Liver specific, systemic and genetic contributors to alcohol-related liver disease progression

Abstract

Alcohol-associated liver disease (ALD) impacts millions of patients worldwide each year and the numbers are increasing. Disease stages range from steatosis via steatohepatitis and fibrosis to cirrhosis, severe alcohol-associated hepatitis and liver cancer. ALD is usually diagnosed at an advanced stage of progression with no effective therapies. A major research goal is to improve diagnosis, prognosis and also treatments for early ALD. This however needs prioritization of this disease for financial investment in basic and clinical research to more deeply investigate mechanisms and identify biomarkers and therapeutical targets for early detection and intervention. Topics of interest are communication of the liver with other organs of the body, especially the gut microbiome, the individual genetic constitution, systemic and liver innate inflammation, including bacterial infections, as well as fate and number of hepatic stellate cells and the composition of the extracellular matrix in the liver. Additionally, mechanical forces and damaging stresses towards the sophisticated vessel system of the liver, including the especially equipped sinusoidal endothelium and the biliary tract, work together to mediate hepatocytic import and export of nutritional and toxic substances, adapting to chronic liver disease by morphological and functional changes. All the aforementioned parameters contribute to the outcome of alcohol use disorder and the risk to develop advanced disease stages including cirrhosis, severe alcoholic hepatitis and liver cancer. In the present collection, we summarize current knowledge on these alcohol-related liver disease parameters, excluding the aspect of inflammation, which is presented in the accompanying review article by Lotersztajn and colleagues.

The need for novel approaches to detect and prevent alcohol-related liver disease

According to the National Survey on Drug Use and Health conducted in 2015, 86.4% of U.S. adults reported having consumed alcohol at some point in their lives [1]. ALD impacts millions of patients worldwide each year and the burden is increasing [2]. The progression of ALD is well-characterized and is actually a spectrum of liver diseases, which ranges from simple steatosis, or fatty liver, to inflammation and necrosis (steatohepatitis), and ultimately, to fibrosis and cirrhosis [3]. Moreover, alcohol-related hepatitis (AH) is an acute clinical syndrome that can occur at any time during the progression of ALD with a dismal survival rate [4,5]. Although the prevalence of subclinical liver damage (e.g., steatosis) in heavy alcohol misusers is nearly 100%, only a fraction of this high-risk population will later develop alcohol-related liver disease (ALD) [4]. Unfortunately, ALD/AH is usually first diagnosed when the patients show symptoms of severe liver dysfunction (i.e., decompensation) at a very late stage of disease progression, where no therapies have been proven effective [5]. As a consequence, the overall prognosis of ALD has not improved in decades. Therefore, a major research goal is to improve diagnosis, prognosis and also treatments for early ALD before it progresses to clinically relevant disease.

The ECM, dynamic remodeling and disease progression

ALD is characterized by significant changes in the extracellular matrix (ECM) that contribute to many complications of advanced disease. The ECM contains a diverse range of components that work dynamically and bidirectionally with surrounding cells to create a microenvironment that regulates cell and tissue homeostasis [6,7]. The original definition of the ECM comprises fibrillar proteins (e.g., collagens, glycoproteins, and proteoglycans). This definition has more recently been expanded to also include ECM-affiliated proteins (e.g., collagen-related proteins, transmembrane proteoglycans), ECM regulators and modifiers (e.g., lysyl oxidases, transglutaminases or matrix metalloproteinases) and secreted factors that bind to the ECM (e.g., TGFβ, and other cytokines) [8]; this expanded definition has been renamed the ‘matrisome’ [9]. The ECM not only provides structure and mechanical support for the cells in a tissue, but also acts as a reservoir for growth factors and cytokines and as a signaling hub through which cells can communicate with their environment and vice versa [7,10,11].

Most chronic diseases are mediated, at least in part, by remodeling of the ECM. End-stage scarring of an organ/tissue is often considered synonymous with ECM remodeling and is, in ALD, associated with clinical complications such as portal hypertension and decreased liver function. However, ECM remodeling occurs in all stages of ALD and represents a key factor in the response to injury and restitution from injury. Indeed, fibrous scar formation is usually the culmination of inappropriate ECM remodeling after chronic injury. This model is likely best described for the balance between healing and scar formation after cutaneous injury [12], but appears broadly applicable to most injuries/diseases. For example, Enos et al. [13] demonstrated in a landmark study that 77% of autopsied Korean War soldiers killed in action already showed significant remodeling of their coronary arteries, indicating early stage atherogenesis. Moreover, a fraction of these young soldiers already had nearly complete vessel occlusion and were at high risk for clinically relevant cardiovascular disease later in life [14]. These findings led to a paradigm shift, whereby atherogenesis and other diseases of ECM remodeling and scarring were no longer considered unavoidable and untreatable diseases of the elderly, but rather chronic lifelong diseases with preventable (and treatable) risk factors [15].

