GENETIC TESTING AND MOLECULAR BIOMARKERS Volume 18, Number 7, 2014 ª Mary Ann Liebert, Inc. Pp. 489–496 DOI: 10.1089/gtmb.2014.0019
Genetic Variants in the PNPLA3 Gene Are Associated with Nonalcoholic Steatohepatitis Eylul Ece Islek,1 Ali Sazci,1 Mavi Deniz Ozel,1 and Cem Aygun 2
In this study, we report the association of the rs738407, rs738409, and rs2896019 variants of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) (adiponutrin) gene with nonalcoholic steatohepatitis (NASH) (v2 = 14.528, p = 0.001; v2 = 18.882, p = 0.000; v2 = 7.449, p = 0.024, respectively) in 80 patients with NASH and 303 healthy controls. We genotyped the subjects using three polymerase chain reaction–restriction fragment length polymorphism methods developed in our laboratory. Our findings confirm the findings of the recent case–control and genome-wide association studies carried out in different populations around the world. Thus, the three variants in PNPLA3 gene may be a genetic risk factor for NASH.
Introduction
N
onalcoholic fatty liver disease (NAFLD) includes a spectrum of conditions ranging from simple hepatic steatosis to potentially fatal nonalcoholic steatohepatitis (NASH) (Matteoni et al., 1999). NASH is characterized by hepatic steatosis, inflammation, ballooning, and fibrosis (Kleiner and Brunt, 2012). NASH may progress to cirrhosis in 15% of subjects (Harrison and Neuschwander-Tetri, 2004). Accumulation of excess fat in the liver is reported to be 20–30% in the USA and European countries in which NASH is observed in 1–3% of the population (Ludwig et al., 1980). Genetics, epigenetics, and environment are underlying factors in the development of NASH (Wilfred de Alwis and Day, 2008; Sazci et al., 2013). Recently, we reported that single-nucleotide polymorphisms (SNPs) in apolipoprotein E (APOE), methylenetetrahydrofolate reductase (MTHFR), and nicotinamide-N-methyltransferase (NNMT) genes are associated with the risk of NASH in the Turkish population (Sazci et al., 2008a, 2008b; Sazci et al., 2013). Genome-wide association studies (GWASs) have recently revealed that SNPs in the patatin-like phospholipase domaincontaining protein 3 (PNPLA3) gene are risk factors for the development of NAFLD (Romeo et al., 2008; Chalasani et al., 2010; Speliotes et al., 2010, 2011; Kawaguchi et al., 2012; Kitamoto et al., 2013). Although the mechanisms leading to NASH still remain unclear, the fact that dysregulation of lipid metabolism was involved (Erickson, 2006; Sazci et al., 2008a) indicated that 30% of the triglyceride (TG) content in NAFLD came from de novo lipogenesis, thus underlying the significance of this pathway in the etiology of the disease (Donnelly et al., 2005; Postic and Girard, 2008).
Hepatic de novo lipogenesis is modulated transcriptionally by the transcription factors, sterol regulatory elementbinding protein 1c (SREBP1c) and carbohydrate response element-binding protein (ChREBP), mediating the effects of insulin and glucose on glycolytic and lipogenic genes (Shimomura et al., 1999; Ishii et al., 2004; Fere and Foufelle, 2007; Postic et al., 2007). The way insulin enhances SREBP1c gene expression is by inducing its binding to the sterol-response element in the promoters of target genes (Fere and Foufelle, 2007), whereas ChREBP gene mediates its activity through the formation of a heterotetramer with Maxlike protein X (Mlx), thus resulting in an efficient binding to ChoRE sequences (Stoeckman et al., 2004; Ma et al., 2005). Inhibition of the expression of SREBP1c and ChREBP genes in the liver of obese ob/ob mice alleviates hepatic steatosis, thus endorsing their role in the lipogenic pathway under physiological and pathophysiological conditions (Yahagi et al., 2002; Dentin et al., 2006). The aim of the current study was to determine whether the three variants, rs738407, rs738409, and rs2896019, of the PNPLA3 gene were associated with the risk of NASH in the Turkish population. Materials and Methods Study population
A total of 80 histologically diagnosed NASH patients were recruited from the University of Kocaeli Hospital Gastroenterology Clinic. Biopsy specimens were stained with hematoxylin and eosin and Masson’s trichrome for morphological evaluation and assessment of fibrosis. The histological grade was evaluated by the classification of Brunt et al. (1999). The demographic data on NASH patients
Departments of 1Medical Biology and Genetics and 2Gastroenterology, Faculty of Medicine, University of Kocaeli, Kocaeli, Turkey.
