Introduction: There is growing evidence that genetic and environmental factors play an important role in the development and progression of non-alcoholic fatty liver disease (NAFLD). We investigated the association of single nucleotide polymorphism (SNP) rs738409 in PNPLA3 gene and TNF-α G238A polymorphism with the development and severity of NAFLD in an overweight and obese Egyptian population. Material and Methods: 100 overweight and obese patients with NAFLD and 30 control subjects were enrolled. All NAFLD patients underwent a confirmatory biopsy. Laboratory investigations included fasting plasma glucose, kidney and liver function tests, liver enzymes, lipid profile and hepatitis markers. Abdominal ultrasound was performed and all subjects were genotyped for (rs738409) PNPLA3 and (rs361525) TNF-α gene polymorphisms using polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Results: The homozygous GG genotype of the PNPLA3 was most frequent among patients with NASH (26%) as compared to borderline NASH (20.5%) and simple steatosis (20%). Higher serum levels of transaminases were observed in NAFLD patients and controls who were carriers of the G allele of rs738409, but this was not statistically significant. Regarding the TNF-α G238A SNP; the frequency of the A allele was significantly higher in NAFLD patients (20%) compared to controls (5%) (p value = 0.006). The highest TNF G allele frequency was observed in the NASH group (88%) and this was statistically significant (p value = 0.009). Conclusion: Our study confirmed the association of the PNPLA3 (rs738409) and TNF-α promoter region G238A polymorphisms with susceptibility to NAFLD and its progression.
NAFLD is increasingly recognized as the leading cause of chronic liver disease worldwide [1] . NAFLD encompasses a wide spectrum of varying liver histology ranging from simple steatosis to non-alcoholic steatohepatitis (NASH), often leading to fibrosis and eventually cirrhosis with a high risk of liver failure and hepatocellular carcinoma. There is growing evidence that genetic as well as environmental factors play an important role in the development and progression of NAFLD [2] - [4] . In recent years, genetic determinants of steatosis are being revealed using genome-wide association studies [5] [6] . These studies have identified patain-like phospholipase domain containing3 (PNPLA3) gene, also called adiponutrin, which encodes a 481 amino acid membrane protein localized in the endoplasmic reticulum and at the surface of lipid droplets [5] .
The first Genome-wide association (GWA) study identified the PNPLA3 gene polymorphism as a major genetic determinant for the predisposition to NAFLD in Hispanic, African American and European Americans populations according to liver fat content [5] , which was subsequently confirmed in Europeans and Asians according to liver biopsy. The association of PNPLA3 gene polymorphisms not only with fatty liver and triglyceride content, but also with histological severity of NAFLD was shown in subsequent studies [7] [8] .
The investigation of the potential role of tumor necrosis factor-α (TNF-α) and other pro-inflammatory cytokines in NASH was first enthused by the obvious similarity between NASH and the effect of these cytokines. The progression of NAFLD from simple steatosis to fibrosis is associated with a significant increase in TNF-α [9] [10] .
The TNF-α gene is polymorphic at different positions including-G308A and -G238A. TNF-α polymorphisms have been shown to be strongly associated with the risk of developing NAFLD especially in Chinese population and this was confirmed in a meta-analysis by Wang et al. [11] .
In the present study we investigated the association of the SNP rs738409 in the PNPLA3 gene and the TNF-α G238A polymorphism with the development and severity of NAFLD in overweight and obese Egyptian population.
2. Patients and Methods
A total of one hundred overweight and obese patients (94 females and 6 males) with NAFLD were enrolled in the study from December 2013 to November 2014. They were prospectively recruited from Gastroenterology and Hepatology outpatient clinic of Kasr El Ainy Hospital. Their age ranged from 29 - 57 years. Thirty healthy, non-obese volunteers who were age- and sex-matched, were included as the control group (all of the control subjects were females). An informed consent was obtained from all participants prior to enrolment. The diagnosis of NAFLD was based on ultrasonographic finding of bright liver, which was defined and graded as: a diffuse hyperechoic echo texture (bright liver) (Grade 1); increased liver echo texture compared to the kidney (Grade 2); vascular blurring (Grade 3) and deep attenuation (Grade 4) [12] . A confirmatory liver biopsy was done after a written consent was obtained. The study was conducted with appropriate approval by the Ethics Committee of Cairo University (N-17-2014) in accordance with the ethical guidelines of the Declaration of Helsinki [13] .
