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Recent advances in clinical practice
Genetic determinants of alcoholic liver disease
  1. Felix Stickel1,
  2. Jochen Hampe2
  1. 1Department of Visceral Surgery and Medicine, Inselspital, University of Berne, Switzerland
  2. 2Department of Internal Medicine I, University Hospital Schleswig-Holstein, Kiel, Germany
  1. Correspondence to Professor Felix Stickel, Department of Visceral Surgery and Medicine, Inselspital, University of Berne, Murtenstrasse 35, 3010 Berne, Switzerland; felix.stickel{at}ikp.unibe.ch

Abstract

Alcoholic liver disease (ALD) accounts for the majority of chronic liver disease in Western countries. The spectrum of ALD includes steatosis with or without fibrosis in virtually all individuals with an alcohol consumption of >80 g/day, alcoholic steatohepatitis of variable severity in 10–35% and liver cirrhosis in approximately 15% of patients. Once cirrhosis is established, there is an annual risk for hepatocellular carcinoma of 1–2%. Environmental factors such as drinking patterns, coexisting liver disease, obesity, diet composition and comedication may modify the natural course of ALD. Twin studies have revealed a substantial contribution of genetic factors to the evolution of ALD, as demonstrated by a threefold higher disease concordance between monozygotic twins and dizygotic twins. With genotyping becoming widely available, a large number of genetic case-control studies evaluating candidate gene variants coding for proteins involved in the degradation of alcohol, mediating antioxidant defence, the evolution and counteraction of necroinflammation and formation and degradation of extracellular matrix have been published with largely unconfirmed, impeached or even disproved associations. Recently, whole genome analyses of large numbers of genetic variants in several chronic liver diseases including gallstone disease, primary sclerosing cholangitis and non-alcoholic fatty liver disease (NAFLD) have identified novel yet unconsidered candidate genes. Regarding the latter, a sequence variation within the gene coding for patatin-like phospholipase encoding 3 (PNPLA3, rs738409) was found to modulate steatosis, necroinflammation and fibrosis in NAFLD. Subsequently, the same variant was repeatedly confirmed as the first robust genetic risk factor for progressive ALD.

  • Alcoholic liver disease
  • genetic polymorphism
  • liver cirrhosis
  • extracellular matrix
  • fibrogenesis
  • genotype
  • ethanol metabolism
  • haemochromatosis
  • fibrosis
  • IBD - genetics
  • genetics
  • gallstones
  • molecular genetics
  • gallstone disease

https://doi.org/10.1136/gutjnl-2011-301239

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    Key messages

    • Alcoholic liver disease (ALD) is a complex trait in which environmental and host factors modulate drinking behaviours and evolution and progression of liver damage.

    • Confirmed environmental risk factors for progressive ALD include quantities of alcohol, obesity and chronic co-infection with hepatitis viruses.

    • There is strong evidence supporting a genetic background as an important modulator of susceptibility to ALD.

    • There is a lack of evidence for a role of HFE mutations.

    • Heterozygous and homozygous carriage of PNPLA3 rs738409 (G) allele is the first confirmed genetic risk factor for progressive ALD.

    Alcoholic liver disease as a complex trait

    Alcoholic liver disease (ALD) is a complex disease whose development depends on long-term excessive drinking and other environmental, individually acquired and inherent modifying factors (figure 1). These factors interact over time to allow for or prevent the development and progression of ALD. While many environmental factors have been identified following careful epidemiology studies in the second half of the 20th century, the more recent advances in our understanding of human genetic variation and the research thereof have prompted us to search for host genetic factors that influence the course of many complex diseases including ALD.

    Figure 1
    Figure 1

    Schematic account of factors of progression in alcoholic liver disease showing the multifactorial pathogenesis of the disease.

    Natural course and pathogenesis of ALD

    Unlike many other chronic liver diseases, ALD is a potentially avoidable disease since excess alcohol consumption is needed for its development. In other words, ALD only becomes a liver disease if interventions against harmful drinking do not take place or fail. However, scientific and common cognition show that alcohol consumption per se is not necessarily sufficient to elicit significant ALD since only a minority of heavy drinkers progress to severe ALD. Studies in humans estimate that liver steatosis evolves in all excessive drinkers, but only about one-third develop significant necroinflammation and fibrosis and only about 10% progress to cirrhosis.1 2 Among the latter, 1–2% per year develop hepatocellular carcinoma (HCC) as a severe complication.3 4 This variable natural course is believed to result from a complex interplay between environmental and host risk factors.

