatosis (graded as 0 to 3), according to the METAVIR1818 The French METAVIR Cooperative Study Group. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 1994;20(1):15–20. classification for the assessment of hepatic fibrosis (graded as F0 to F4) and hepatic inflammatory activity (graded as A0 to A4), and according to Perls’ staining criteria (graded as 0 to 4) for the assessment of hepatic siderosis.

Single nucleotide polymorphism genotyping

Peripheral blood (10 mL) was collected from patients with chronic hepatitis C from 2010 to 2012 and was stored at -80°C until processing. To genotype the -493G/T (rs1800591), I128T (rs3816873), Q95H (rs61733139) and Q244E (rs17599091) MTTP SNPs, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed. First, DNA was isolated using the ReliaPrep Blood gDNA Miniprep System according to the manufacturer's instructions (Promega, USA). The primers used during PCR to amplify the MTTP gene fragments have been described by Karpe et al.1919 Karpe F, Lundahl B, Ehrenborg E, Eriksson P, Hamsten A. A common functional polymorphism in the promoter region of the microsomal triglyceride transfer protein gene influences plasma LDL levels. Arterioscler Thromb Vasc Biol 1998;18(5):756–61. and Ledmyr et al.2020 Ledmyr H, Karpe F, Lundahl B, McKinnon M, Skoglund-Andersson C, Ehrenborg E. Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index. J Lipid Res 2002;43(1):51–8. PCR was performed in a 16.7 μL reaction mix that included 5.3 μL of ultrapure water, 8.3 μL of GoTaq Green Master Mix (Promega, USA), 1.7 μL (30-50 ng/μL) of isolated DNA and 0.7 μL (7 pmoL) of each primer. The number of PCR cycles was 40, and the annealing temperature varied between 53° and 60°C, according to the primer pair size and composition. PCR amplification was confirmed under Ultraviolet (UV) light by 3% agarose gel electrophoresis, and a 50 bp DNA molecular weight marker was utilized to validate the amplicon size.

The amplified PCR product was digested with a specific endonuclease restriction enzyme that recognizes one of the SNP alleles to cleave the amplified fragment at a specific site for subsequent differentiation of the alleles by size. The RFLP assay was performed in a 20 μL reaction mix containing 15 µL of PCR product, 2 µL of ultrapure water, 2 µL of the corresponding buffer of each enzyme, and 0.5-1 µL of restriction enzyme according to the concentration used. The temperature and incubation time varied depending on the enzyme and the manufacturer's instructions. The restriction enzymes that were utilized have been previously described by Karpe et al.1919 Karpe F, Lundahl B, Ehrenborg E, Eriksson P, Hamsten A. A common functional polymorphism in the promoter region of the microsomal triglyceride transfer protein gene influences plasma LDL levels. Arterioscler Thromb Vasc Biol 1998;18(5):756–61. and Ledmyr et al.2020 Ledmyr H, Karpe F, Lundahl B, McKinnon M, Skoglund-Andersson C, Ehrenborg E. Variants of the microsomal triglyceride transfer protein gene are associated with plasma cholesterol levels and body mass index. J Lipid Res 2002;43(1):51–8. Enzymatic digestion was confirmed under UV light by 3% agarose gel electrophoresis. The genotypes expected for each MTTP SNP were as follows: -493G/T SNP, GG: 89 bp and 20 bp, GT: 109 bp, 89 bp, and 20 bp, TT: 109 bp; I128T SNP, II: 167 bp, IT: 167 bp, 138 bp, and 29 bp, TT: 138 bp and 29 bp; Q95H SNP, QQ: 148 bp and 35 bp, QH: 183 bp, 148 bp, and 35 bp, HH: 183 bp; Q244E SNP, QQ: 201 bp, QE: 201 bp, 149 bp, and 52 bp, EE: 149 bp and 52 bp. Quality control was used to verify the reproducibility of the results.

Statistical analysis

For statistical analysis, IBM-SPSS version 20 software (IBM Corp., USA) and Hosmer; Lemeshow2121 Hosmer DW, Lemeshow S. Applied logistic regression. 2nd ed. New York: Wiley; 2000. were used. The analysis of the Hardy-Weinberg equilibrium was performed using the Chi-Square test (p ≥ 0.05). All variables were categorized, and their frequencies were described. Bivariate and multiple logistic regression was used to determine the individual variables that could influence the presence of steatosis in patients with chronic hepatitis C. The Odds Ratio (OR) of each variable with the presence of hepatic steatosis was estimated with the respective 95% Confidence Interval (95% CI). The associations were evaluated in three different genetic models (codominant, dominant and recessive models) because the optimal genetic model of inheritance in genes in complex diseases has not been well established.