Inclusion criteria for NAFLD patients were age above 18 years, overweight or BMI >25 kg/m2, and bright liver on abdominal ultrasound with or without elevated liver enzymes. Patients were excluded from the study if one of the following criteria were present: any liver disease other than NAFLD such as hepatitis B or C, autoimmune hepatitis, alpha one antitrypsin deficiency or Wilson’s disease, alcohol consumption, history of drug intake (such as use of amiodarone, corticosteroids, tamoxifen, methotrexate, oral contraceptives), pregnancy, diabetes, hypertension, thyroid disease, malignancy and decompensated liver disease. Any subjects with evidence of local or systemic infection on physical examination were excluded from the study. In all controls, the absence of any current or past liver disease was established based on the presence of normal liver function tests and the presence of a normal abdominal ultrasound.
All subjects included in the study were subjected to detailed history taking, complete clinical examination including anthropometric evaluation (weight, height, waist circumference, and BMI was calculated). BMI between 25 - 30 kg/m2 (<30) and ≥30 kg/m2 were defined as overweight and obesity respectively. Laboratory investigations included fasting plasma glucose, liver function tests, serum transaminases (ALT, AST), GGT, urea and creatinine, lipid profile, hepatitis markers including hepatitis B surface antigen and hepatitis C virus antibodies. All the patients as well as the controls were genotyped for Rs738409 PNPLA3 gene and Rs361525 TNF gene polymorphisms by polymerase chain reaction and restriction fragment length polymorphism (PCR/RFLP) using specific primer sequence and restriction enzyme.
Abdominal ultrasonography was performed for all subjects using a Toshiba Apilo XV scanner equipped with a broad band 3.5 MHz curved array probe to assess the presence of liver steatosis (bright liver) and by a single operator to avoid inter-observer variability. Patients were examined after at least 8 hours fasting and were examined in the supine, right and left lateral positions.
Liver biopsies were obtained from all patients with NAFLD (diagnosis based on the finding of bright liver on abdominal ultrasound) after a written consent, using an automated gun device and under complete aseptic precautions. The histological features of the biopsies were graded according to the NAFLD scoring system proposed by the National Institute of Diabetes and Digestive and Kidney Diseases NASH Clinical Research Network and reported as NAFLD activity score (NAS) [14] .
Total NAS score represents the sum of scores for steatosis (0 - 3), lobular inflammation (0 - 3), and ballooning (0 - 2), and ranges from 0 - 8. Diagnosis of NAFLD was made first, then NAS was used to grade activity. In patients with NAFLD, NAS score of ≥5 strongly correlated with a diagnosis of “definite NASH”, 3 or 4 correlated with ‘‘borderline NASH’’, whereas NAS ≤2 correlated with a diagnosis of “not NASH” [14] . The stage of fibrosis was assessed separately from NAS using a four-point scale: 0 = no fibrosis; 1 = mild/moderate zone 3 perisinusoidal fibrosis or portal/periportal fibrosis only; 2 = perisinusoidal and portal/periportal fibrosis; 3 = bridging fibrosis and 4 = cirrhosis. Significant fibrosis was defined as fibrosis grade ≥2 [15] .
3. DNA Preparation and Genetic Analysis
The genomic DNA was extracted from whole blood samples using a Qiagen amp DNA mini kit (USA) extraction kit, according to manufacturer instruction. The purity and concentration of DNA was determined using spectrophotometry, quality of DNA was also determined on 0.8% agarose gel electrophoresis and DNA was stored at −20˚C until further analyses.
Amplification was carried out in a final volume of 50 μL reaction containing 100 ng genomic DNA, 200 mM each dNTP, 1 mM MgCl, 10 mM tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 1.5 units of Taq DNA polymerase (MBI fermentas, Canada), and 1 mM of each of the primers with the following sequence:
Rs361525 TNF with Forward Primer: 5’-AGAAGACCCCCCTCGGAACC-3’ and
Reverse Primer: 5’-ATCTGGAGGAAGCGGTAGTG-3’ [16] and Rs738409 PNPLA3 gene with Forward primer: 5’-TGGGCCTGAAGTCCGAGGGT-3’ and Reverse primer: 5’-CCGACACCAGTGCCCTGCAG-3’ [17] .