    Various mechanisms are implicated in alcohol-associated acute and chronic liver injury. Among these, experimental and human data suggest a primary role for cytochrome P450 2E1 (CYP2E1), a microsomal enzyme which can be induced 10–20-fold by chronic alcohol consumption.5 CYP2E1 metabolises ethanol to acetaldehyde, a highly toxic and mutagenic molecule, and increases oxidative stress by the production of reactive oxygen species (ROS) and lipid peroxides such as 4-hydroxy 2,3-nonenal, 4-hydroxy-2,3-alkenals and malondialdehyde.6 CYP2E1 induction is associated with fat accumulation,7 inflammation and fibrosis8 and DNA lesions.5 Furthermore, excessive alcohol consumption can lead to an increased portosystemic uptake of endotoxins from gut bacteria which contribute to necroinflammation and fibrosis progression via various molecular mechanisms including tumour necrosis factor α (TNFα) and the CD14/toll-like receptor-4 complex to produce ROS via NADPH oxidase.9

    Patients with ALD often present with hyperhomocysteinaemia resulting from impaired methyl group transfer due to deficiencies of folate, vitamins B12 and B6 as well as acetaldehyde-mediated interception with enzymes involved in one-carbon metabolism.10 On the other hand, elevated hepatic homocysteine results in endoplasmic reticulum stress by causing malfolding of endoplasmic reticulum proteins and promotes necroinflammation.11

    Progressive fibrosis is crucial in chronic ALD as it may eventually result in cirrhosis, liver failure and associated clinical complications. In response to acetaldehyde, ROS, lipid peroxides and endotoxins, Kupffer cells and other inflammatory cells become activated to secrete numerous growth factors and cytokines including TNFα, platelet-derived growth factor and transforming growth factor β1 (TGFβ1), both pivotal stimuli of hepatic stellate cell (HSC) and portal myofibroblast (MFB) proliferation and secretory function.12 13 Activated HSCs/MFBs markedly increase the production of fibrous matrix, particularly collagens, and downregulate their degradation. The latter is mediated by collagenases termed matrix metalloproteinases (MMPs) which are under the control of the corresponding tissue inhibitors of matrix metalloproteinases.14 Based on this multifactorial concept of ALD, polymorphic variants of genes coding for proteins involved in these processes have been selected for genetic case–control studies, as described below.

    Host versus environmental factors modulating ALD progression

    Risk factors for the progression of alcoholic fibrosis are commonly stratified into host or environmental modifiers, or genetic and non-genetic factors, respectively. By far the most crucial environmental factor is alcohol, and there is a clear relationship between the amount of alcohol consumed and the likelihood of developing ALD (figure 2). Alcoholic steatosis occurs in approximately 60% of subjects who drink >60 g/day, and the risk of developing cirrhosis is highest among those with a consumption of >120 g/day.1 2 Other undisputed risk factors for progression of fibrosis in ALD are being overweight15 and co-infection with hepatitis C virus.16

    Figure 2
    Figure 2

    Relationship between the amount of alcohol consumed and the likelihood of developing cirrhotic alcoholic liver disease (ALD).1

    Three lines of evidence are indicative of at least a partial genetic background of the ALD phenotype: (1) women are more susceptible to ALD following the consumption of similar amounts of alcohol; (2) Hispanic subjects are more prone to developing ALD than black and white subjects; and (3) twin studies have shown that monozygotic twins have a higher prevalence of alcoholic cirrhosis than dizygotic twins.

    Gender-specific susceptibility to alcohol has long been recognised. Studies in humans have shown that women are more susceptible to the hepatotoxic effects of alcohol and develop ALD more quickly than men with the same daily alcohol consumption.2 17 18 The pathophysiology behind this increased sensitivity to alcohol is not yet fully understood but—among other factors—could be related to hormonal differences such as oestrogens and their synergistic impact on oxidative stress and inflammation.19 Gender-related metabolic differences also exist. Women who drink the same amount of alcohol as men have higher blood ethanol levels due to higher gastric alcohol dehydrogenase levels resulting in a faster first-pass metabolism of alcohol in men20 or a lower volume of distribution of alcohol in women.

