Sociodemographic and genetic determinants of nonalcoholic fatty liver disease in type 2 diabetes mellitus patients

Background Nonalcoholic fatty liver disease (NAFLD) showed significant association with PNPLA3 rs738409 polymorphism in unrelated individuals. However, it is still unknown whether the relationship of NAFLD with PNPLA3 variant exists or not among subjects with type 2 diabetes mellitus (T2DM). Therefore, the study aimed to evaluate sociodemographic and genetic determinants of NAFLD in type 2 diabetics. Methods The cross-sectional analytical study was conducted at the Department of Molecular Biology, Virtual University of Pakistan, Lahore, Pakistan, during 2019–2020. A total of 153 known cases of T2DM were enrolled using convenience sampling. After excluding patients (n = 24) with HCV, alcoholism, or missing information, data from 129 eligible diabetics with and without NAFLD were analyzed using SPSS. Odds ratios using crosstabs and adjusted odds ratios using binary and multinomial logistic regression were calculated to measure the risk of NAFLD. Results Adults 18–35 years were 7.0%, 36–55 years were 64.3%, ≥ 56 years were 28.7%, and females were 66.7%. A total of 41.1% of patients had obesity, 52.7% had NAFLD, and 29.05% carried mutant G allele of rs738409 polymorphism. Among overall diabetics, NAFLD showed association with female (OR = 2.998, p = 0.007), illiterate (OR = 3.067, p = 0.005), and obese (OR = 2.211, p = 0.046) but not with PNPLA3 genotype under any model (all p = > 0.05). Among obese diabetics, NAFLD showed association with female (AOR = 4.010, p = 0.029), illiterate (AOR = 3.506, p = 0.037), GG + CG/CC (AOR = 3.303, p = 0.033), and GG/CG + CC (AOR = 4.547, p = 0.034) using binary regression and with female (AOR = 3.411, p = 0.051), illiterate (AOR = 3.323, p = 0.048), GG + CG/CC (AOR = 3.270, p = 0.029), and GG/CG + CC (AOR = 4.534, p = 0.024) using multinomial regression. Conclusions NAFLD and obesity were the most common comorbid diseases of T2DM. Gender female, being illiterate, and PNPLA3 rs738409 polymorphism were significant risk factors of NAFLD among obese diabetic patients.


Background
Nonalcoholic fatty liver disease (NAFLD) is a polygenic and heritable disorder [1], characterized by excess accumulation of fat in the liver parenchyma without history of alcoholism and hepatitis [2]. Its prevalence is 25.0% in the general adult population [3] and 50.0 to 70.0% in patients with type 2 diabetes mellitus (T2DM) [4]. NAFLD increases the risk of developing T2DM, whereas diabetes increases the progression of NAFLD to nonalcoholic steatohepatitis (NASH) and the risk of cirrhosis and hepatocellular carcinoma (HCC). Hence, a two-way relationship is present between NAFLD and T2DM [5].
Various demographic and genetic factors demonstrated greater risk for NAFLD. Age [6,7], gender [6][7][8], ethnicity [9], metabolic syndrome (MetS), and its components including dyslipidemia, obesity, hypertension (HTN), and T2DM [10] were associated with the risk of NAFLD. Genetic factors such as the patatin-like phospholipase domain containing 3 (PNPLA3), the transmembrane 6 superfamily member 2 (TM6SF2), the membrane-bound O-acyltransferase domain containing 7 (MBOAT7), and the glucokinase regulator (GCKR) [11] were also associated with the risk of NAFLD. The product of human PNPLA3 gene, i.e., triacylglycerol lipase enzyme mediates hydrolysis of triacylglycerol (TAG) in adipocytes. However, the substitution of isoleucine with methionine at position 148 (I148M) causes a loss of function [12]. Genome-wide association studies (GWAS) identified several genes such as the glucokinase (GCK), the glucokinase regulator (GCKR), the transcription factor 7-like 2 (TCF7L2), the hepatocyte nuclear factor-1A (HNF1A), and fat mass and obesity-associated (FTO) gene playing a role in developing T2DM [13]. In people with T2DM, PNPLA3 I148M or rs738409 polymorphism showed significant association with liver fibrosis independent of body mass index (BMI) [14] and with the risk of increased liver fat content (LFC) independent of serum lipids [15], but not with susceptibility of NAFLD [16]. However, it revealed association with the risk and severity of NAFLD in meta-analyses of case-control studies on unrelated individuals [17][18][19]. The variations across studies in terms of characteristics of study population, selection of controls, laboratory methods, and statistical approaches lead to the ambiguity, whether or not these demographic and genetic factors, particularly rs738409 polymorphism, are associated with NAFLD in T2DM cases. Therefore, the present study aimed to determine the sociodemographic and genetic determinants of NAFLD in T2DM patients.

Ethical approval
The study was approved by the subcommittee of Advanced Studies and Research Board (ASRB) of the Faculty of Science and Technology, Virtual University of Pakistan, Lahore, Pakistan, vide letter no.VU/ ASRB/214-5 dated December 02, 2019. Written informed consent was sought from all volunteer patients.

Design, setting, and duration of study
The cross-sectional analytical study was conducted at the Department of Molecular Biology, Virtual University of Pakistan, Lahore, Pakistan, during 2019-2020.

Characteristics, size, and selection of sample
A total of 153 known T2DM patients, of age 18-90 years, both male and female patients, belonging to any income group, caste or area of Pakistan, were enrolled by nonprobability convenience sampling technique. None of 153 patients was reactive to hepatitis B surface antigen (HBsAg); however, patients reactive to anti-HCV antibodies (6.5%), patients with γ-GT levels ≥ 55 IU/L or history of alcoholism (8.5%), and patients with incomplete data (0.7%) were excluded.