A bivariate and a multiple logistic regression model were used to determine which host and viral characteristics combined with each SNP (-493G/T, I128T, Q95H and Q244E) to influence the presence of steatosis in patients with chronic hepatitis C according to different genetic models. All variables independently associated with hepatic steatosis (p < 0.05) in multiple logistic regression and interactions that presented a significance level of 0.20 (p < 0.2) in the bivariate tests were included in the final multiple logistic regression model. In the multiple logistic regression model, the significance level was 0.05 (p < 0.05).

Results

The study group consisted of 236 patients with chronic hepatitis C infection, 56.4% of whom were females, and the most frequent infection was by genotype non 3 (81.8%). Additionally, the most frequent self-reported ethnicity was white (81.0%). The distribution of patients with chronic hepatitis C considering the Body Mass Index (BMI) classification was as follows: 39.8% of the patients presented BMI < 25 kg/m2, which is considered a normal BMI and 60.2% of the patients presented BMI ≥ 25 kg/m2, which can be classified as overweight or obese. A total of 57.6% of patients exhibited elevated levels of ALT, 51.3% exhibited elevated levels of AST, and 44.1%. exhibited elevated levels of GGT. According to liver histology findings, 53% of the patients exhibited hepatic steatosis, 20.8% exhibited hepatic fibrosis stages of F3 and F4 and 66.1% exhibited hepatic inflammatory activity grades of A2 and A3 (Table 1).

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Table 1
General characteristics of patients with chronic hepatitis C included in the study (total) and split according to the presence of hepatic steatosis, n (%).

The four SNPs (-493G/T, I128T, Q95H, and Q244E) evaluated in the MTTP gene in all patients were genotyped. For the -493G/T SNP, 109 (46.2%) patients exhibited the GG genotype (wild-type homozygous), 102 (43.2%) patients exhibited the GT genotype (heterozygous) and 25 (10.6%) patients exhibited the TT genotype (mutated homozygous). For the I128T SNP, 117 (49.6%) patients exhibited the II genotype (wild-type homozygous), 97 (41.1%) patients exhibited the IT genotype (heterozygous) and 22 (9.3%) patients exhibited the TT genotype (mutated homozygous). For the Q95H SNP, 196 (83.1%) patients exhibited the QQ genotype (wild-type homozygous), 39 (16.5%) patients exhibited the QH genotype (heterozygous), and 1 (0.4%) patient exhibited the HH genotype (mutated homozygous). For the Q244E SNP, 209 (88.6%) patients exhibited the QQ genotype (wild-type homozygous), and 27 (11.4%) patients exhibited the QE genotype (heterozygous); the EE genotype (mutated homozygous) was not detected in any participant included in the present study. Therefore, the frequency of mutated alleles of each SNP was > 5%, and the genotypes were distributed according to Hardy-Weinberg equilibrium (-493G/T: p= 0.875, I128T: p = 0.770, Q95H: p = 0.521 and Q95H: p = 0.351).

Bivariate and multivariate analyses were performed to individually assess which characteristics were independently associated with hepatic steatosis in patients with chronic hepatitis C (Supplementary file ‒ Tables S1 and S2). The multivariate analysis indicated that HCV genotype 3 infection (OR = 2.74, 95% CI 1.24‒6.06, p = 0.013) and female sex (OR = 2.28, 95% CI 1.21‒4.28, p = 0.011) were associated with a higher risk of hepatic steatosis. Additionally, patients with moderate-and high-intensity liver inflammatory activity (A2 and A3) exhibited a higher risk of hepatic steatosis than patients without or with low-intensity inflammatory activity (A0 and A1) (OR = 3.61, 95% CI 1.86‒7.01, p < 0.001). The variables that had a significant p-value were included in the interaction analysis (HCV genotype 3, female sex, and inflammatory activity A2-A3). In addition to these variables, HOMA-IR was included in the bivariate and multivariate analyses to assess the association of SNPs in the MTTP gene combined with different characteristics in the presence of hepatic steatosis (interaction analysis) due to its relevance to steatosis in chronic hepatitis C.