    Differences in the prevalence of ALD and associated mortality have been reported in different ethnic groups.21 22 Stinson et al analysed the mortality of subjects with alcoholic cirrhosis across races and found the highest mortality rates for men in white Hispanics, followed by black non-Hispanics, white non-Hispanics and black Hispanics. For women the order was black non-Hispanics, white Hispanics, white non-Hispanics and black Hispanics.22 However, such seemingly ethnic differences in the rates of alcoholic cirrhosis and ALD could also be related to the amount and type of alcohol consumed, dietary preferences, differences in socioeconomic status, access to medical care and/or differences in attitudes towards a healthy lifestyle.

    Alcoholics often show biochemical and histological signs of iron overload reflected by raised serum ferritin levels and accentuated iron staining on liver biopsies, respectively.23 The identification of the genetic background of iron overload by detection of mutations within the haemochromatosis (HFE) gene gave rise to the hypothesis that their presence may also affect iron storage in alcohol abusers. However, genetic case–control studies with adequate matching of cases and controls for age, sex and alcohol consumption found no increased prevalence of HFE mutations in alcoholics with liver disease, including cirrhosis.24 25

    The search for genetic risk factors of ALD

    Candidate gene case–control studies

    Until recently, the most extensively applied methodology to identify genetic risk factors of ALD (and many other complex diseases) was through genetic case–control studies. With simple and reliable genotyping methods becoming widely available, the attempt to identify possible genetic modifiers of the risk of ALD prompted a large number of hypothesis-driven candidate gene case–control studies comparing allelic and/or genotypic frequencies of certain genetic variants (ie, single nucleotide polymorphisms, SNPs) between individuals with alcoholic cirrhosis or alcoholic hepatitis, alcoholics without apparent liver disease and healthy controls.26 SNPs are the most common type of allelic variation (∼90%) and can be found throughout the human genome at a frequency of 1 every 1000–2000 base pairs (http://www.ncbi.nlm.nhi.gov/SNP). They refer to a relatively frequent nucleotide sequence variation (arbitrary cut-off for minor allele frequency >1%) in which a single base is replaced by another leading to an altered base triplet that potentially could code for a different amino acid when located in the coding sequence (non-synonymous SNP). If located within the coding region of a gene, such coding SNP could potentially alter the function of the generated protein (eg, enzyme activity, substrate specificity). If located in the promoter sequence, SNPs may result in altered quantitative expression of the respective gene. The large majority of SNPs (>99%) are situated in non-coding regions of genes and therefore their functional implication is often unclear, but may still affect gene splicing or transcription factor binding.27 This must be considered when choosing candidate polymorphisms for association studies. In the majority of studies, genetic variants were chosen based on the known pathophysiology of ALD, assuming that genetic variation of genes involved in key events of the evolution of chronic alcoholic liver damage such as oxidative degradation of alcohol, tissue remodelling or inflammation would render their carriers either resistant or susceptible to the hepatotoxic effects of alcohol (table 1). This strategy was obviously restricted to genes selected from a panel of candidates considered important for ALD, but failed to detect as yet disregarded genetic variants that may affect the natural course of ALD even more significantly. The potential of this approach to create novel hypotheses is therefore low. Overall, despite a large number of case–control studies since the late 1980s, no confirmed genetic factor for ALD had been established until recently.28 29 Failure to replicate findings from index studies was often due to inherent methodological limitations, mostly with respect to sample size and characterisation/selection of cases and controls (box 1). Thus, future candidate gene case–control studies should seek to avoid these pitfalls by assuring statistical power via large-scale multicentre cooperations, careful phenotyping of cases and controls, and the selection of promising candidate variants derived from hypothesis-generating genome-wide analyses.

    View this table:
    Table 1

    Genes tested for association with alcoholic liver disease (ALD)

    Box 1

    Methodological limitations in genetic case–control studies

    • Low sample size (type I and II errors)

    • Lack of adequate statistical power (should be at least 80% for minor allele frequency)

    • Single-centre studies (selection bias)

    • Insufficient characterisation of cases (assessment of alcohol consumption, comorbidities)

    • Inappropriate selection of controls (eg, blood donors, healthy individuals)

    • Lack of control for confounding factors (eg, overweight, coffee consumption, smoking, cannabis consumption)

    • No replication of association in an independent cohort

    • Population admixture (mixed ethnicity)

    • Deviation from Hardy-Weinberg equilibrium, population stratification

    • Lack of functional characterisation of the tested SNP (eg, intronic SNPs, no expression in liver tissue)

    • Publication and time-lag bias (reluctance of researchers and editors to publish negative but equally meaningful results)

    Genetic case–control studies testing various genetic variants for a possible association with ALD are described below and judged for their strengths and weaknesses.

