A Meta-Analysis of the Association between PNPLA3 rs738409 C>G Polymorphism and Non-alcoholic fatty liver disease

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A Meta-Analysis of the Association between PNPLA3 rs738409 C>G Polymorphism and NAFLD in Han Chinese Population

Abstract

Objective: To evaluate the relationship between regular PNPLA3 rs738409 C>G Polymorphism and Non-alcoholic fatty liver disease (NAFLD) by a meta-analysis.

Odds ratios (ORs) and 95% confidence intervals (CIs) were appropriately derived for each gene–disease association using fixed and random effect models.

Methods: Studies were identifide by searches of the Medline database , Embase, China National Knowledge Infrastructure (CNKI) database, Chinese VIP database, Chinese Wanfang database, up to December 2013. Hardy-Weinberg equilibrium (HWE) was employed to test genetic equilibrium among the genotypes of the selected literature. Power analysis was performed with the Power and Sample Size Calculation (PS) program. A fixed or random effect model was used on the basis of heterogeneity. Publication bias was quantified and examined with the Begg’s funnel plot test and Egger’s linear regression test. The meta-analysis was performed with Review Manager 5.2 and Stata11.0

Results: There were 5 studies including 1187 NAFLD patients and 2153 healthy control subjects involved in this meta-analysis. The pooled ORs (95% CIs) for PNPLA3 rs738409 C>G of allelic, dominant and recessive genetic models were 1.67 [1.33, 2.10], 1.66 [1.41, 1.94] and 2.12 [1.49, 3.02] respectively, With statistically significantdifference among them(P<0.0001). Publication bias might exist in the involved studies. After corrected by trim and fill method, Meta-analysis results from two effect models indicated that there was significant difference between the two groups, which indicating that the result was stable.

Conclusions: This meta-analysis result suggested that PNPLA3 rs738409 C>G polymorphism was strongly associated with NAFLD, and G allele at this locus might be a susceptibility allele for increasing the development of NAFLD in Han Chinese population. Meta-analysis, of course, on a larger scale with associated analysis of other ethnic groups and larger sample size of updated studies in future studies are essential.

关键词:PNPLA3; Polymorphism; NAFLD; Meta-analysis; Chinese Han population

Introduction

Non-alcoholic fatty liver disease (NAFLD) comprehends a wide range of conditions, encompassing from fatty liver or steatohepatitis with or without fibrosis, to cirrhosis and its complications. NAFLD has become the most common form of liver disease in childhood and a major cause of liver disease in Asia as its prevalence has more than doubled over the past 20 years, paralleling the increased prevalence of childhood obesity.(07P)The pathogenesis of NAFLD is multifactorial; it is well-recognized that obesity, insulin resistance, hyperlipidemia, diabetes, and environmental factors influence the development and progression of NAFLD [4,5,9,10,11](peng XE)]. In the past decade new techniques have been developed to investigate the causes of NAFLD. In addition, genetic factors have been suspected to play a role in NAFLD development [6,7,9,10,11](peng XE)], and several genes, including PPARa, PEMT, STAT3andABCC2, have been suggested as potential candidates for either NAFLD susceptibility or disease progression [8,9,10,11](peng

Patatin like phospholipase-3 (PNPLA3), also known as adiponutrin, belongs to the patatin-like phospholipase family of proteins that contain adipose triglyceride lipase (PNPLA2), the key protein involved in the hydrolysis of triglycerides to diglycerides in adipocytes. PNPLA3 is a transmembrane protein which, in humans, is prominently expressed in hepatocytes and is highly responsive to changes in energy balance [12(9G)]. PNPLA3 exhibits hydrolase activity against triglycerides, and variations in PNPLA3 alter its catalytic activity and can lead to triglyceride accumulation in hepatocytes [13, (9G)14]. (9G)

PNPLA3 substitutes methionine for isoleucine at residue 148 (I148M) was first reported to be associated with hepatic triglyceride content in a genome-wide screen [11(Li Y]; subsequently its association with NAFLD was confirmed in multiple ethnic and geographic groups. (for a review see [14(Li Y]) The I148M variant was also reported to be associated with the histological severity of NAFLD (for a review see [20(Li Y]);. The I148M allele (rs738409: C>G) of the PNPLA3 gene represents a cytosine to guanine substitution, resulting in an isoleucine to methionine switch at codon 148. Individuals who were homozygous for the G allele had a higher hepatic triglyceride level [1104G] and an elevated serum level of ALT [1204G]. Moreover, PNPLA3 (rs738409: C>G) may influence fibrosis severity in patients with fatty liver [13–16].(04G) However, inconsistent opinions still exist in different studies on the association between PNPLA3 rs738409 C>G and NAFLD in Han Chinese Population. A study with a group of 203 NAFLD patients found that the PNPLA3 rs738409 polymorphism was associated with steatosis [Peng22].