The results of the bivariate analysis of the characteristics combined with SNPs in the MTTP gene are presented in Tables 2,3,4,5. The characteristics combined with SNPs that had a significant statistical association with the presence of hepatic steatosis in this analysis were also included in the multiple logistic regression analysis for the MTTP -493G/T, I128T, Q95H and Q244E SNPs, and the results are described in Tables 6,7,8,9. Therefore, all variables associated alone with hepatic steatosis (p<0.05) in multiple logistic regression (Tables S2) and variables combined with SNPs (Tables 2,3,4,5) presented a significance level of 0.20 (p < 0.2) in the bivariate tests were included in the final multiple logistic regression model (interaction analysis) according to different genetic models (dominant, codominant and recessive models).

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Table 2
Result of bivariate tests for interactions of the -493G/T SNP in the MTTP gene in different genetic models with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 3
Result of bivariate tests for interactions of the I128T SNP in the MTTP gene in different genetic models with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 4
Result of bivariate tests for interactions of the Q95H SNP in the MTTP gene with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 5
Result of bivariate tests for interactions of the Q244E SNP in the MTTP gene with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 6
Result of multivariate tests for interactions of the ‒493G/T SNP in the MTTP gene (in different genetic models) with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 7
Result of multivariate tests for interactions of the I128T SNP in the MTTP gene (in different genetic models) with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 8
Result of multivariate tests for interactions of the Q95H SNP in the MTTP gene (in different genetic models) with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.
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Table 9
Result of multivariate tests for interactions of the Q244E SNP in the MTTP gene (in different genetic models) with characteristics of interest that influence the presence of hepatic steatosis in patients with chronic hepatitis C.

In the final multiple logistic regression model used to determine which characteristics combined with each SNP to influence the presence of hepatic steatosis in patients with chronic hepatitis C, it was observed that in the dominant genetic model (GG × GT/TT), the GT/TT genotype of the -493G/T SNP in the MTTP gene combined with HCV genotype 3 infection presented an 11.51-fold higher risk of hepatic steatosis than that observed in carriers of the GG genotype without HCV genotype 3 infections (95% CI 2.08-63.59, p = 0.005). Similar results were observed in the codominant model (GG × GT × TT) of the -493G/T SNP, in which carriers of the GT genotype combined with HCV genotype 3 infection presented a 15.69-fold higher risk of hepatic steatosis than that observed in carriers of the GG genotype without HCV genotype 3 infections (95% CI 2.46‒99.85, p = 0.004) (Table 6).

In the dominant genetic model (II × IT/TT), the IT/TT genotype of the I128T SNP in the MTTP gene combined with HCV genotype 3 infection presented an 8.51-fold higher risk of hepatic steatosis than that observed in carriers of the II genotype without HCV genotype 3 infections (95% CI 1.59–45.54, p = 0.012). In the codominant model (II × IT × TT), carriers of the IT genotype of the I128T SNP combined with HCV genotype 3 infection presented an 8.40-fold higher risk of hepatic steatosis than that observed in carriers of the II genotype without HCV genotype 3 infections (95% CI 1.51-46.91, p = 0.015) (Table 7). The Q95H and Q244E SNPs in the MTTP gene did not influence the presence of hepatic steatosis in this group of patients when combined with other variables (Tables 8 and 9).

Discussion

Hepatitis C is a frequent liver disease found worldwide. Even in the era of DAAs, the determination of genetic markers is important to identify individuals at higher risk of severe disease and to guide therapeutic decisions prior to therapy in patients with chronic hepatitis C.2222 Hemeda AA, Mohamed AA, Aziz RK, Abdel-Hakeem MS, Ali-Tammam M. Impact of IL10, MTP, SOD2, and APOE gene polymorphisms on the severity of liver fibrosis induced by HCV genotype 4. Viruses 2021;13(4):1–18. In the present study, the authors analyzed the effect of four candidate SNPs in the MTTP gene combined with host and viral characteristics on hepatic steatosis in a group of chronic hepatitis C patients. Multiple logistic regression analysis for interaction showed an 11.51-fold higher risk of steatosis in patients with the GT/TT genotype of the -493G/T SNP and HCV genotype 3 infections. The same results were observed when another genetic model, the codominant model, was analyzed, in which carriers of the GT genotype combined with HCV genotype 3 infection presented a 15.69-fold higher risk of steatosis. The present study also investigated the I128T SNP of the MTTP gene, and the risk of steatosis was 8.51-fold higher in patients with the IT/TT genotype of the I128T SNP when combined with HCV genotype 3 infections. In another genetic model, the codominant model, carriers of the IT genotype combined with HCV genotype 3 infection presented an 8.40-fold higher risk of steatosis.