Hepatic fibrosis caused by ALD is a canonical example of ECM dyshomeostasis, leading to accumulation of fibrillary ECM. Although hepatic fibrosis is considered almost synonymous with collagen accumulation [16], the qualitative and quantitative alterations to the hepatic ECM during fibrosis are much more diverse than simple collagen accumulation [17]. The hepatic ECM also responds rapidly and dynamically to insult, even after acute injury. Indeed we have shown dynamic transitional changes in the hepatic ECM that appear to play a key role in the normal response to acute injury and recovery, as well as setting the stage for chronic disease [6]. Homeostasis in ECM is mediated by a balance in the production of ECM, as well as in the degradation of existing ECM by matrix metalloproteinases [18]. Even in cases where there is a net increase in ECM in liver (e.g. fibrosis), overall turnover is also increased [19]. Importantly, we and others have demonstrated that even acute alcohol-mediated experimental liver injury causes a robust change to the hepatic ECM [11,20].

Hepatic stellate cells in alcoholic liver disease

Hepatic stellate cells (HSCs) are considered the main fibrogenic cell type of the liver [21]. Most insight on HSCs are derived from mouse models and cell culture experiments. As such, we have shown that HSCs constitute 80–95% of fibrogenic cells in models of toxic, biliary and non-alcoholic fatty liver disease [22]. Although it is likely that HSCs represent the main fibrogenic cell types in ALD, mice do not represent an appropriate model to study this question, as they do not develop advanced fibrosis in response to alcohol-containing diets. Moreover, recent single cell RNA-sequencing studies have revealed multiple fibrogenic/mesenchymal cell types in human liver that do not all clearly align with a clear HSCs phenotype [23]. As such, human cirrhotic liver contains four mesenchymal cell populations including HSCs, vascular smooth muscle cells, scar-associated mesenchymal cells and mesothelia [23]. In view of the difficulties in inducing alcoholic liver fibrosis in mice and potential differences between mouse and human fibrogenic cells, single cell RNA-sequencing in different stages of human ALD provides an exciting opportunity for better understanding of the regulation and role of fibrogenic cell types including HSCs in ALD. Given that the activation of fibrogenic cells and ECM remodeling exert a major impact on injury responses, it is conceivable that they not only contribute to fibrosis-associated complications in end-stage ALD but also mediate beneficial responses in earlier disease stages, as described above. Single cell RNA-sequencing and spatial transcriptomics may also provide novel insights into the interaction of HSCs with other cell types and identify transcriptional regulators that drive disease progression and the development of fibrosis in ALD. Better understanding of these processes may provide novel therapeutic targets for the treatment of ALD.

Besides liver cirrhosis and AH, the development of liver cancer, in particular hepatocellular carcinoma (HCC), represents a major cause for mortality in patients with ALD. HCC causes around 810,000 deaths annually and ALD is responsible for approximately 30% of these HCC-related deaths [24], which far exceeds the number of HCC deaths caused by non-alcoholic fatty liver disease at this time, however it receives only little attention in basic research. There is increasing evidence that the accumulation of activated HSCs and alterations of the ECM contribute to increased risk for HCC development in chronically injured and cirrhotic liver [25,26]. However, the mechanisms of alcohol-related HCC remain poorly understood and it is not clear if they are similar to HCC caused by other liver diseases, which is partly due to the paucity of mouse models for alcohol-related HCC.