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and controls were previously described (Sazci et al., 2008a). Inclusion criteria for NASH patients were as follows: (1) no history of alcohol use, (2) no history of HBV/HCV/HIV infection, (3) diagnosis of liver biopsy, and (4) availability of other demographic data. The control population shared the same genetic and social background. We had 303 wellmatched controls who were described previously (Sazci et al., 2008a). The institutional review board approved the study, and informed consent was obtained from each subject who participated in the study. Genotyping
Genomic DNA was isolated from subjects using a conventional salting-out method (Miller et al., 1988). Genotypes of the subjects were determined by the polymerase chain reaction–restriction fragment length polymorphism (PCRRFLP) method developed in our laboratory (Figs. 1–3). The PCR cycling conditions for the rs738407 variant were as follows: briefly, the genomic DNA was denatured at 95C for 5 min, followed by 35 cycles at 95C for 1 min, 56C for 1 min, 72C for 1 min, and a final extension step at 72C for 10 min. The digestion of the amplified 235 bp fragment with the restriction endonuclease MluC1 was carried out at 37C overnight. Subsequently, the digested fragment was run on an 8% polyacrylamide gel electro-
ISLEK ET AL.
phoresis (PAGE) at 20 W for 35 min, followed by silver staining and scanning (Fig. 1). The PCR cycling conditions for the rs738409 variant were as follows: initial denaturation at 95C for 5 min, followed by 35 cycles at 95C for 1 min, 58C for 30 s, 72C for 1 min, and a final extention step at 72C for 10 min. The digestion of the amplified 139 bp fragment with the restriction endonuclease NlaIII was carried out at 37C overnight. Subsequently, the digested fragment was run on an 8% PAGE at 20 W for 30 min followed by siver staining and scanning (Fig. 2). The PCR cycling conditions for the rs2896019 variant were as follows: initial denaturation at 95C for 5 min, followed by 35 cycles at 95C for 1 min, 55C for 30 s, 72C for 1 min, and a final extention step at 72C for 10 min. The digestion of the amplified 206 bp fragment with the restriction endonuclease SfcI was carried out at 37C overnight. Subsequently, the digested fragment was run on an 8% PAGE at 20 W for 30 min followed by silver staining and scanning (Fig. 3). Statistical analysis
A case–control association analysis was carried out using SPSS v21 (Chicago, IL). The association of alleles and genotypes was carried out using a logistic regression analysis.
FIG. 1. A schematic illustration of the patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene showing the location of MluCI recognition sequences (5¢.AATT.3¢) relative to the primers annealing sites. The forward and reverse primers depicted on the gene after amplification can create a fragment of 235 bp, of which there is always an MluCI site at the position of 44323955 bp. Below is the polyacrylamide gel electrophoresis (PAGE) of PNPLA3 gene. The amplified 235 bp fragment was cut with the restriction endonuclease and run on an 8% PAGE at 20 W for 30 min followed by silver staining. Lane M showing the marker (M); lane GG showing the GG genotype with two fragments of 204 and 31 bp; lane AG showing the AG genotype with four fragments of 204, 179, 31, and 25 bp; lane AA showing the AA genotype with three fragments of 179, 31, and 25 bp.
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FIG. 2. A schematic illustration of the PNPLA3 gene showing the location of NlaIII recognition sequences (5¢.CATG.3¢) relative to the primers annealing sites. The forward and reverse primers depicted on the gene after amplification can create a fragment of 139 bp, of which there is an NlaIII site at the position of 44324727 bp. Below is the PAGE of PNPLA3 gene. The amplified 139 bp fragment was cut with the restriction endonuclease and run on an 8% PAGE at 20 W for 30 min followed by silver staining. Lane M showing the marker (M); lane CC showing the CC genotype with one fragment of 139 bp; lane GC showing the GC genotype with three fragments of 139, 112, and 27 bp; lane GG showing the GG genotype with two fragments of 112 and 27 bp.