    Genetic variants modulating addiction to alcohol

    Genetic risk factors which increase the likelihood of alcohol dependence or a strong preference for alcohol consumption are likely to influence the risk of developing ALD as they increase alcohol consumption as a prerequisite for liver injury. Combined analyses of twin studies show that the overall heritability for alcoholism is approximately 50%,30 and members of families with a history of alcoholism carry a significantly increased risk of developing alcohol dependence than individuals without a history of alcoholism.31 Numerous candidate gene case–control studies and genome-wide analyses have been performed, and the genes associated with alcoholism in these studies belong either to neurotransmission or alcohol-metabolising genes. Of particular interest for modulating the preference for and withdrawal of alcohol are the inhibitory neurotransmitter γ-aminobutyric acid (GABA) and its corresponding receptors located on chromosomes 4, 5, 15 and X. In this regard, several studies—including a Collaborative Study on the Genetics of Alcoholism (COGA) launched by the National Institute on Alcohol Abuse and Alcoholism (NIAAA) and two genome-wide association studies—have identified and independently confirmed the association of alcohol dependence with a chromosome 4q13-q11 GABAA cluster32 33 which was further refined to the GABRA2 gene.34 Polymorphic variation of GABRA2 was also linked to alcohol withdrawal symptoms, daily alcohol consumption and the extent of positive reinforcement by alcohol consumption among non-dependent alcohol consumers.35 36 Conceivably, genetic risk factors modulating preference and aversion towards alcohol may well affect ALD evolution and other alcohol-related organ dysfunctions, although their functional role is unrelated to liver pathophysiology. Variants of genes expressing alcohol-degrading enzymes were found to be associated with alcoholism and also with alcohol-related organ damage, and will therefore be addressed in more detail below.

    Genetic variation of alcohol-metabolising enzymes

    Cytosolic alcohol dehydrogenase (ADH) and mitochondrial aldehyde dehydrogenase (ALDH) are responsible for metabolising the magnitude of ingested ethanol, and their expression is highest in the liver but also detectable in the surface epithelia of luminal organs such as the gastrointestinal tract. Following absorption, alcohol is degraded by ADH in the cytosol and CYP2E1 in microsomes to acetaldehyde which is further converted by ALDH to acetate. After release from the liver, acetate is metabolised by cardiac and skeletal muscle tissue. All three genes for ADH, ALDH and CYP2E1 have polymorphisms that change their maximum (reaction) velocity (Vmax) and Michaelis constant, and thus their ability to degrade their substrates alcohol and acetaldehyde, respectively. A novel classification of ADH isoenzymes has been proposed which unfortunately produces some confusion as many case–control studies that have tested a possible association of ADH polymorphisms with ALD and other alcohol-related organ disorders have applied the previous terminology. This review uses the novel nomenclature which dissects six classes of different ADHs of which classes I, II and IV participate in ethanol oxidation in humans.37

    Genetic variation of ADH and ALDH has consistently been found to be associated with alcohol dependence. Of note, coding genes of all class I ADH isoenzymes (ADH1A-C) are located on chromosome 4q21-23 within the same linkage peak which was associated with alcoholism in the initial COGA study.32 A recent meta-analysis including 50 genetic case–control studies investigated whether carriage of ADH, CYP2E1 and ALDH variants was associated with alcoholism.38 The heterogeneity between studies, confounding factors, ethnic/gender subgroups, deviation from Hardy-Weinberg equilibrium and statistical power were carefully analysed. Significant associations were found between carriage of ADH1B*1, ADH1C*2 and ALDH2*1 and the risk of alcoholism (ORs 1.89, 1.32 and 4.35, respectively) in Asian populations but not in Caucasians. In subgroup analyses, associations of ALDH2*2 and ADH1C*2 for alcoholism were restricted to Asian men while there was also a weak association of ADH1B*1 in Caucasians. However, no association for any of the tested genetic variants was found with ALD. Of particular interest is the ALDH2*2 allele which encodes an enzyme with low activity to degrade acetaldehyde, leading to a sudden accumulation thereof upon alcohol exposure if carriers are homozygous for this allele. ALDH2*2 is found in 30–50% of Asians but is almost absent in Caucasians and African-Americans. Homozygotes experience unpleasant adverse effects from alcohol drinking commonly referred to as ‘flush syndrome’ with facial reddening, tachycardia, nausea and vomiting. However, this reaction to alcohol in ALDH2*2 homozygotes results in a reluctance to drink alcohol and protection from ALD and alcohol-associated cancers. Regarding the latter, it would be extremely helpful to identify the genetic markers that render carriers at risk for alcohol-associated cancers once a predisposing precancerous lesion has evolved, such as alcoholic cirrhosis. Case–control studies comparing subjects with alcoholic cirrhosis who developed HCC with those with alcoholic cirrhosis without HCC matched for age, alcohol consumption, body weight and other HCC risk factors could identify such genetic risk factors. A large population-based case–control study from Germany assessed the role of ADH1C polymorphism in patients with alcohol-associated cancers including HCC and found a significant association of genotype ADH1C*1/1 with HCC (OR 3.53, 95% CI 1.31 to 9.47; p=0.012) compared with subjects with alcoholic cirrhosis.39