In order to address this issue, we investigated the association of polymorphisms inPNPLA3and their predicted haplotypes with the risk for NAFLD in a Han Chinese population. Furthermore, bytaking into consideration the fact that NAFLD is a multifactorial disorder, we also studied the possible interactions with environmental and ‘internal’ exposures, such as tea drinking, body mass and other components of the metabolic syndrome.(9G)

Individual studies and previous meta-analyses of such studies included too few subjects to provide conclusive evidence for or against an association of this polymorphism and NAFLD. The aim of the study was to fully address this issue and to minimise genetic background noise and importantly assess the relation of PNPLA3 rs738409 C>G polymorphism with NAFLD by conducting a meta-analysis of individual participant data from all case-contaol obsernational studies that had data on this polymorphism and NAFLD, while addressing heterogeneity, as well as publication bias. (重点阅读)

Methods

Data Sources

For the electronic searches, published studies were found through Medline database (http://www.ncbi.nlm.nih.gov/pubmed) , Embase(http://www.embase.com), China National Knowledge Infrastructure (CNKI) database (http://www.cnki.net/), Chinese VIP database (http://www.cqvip.com/), Chinese Wanfang database (http://g.wanfangdata.com.cn/), up to December 2013, for all publications about the association between TNF-a 2308G.A polymorphism and T2DM in Han Chinese population. The keywords in this search were consisted of patatin-like phospholipase domain containing 3 (PNPLA3)、polymorphism/genetic、non-alcoholic fatty liver disease (NAFLD)、China/Chinese et al.

Selection Criteria

To be included in this meta-analysis, studies must meet the following criteria: (1) case-control studies, available genotypic and allelic data or summarized frequencies; (2) Chinese Han as the studied subjects; (3) the diagnosis of NAFLD patients based on the1999 WHO Diabetes Criteria [13]. However, studies were excluded if one of the following existed: (1) the study was a review, lecture, editorial or correspondence letter; (2) studies whose healthy control groups failed Hardy-Weinberg equilibrium (to limit heterogeneity and ensure quality of data). Study quality was assessed using the guidelines for case-control studies [14]. Due to the criteria developed by Clark et al [15], a quality assessment score (as implemented in [16–18]) was estimated for the quality of each association study based on traditional epidemiological and genetic considerations. Using a 10-point scoring system, studies were scored as ‘‘good’’ if the score was 8 to 10, ‘‘moderate’’ if the score was 5 to 7 and ‘‘poor’’ if the score was less than 4 [19](5)

Data Extraction and Study quality assessment(1 ,2)

Two authors extracted data independently and in duplicate, and reached on all items, including: the first author’s name, year of publication, region and duration of the study carried out, (1). follow-up period, research outcomes’ genotype and allele frequency ,confirmation method, age and sex of the participants, the number of cases and the total number of follow-ups (cohort studies), sample sizes of the case and the control group (case–control studies), corrected OR/RR/HR and 95% CI, and the correction factor. After completion of data extraction, the investigators exchanged the data sheets and inconsistency was resolved by discussion.(公司到2). The authors developed a 10-point scoring sheet, based on criteria adopted from published recommendations on the assessment of the quality of genetic association studies [5, 6, 7](1de 5.6.7). Studies were scored as “good” if the score was 8 to 10, “fair” if the score was 5 to 7 and “poor” if the score was <4.( 2) æ-‡çŒ®è´¨é‡è¯„价为适中æˆ-以上,则入选æ-‡çŒ®è´¨é‡è¾ƒé«˜ã€‚

Statistical Analysis

To ensure that the quality of the selected literature, Hardy - Weinberg equilibrium test was performed.This meta-analysis was performed with Review Manager 5.2 and Stata 11.0. Power analysis was performed using the Power and Sample Size Calculation (PS) program (http://biostat.mc.vanderbilt.edu/wiki/Main/PowerSample Size) (3) Heterogeneity among studies was examined with x 2-based Q statistic.If there was no statistical heterogeneity among studies (P>0.05, I2<50%), the ORs and 95% CI would be estimated for each study in a fixed-effects model. Otherwise, a random-effect model should be employed. In sensitivity analysis, relative influence of each study on the pooled estimate was assessed by omitting one study at a time. Funnel plots were used to evaluate publication bias. AllP-values were two-tailed(6)。Then, publication bias was estimated by Begg’s funnel plot test and Egger’s linear regression test. Finally, sensitivity analysis was performed to assess the influence of each individual study.(5)