FIG. 3. A schematic illustration of the PNPLA3 gene showing the location of SfcI recognition sequences (5¢.CTRYAG.3¢) relative to the primers annealing sites. The forward and reverse primers depicted on the gene after amplification can create a fragment of 206 bp, of which there is an SfcI site at the position of 44333694 bp. Below is the PAGE of PNPLA3 gene. The amplified 206 bp fragment was cut with the restriction endonuclease SfcI and run on an 8% PAGE at 20 W for 30 min followed by silver staining. Lane M showing the marker (M); lane TT showing the TT genotype with one fragment of 206 bp; lane TG showing the TG genotype with three fragments of 206, 163, and 43 bp; lane GG showing the GG genotype with two fragments of 163 and 43 bp.
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A p-value < 0.05 was considered statistically significant. Hardy–Weinberg equilibrium was verified for both the groups (http://ihg.gsf.de/cgi-bin/hw/hwa2.pl). Results
The demographic and clinical data of the NASH and control groups were previously described (Sazci et al., 2008a). In the present study, we determined alleles and genotypes of three SNPs, namely rs738407, rs738409, and rs2896019, using the PCR-RFLP methods described in the Materials and Methods section. Genotype and allele frequencies of PNPLA3 gene rs738407 polymorphism
MluCI restriction endonuclease was used for the determination of genotypes of the rs738407 variant of PNPLA3 gene (Fig. 1). The rs738407 variant of PNPLA3 gene was associated with the risk of NASH (v2 = 14.528, p = 0.001). Individuals with the AA genotype had protection against NASH (v2 = 14.484, p = 0.000, odds ratio [OR] = 0.304, 95% confi-
dence interval [95% CI] = 0.161–0.575), whereas individuals with the AG genotype had a 2.020-fold increased risk of NASH (v2 = 7.644, p = 0.006, OR = 2.020, 95% CI = 1.221– 3.344). The G allele was statistically significantly associated with the risk of NASH (v2 = 14.484, p = 0.000, OR = 3.287, 95% CI = 1.738–6.218). Stratification analysis according to the gender revealed that the rs738407 variant in male patients with NASH was significantly associated with the risk of NASH (v2 = 9.489, p = 0.009). The AA genotype was protective against NASH in female patients with NASH (v2 = 5.299, p = 0.021, OR = 0.381, 95% CI = 0.164–0.884). The G allele showed a 2.626-fold increased risk of NASH (v2 = 5.299, p = 0.021, OR = 2.626, 95% CI = 1.131–6.099). There were also allelic and genotypic associations with NASH (Table 1). Genotype and allele frequencies of PNPLA3 gene rs738409 polymorphism
NlaIII restriction endonuclease was used for the determination of genotypes of the rs738409 variant of PNPLA3 gene (Fig. 2). The rs738409 variant of PNPLA3 gene was
Table 1. Allele and Genotype Frequencies of PNPLA3 Gene rs738407 Variant in Patients with NASH and Controls, and in Female and Male Patients with NASH and in Female and Male Controls Gene PNPLA3 rs738407 AA AG GG Allele frequency A allele G allele HWE (exact)
w2
p-Value
OR (95% CI)
(100.0) (38.9) (43.9) (17.2)
14.528 14.484 7.644 1.208
0.001 0.000 0.006 0.272
0.304 (0.161–0.575) 2.020 (1.221–3.344) 1.401 (0.766–2.563)
75 (46.87) 85 (53.13) 0.070
369 (60.89) 237 (39.11) 0.185
1.208 14.484
0.272 0.000
0.714 (0.390–1.305) 3.287 (1.738–6.218)
Cases
Controls
Male
Male
Cases 80 13 49 18
Gene PNPLA3 rs738407 AA AG GG Allele frequency A allele G allele HWE (exact)
303 118 133 52
w2
p-Value
OR (95% CI)
(100.0) (37.5) (44.7) (17.8)
9.489 9.483 4.509 0.915
0.009 0.002 0.034 0.339
0.231 (0.086–0.624) 2.141 (1.051–4.361) 1.493 (0.654–3.409)
36 (43.90) 46 (56.10) 0.113
182 (59.87) 122 (40.13) 0.401
0.915 9.483
0.339 0.002
0.670 (0.293–1.528) 4.320 (1.603–11.642)
Cases
Controls
Female
Female
Gene PNPLA3 rs738407 AA AG GG Allele frequency A allele G allele HWE (exact)
(100.0) (16.3) (61.3) (22.5)
Controls
41 5 26 10
(100.0) (12.2) (63.4) (24.4)
39 8 23 8
(100.0) (20.5) (59.0) (20.5)
39 (50.00) 39 (50.00) 0.347
152 57 68 27
w2
p-Value
OR (95% CI)
(100.0) (40.4) (43.0) (16.6)
5.352 5.299 3.163 0.338
0.069 0.021 0.075 0.561
0.381 (0.164–0.884) 1.902 (0.931–3.887) 1.301 (0.535–3.160)
187 (61.92) 115 (38.08) 0.3013
0.338 5.299
0.561 0.021
0.769 (0.316–1.868) 2.626 (1.131–6.099)
151 61 65 25
95% CI, 95% confidence interval; HWE, Hardy–Weinberg equilibrium; NASH, nonalcoholic steatohepatitis; OR, odds ratio; PNPLA3, patatin-like phospholipase domain-containing protein 3.