Interdependency of HSCs and matrisome

HSCs as well as the hepatic matrisome’s dynamism represent potential therapeutic targets for different stages of ALD. Although the most profound changes in both, HSCs fate and the matrisome are found in late disease stages, we propose that they exert key functions even in early disease stages which deserves further investigation. It is also conceivable that the role of HSCs and/or the matrisome changes from restorative in early stages to disease-promoting in later stages. Further investigations are needed to explore the hepatic matrisome’s functions in the context of inflammation, adhesion, structure, presentation and storage, as well as sensing (Figure 1). Likewise, the role of fibrogenic cells, including HSCs, in ALD needs to be investigated in all disease stages and also fibrosis-independent as well as protective functions of HSCs need to be considered (Figure 2). Furthermore, the role of HSCs and the matrisome in ALD-associated HCC needs to be better understood. It is likely that there is a bidirectional crosstalk between the matrisome and HSCs. As such, HSCs are impacting the matrisome composition through the secretion of ECM and growth factors. Vice versa, the matrisome is modulating HSCs activation and fate in both positive and negative manner through changes in stiffness and physical properties, matrix degradation products and the release of growth factors. Better understanding of the underlying pathobiology, the complex molecular interactions and alterations in the matrisome as well as disease-promoting and restorative functions of HSCs may provide novel therapeutic options for the treatment of ALD at different disease stages.

Figure 1. Functions of the hepatic extracellular matrix (ECM) that contribute to inflammation.

The ECM plays multiple roles that contribute to the regulation of inflammation and injury in the liver. These roles are related to adhesion, structure, presentation of signaling molecules and/or receptors, storage of signaling molecules, and sensing. Figure modified with permission from Dolin and Arteel [11].

Figure 2. Roles of hepatic stellate cells in ALD.

Hepatic stellate cells and other types of fibrogenic cells are present and/or activated in early and late-stage ALD. Main functions include the secretion of ECM and growth factors (GF), the latter often being bound to ECM for long-term storage. While the disease-promoting role of HSC/fibroblast in advanced disease, leading to decreased liver function and increased risk for HCC, are increasingly established, their role in early disease remain poorly understood and could also include protective functions that either promote regeneration or protect from injury.

Impact of the gut on alcohol-related liver disease progression and alcoholic hepatitis

The human body developed a symbiotic relationship with microbes in the gut. The intestinal microbiota contains bacteria, archaea, viruses and fungi. Changes in the bacterial microbiota in patients with alcohol use disorder and alcohol-associated liver disease have been well characterized and described elsewhere in great detail [27]. The importance of the intestinal microbiota for alcohol-associated liver disease has been demonstrated in several preclinical and clinical studies. In a landmark study, mice transplanted with stool from a patient with severe alcoholic hepatitis developed more severe steatohepatitis than mice transplanted with stool from a patient without alcoholic hepatitis [28].

Patients with alcoholic hepatitis have a significantly lower bacterial diversity than patients with alcohol use disorder or non-alcoholic controls [29]. Patients with alcoholic hepatitis were compared based on the median bilirubin level. The high bilirubin patient group had a different fecal microbiota composition than the low bilirubin group. The patient group with more cholestasis was characterized by an increase in Veillonella and Enterococcus, and a reduction in Akkermansia [30]. A significant negative correlation between MELD scores and bacterial diversity was found in patients with alcoholic hepatitis, which was independent of antibiotic or steroid treatment [30]. Patients with alcoholic hepatitis and severe disease, as defined by a histologic scoring system, showed a higher proportion of Streptococcus, Bifidobacterium and Enterobacteria in the fecal microbiota [28]. Patients with alcoholic hepatitis exhibited lower proportions of Akkermansia muciniphila as compared with healthy controls [31].

Using qPCR it was recently demonstrated that fecal samples from patients with alcoholic hepatitis had about 2,700-fold more Enterococcus faecalis (E. faecalis) than samples from non-alcoholic control subjects[29]. The total amount of fecal E. faecalis or fecal E. faecalis positivity did not correlate with disease severity or mortality in patients with alcoholic hepatitis[29]. Cytolysin is a bacterial exotoxin produced and secreted by E. faecalis. Genomic DNA of the two subunits of cytolysin was detected in fecal samples from 30% of patients with alcoholic hepatitis (cytolysin-positive), but not in non-alcoholic controls and only in samples from one patient with alcohol use disorder[29]. The majority (89%) of cytolysin-positive, but only 3.8% of cytolysin-negative patients with alcoholic hepatitis died within 180 days following hospital admission [29]. The presence of cirrhosis was not associated with cytolysin-positivity or the total amount of fecal E. faecalis [29]. Only seven out of 96 patients with non-alcoholic fatty liver disease (NAFLD) were cytolysin-positive, and cytolysin-positivity was not associated with increased liver disease activity [32]. These results indicate that the presence of cytolysin producing E. faecalis determines the severity of alcoholic hepatitis and mortality, but is not a general biomarker for severity of any chronic liver disease.