    Thus, polymorphisms of genes coding enzymes involved in alcohol metabolism play a role in modulating alcohol dependence and tolerance (and possibly cancer), but there is no evidence of their impact on the risk of genuine ALD.

    Variants of genes involved in the generation and counteraction of oxidative stress

    Several enzyme systems including CYP2E1, the mitochondrial respiratory chain and the cytosolic enzymes xanthine oxidase and aldehyde oxidases are important sources of O2− and H2O2 in hepatocytes during ethanol oxidation.40 Antagonistic to these are mechanisms which counteract oxidative stress generated in ALD such as glutathione-S-transferases (GSTs) and superoxide dismutases (SODs) for which numerous SNPs have been described. While enzymes increasing oxidative pressure were not found to be associated with ALD, some controversy prevails regarding the effect of GST and SOD genetic variants. GSTs are enzymes comprising four subfamilies (GSTA, GSTM, GSTT, and GSTP) which conjugate ROS and many toxic and carcinogenic xenobiotics with glutathione.41 Interestingly, the isoenzyme GSTP1 is expressed in biliary epithelial cells and HSCs, and polymorphic variation at base pair 313 (A→G) within the coding region leads to an amino acid substitution (Ile→Val) at codon 105 and threefold decreased capability of GSTP1 to detoxify 4-hydroxynonenal, a highly reactive lipid peroxide promoting fibrogenesis.42 Genotype GSTP1 Val/Val is associated with cirrhosis development in hereditary haemochromatosis, a disease in which oxidative stress is the crucial pathogenic trigger.43 However, the findings of >10 case–control studies investigating GST SNPs in patients with ALD from different ethnicities have remained inconsistent and all positive findings in ‘index studies’ were later challenged by negative results in independent cohorts.26 44

    Similarly, results on the role of mitochondrial manganese SOD (MnSOD) SNPs in modifying the risk of ALD are controversial. Mitochondria-derived ROS are detoxified to hydrogen peroxide and water by the successive action of MnSOD and glutathione peroxidase.45 MnSOD is expressed with a cleavable N-terminal target sequence that facilitates its import into mitochondria. A variation (C→T) within codon 16 of the precursor protein leads to either alanine (Ala) or valine (Val) at amino acid position −9 of the target sequence which results in its enhanced translocation into mitochondria and a 40% higher concentration of active MnSOD in case of the Ala sequence.46 A French study of 71 patients with ALD and 79 healthy blood donors found that homozygosity for Ala in the mitochondrial targeting sequence of MnSOD increased the risk of microvesicular steatosis threefold and that of alcoholic hepatitis and cirrhosis sixfold and 10-fold, respectively.47 However, in a larger study from the UK, genotyping for MnSOD in 281 patients with advanced ALD (cirrhosis/fibrosis) and 218 drinkers without liver disease found no differences in heterozygote (55% vs 50%) or homozygote (19% vs 23%) Ala allele frequency.48 The negative findings of the latter study illustrate the problems of the low sample size and inappropriate controls included in the earlier study. As yet, confirmed associations between GST and SOD variants with ALD have not been established.

    Variations of genes controlling hepatic lipid storage

    The initial and uniform alcohol-associated liver abnormality is steatosis in almost all chronic drinkers. Variants of genes that govern hepatic fat storage and mobilisation are therefore plausible modifiers of this early alcohol-related liver lesion. However, surprisingly few case–control studies have been carried out testing genetic variants of modifiers of hepatic lipid turnover in the context of ALD.