The PCR conditions were as follows: 95˚C for 5 min, and then 37 cycles of 94˚C for 30 sec, 66˚C for 30 sec, and 72˚C for 40 sec and a final extension step of 72˚C for 5 minutes. Then PCR products were digested overnight at 65˚C with Taq1 restriction enzyme and BstF5 I (Fermentas, Canada) for PNPLA3 and TNF alpha respectively. Digested PCR products were subjected to horizontal electrophoresis in 1.5% ethidium bromide- stained agarose gels in 1X TBE buffer at 120 V for 1 hr and were visualized using WiseDoc WGD-30 (DAI- HAN, Korea).
4. Statistical Methods
Data were coded and entered using the statistical package SPSS version 21. Data was summarized using mean, standard deviation, median, minimum and maximum for quantitative variables and frequencies (number of cases) and relative frequencies (percentages) for categorical variables. Comparison of quantitative variables was done using the nonparametric Kruskal-Wallis when comparing more than 2 groups and using the nonparametric Mann-Whitney U test when comparing 2 groups. For comparing categorical data, Chi square (c2) test was performed. Exact test was used instead when the expected frequency was less than 5. Genotype frequencies were compared between the different study groups using chi-square tests. Odds ratio (OR) with 95% confidence intervals was calculated. P value < 0.05 was taken as statistically significant [18] [19] .
5. Results
The demographic, anthropometric and laboratory data of NAFLD patients and their age- and sex-matched controls are shown in Table 1. NAFLD patients showed a statistically significant higher BMI, waist circumference, serum levels of AST and ALT, fasting blood glucose, serum levels of triglycerides, total cholesterol, HDL-C and LDL-C. According to the results of liver biopsy, the NAFLD group was divided into three subgroups by the NAS score: Group 1 (Not NASH or Simple Steatosis); included 10 patients (10%) (9 females and 1 male) with mean age 45.50 ± 4.58 years, Group 2 (Borderline NASH); included 44 patients (44%) (42 females and 2 males) with mean age 43.09 ± 7.89 years, Group 3 (NASH) (3 of them had fibrosis); included 46 patients (46%) (43 females and 3 males) with a mean age 44.07 ± 7.07 years. Comparison of clinical and laboratory characteristics of the three NAFLD subgroups (Table 2), revealed that the Simple Steatosis group showed a statistically significant lower mean ALT level (p value = 0.025) as compared to the borderline NASH and NASH groups.
Clinical and laboratory data of the NAFLD patients and control participants
Parameters
NAFLD (N = 100)
Control (N = 30)
p value
Age (years)
43.78 ± 7.23
43.03 ± 7.07
0.613
BMI (kg/m2)
34.88 ± 3.80
22.04 ± 1.56
<0.001
Waist Circumference (cm)
106.66 ± 15.22
72.53 ± 4.53
<0.001
ALT (IU/L)
32.74 ± 19.03
16.50 ± 5.08
<0.001
AST (IU/L)
33.15 ± 20.47
17.23 ± 4.36
<0.001
GGT (IU/L)
42.23 ± 20.00
31.67 ± 8.79
0.012
FBS (mg/dl)
105.96 ± 15.84
97.50 ± 8.48
0.001
T-CHOL (mg/dl)
206.07 ± 33.05
156.87 ± 7.65
<0.001
LDL-C (mg/dl)
103.41 ± 19.50
86.57 ± 6.59
<0.001
HDL-C (mg/dl)
46.15 ± 9.46
51.83 ± 7.20
0.003
TGs (mg/dl)
166.63 ± 39.42
111.97 ± 15.27
<0.001
All data are expressed as mean ± SD. BMI; body mass index, AST: aspartate transaminase, ALT: alanine aminotransferase, GGT: Gamma-glutamyl transpeptidase, FBS: fasting blood sugar, T-CHOL: total cholesterol, LDL-c: low density lipoprotein, HDL-c: high density lipoprotein, TG: triglycerides.