Several studies have explored the association between the -493G/T and I128T SNPs of the MTTP gene and NAFLD. However, the effect of these polymorphisms on NAFLD remains uncertain due to the inconsistent results of different studies. A recent meta-analysis evaluated these SNPs under different genetic models on NASH and NAFLD, demonstrating that the -493G/T SNP is associated with NASH susceptibility (determined by liver biopsy).2323 Tan J, Zhang J, Zhao Z, Zhang J, Dong M, Ma X, et al. The association between SNPs rs1800591 and rs3816873 of the MTTP gene and nonalcoholic fatty liver disease: a meta-analysis. Saudi J Gastroenterol 2020;26(4):171–8.

The role of the -493G/T SNP in the MTTP gene associated with hepatitis C has been thoroughly studied, but different results have been reported.2425 Zampino R, Ingrosso D, Durante-Mangoni E, et al. Microsomal triglyceride transfer protein (MTP) -493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients. J Viral Hepat 2008;15:740–6.,2525 Zampino R, Ingrosso D, Durante-Mangoni E, et al. Microsomal triglyceride transfer protein (MTP) -493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients. J Viral Hepat 2008;15:740–6.,2626 Mirandola S, Osterreicher CH, Marcolongo M, Datz C, Aigner E, Schlabrakowski A, et al. Microsomal triglyceride transfer protein polymorphism (-493G/T) is associated with hepatic steatosis in patients with chronic hepatitis C. Liver Int 2009;29(4):557–65.,2727 Cai T, Dufour JF, Muellhaupt B, Gerlach T, Heim M, Moradpour D, et al. Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis. J Hepatol 2011;55(3):529–35.,2828 Siqueira ER, Oliveira CP, Correa-Giannella ML, et al. MTP -493G/T gene polymorphism is associated with steatosis in hepatitis C-infected patients. Braz J Med Biol Res 2012;45:72–7. Akgöllü and Akkız2929 Akgöllü E, Akkız H. Association between hepatic steatosis and MTP gene -493G/T polymorphism in the patients with HCV genotype 1 infection. Infect Genet Evol 2019;70:101–6. evaluated the relationship of the -493G/T SNP with hepatic steatosis in a Turkish population with HCV genotype 1 infection. Despite finding a statistically significant association between levels of triglycerides, total cholesterol, LDL, and VLDL and the number of patients with steatosis, these researchers reported that the studied polymorphism is not associated with the presence of steatosis in individuals who exhibited HCV genotype 1 infection.2929 Akgöllü E, Akkız H. Association between hepatic steatosis and MTP gene -493G/T polymorphism in the patients with HCV genotype 1 infection. Infect Genet Evol 2019;70:101–6. A previous Swiss cohort study, including 443 patients infected with HCV genotype non 3 and 183 patients infected with HCV genotype 3, reported that the -493G/T SNP is associated with the presence of steatosis in multivariate analysis only in patients with HCV genotype non 3.2727 Cai T, Dufour JF, Muellhaupt B, Gerlach T, Heim M, Moradpour D, et al. Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis. J Hepatol 2011;55(3):529–35. However, in another study evaluating 102 treatment-naïve patients for hepatitis C it was reported that patients infected with HCV genotype 3 and with the T (mutated) allele of the -493G/T SNP are associated with hepatic steatosis, which corroborates the present findings. Furthermore, these patients also present a higher grade of inflammation in the liver and more liver fibrosis as well as higher HCV-RNA serum levels than those observed in carriers of the wild-type allele (G).2525 Zampino R, Ingrosso D, Durante-Mangoni E, et al. Microsomal triglyceride transfer protein (MTP) -493G/T gene polymorphism contributes to fat liver accumulation in HCV genotype 3 infected patients. J Viral Hepat 2008;15:740–6.