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statistically significantly associated with the risk of NASH (v2 = 18.882, p = 0.000). Individuals with the CC genotype had protection against NASH (v2 = 13.141, p = 0.000, OR = 0.380, 95% CI = 0.223–0.649), whereas individuals with the GG genotype had a 3.359-fold increased risk of NASH (v2 = 11.044, p = 0.001, OR = 3.359, 95% CI = 1.590–7.096). The C allele was protective against NASH (v2 = 11.044, p = 0.001, OR = 0.298, 95% CI = 0.141–0.629), whereas the G allele was a genetic risk factor for NASH (v2 = 13.141, p = 0.000, OR = 2.630, 95% CI = 1.542–4.486). Both cases and controls were in Hardy–Weinberg equilibrium (Table 2). Stratification analysis according to the gender suggested that the rs738409 variant was associated with NASH in both male and female NASH patients (v2 = 10.585, p = 0.005; v2 = 8.568, p = 0.014, respectively) (Table 2). There were allelic and genotypic associations in both male and female patients with NASH (Table 2). Genotype and allele frequencies of PNPLA3 gene rs2896019 polymorphism
SfcI restriction endonuclease was used for the determination of genotypes of the rs2896019 variant of the PNPLA3 gene (Fig. 3). The rs2896019 variant of the PNPLA3 gene was associated with the risk of NASH (v2 = 7.449, p = 0.024).
Individuals with the TT genotype had protection against NASH (v2 = 6.804, p = 0.009, OR = 0.519, 95% CI = 0.316– 0.853). Individuals with the G allele had a 1.927-fold increased risk of NASH (v2 = 6.804, p = 0.009, OR = 1.927, 95% CI = 1.172–3.168). Stratification analysis according to the gender revealed that male patients with NASH had the TT genotype (v2 = 6.262, p = 0.012, OR = 0.413, 95% CI = 0.205– 0.835) and the G allele (v2 = 6.262, p = 0.012, OR = 2.420, 95% CI = 1.198–4.890) associated with NASH, whereas there was no association in female patients with NASH (v2 = 2.927, p = 0.231) (Table 3). Both cases and controls were in Hardy– Weinberg equilibrium (Table 3). Discussion
In the present study, we show the association of rs738407, rs738409, and rs2896019 variants of the PNPLA3 gene with the risk of NASH as it has been shown in case–control and GWASs worldwide (Romeo et al., 2008; Hotta et al., 2010; Rotman et al., 2010; Speliotes et al., 2010, 2011; Sookoian and Pirola, 2011; Kawaguchi et al., 2012; Peng et al., 2012; Zain et al., 2012; Guyot et al., 2013; Kitamoto et al., 2013). The PNPLA3 (NM 025225.2) gene is located in the long arm of chromosome 22 at 22q13.31 between the 44.319.619 bp and 44.360.368 bp and has a sequence of 40.750 bp in length.