Results

Characteristics of Eligible Studies

After HWE(P>0.05), there were actually 5 studies (7-11) to be used in this meta-analysis, all of which were English studies, with a total of 1187 patients of NAFLD and 2153 healthy control subjects. The characteristics of the included studies were summarized in Table 1(5). The details for the study searching were shown in Figure S1. Moreover, not all of the included studies were adjusted for covariates, such as body mass index(BMI), age, sex and so on. As a result, the information for these covariates was not shown in Table 1. The quality for these studies was rated to be moderate or above according to the quality assessment scores calculated,which is 6-8.

Power analysis

Before this meta-analysis, power analysis was performed by using the PS program to prove whether the selected studies could offer adequate power(5). With data provided by the 5 studies, Mantel-Haenszel test, assuming that α=0.05,OR=1.5,each 2 x 2 table consists of cases and controls selected from a different stratum that is defined by studies, could provide the power of 1 , 1 and 0.96 under the allelic, dominant and recessive genetic models, respectively.

定量数据合并

The Cochran’s χ2 test (Q test) was used to evaluate heterogeneity between studies, and a threshold P-value of 0.1 was considered statistically significant. The I2 test was

also conducted to evaluate heterogeneity between studies [19]. Begg’s and Egger’s tests were both used to test the significance of the publication bias, with aP-value < 0.1 considered as significant. We used the software developed by Guoet al. [20] to test (using exact test [21]) the significance of the HWE in control subjects, with a threshold P-value of 0.05 and 0.001 considered statistically significant for candidate gene association studies and GWAS [22,23], respectively. Heterogeneity is considered higher when I2>50% and much higher when I2>75% [24], and higher heterogeneity is a common phenomenon in genetic association studies [25,26]. To estimate heterogeneity, meta-regression was performed with the mean age of control subjects (Age_Control) and the mean BMI of control subjects (BMI_Control) as the covariates [27].( FTO gene polymorphisms and obesity risk:a meta-analysis)

Study examined the association between PNPLA3 rs738409 C>G A Polymorphism and NAFLD in Han Chinese Population ,under the allelic, dominant and recessive genetic models, respectively.

According to the heterogeneity test , a random effect model was selected to merge data both in the allelic and recessive genetic models,since P<0.05, I2>50%. However a mixed effect model was selected to merge data in dominant genetic model,whose result was P=0.07,I2=53% , considerating that mixed effect model possessing smaller scope and more stable detection compared with therandom effect model with 95% CI(8).

The overall allelic ,dominant and recessive OR(95% CI) was 1.67 [1.33, 2.10] (Fig 2),1.66 [1.41, 1.94](Fig 3)and 2.12 [1.49, 3.02] (Fig 4)respectively, with a significant difference (P<0.0001)

Sensitivity Analysis

表明该æ-‡çŒ®å¯èƒ½æ˜¯å¯¼è‡´å¼‚质性产生的直接æˆ-根本原因。

To further strengthen the confidence for the results, we conducted a sensitivity analysis. This analysis confirmed the stability of the null association between PNPLA3 rs738409 C>G A Polymorphism and NAFLD in Han Chinese Population. Exclusion of individual studies did not modify the estimates much, with pooled ORs ranging from 1.02 to 1.04. (Fixed effect OR range= 1.12-1.14; Random effect OR range= 1.13-1.15) .(6).Although pooled ORs did not reverse, heterogeneity was significantly reduced and turned into no statistical difference in all of the three models after omitting Xu J et al.(11])Therefore,it might lead to the heterogeneity.

we performed sensitivity analysis by omitting individual studies one by one. The sensitivity analysis indicated that none of the individual studies greatly influenced the overall pooled RR(3.7分)

Assessment of publication bias

Begger’s funnel plot and Egger’s test were performed to assess the publication bias.The Funnel plot (Fig. 5) showed a relatively asymmetrical distribution; the Begg test (P= 0.027) and the Egger test (P= 0.031) showed a publication bias among studies.(公司033)

Duvall and Tweedietrim and fill procedure resulted in an effect size of d= 0.88 (95% CI = 0.68-1.09; number of imputed studies: 2), which suggesting that the results of the study were not significantly altered after adjusting for the publication bias.(2.2分)