Clinical and laboratory data in subgroups of NAFLD patients
ReferencesMarchesini, G., Brizi, M., Bianchi, G., et al. (2001) Nonalcoholic Fatty Liver Disease: A Feature of the Metabolic Syndrome. Diabetes, 50, 1844-1850. http://dx.doi.org/10.2337/diabetes.50.8.1844Gambino, R., Cassader, M., Pagano, G., Durazzo, M. and Musso, G. (2007) Polymorphism in Microsomal Triglyceride Transfer Protein: A Link between Liver Disease and Atherogenic Postprandial Lipid Profile in NASH? Hepatology, 45, 1097-1107. http://dx.doi.org/10.1002/hep.21631Tokushige, K., Takakura, M., Tsuchiya-Matsushita, N., Taniai, M., Hashimoto, E. and Shiratori, K. (2007) Influence of TNF Gene Polymorphisms in Japanese Patients with NASH and Simple Steatosis. Journal of Hepatology, 46, 1104- 1110. http://dx.doi.org/10.1016/j.jhep.2007.01.028Wilfred de Alwis, N.M. and Day, C.P. (2008) Genes and Nonalcoholic Fatty Liver Disease. Current Diabetes Reports, 8, 156-163. http://dx.doi.org/10.1007/s11892-008-0027-9Romeo, S., Kozlitina, J., Xing, C., et al. (2008) Genetic Variation in PNPLA3 Confers Susceptibility to Nonalcoholic Fatty Liver Disease. Nature Genetics, 40, 1461-1465. http://dx.doi.org/10.1038/ng.257Kamatani, Y., Matsuda, K., Okada, Y., et al. (2010) Genome-Wide Association Study of Hematological and Biochemical Traits in a Japanese Population. Nature Genetics, 42, 210-215. http://dx.doi.org/10.1038/ng.531Sookoian, S., Castano, G.O., Burgueno, A.L., Gianotti, T.F., Rosselli, M.S. and Pirola, C.J. (2009) A Nonsynonymous Gene Variant in the Adiponutrin Gene Is Associated with Nonalcoholic Fatty Liver Disease Severity. The Journal of Lipid Research, 50, 2111-2116. http://dx.doi.org/10.1194/jlr.P900013-JLR200Sookoian, S. and Pirola, C.J. (2011) Metabolic Syndrome: From the Genetics to the Pathophysiology. Current Hypertension Reports, 13, 149-157. http://dx.doi.org/10.1007/s11906-010-0164-9Bayley, J.P., Ottenhoff, T.H. and Verweij, C.L. (2004) Is There a Future for TNF Promoter Polymorphisms? Genes and Immunity, 5, 315-329. http://dx.doi.org/10.1038/sj.gene.6364055Elahi, M.M., Asotra, K., Matata, B.M. and Mastana, S.S. (2009) Tumor Necrosis Factor Alpha-308 Gene Locus Promoter Polymorphism: An Analysis of Association with Health and Disease. Biochimica et Biophysica Acta, 1792, 163-172. http://dx.doi.org/10.1016/j.bbadis.2009.01.007Wang, J.K., Feng, Z.W., Li, Y.C., Li, Q.Y. and Tao, X.Y. (2012) Association of Tumor Necrosis Factor-Alpha Gene Promoter Polymorphism at Sites-308 and -238 with Non-Alcoholic Fatty Liver Disease: A Meta-Analysis. Journal of Gastroenterology and Hepatology, 27, 670-676. http://dx.doi.org/10.1111/j.1440-1746.2011.06978.xRicci, C., Longo, R., Gioulis, E., et al. (1997) Noninvasive in Vivo Quantitative Assessment of Fat Content in Human Liver. Journal of Hepatology, 27, 108-113. http://dx.doi.org/10.1016/S0168-8278(97)80288-7World Medical Association (2013) Declaration of Helsinki: Ethical Principles for Medical Research Involving Human Subjects. JAMA, 310, 2191-2194. http://dx.doi.org/10.1001/jama.2013.281053Kleiner, D.E., Brunt, E.M., Van Natta, M., et al. (2005) Design and Validation of a Histological Scoring System for Nonalcoholic Fatty Liver Disease. Hepatology, 41, 1313-1321. http://dx.doi.org/10.1002/hep.20701Brunt, E.M., Kleiner, D.E., Wilson, L.A., et al. (2009) NASH Clinical Research Network. Portal Chronic Inflammation in Nonalcoholic Fatty Liver Disease (NAFLD): A Histologic Marker of Advanced NAFLD-Clinicopathologic Correlations from the Nonalcoholic Steatohepatitis Clinical Research Network. Hepatology, 49, 809-820. http://dx.doi.org/10.1002/hep.22724Hedayati, K., Sharifi, F., Rostami, M.S., et