Research Article
Carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of non-alcoholic fatty liver disease associated hepatocellular carcinoma

https://doi.org/10.1016/j.jhep.2014.02.030Get rights and content

Background & Aims

Subtle inter-patient genetic variation and environmental factors combine to determine disease progression in non-alcoholic fatty liver disease (NAFLD). Carriage of the PNPLA3 rs738409 c.444C >G minor allele (encoding the I148M variant) has been robustly associated with advanced NAFLD. Although most hepatocellular carcinoma (HCC) is related to chronic viral hepatitis or alcoholic liver disease, the incidence of NAFLD-related HCC is increasing. We examined whether rs738409 C >G was associated with HCC-risk in patients with NAFLD.

Methods

PNPLA3 rs738409 genotype was determined by allelic discrimination in 100 European Caucasians with NAFLD-related HCC and 275 controls with histologically characterised NAFLD.

Results

Genotype frequencies were significantly different between NAFLD-HCC cases (CC = 28, CG = 43, GG = 29) and NAFLD-controls (CC = 125, CG = 117, GG = 33) (p = 0.0001). In multivariate analysis adjusted for age, gender, diabetes, BMI, and presence of cirrhosis, carriage of each copy of the rs738409 minor (G) allele conferred an additive risk for HCC (adjusted OR 2.26 [95% CI 1.23–4.14], p = 0.0082), with GG homozygotes exhibiting a 5-fold [1.47–17.29], p = 0.01 increased risk over CC. When compared to the UK general population (1958 British Birth Cohort, n = 1476), the risk-effect was more pronounced (GC vs. CC: unadjusted OR 2.52 [1.55–4.10], p = 0.0002; GG vs. CC: OR 12.19 [6.89–21.58], p <0.0001).

Conclusions

Carriage of the PNPLA3 rs738409 C >G polymorphism is not only associated with greater risk of progressive steatohepatitis and fibrosis but also of HCC. If validated, these findings suggest that PNPLA3 genotyping has the potential to contribute to multi-factorial patient-risk stratification, identifying those to whom HCC surveillance may be targeted.

Introduction

Non-alcoholic fatty liver disease (NAFLD) describes a spectrum ranging from simple steatosis, through steatohepatitis (NASH) to fibrosis, in the absence of excessive alcohol consumption [1]. NAFLD is strongly associated with features of the metabolic syndrome, including obesity, insulin resistance or type 2 diabetes mellitus (T2DM) and dyslipidaemia [1]. As lifestyles have become increasingly sedentary, NAFLD has rapidly become one of the most common causes of liver disease worldwide [1], [2], [3].

The rise in the burden of NAFLD coincides with a marked increase in the incidence of HCC in many countries [4], [5], [6], [7]. Worldwide, most HCC are related to chronic viral hepatitis; however, more than half of all HCC cases in developed countries occur in the viral hepatitis negative population [8], [9]. Features of the metabolic syndrome including obesity and T2DM (with gender, age, and ethnicity) are independent risk factors for HCC [10], [11] whilst the pathogenic processes that favour progression from steatosis to steatohepatitis are also pro-carcinogenic [4]. Longitudinal studies indicate HCC prevalence to be ∼0.5% in steatosis and ∼2.8% in NASH and so, whilst HCC remains an infrequent complication, the high prevalence of NAFLD means that NAFLD-related HCC contribute significantly to the disease burden [10]. The majority of NAFLD-related HCC are thought to develop on a background of cirrhosis with an estimated incidence of 2.6%/year, although recent studies suggest that in this context a substantial proportion of HCC occur in the absence of advanced fibrosis [12], [13], [14].

Despite its high prevalence, only a minority of NAFLD patients exhibit steatohepatitis, progress to significant fibrosis or experience associated morbidity and mortality [1]. The reasons for the apparent variation in individual susceptibility to progressive NAFLD in general and NAFLD-related HCC in particular are incompletely understood [9], [15]. NAFLD-related HCC develops through the complex interplay of environmental and genetic factors that determine individual risk [6], [16]. The role of the non-synonymous patatin-like phospholipase domain-containing 3 (PNPLA3) c.444C >G single nucleotide polymorphism (rs738409) that encodes the p.I148M (isoleucine-to-methionine substitution at residue 148) variant is well recognized as a modifier of hepatic triacylglycerol (TAG) accumulation and NAFLD progression [15], [17], [18]. This variant has been associated with increased HCC risk in alcohol-related liver disease [19], [20], [21], [22] and, more variably, in chronic viral hepatitis [19], [20], [22], [23], [24]. Data has also been presented showing an association with HCC in morbidly obese patients [25] and a mixed-aetiology cohort [26]. Although it may be hypothesised that these latter associations are related to underlying NAFLD, to date no studies have specifically addressed the effect of PNPLA3 rs738409 C >G minor allele carriage on HCC risk in a NAFLD cohort.

The aim of the current study was to determine whether carriage of the PNPLA3 rs738409 C >G polymorphism confers an increased risk of NAFLD-associated HCC, and whether that effect is independent of presence of cirrhosis. To address this, we performed a case-control associated study in a well-characterised Northern European cohort with established NAFLD.

Section snippets

Patients

Patients were recruited from hepatology clinics at two European specialist centres, the Freeman Hospital, Newcastle-upon-Tyne, UK and Inselspital Hospital, Bern, Switzerland. The study had all the necessary ethical approvals in both the countries and all participants gave informed consent.

A cohort of 100 consecutive Northern European Caucasian patients with primary HCC arising on a background of NAFLD was identified (UK 82, Switzerland 18 patients). The diagnosis of HCC was established

Cohort characteristics

One hundred NAFLD-HCC patients and 275 histologically characterised NAFLD control patients were recruited. Characteristics of the NAFLD-HCC and NAFLD control populations are provided in Table 1. Patients with NAFLD-HCC were significantly older (mean age 70.3 ± 8.0 vs. 50.9 ± 12.4 years, p <0.0001) than a general NAFLD cohort. NAFLD-HCC patients were also significantly more likely to be male (82% vs. 59%, p <0.0001) and to be diabetic (68% vs. 43%, p <0.0001). The presence of NAFLD-HCC was also

Discussion

Using a large, well-characterised Northern European cohort with biopsy-proven NAFLD we show a strong association between PNPLA3 rs738409 genotype and HCC risk. This highly significant effect was independent of potentially confounding factors including age, gender, co-existent diabetes, obesity, and the presence of cirrhosis. Although PNPLA3 rs738409 is principally recognised as a disease modifier in NAFLD [15], studies thus far reporting associations with HCC have been in individuals with viral

Financial support

This study was supported by the ‘Fatty Liver Inhibition of Progression’ (FLIP) project funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement Health-F2-2009-241762. QMA is the recipient of a Clinical Senior Lectureship Award from the Higher Education Funding Council for England (HEFCE). Aspects of this work have been supported by the facilities of the Newcastle NIHR Biomedical Research Centre.

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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