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.

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] .

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).

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] .

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.

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.