Data collection procedure
An interviewer-administered close-ended proforma was used to record age, sex, education, family income, cigarette smoking, co-illness, duration of diabetes, and family history of diabetes.

PCR-RFLP
The genomic deoxyribonucleic acid (DNA) was extracted by using the GeneJET Whole Blood Genomic DNA Purification Mini Kit. The amplification of PNPLA3 gene was performed by PCR using the forward primer (5′-TGG GCC TGA AGT CCG AGG GT-3′) and the reverse primer (5′-CCG ACA CCA GTG CCC TGC AG-3′) as reported by Dutta (2011) [20]. The composition of PCR reaction mix and the optimized conditions are shown in Table 1. The restriction of amplified PNPLA3 gene product (333 bp) was performed by using the BtsCI enzyme. The composition of the RFLP reaction mix is also shown in

Discussion
NAFLD is a multisystem disease that not only results in progressive liver disease but also affects extrahepatic organs [21]. Various demographic [6][7][8][9], clinical [10], and genetic factors [11] showed association with the risk of NAFLD. Among genetic risk factors, PNPLA3 Table 3 Factors associated with NAFLD in T2DM patients (n = 129) a n = 129; b n = 108; c n = 102; OR, odds ratio; CI, confidence interval; NAFLD, nonalcoholic fatty liver disease; T2DM, type 2 diabetes mellitus. rs738409 polymorphism demonstrated significant association with NAFLD. However, the characteristics of the study population varied across studies [17][18][19]. It is still unknown whether the association of PNPLA3 rs738409 polymorphism with NAFLD exists or not among type 2 diabetic patients. Therefore, the present study aimed to evaluate the sociodemographic and genetic determinants of NAFLD in T2DM patients. The results showed that PNPLA3 rs738409 polymorphism had higher risk for NAFLD under codominant, dominant, and recessive models, but was not associated with NAFLD in Pakistani adults with T2DM (all p > 0.05). It is in agreement with the results of Hsieh et al. (2015) who reported that rs738409 polymorphism had no association with NAFLD in Taiwanese patients with T2DM (p = 0.344) [16].
The prevalence of NAFLD is 50.0 to 70.0% in diabetic subjects [4]. An equivalent higher frequency of NAFLD 52.7% is obtained in the present study. Among sociodemographic factors, age, gender, obesity, and ethnicity are the most frequently reported risk factors for NAFLD. Hu et al. (2018) observed an increasing trend between advance age and occurrence of NAFLD (OR = 1.049; p = 0.607), but after adjustment, age had an inverse relation with NAFLD in adult Chinese (OR = 0.844; p = 0.157) [7]. Ferreira et al. (2010) also reported that age was not related with NAFLD in adults with T2DM (57.1 ± 10.9 vs. 57.6 ± 9.5 years; p = 0.818) [6]. In the same way, the present study found no significant relation between age and NAFLD (OR = 1.459; p = 0.382); however, an opposite trend was observed, where the highest frequency of NAFLD 57.1% was in age < 40 years and the lowest 42.9% in age > 60 years. Hu et al. (2018) reported that gender male was significantly associated with NAFLD in Chinese adults (OR = 3.484; p = < 0.001) [7]. Oppositely, Summart et al. (2017) reported that females had higher risk (OR = 1.3, 1.2-1.4) for NAFLD in Thai adults (> 40 years) [8]. Differently, Ferreira et al. (2010) reported that gender female was not associated with NAFLD in adults with T2DM (p = 0.939) [6], whereas gender female revealed significantly higher risk for NAFLD in the present study  Education status also showed a significant relationship with the risk for NAFLD in the present study. Illiterates had significantly higher risk for NAFLD (OR = 3.067; p = 0.005); and after adjustment, risk for NAFLD was further increased in obese illiterates (AOR = 3.506; p = 0.037). Similar significant relation between low education level and FLD (OR = 0.704; p = 0.001) or NAFLD had been reported in other studies [22,23]. The present study also showed association of elevated ALT levels with PNPLA3 genotype GG/CC (p = 0.024), GG + CG/CC (p = 0.054), and GG/CG + CC (p = 0.018), which is consistent with other studies, where rs738409 polymorphism was associated with elevated AST levels (p = 0.039) [24] and ALT levels (d = 0.47) [17]. The human PNPLA3 gene is expressed in various tissues of the body mainly in the liver. Its gene product, i.e., triacylglycerol lipase enzyme, mediates hydrolysis of TAG in adipocytes. However, PNPLA3 rs738409 polymorphism causes loss of enzyme function resulting in the accumulation of TAG in the liver [12]. PNPLA3 variants are the most common genetic risk factors leading to NAFLD in obese across different ethnic groups [25]. PNPLA3 rs738409 variant had been reported as a significant risk factor for NAFLD in all genetic models [19] and in codominant model [17,24,26] among different study populations. However, the present study found no relationship between rs738409 polymorphism and risk of NAFLD in the whole T2DM group, which is in agreement with the findings of Hsieh et al. (2015) [16]. Differently, both binary and multinomial logistic regression analyses in the present study revealed that rs738409 polymorphism was significantly associated with NAFLD in obese diabetics, under dominant and recessive models (all p < 0.05).

Conclusions
NAFLD and obesity were the most common comorbid diseases of T2DM in the setting. Gender female, being illiterate, and PNPLA3 rs738409 polymorphism were significant risk factors of NAFLD among obese diabetic patients. Further research studies are needed to evaluate the association of PNPLA3 rs738409 polymorphism and other genetic factors with the NAFLD particularly among obese diabetics.