Table 2. Allele and Genotype Frequencies of PNPLA3 Gene rs738409 Variant in Patients with NASH and Controls, and in Female and Male Patients with NASH and in Female and Male Controls Gene PNPLA3 rs738409 CC CG GG Allele frequency C Allele G Allele HWE (exact)
Cases 80 23 43 14
Gene PNPLA3 rs738409 CC CG GG Allele frequency C Allele G Allele HWE (exact)
303 156 129 18
(100.0) (51.5) (42.6) (5.9)
89 (0.56) 71 (0.44) 0.501
441 (0.72.77) 165 (0.27.23) 0.246
Cases
Controls
Male
Male
Gene PNPLA3 rs738409 CC CG GG Allele frequency C Allele G Allele HWE (exact)
(100.0) (28.8) (53.8) (17.5)
Controls
41 12 22 7
(100.0) (29.3) (53.7) (17.1)
46 (0.56) 36 (0.44) 0.753
152 83 60 9
(100.0) (54.6) (39.5) (5.9)
226 (0.74.34) 78 (0.25.66) 0.832
Cases
Controls
Female
Female
39 11 21 7
(100.0) (28.2) (53.8) (17.9)
43 (0.55) 35 (0.45) 0.749
151 73 69 9
(100.0) (48.3) (45.7) (6.0)
215 (0.71.19) 87 (0.28.81) 0.232
w2
p-Value
OR (95% CI)
18.882 13.141 3.195 11.044
0.000 0.000 0.074 0.001
0.380 (0.223–0.649) 1.568 (0.956–2.572) 3.359 (1.590–7.096)
11.044 13.141
0.001 0.000
0.298 (0.141–0.629) 2.630 (1.542–4.486)
w2
p-Value
OR (95% CI)
10.585 8.294 2.659 5.282
0.005 0.004 0.103 0.022
0.344 (0.163–0.724) 1.775 (0.886–3.556) 3.271 (1.138–9.406)
5.282 8.294
0.022 0.004
0.306 (0.106–0.879) 2.907 (1.381–6.121)
w2
p-Value
OR (95% CI)
8.568 5.097 0.826 5.776
0.014 0.024 0.363 0.016
0.420 (0.195–0.904) 1.386 (0.684–2.810) 3.451 (1.196–9.958)
5.776 5.097
0.016 0.024
0.290 (0.100–0.836) 2.382 (1.106–5.130)
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Table 3. Allele and Genotype Frequencies of PNPLA3 Gene rs2896019 Variant in Patients with NASH and Controls, and in Female and Male Patients with NASH and in Female and Male Controls w2
P-Value
OR (95% CI)
(100.0) (62.4) (34.7) (3.0)
7.449 6.804 4.464 1.933
0.024 0.009 0.035 0.164
0.519 (0.316–0.853) 1.706 (1.036–2.809) 2.178 (0.709–6.689)
112 (70.0) 48 (30.0) 0.298
483 (79.70) 123 (20.30) 0.286
1.933 6.804
0.164 0.009
0.459 (0.149–1.410) 1.927 (1.172–3.168)
Cases
Controls
Male
Male
w2
p-Value
OR (95% CI)
(100.0) (63.2) (33.6) (3.3)
6.271 6.262 5.550 0.233
0.043 0.012 0.018 0.629
0.413 (0.205–0.835) 2.293 (1.139–4.618) 1.508 (0.282–8.069)
56 (68.29) 26 (31.71) 0.274
243 (79.93) 61 (20.07) 0.799
0.233 6.262
0.629 0.012
0.663 (0.124–3.550) 2.420 (1.198–4.890)
Cases
Controls
Female
Female
Gene PNPLA3 rs2896019 TT TG GG Allele frequency T allele G allele HWE (exact)
Cases 80 37 38 5
Gene PNPLA3 rs2896019 TT TG GG Allele frequency T allele G allele HWE (exact)
Gene PNPLA3 rs2896019 TT TG GG Allele frequency T allele G allele HWE (exact)
41 17 22 2
39 20 16 3
(100.0) (46.3) (47.5) (6.3)
(100.0) (41.5) (53.7) (4.9)
(100.0) (51.3) (41.0) (7.7)
56 (71.79) 22 (28.21) 1.0
Controls 303 189 105 9
152 96 51 5
w2
p-Value
OR (95% CI)
(100.0) (61.6) (35.8) (2.6)
2.927 1.366 0.369 2.222
0.231 0.242 0.543 0.136
0.656 (0.323–1.333) 1.250 (0.608–2.566) 3.063 (0.656–14.295)
240 (79.47) 62 (20.53) 0.322
2.222 1.366
0.136 0.242
0.344 (0.070–1.524) 1.523 (0.750–3.093)
151 93 54 4
The mature protein is 481 amino acids in length and 52.865 kDa. It is a single-pass type II membrane protein with both triacylglycerol lipase and acylglycerol o-acyltransferase activities. It is involved in the triacylglycerol hydrolysis in adipocytes. The expression of PNPLA3 gene is under metabolic control in adipocytes in the liver. Under fasting conditions, mRNA levels are low and increase dramatically with carbohydrate feeding (Baulande et al., 2001; Lake et al., 2005). The PNPLA3 gene has activity against TG in vitro and can also transfer fatty acids to and from mono- and diacylglycerol ( Jenkins et al., 2004). The isoleucine to methionine substitution at position 148 (I148M) of PNPLA3 gene is the most replicated genetic variant (rs738409) strongly associated with the entire spectrum of chronic liver disease (Romeo et al., 2008, 2010; Tian et al., 2010; Valenti et al., 2010, 2011). PNPLA3 has a patatin-like domain at the N terminus (residues 10–179, UniProt http://pir.uniprot.org/uniprot/) with consensus sequences for a Ser-Asp catalytic dyad (Gly-X-Ser-X-Gly and Asp-X-Gly/Ala) (Wilson et al., 2006). The I148M substitution (rs738409) is located between the consensus sequences for the catalytic dyad and is highly conserved. The PNPLA3 148M allele is associated with higher liver fat content when compared with the 148I allele (Romeo et al., 2008; Kotronen