al. (2012) Association between TNF-a Promoter G-308A and G-238A Polymorphisms and Obesity. Molecular Biology Reports, 39, 825-829. http://dx.doi.org/10.1007/s11033-011-0804-4Dutta, A.K. (2011) A New PCR-RFLP Method for Diagnosing PNPLA3 rs738409 Polymorphism. Webmed Central Genetics, 2, 11.Chan Y.H. ,et al. (2003)Biostatistics 102: Quantitative Data—Parametric & Non-Parametric Tests 44, 391-396.Chan Y.H. ,et al. (2003)Biostatistics 103: Qualitative Data—Tests of Independence 44, 498- 503.Angulo, P. (2002) Nonalcoholic Fatty Liver Disease. The New England Journal of Medicine, 18, 1221-1231. http://dx.doi.org/10.1056/NEJMra011775Farrell, G.C. (2003) Non-Alcoholic Steatohepatitis: What Is It, and Why Is It Important in the Asia-Pacific Region? Journal of Gastroenterology and Hepatology, 18, 124-138. http://dx.doi.org/10.1046/j.1440-1746.2003.02989.xWilfred de Alwis, N.M. and Day, C.P. (2007) Genetics of Alcoholic Liver Disease and Nonalcoholic Fatty Liver Disease. Seminars in Liver Disease, 27, 44-54. http://dx.doi.org/10.1055/s-2006-960170Day, C.P. and James, O.F. (1998) Steatohepatitis: A Tale of Two “Hits”? Gastroenterology, 114, 842-845. http://dx.doi.org/10.1016/S0016-5085(98)70599-2Kim, H.J., Kim, H.D., Lee, K.E., et al. (2004) Metabolic Significance of Nonalcoholic Fatty Liver Disease in Nonobese, Nondiabetic Adults. Archives of Internal Medicine, 164, 2169-2175. http://dx.doi.org/10.1001/archinte.164.19.2169Sorrentino, P., Tarantino, G., Conca, P., et al. (2004) Silent Non-Alcoholic Fatty Liver Disease a Clinical-Histological Study. Journal of Hepatology, 41, 751-757. http://dx.doi.org/10.1016/j.jhep.2004.07.010Amarapurkar, D., Kamani, P., Patel, N., et al. (2007) Prevalence of Non-Alcoholic Fatty Liver Disease: Population Based Study. Annals of Hepatology, 6, 161-163.Duseja, A., Das, A., Das, R., et al. (2007) The Clinicopathological Profile of Indian Patients with Nonalcoholic Fatty Liver Disease (NAFLD) Is Different from That in the West. Digestive Diseases and Sciences, 52, 2368-2374. http://dx.doi.org/10.1007/s10620-006-9136-yDixon, J.B., Bhathal, P.S. and O’Brian, P.E. (2001) Non-Alcoholic Fatty Liver Disease: Predictors of Non-Alcoholic Steatohepatitis and Liver Fibrosis in the Severely Obese. Gastroenterology, 121, 91-100. http://dx.doi.org/10.1053/gast.2001.25540Marchesini, G., Bugianesi, E., Forlani, G., et al. (2003) Non Alcoholic Fatty Liver, Steatohepatitis, and the Metabolic Syndrome. Hepatology, 37, 917-923. http://dx.doi.org/10.1053/jhep.2003.50161Harnois, F., Msika, S., Sabaté, J.M., et al. (2006) Prevalence and Predictive Factors of NASH in Morbidly Obese Patients Undergoing Bariatric Surgery. Obesity Surgery, 16, 183-188. http://dx.doi.org/10.1381/096089206775565122Caldwell, S.H. and Argo, C.K. (2011) Non-Alcoholic Fatty Liver Disease and Nutrition. In: Sherlock’s Diseases of the Liver and Biliary System, 12th Edition, 28, 546-561. http://dx.doi.org/10.1002/9781444341294.ch28Valenti, L., Al-Serri, A., Daly, A.K., et al. (2010) Homozygosity for the Patatin-Like Phospholipase-3/Adiponutrin I148 M Polymorphism Influences Liver Fibrosis in Patients with Nonalcoholic Fatty Liver Disease. Hepatology, 51, 1209-1217.Goran, M.I., Walker, R., Le, K.A., et al. (2010) Effects of PNPLA3 on Liver Fat and Metabolic Profile in Hispanic Children and Adolescents. Diabetes, 59, 3127-3130. http://dx.doi.org/10.2337/db10-0554Li, Y., Xing, C., Tian, Z. and Ku, H.C. (2012) Genetic Variant I148M in PNPLA3 Is Associated with the Ultrasonography-Determined Steatosis Degree in a Chinese Population. BMC Medical