Meta-analysis from random-effect model and fixed-effect model revealed no difference between the two groups (logOR=0.377, 95%CI=-0.249~1.003, P=0.238). After corrected by trim and fill method, Meta-analysis results from two effect models indicated that there was significant difference between the two groups (logOR=0.368, 95% CI=-0.255~0.991,P=0.247)(0.58)The point estimate of the study did not deviate with the addition of smaller studies, ruling out the possibility of publication bias.(2)

Discussion

NAFLD, the most common liver disorder in the world, is a clinico-histopathological entity in which excessive triglyceride accumulation in the liver occurs. New gene polymorphisms increasing the risk of fatty liver, namely PNPLA3, have been lately identified allowing further insights into the pathogenesis of this condition. PNPLA3, also referred to as adiponutrin, which is expressed in the primary metabolic cell type in the liver [17(xuj)], namely the hepatocytes, has triglyceride lipidase and acylglycerol acyl-enzymic activity. To speculate it likely has the regulation function both adipolysis and fat synthesis. He had found the I148M mutation altered the protein structure of PNPLA3,and enzyme's active center were covered up, which suggesting that its function changed[18(xuj)]. Chen have found that siRNA-mediated knockdown of PNPLA3 in 3T3-L1 adipocytes had no effect on triglyceride hydrolyse, aminotransferase raise in liver or insulin resistance [13(xuj)]. But He expressed wild type and mutantforms of the PNPLA3 protein in Sf9cells, in cultured hepatocytes, and in livers of mice,they have found the expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content [(xuj)18]. This shows that the mechanism of PNPLA3 gene polymorphisms in NAFLD is more complicated than what people generally think.

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Meta regression also showed study size to be a potential source of between study heterogeneity. Our results need to be interpreted with caution as they are based on unadjusted estimates. (2candi) To avoid potential racial admixture, we restricted our analysis to Han Chinese in this study. All of the 5 included studies were of Han Chinese ancestry. Nevertheless, for Han Chinese population who live in a vast country, geographical and culture differences might be additional factors causing genetic heterogeneity.(5meta) The culture and habits, such as personality, diet, living environment, customs and so on, were very different, and the genetic communications may lead to population stratification. The fact that significant between-study heterogeneity in allelic association and marginal significance in genotypic association across the 5 studies might support our perspective. Other factors (e.g., age, sex, sample size, genotyping method, gene-gene and gene-environment interactions) might contribute to heterogeneity as well.(5Mete)

This is the first meta-analysis to assess the association between PNPLA3 rs738409 C>G Polymorphism and NAFLD in Han Chinese Population. The main finding of this study is the strong, independent association between NAFLD patients and healthy control subjects. This collaborative work involved all of the researchers that have been studying the link between PNPLA3 rs738409 C>G and NAFLD preceding and through the third quarter of 2013.

The study focuseson Chinese Han population to explore the association between PNPLA3 rs738409 C>G Polymorphism and NAFLD, avoiding the impact of ethnic differences, thus provides a new direction for future research. This study can be compared with the results associated with other ethnic groups, analyzing the differenceandrelationbetweenthem. Limitations Inherent limitations of this meta-analysis should be pointed out, including the following: First, Based on the statistical tests, the subgroup analyzes were not conducted of this meta-analysis, due to fewer selected literature. There may be many potential reasons for the higher heterogeneities, including differences in genetic susceptibility across ethnic groups and measurement error of BMI, age and sex. Second, Gene-gene and gene-environment interactions could not be addressed in this meta-analysis. Actually, NAFLD is a complex trait, and many genes are related to NAFLD, such as PPARa, PEMT, STAT3andABCC2(Peng). On the other hand, lifestyle factors, including diet and physical inactivity, are important contributors to NAFLD. ( FTO gene polymorphisms and obesity risk:a meta-analysis) Thus, this study, losed sight of other genetic mutations, only selected one gene polymorphisms (PNPLA3 rs738409 C> G),which might underestimate the effects of the overall genetic susceptibility to NAFLD.

Overall, our results provide a strong argument for performing further mechanistic studies to better understand the role of PNPLA3 in NAFLD development.

This meta-analysis result suggested that PNPLA3 rs738409 C>G polymorphism was strongly associated with NAFLD, and G allele at this locus might be a susceptibility allele which contributes to the development of NAFLD in Han Chinese population. Meta-analysis, of course, on a larger scale with associated analysis of other ethnic groups and larger sample size of updated studies in future studies are essential.

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