Cytolysin directly killed primary mouse hepatocytes in culture, and cytolysin-positive E. faecalis exacerbated ethanol-induced liver disease in mice [29]. Cytolysin did not affect gut barrier dysfunction in mice, indicating that cytolytic E. faecalis is sufficient for ethanol-induced liver disease acting directly on the liver rather than the intestine. Bacteriophages (phages) are small viruses that can recognize, infect, replicate inside and lyse specific bacteria. Oral gavage of phages reduced ethanol-induced liver disease in gnotobiotic mice colonized with feces from cytolysin-positive patients with alcoholic hepatitis [29]. Reducing cytolysin-negative E. faecalis in gnotobiotic mice did not decrease ethanol-induced liver disease, indicating that cytolysin-positive E. faecalis are necessary for alcohol-associated liver disease. This novel mechanism demonstrates the importance of the pathobiont for alcoholic hepatitis. Pathobionts are commensal bacteria that are not pathogenic under homeostatic conditions. They can cause disease onset or progression when their genetic condition or immune surveillance of the host are altered. Future studies are required to determine whether other virulence factors of pathobionts are increased and contribute to disease progression in patients with alcohol-associated liver disease.

Research in the field of gut microbiota has largely focused on bacteria. Fungi are an integral part of the human gut microbiota. Although bacteria outnumber fungi in the human microbiome (10¹³–10¹⁴ for bacteria, 10¹²–10¹³ for fungi), the cell size of fungi is much larger than that of bacteria [33]. The fungal microbiome (also called mycobiome) consists mainly of members of the phylum Ascomycota (Candida, Saccharomyces, Aspergillus, and Malassezia) [33]. We observed a significant increase of fungal colony forming units in feces from wild type C57BL/6 mice fed a chronic Lieber DeCarli diet for 8 weeks compared with mice fed an isocaloric control diet as determined by culture dependent and culture independent techniques [34,35]. Similarly, we found fungal overgrowth in stool from patients with alcoholic hepatitis compared with fecal cultures from patients with alcohol use disorder and non-alcoholic controls [34]. Using single colony PCR, a significantly higher proportion of patients with alcoholic hepatitis had positive cultures for Candida albicans (C. albicans) than patients with alcohol use disorder [34]. Using culture independent internal transcribed spacers (ITS)-sequencing, patients with alcohol use disorder and alcoholic hepatitis had a lower fungal diversity in the fecal mycobiome than non-alcoholic controls. The genus Candida was significantly higher in patients with alcohol use disorder and alcoholic hepatitis, whereas the genus Penicillium dominated the mycobiome of nonalcoholic controls [36]. Two weeks of alcohol abstinence resulted in lower abundance of the genera Candida, Malassezia, Pichia, Kluyveromyces, Issatchenkia, and the species Candida albicans and Candida zeylanoides in patients with alcohol use disorder [37]. The specific anti-Candida albicans immunoglobulin G (IgG) expression was increased in patients with alcohol use disorder as compared with non-alcoholic controls. Abstinence for two weeks resulted in a significant reduction of anti-Candida albicans IgG levels [37]. Thus, alcohol use disorder alone is associated with a significant absolute and relative abundance of C. albicans in the fecal mycobiota and an increased immune response.

Do fungi and in particular C. albicans contribute to ethanol-induced liver disease? Reducing intestinal fungi with the poorly absorbable amphotericin B reduces ethanol-induced liver disease in mice. The disease exacerbating effect is partly mediated via the fungal cell wall component β-glucan, which binds to the pattern recognition receptor C-type lectin domain family 7 member A (Clec7a; also called dectin-1) and activates the inflammasome in Kupffer cells and possibly other bone marrow-derived cells [35]. Candidalysin is a peptide toxin secreted by Candida albicans [38]. Candidalysin increases ethanol-induced liver disease independently of the β-glucan receptor Clec7a in mice without affecting intestinal permeability. Candidalysin can directly cause cell death of primary mouse hepatocytes, indicating a direct role of candidalysin on ethanol-induced liver disease [34]. The detection of the candidalysin encoding gene, ECE1, was further associated with increased liver disease severity and mortality in patients with alcoholic hepatitis [34].

Intestinal fungi and bacteria show positive and negative associations in patients with alcoholic hepatitis [39], thus mechanistic studies are required to determine the contribution of bacterial and fungal interaction for the progression of alcohol-associated liver disease.