Genetics, 13, 113. http://dx.doi.org/10.1186/1471-2350-13-113Xu, R., Tao, A., Zhang, S., Deng, Y. and Chen, G. (2015) Association between Patatin-Like Phospholipase Domain Containing 3 Gene (PNPLA3) Polymorphisms and Nonalcoholic Fatty Liver Disease: A HuGE Review and Meta-Analysis. Scientific Reports, 5, 9284. http://dx.doi.org/10.1038/srep09284Zain, S.M. and Mohamed, R. (2012) A Multi-Ethnic Study of a PNPLA3 Gene Variant and Its Association with Disease Severity in Non-Alcoholic Fatty Liver Disease. Human Genetics, 131, 1145-1152. http://dx.doi.org/10.1007/s00439-012-1141-yHotta, K., Yoneda, M., Hyogo, H., et al. (2010) Association of the rs738409 Polymorphism in PNPLA3 with Liver Damage and the Development of Nonalcoholic Fatty Liver Disease. BMC Medical Genetics, 11, 172. http://dx.doi.org/10.1186/1471-2350-11-172Lin, Y.C., Chang, P.F., Hu, F.C., Yang, W.S., Chang, M.H. and Ni, Y.H. (2011) A Common Variant in the PNPLA3 Gene Is a Risk Factor for Non-Alcoholic Fatty Liver Disease in Obese Taiwanese Children. The Journal of Pediatrics, 158, 740-744. http://dx.doi.org/10.1016/j.jpeds.2010.11.016Romeo, S., Sentinelli, F., Cambuli, V.M., et al. (2010) The 148M Allele of the PNPLA3 Gene Is Associated with Indices of Liver Damage Early in Life. Journal of Hepatology, 53, 335-338. http://dx.doi.org/10.1016/j.jhep.2010.02.034Valenti, A., Alisi, E., Galmozzi, A., et al. (2010) I148M Patatin-Like Phospholipase Domain Containing 3 Gene Variant and Severity of Pediatric Nonalcoholic Fatty Liver Disease. Hepatology, 52, 1274-1280.Speliotes, E.K., Butler, J.L., Palmer, C.D., Voight, B.F. and Hirschhorn, J.N. (2010) PNPLA3 Variants Specifically Confer Increased Risk for Histologic Nonalcoholic Fatty Liver Disease But Not Metabolic Disease. Hepatology, 52, 904-912. http://dx.doi.org/10.1002/hep.23768Jou, J., Choi, S.S. and Diehl, A.M. (2008) Mechanisms of Disease Progression in Nonalcoholic Fatty Liver Disease. Seminars in Liver Disease, 28, 370-379. http://dx.doi.org/10.1055/s-0028-1091981Valenti, L., Fracanzani, A.L., Dongiovanni, P., et al. (2002) Tumor Necrosis Factor Alpha Promoter Polymorphisms and Insulin Resistance in Nonalcoholic Fatty Liver Disease. Gastroenterology, 122, 274-280. http://dx.doi.org/10.1053/gast.2002.31065Tokushige, K., Takakura, M., Tsuchiya-Matsushita, N., et al. (2007) Influence of TNF Gene Polymorphisms in Japanese Patients with NASH and Simple Steatosis. Journal of Hepatology, 46, 1104-1110. http://dx.doi.org/10.1016/j.jhep.2007.01.028Yang, H.R., Ko, J.S. and Seo, J.K. (2012) Role of Tumor Necrosis Factor-α Promoter Polymorphism and Insulin Resistance in the Development of Non-Alcoholic Fatty Liver Disease in Obese Children Pediatric Gastroenterology. Hepatology & Nutrition, 15, 44-51.Chowdhury, S.D., Ramakrishna, B., Eapen, C.E., et al. (2013) Fibrosis in Non-Alcoholic Fatty Liver Disease: Correlation with Simple Blood Indices and Association with Tumor Necrosis Factor-Alpha Polymorphisms. Tropical Gastroenterology, 34, 31-35. http://dx.doi.org/10.7869/tg.2012.88Trujillo-Murillo, K., Bosques-Padilla, F.J., Calderón-Lozano, I., et al. (2011) Association of Tumor Necrosis Factor α and Manganese Superoxide Dismutase Polymorphisms in Patients with Non-Alcoholic Steatohepatitis from Northeast Mexico. The Open Hepatology Journal, 3, 1-6. http://dx.doi.org/10.2174/1876517301103010001Cheng, Y., An, B., Jiang, M., Xin, Y. and Xuan, S. (2015) Association of Tumor Necrosis Factor-Alpha Polymorphisms and Risk of Coronary Artery Disease in Patients with Non-Alcoholic Fatty Liver Disease. Hepatology, 15, e26818. http://dx.doi.org/10.5812/hepatmon.26818