Regarding the I128T SNP in the MTTP gene, previous multivariate analyses conducted in a Han Chinese population have indicated that this SNP is not associated with NAFLD.3030 Peng XE, Wu YL, Lu QQ, Hu ZJ, Lin X, et al. MTTP polymorphisms and susceptibility to non-alcoholic fatty liver disease in a Han Chinese population. Liver Int 2014;34(1):118–28. However, this polymorphism has been associated with the presence of central obesity, elevated liver enzymes, and alcoholic fatty liver disease in Koreans.3131 Jun DW, Han JH, Jang EC, Kim SH, Kim SH, Jo YJ, et al. Polymorphisms of micro-somal triglyceride transfer protein gene and phosphatidylethanolamine N-methyl-transferase gene in alcoholic and nonalcoholic fatty liver disease in Koreans. Eur J Gastroenterol Hepatol 2009;21(6):667–72. Hashemi et al.3232 Hashemi M, Hoseini H, Yaghmaei P, Moazeni-Roodi A, Bahari A, Hashemzehi N, et al. Association of polymorphisms in glutamate-cysteine ligase catalytic subunit and microsomal triglyceride transfer protein genes with nonalcoholic fatty liver disease. DNA Cell Biol 2011;30(8):569–75. investigated the association between the I128T and Q95H SNPs in the MTTP gene in a sample of Iranian patients with NAFLD, and they observed that the IT genotype and the IT+TT genotype of the I128T SNP increase susceptibility to NAFLD. In addition, it has been reported that there is no association between the Q95H SNP and NAFLD.3232 Hashemi M, Hoseini H, Yaghmaei P, Moazeni-Roodi A, Bahari A, Hashemzehi N, et al. Association of polymorphisms in glutamate-cysteine ligase catalytic subunit and microsomal triglyceride transfer protein genes with nonalcoholic fatty liver disease. DNA Cell Biol 2011;30(8):569–75.

Hepatic steatosis is a frequent histological feature among patients with chronic hepatitis C, and it significantly affects disease progression.3333 Mirandola S, Bowman D, Hussain MH, Alberti A. Hepatic steatosis in hepatitis C is a storage disease due to HCV interaction with microsomal triglyceride transfer protein (MTP). Nutr Metab (Lond) 2010;7:13. In the present study, hepatic steatosis was observed in 53% of the patients, which was similar to the mean prevalence of 55% previously described by Asselah et al.3434 Asselah T, Rubbia-Brandt L, Marcellin P, Negro F. Steatosis in chronic hepatitis C: why does it really matter? Gut 2006;55(1):123–30. Host and viral factors are involved in the development and severity of HCV-associated steatosis. Recently, several SNPs have been reported to be associated with alterations in hepatic fibrosis and steatosis even after DAA therapy for HCV infection, indicating that they may have prognostic value for the assessment of post-SVR evolution.3535 Gavril OI, Arhire LI, Gavrilescu O, Dranga M, Barboi O, Gavril RS, et al. Role of PNPLA3 in the assessment and monitoring of hepatic steatosis and fibrosis in patients with chronic hepatitis C infection who achieved a sustained virologic response. Medicina 2021;57(11):1153.,3636 Matsumoto K, Miyaaki H, Fukushima M, Sasaki R, Haraguchi M, Miuma S, et al. The impact of single-nucleotide polymorphisms on liver stiffness and controlled attenuation parameter in patients treated with direct-acting antiviral drugs for hepatitis C infection. Biomed Rep 2022;16(2):1–8.

The present study had several limitations. The sample size was limited, and the study was conducted in a single center. To validate the present findings, a study including a larger number of samples should be performed in the future. However, the present results may aid in the establishment of genetic markers to predict hepatic steatosis.

Conclusions

In summary, the present study highlighted the significant role of MTTP SNPs in the pathogenesis of hepatic steatosis in hepatitis C. The association analysis of the group of patients with chronic hepatitis C provides useful information on the effect of HCV genotype 3 infections combined with the -493G/T and I128T SNPs in the MTTP gene on hepatic steatosis. These findings may have prognostic importance in patients with chronic hepatitis C and help guide decision-making for appropriate follow-up and treatment for those at increased risk of liver disease progression.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.clinsp.2022.100094.

  • Funding
    This work was supported by the Sao Paulo Research Foundation (FAPESP) via grant #2016/19690-5.

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Publication Dates

  • Publication in this collection
    10 Oct 2022
  • Date of issue
    2022

History

  • Received
    15 Mar 2022
  • Reviewed
    21 June 2022
  • Accepted
    12 July 2022
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