The biliary system in liver fibrosis

Ductular reaction in fibrogenesis -

The biliary tract of the liver plays an important role in the formation and secretion of bile as well as excretion of toxic substances [40]. It adapts to chronic liver disease by complex morphological and functional changes, with ductular reaction (DR) representing one of the most obvious features [41,42]. DR is defined as an increased occurrence of structures with a ductular phenotype, possibly but not necessarily of ductular origin [43,44]. A well-established model to study DR is ligation of the common bile duct in mice, which induces a highly reproducible sequence of changes in the architecture of interlobular bile ducts. Initially, cholangiocytes proliferate, followed by corrugation of the inner surface of the ducts, branching and elongation of the new branches and finally loop formation of the new ducts by self-joining [40]. By this process, an initially relatively sparce mesh of interlobular bile ducts surrounding the portal vein becomes much denser.

The lobular zonation of the DR depends on the damage pattern of the hepatotoxic insult. After BDL that induces major periportal damage, we observed exclusively new bile duct branches arising from preexisting ducts [40]; up to 28 days after BDL, the newly formed ducts remained periportal and never infiltrated into the pericentral lobular zone. In contrast, CCl₄, thioacetamide or alcohol exposure to mice cause damage to the pericentral region; after chronic exposure to these compounds, new bile ducts infiltrate into the damaged central lobular zone [41,45]. Intravital imaging with fluorescent bile salt analogues demonstrated that the newly formed ducts efficiently drain bile. It still remains an open question if DR or fibrotic streets appear first. The concept of cholangiocytes as fibrogenic drivers is supported by reports that cholangiocytes of DR activate portal fibroblasts and stellate cells by secreting platelet-derived growth factor, vascular endothelial growth factor and TGFβ [46–48]. Moreover, the DR cell population may attract immune cells by chemokines, such as the B-cell chemoattractant Ccl 20 [49]. These observations suggest that the proliferating cholangiocytes may play a causal role in fibrogenesis and may recapitulate features of the process by which they induce the embryonic development of periportal fields [50]. As a further mechanism, bile ducts may become leaky in inflamed hepatic tissue and the released bile acids may cause cytotoxicity to adjacent hepatocytes, further enhancing the inflammatory, pro-fibrotic microenvironment [51]. Although evidence has been presented it remains to be systematically studied if DR really is an initial event that induces all further processes in the formation of fibrotic streets.

Ductular reactions compensate for the compromised diffusion dominated zone -

The pathophysiological relevance of DR is currently controversially discussed. On one hand, it represents a tissue level adaptive response to cholestatic situations [40,43,52]. On the other hand, it has been shown that the extent of DR is closely related to the progression of liver fibrosis [42,47,53–56]. In support of the concept of an adaptive response, bile ducts infiltrate into tissue regions with compromised canalicular flux. Recently, imaging techniques have been established that allow the direct flux analysis in bile canaliculi and interlobular bile ducts in intact livers of anaesthetized mice [57,58]. In contrast to the prevailing osmotic concept, these analyses have shown that the transport of solutes in the liquid of bile canaliculi is diffusion dominated, while flow is negligibly small; in contrast, diffusion is augmented by flow in the interlobular ducts [57]. This framework of bile flux becomes relevant to understand under which conditions bile ducts infiltrate into liver tissue. A recent landmark study demonstrated that ductular reactions occur as soon as bile canaliculi have been structurally and functionally compromised, which can e.g. be induced by knockdown cytoskeletal protein radixin [59]. Thus, on the background of the novel concept of bile flux (Figure 3), ductular reactions can be interpreted as an expansion of the flow augmented zone (interlobular bile ducts) to compensate for the compromised diffusion dominated zone (bile canaliculi) [57]. Many damage types may compromise the diffusion dominated zone, including hepatotoxic compounds such as alcohol, obstructive cholestasis, and NASH [52,57,60]. A typical morphological feature of the damaged diffusion dominated zone is fragmented and/or widened bile canaliculi due to compromised hepatocyte polarity [52]. If bile ducts infiltrate into such regions, they may support clearance of bile acids from the compromised diffusion dominated zone which may ameliorate cholestasis [58]. On the other hand, a long-term complication of ductular reactions is the above-described fibrotic streets that are associated with the new ducts. This Dr. Jekyll and Mr. Hyde situation concerning ductular reaction illustrates the therapeutic dilemma, since antagonization of DR on the one hand ameliorates long-term fibrogenesis, but on the other hand aggravates cholestasis, at least if the pathogenic cause has not yet been eliminated.

Figure 3.