    Particularly appealing as candidate genes appear to be peroxisome proliferator-activated receptors (PPARs), a group of nuclear receptors which act as transcription factors in the regulation of cell differentiation, development, tumour growth and fat metabolism. The family of PPAR comprises PPARα, PPARδ and PPARγ nuclear receptors which mediate transcriptional responses to insulin resulting in cellular glucose uptake, increased fatty acid oxidation, lipogenesis and lipid storage.49 A polymorphic splice variant with an amino acid substitution (Pro12Ala) following a nucleotide substitution (C→G) in the PPARγ2 gene leads to 5–6-fold increased transcriptional activity of PPARγ. Rey et al genotyped 259 healthy blood donors and 263 patients with histologically-proven non-alcoholic fatty liver disease (NAFLD) and 100 patients with alcoholic steatosis for this locus.50 Neither NAFLD nor ALD was associated with PPARγ genotypes, but carriage of the mutated PPARγ Ala12 allele was associated with histological necroinflammation in patients with alcoholic steatosis (OR 2.50, 95% CI 1.05 to 5.90; p=0.028).

    A possible association of polymorphisms in the genes coding for apolipoprotein E (ApoE) and microsomal triglyceride transfer protein (MTP) with ALD has been tested in insufficiently powered case–control studies with inconsistent results.51–53

    Genetic polymorphisms modulating endotoxin-mediated inflammation

    ALD, and in particular alcoholic hepatitis, can be complicated by marked necroinflammation characterised by cellular ballooning, cytoskeleton aggregates (Mallory-Denk bodies), apoptosis, decay of liver cells and neutrophilic infiltrates. These severe histological changes are associated with significant clinical manifestations such as subacute liver failure, massive jaundice, portosystemic encephalopathy, infections and death.54 The triggers of this worrisome sequel are endotoxins—that is, lipopolysaccharides (LPS) derived from intestinal Gram-negative bacteria shifted from the gut to the liver following alcohol-mediated disruption of the mucosal barrier function and changes in the gastrointestinal microbiota.55 LPS stimulate Kupffer cells (liver macrophages) to secrete proinflammatory cytokines including TNFα, a proinflammatory cytokine which can stimulate hepatocyte regeneration but also hepatocyte death by eliciting apoptosis and necrosis. Functionally, endotoxin receptors are attractive candidates for genetic case–control studies and, recently, a C→T (−159) polymorphism in the promoter region of the CD14 endotoxin receptor gene was detected and found to result in increased CD14 expression. In total, four studies have tested a possible association of CD14 genetic variants and ALD, of which the largest found an OR of 4.17 (95% CI 1.56 to 11.16, p=0.005) for developing alcoholic cirrhosis in CD14 -159TT homozygous heavy drinkers compared with CC homozygotes.56 This association was confirmed in a second smaller study which also showed that CD14 -159TT homozygotes have higher levels of the LPS-binding acute phase proteins LBP and soluble CD14 than carriers of the C allele.57 A third study also found a higher frequency of the CD14 -159TT in subjects with alcoholic cirrhosis compared with those without, but the sample size was relatively low with only 62 cases (118 controls).58 Finally, a small genetic study from Portugal could not find an association between carriage of the CD14 -159T allele and ALD.59

    Several studies have selected a promoter polymorphism (C→A at position −627) in the gene coding for interleukin 10 (IL-10) which results in decreased IL-10 production to test its association with ALD.60–62 IL-10 acts as an anti-inflammatory cytokine by downregulating proinflammatory cytokines including IL-1, TNFα, IL-6, IL-8 and IL-12 and upregulation of the IL-1R antagonist to inhibit collagen I gene transcription and increase collagenase expression. Grove and coworkers genotyped 287 heavy drinkers with biopsy-proved advanced ALD, 107 alcoholics without histological evidence of ALD and 227 local healthy volunteers for the IL-10 promoter SNP and found a significantly higher fraction of the IL10 −627 A allele among those with advanced ALD (50%) compared with drinkers without histological evidence of ALD (34%) and healthy controls (33%). However, this association was not confirmed in two similar studies which might have missed an association due to small sample sizes.61 62

    Among the few associations between genetic variants and ALD that have been replicated independently are SNPs in the promoter of the TNFα gene, for which a number of SNPs have been described. The most common variants are upstream of the gene at positions −1031, −863, −857, −851, −376, −308 and −238, of which the latter two are associated with higher TNFα production. Eleven studies investigating the roles of −308 and −238 TNFα variants were recently included in a meta-analysis which found a significant overall association (OR 1.47; 95% CI 1.05 to 2.07) of variant −238A with alcoholic cirrhosis.63

    Other studies tested a possible association of ALD with carriage of variants in genes coding for the IL-1 receptor antagonist IL-1β and the cytotoxic T lymphocyte antigen-4 gene (CTLA-4), but their positive associations are as yet unconfirmed.64–67