et al., 2009). The G allele of rs738409 in the PNPLA3 gene is associated with histological steatosis as well as NASH, fibrosis, and cirrhosis (Speliotes et al., 2011). The rs2896019 and rs738407 variants of the PNPLA3 gene are located in the noncoding region of the gene and are associated with NAFLD (Kawaguchi et al., 2012; Kitamoto et al., 2013). rs738409 and rs2896019 are associated with fibrosis (Kitamoto et al., 2013). Among the three SNPs we studied, the rs738409 variant most robustly associated with NASH (Table 2). The risk G allele of PNPLA3 rs738409 is strongly associated with the risk of NAFLD and also with increases in aspartate transaminase (AST) and alanine transaminase (ALT), ferritin levels, and fibrosis stage in the patients with NAFLD in the Japanese population (Hotta et al., 2010). The G allele frequency of the rs738407 was significantly higher in the patients with NASH (53.13%) when compared with the controls (39.11%). The G allele frequency in Japanese is 72% in NAFLD and 63% in controls (Kawaguchi et al., 2012). The G allele frequency of the rs738409 variant was 27% in controls and 44% in cases. There seem to be differences in the allelic distributions of rs738409 among ethnic groups, such as G allele frequencies in a decending order in Mexican children: 61% (Larrieta-Carrasco et al., 2013), in American Hispanics: 49% (Romeo et al., 2008), in Japanese: 48% (Kawaguchi
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et al., 2012), Han Chinese: 34% (Peng et al., 2012), in Turkish: 27% (Table 2), in obese Italians: 26% (Romeo et al., 2010), in Malays: 24% (Zain et al., 2012), in European Americans: 23% (Romeo et al., 2008; Rotman et al., 2010), in Indians: 18% (Zain et al., 2012), and African Americans: 17% (Romeo et al., 2008). There seem to be differences in the allelic distributions of rs2896019 among ethnic groups: the G allele frequency in Japanese: 45% (Kitamoto et al., 2013) and 48% (Kawaguchi et al., 2012), in Turkish: 20% (Table 3). The PNPLA3 gene is believed to be involved in the abnormal lipid metabolism in the liver of NAFLD patients. Mice deficient in the PNPLA3 gene and transgenic mice did not develop fatty liver (Chen et al., 2010; Basantani et al., 2011; Li et al., 2012). However, overexpression of the PNPLA3 I148M variant in mouse liver resulted in fatty liver (Li et al., 2012). Therefore, the PNPLA3 gene variants may play an important role in the development of NASH. The SAMM50 and PARVB genes have been recently reported to be associated with NAFLD (Kitamoto et al., 2013). The SAMM50 gene may be involved in mitochondrial dysfunction and inability to remove reactive oxygen species, thus resulting in the development of NAFLD (Kitamoto et al., 2013). The PARVB gene is thought to play a role in lipid accumulation and fibrosis in the liver, thus leading to NAFLD (Desgrosellier and Cheresh, 2010; Kimura et al., 2010; Patsenker and Stickel, 2011; Kitamoto et al., 2013). In conclusion, we report the association of the rs738407, rs738409, and rs2896019 variants of the PNPLA3 gene with the risk of NASH in overall subjects as well as genders on the genotypic and allelic levels. The degree of variable association may also reveal the stages in which the variants were involved in NASH. In other words, staging in NASH may be created by differential expression of SNPs. Acknowledgment
The study was supported by a Grant from the University of Kocaeli Research Fund to A.S. Author Disclosure Statement
The authors declare no conflicts of interest. References
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Address correspondence to: Ali Sazci, PhD Department of Medical Biology and Genetics Faculty of Medicine University of Kocaeli Kocaeli 41380 Turkey E-mail:
[email protected] 