The liver lobule consists of the diffusion dominated canalicular zone and the flow augmented network of interlobular ducts [57,58]. When the canalicular network is compromised in chronic liver disease, new ducts infiltrate into the diffusion dominated zone to support the clearance of bile acids. While this process may ameliorate cholestasis, the new ducts may also induce the formation of fibrotic streets, although a causal relationship still has to be analyzed.

Host genetics of alcohol-related liver disease

In addition to environmental factors such as drinking patterns, coexisting non-alcoholic liver disease, obesity, dietary habits, and certain co-medications, a plethora of evidence suggests a relevant contribution of host genetic factors to the predisposition of developing alcohol-related liver disease (ALD). Twin studies have convincingly demonstrated a significant contribution of hereditary factors to the evolution of ALD as demonstrated by a three-fold higher disease concordance between monozygotic twins and dizygotic twins [61]. In addition, there are ethnic and gender differences in the tolerance towards alcohol, showing a higher risk of developing ALD in Asians and women when exposed to similar amounts of alcohol, indicating that the genetic background is pivotal [62–64].

The quest for robust genetic modifiers of ALD was strongly fostered by the first genome-wide association study in subjects with non-alcoholic fatty liver disease (NAFLD), in which a sequence variation within the gene coding for patatin-like phospholipase encoding 3 (PNPLA3, rs738409) increased liver fat content two-fold [65]. Shortly thereafter, this risk variant also showed significant association with progressive ALD including cirrhosis, which has since then been robustly confirmed by numerous candidate association studies in almost all ethnicities around the globe [66].

Apart from PNPLA3, several additional risk loci have been confirmed which modulate the risk of ALD progression, albeit with lower effect sizes. These include genetic variants in the genes coding for transmembrane 6 superfamily member 2 (TM6SF2) [67], membrane bound O-acyltransferase domain containing 7 (MBOAT7) [67], hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) [68,69], mitochondrial amidoxime reducing component 1 (MARC1) [70], heterogeneous nuclear ribonucleoprotein U like 1 (HNRNPUL1) [70], and Fas-associated factor family member 2 (FAF2) [71].

While risk loci in PNPLA3, TM6SF2, MBOAT7, and HNRNPUL1 increase the risk of ALD progression, risk variants in HSD17B13, MARC1 and FAF2 confer protection towards progressive ALD (Figure 4). Some variants were also found to be associated with other chronic liver diseases such as haemochromatosis (PNPLA6, TM6SF2) [72], chronic hepatitis C (PNPLA3) [73], or to variably modulate the course of other diseases, such as cardiovascular diseases (TM6SF2) [74] or COVID-19 (PNPLA3) [75].

Figure 4. Genetic loci modulating risk for onset and progression of ALD.

Both risk-increasing and protective loci with different effect sizes have been identified.

Importantly, some variants were also found to modulate the risk of hepatocellular carcinoma (HCC) on the background of ALD (PNPLA3, TM6SF2, HSD17B13) [69,76–79], which may serve the clinical task of stratifying alcoholic cirrhosis at risk of HCC according to their genetic risk profile. Patients identified as being at particular risk of developing HCC based on a polygenic risk score could thus be screened more carefully (e.g. by MRI scanning) and frequently (3-monthly instead of 6-monthly) to allow for timely detection and better treatment outcomes [69].

Interestingly, none of the above-mentioned modulator genes are related to molecular mechanisms that are strongly specific to alcohol-mediated hepatotoxicity, alcohol metabolism or patterns of alcohol dependence. In fact, none of the genetic polymorphisms implicated in earlier candidate gene association studies (e.g. TGFβ1, TNFα, alcohol-metabolizing enzymes) were found significantly associated with ALD in genome-wide scans [67,68,70,71]. Instead, several genes with significant risk loci are closely to lipid turnover and transport (PNPLA3, TM6SF2, MBOAT7, HSD17B13), providing an explanation for the strikingly similar genetic background of ALD and non-alcoholic fatty liver disease (NAFLD) [80]. While human association data is undisputed, the functional implications of the identified genes still remain incompletely understood. Experimental translation in mice genetically modified for respective genes also remains inconclusive, as mice implicated in risk loci for human diseases develop only mild or no hepatic disease phenotypes, similar to that of human ALD [81–83].

Clinical translation of genetic insight into useful biomarkers is under way, although effect sizes of individual risk loci are small and far from adequate as a diagnostic or prognostic test. However, genotypes could be computed within polygenic risk scores that more closely reflect an individual’s risk of developing cirrhosis or HCC, making personalized medicine an imaginable scenario in the near future.