    Polymorphic variants of fibrosis-associated genes

    Progressive fibrosis is the hallmark of most chronic liver diseases including ALD and resembles the process of wound healing. Considering the importance of matrix accumulation in ALD progression, surprisingly few case–control studies have selected variants of fibrosis-associated genes. In this regard, the only two candidate genes selected are those coding for TGFβ1, which activates HSCs to produce collagens, and MMP3, a matrix-degrading (fibrolytic) enzyme. Two relatively large studies included subjects with alcoholic cirrhosis as cases and alcoholics without apparent liver disease as controls matched for age, gender and cumulative alcohol consumption, but found no association of MMP3 and TGFβ1 genotypes with ALD.68 69

    Role of PNPLA3 polymorphism in ALD

    The identification and repeated confirmation of a genetic polymorphism in the gene coding for patatin-like phospholipase domain-containing 3 (PNPLA3; adiponutrin; rs738409 C/G, M148I) as a genetic risk factor for increased fat storage in patients with NAFLD has stimulated similar genetic analyses in patients with ALD.70 Mindful of the multiple similarities shared by NAFLD and ALD, Tian and coworkers investigated a possible relation with ALD using 17 variants in PNPLA3 and a panel of 306 ancestry-informative SNPs in a Mestizo (mixed European and Native American ancestry) population with a history of alcohol abuse.71 A significant association of PNPLA3 rs738409 GG with cirrhosis was detected (OR 2.25, p=1.7×10−10). Owing to marked differences in allele frequencies of PNPLA3 rs738409 G across populations, an ancestry-adjusted analysis was performed which confirmed the initial observation (adjusted OR 1.79, p=1.9×10−5). This exciting revelation has since been replicated independently in three different genetic case–control studies in the UK, Belgium and Germany (table 2). First confirmation of a role for PNPLA3 rs738409 came from a report by Seth and coworkers who studied 266 subjects with alcoholic cirrhosis and 182 heavy drinkers with normal liver enzymes and no clinical evidence of liver damage.72 Carriage of PNPLA3 rs738409 G allele and GG genotype were significantly associated with alcoholic cirrhosis. Detailed analyses such as a possible association with liver enzyme levels or Child–Pugh scores were not provided in this research letter. More details were reported in a comparably sized case–control study conducted in Belgium in which patients with ALD were compared with healthy controls who did not drink significant amounts of alcohol.73 Although the control group poses a limitation to this study, again PNPLA3 rs738409 G was significantly associated with alcoholic liver injury and cirrhosis. The selection of healthy controls may explain why the ORs were slightly lower than in other case–control studies.

    View this table:
    Table 2

    Genetic case–control studies investigating PNPLA3 rs738409 in alcoholic liver disease (ALD)

    The largest study to date on PNPLA3 variation in alcoholic Caucasians was conducted in Germany and included 1043 alcoholic cases and controls from cohorts recruited in eight different German tertiary hospitals and an independent replication cohort (n=376) from a population-based sample.74 rs738409 GG was significantly more frequent in patients with alcoholic liver cirrhosis and in non-cirrhotic alcoholics with elevated liver enzymes than in alcoholics without liver damage (n=439). The latter association was confirmed in an independent population-based cohort of drinkers at risk of ALD with a median alcohol intake of 300 g/week and elevated aspartate aminotransferase levels. Of note, the population-attributable risk from PNPLA3 rs738409 G—that is the reduction in incidence of ALD that would be observed if all drinkers were homozygous carriers of the protective rs738409 C allele—was calculated at 26.6%.

    Despite the undoubted evidence of its important role in conferring genetic risk to develop progressive ALD, the functional implications of PNPLA3 variation remain unclear as yet, and belief among researchers prevails that its molecular role goes beyond that of mere modulation of fat storage. PNPLA3 is located on chromosome 22 and encodes a 481-amino acid protein. It shares similarities with PNPLA2, the major triglyceride hydrolase in peripheral fat tissue.75 Expression of PNPLA3 among species is distinct, with high hepatic expression in humans whereas expression is low in mice and rat livers from normal animals in which adipose tissues account for the magnitude of PNPLA3.76 77 However, fat challenge upregulates hepatic PNPLA3 expression in mice whereas starving leads to its downregulation.78 79 In cells, PNPLA3 is localised on membranes rather than adjacent to lipid droplets as one would expect, suggesting that its function may also involve receptor-like interactions with extracellular signals.79 Interestingly, the lengths and types of fatty acids to which cells are exposed lead to different PNPLA3 responses with saturated (palmitate), monounsaturated (oelate) and polyunsaturated fatty acids (linoleic acid) causing increased PNPLA3 expression, whereas long chain fatty acids such as arachidonic or eicosapentanoic acid had no such effect.76 In vitro studies with human PNPLA3 showed that PNPLA3 hydrolyses triglycerides and partitions between cell membranes and lipid droplets. Stable transfection of mutant PNPLA3 rs738409 G into HuH-7 hepatoma cells led to a marked reduction of its hydrolase activity in mutant cells compared with wild type PNPLA3, consistent with the concept of a ‘loss of function’ polymorphism allowing for ‘lipid trapping’ in the liver in case of the polymorphic variant.80 However, modelling the effect of PNPLA3 on disease phenotypes proved difficult as mice deleted for Pnpla3 by gene targeting showed no propensity to increased body weight, insulin resistance, elevated liver enzyme levels or steatosis on histology.81 82 Explanations for this obvious lack of in vivo relevance in mice could be species-related differences of the function of PNPLA3 in the evolution of murine and human steatosis; or it could be a hint towards a specific effect on inflammatory and/or fibrogenesis-associated downstream signalling pathways of PNPLA3 distinct from mere fat storage. To clarify these unresolved questions, models with Pnpla−/− mice suited to study aspects of fibrosis and necroinflammation would be helpful, particularly since it remains so far unexplained why there are clear associations of PNPLA3 rs738409 G carriage with progressive fibrosis in patients with ALD and also in patients with chronic hepatitis C who drink alcohol.83 84 This phenomenon appears to be a good example of how host-environment interactions modulate the phenotype of a disease.

    Conclusion and outlook

    In the search for genetic risk factors for ALD, much work has been carried out but most investigated genetic variants have not been confirmed in independent case–control studies. If commonly accepted quality criteria for candidate gene case–control studies are applied, only a few studies bear a certain degree of scrutiny as many of them are hampered by one or several limitations (see box 1). While genetic variation of the ADH, ALDH and GABA system have consistently been found to be associated with alcohol dependence, this risk is not further conveyed to developing ALD. In fact, only two candidate gene polymorphisms have sufficient data to consider them highly suspicious as genetic risk factors for ALD. These are TNFα −238A and PNPLA3 rs738409 G, of which only the latter is supported by robust and unequivocal data (figure 3). The data on PNPLA3 allow for the conclusion that PNPLA3 rs738409 GG carriers represent a genetically-defined subpopulation of high-risk subjects susceptible to progression of clinically unapparent disease to overt ALD. It remains to be established whether the PNPLA3 genotype represents a marker that will assist decision-making in clinical practice and whether it could serve as a therapeutic target.

    Figure 3
    Figure 3

    Schematic overview of the pathogenesis of alcoholic liver disease (ALD) and gene variants implicated in its aetiology. Confirmed or likely variants supported by consistent meta-analysis results and multiple candidate gene studies are shown in dark blue boxes (PNPLA3, TNFα). As yet unconfirmed or controversial risk genes with conflicting evidence are shown in light blue boxes with dashed lines. Mediators of alcohol-related hepatotoxicity are shown in red boxes. Genes are printed in italics. ADH, alcohol dehydrogenase; ALDH, aldehyde dehydrogenase; ApoE, apolipoprotein E; CYP2E1, cytochrome P450 2E1; FFA, free fatty acids; LSEC, liver sinusoidal endothelial cells; PPARγ, peroxisome proliferator-activated receptor γ; ROS, reactive oxygen species; TG, triglycerides; TNFα, tumour necrosis factor α.

    However, data on ALD from genome-wide analyses similar to those generated for other chronic liver diseases are still lacking.85 Conducting this kind of global scan would help to confirm PNPLA3 rs738409 as an important susceptibility gene of ALD and also potentially identify novel as yet unknown genetic variants for better screening of patients, and subsequent clinical and experimental research. Despite these limitations, functional pathway information and the genetic risk factors of ALD start to converge into a common aetiopathogenetic concept, allowing for a better understanding of this serious disorder in the future.

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    Footnotes

    • Funding Research into the genetics of ALD carried out by FS was in part funded by the Swiss National Fund (grant no. 310030_138747/1).

    • Competing interests None.

    • Patient consent Obtained.

    • Provenance and peer review Commissioned; externally peer reviewed.