Combined Analysis of Gut Microbiota, Diet and PNPLA3 Polymorphism in Biopsy-proven Non-alcoholic Fatty Liver Disease

Sonja Lang; Anna Martin; Xinlian Zhang; Fedja Farowski; Hilmar Wisplinghoff; Maria J.G.T. Vehreschild; Marcin Krawczyk; Angela Nowag; Anne Kretzschmar; Claus Scholz; Philipp Kasper; Christoph Roderburg; Raphael Mohr; Frank Lammert; Frank Tacke; Bernd Schnabl; Tobias Goeser; Hans-Michael Steffen; Münevver Demir


Liver International. 2021;41(7):1576-1591. 

In This Article


NAFLD is considered a multisystem disease. Due to its close interconnection with other metabolic diseases and shared risk factors with them (eg, obesity and overnutrition), it is inherently difficult to dissect the impact of distinct pathogenic pathways to the overall phenotype. However, in order to therapeutically target the most relevant of these pathways, to adapt targeted interventions to the stage of NAFLD and to reduce the risk of non-efficacious treatment strategies, it is of utmost importance to understand which of the manifold factors promote features of NAFLD progression in the clinical setting of a real-life patient cohort. To our knowledge, this is the first study investigating associations between the gut microbiome, the PNPLA3 polymorphism, dietary factors and clinical features in one well-described NAFLD cohort. We have chosen stepwise multiple ordinal regression analyses as a powerful approach assessing associations across a relevant range of outcomes. Dichotomizing outcomes that are originally measured on an ordinal scale is a common practice but results in loss of information.[23]

We found that covariates that we investigated were differently associated with specific features of liver histology, which supports the multifactorial pathogenesis of NAFLD severity. Given all investigated variables, the BMI had the strongest association with liver fibrosis and was also associated with inflammation, ballooning and the NAS in the multiple regression analyses. This indicates that a higher BMI is one of the most important modifiers of NAFLD severity and underlines that lifestyle modification to achieve weight loss remains the first-line intervention in patients with NAFLD, as recently emphasized in a clinical practice guideline update of the American Gastroenterological Association.[27] The presence of type 2 diabetes was one of the most important factors associated with liver inflammation, ballooning, the NAS and stages of fibrosis in the simple regression analysis but seems to be dependent from other clinical factors such as age, the BMI and the presence of dyslipidemia in the multiple regression analysis. This indicates that those clinical factors that typically co-occur with the presence of type 2 diabetes are potentially moderating associations with liver disease severity.

The carriage of the PNPLA3 p.I148M polymorphism was the most important feature independently associated with higher histological degrees of steatosis and NAS, which is in line with other studies showing an increased risk for disease progression, the occurrence of liver-related events[28] and hepatocellular cancer[29] in patients with either a heterozygote or homozygote variant. In our study, this association remained significant even after taking additional potential synergistic cofactors such as an increased BMI or dyslipidemia into account, indicating a pivotal role of this variant as endogenous determinant of severe NAFLD.

Whereas several studies investigate associations between the gut microbiota composition and disease severity in NAFLD individually, we further demonstrate in our analysis that specific gut bacterial features are associated with NAFLD severity even after considering other cofactors that might otherwise bias the results. The strongest associations were observed for liver inflammation, hepatocellular ballooning and the fibrosis stage. Translocation of gut bacteria through the portal vein to the liver is one mechanism of how the gut bacterial microbiota might mediate disease. Once having reached the liver, microbial-derived pathogen associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) are recognized by specific receptors such as toll-like receptors, where their activation can mediate hepatic inflammation. Further, activation of the NOD-like receptor protein 3 (NLRP3) inflammasome has been identified as another trigger for liver inflammation responding to PAMPs in the setting of NAFLD. NLRP3 is significantly upregulated in the liver of patients with NASH compared with simple steatosis, and preclinical data have demonstrated that NLRP3 activation results in the activation of caspase 1 as well as in the production of several inflammatory cytokines, which ultimately results in programmed cell death, inflammation and fibrosis.[30] Of the gut microbial taxa at genus level, lower abundances of Faecalibacterium were associated with higher stages of fibrosis, a higher NAS and higher degrees of ballooning in the multiple regression analyses. As a member of the Ruminococcaceae family, Faecealibacterium prausnitzii (F prausnitzii) is the only species within the genus of Faecalibacterium that has been successfully isolated.[31]F prausnitzii is an important short-chain fatty acid producer and has anti-inflammatory properties.[32] A lower abundance of Faealibacterium in more advanced NAFLD has been described before.[33,34] In a randomized-controlled trial, treatment of obese women with prebiotics led to an increase of F prausnitzii, which was associated with lower circulating levels of LPS.[35] Overall, our data indicate that a depletion of intestinal Faecalibacterium might play an important role in NAFLD disease severity.

A higher relative abundance of Gemmiger, on the other hand, was associated with more severe disease in our patients. Gemmiger is a gramme-negative bacterium that also belongs to the Ruminococcaceae family and has been associated with the presence of early hepatocellular carcinoma[36] and Crohn's disease.[37] We have shown previously that the relative Gemmiger abundance is positively correlated with increased liver enzymes, ferritin and overweight.[17] Overall, further mechanistic studies are needed to clarify a potential detrimental role of Gemmiger in the setting of NAFLD. Our study revealed associations between the gut microbiome and NAFLD disease severity that remained significant even after taking other cofactors into account. Microbe-based treatments such as administration of prebiotics, probiotics or a combination of both (synbiotics) might be an attractive treatment approach and should be studied in clinical trials.

Our study has several limitations. Although we found associations between the gut microbiome, the PNPLA3 polymorphism, dietary factors and clinical features, we cannot make causal conclusions due to the cross-sectional study design. Particularly when considering factors with high colinearity such as conditions of the metabolic syndrome, it is important to bear in mind that failing to be included in the multiple regression models does not necessarily indicate that those factors are not of relevance. We have chosen a forward selection process in which a choice is made over two variables that are correlated with each other and where the variable with the higher importance over the other variable regarding the respective outcome is included in the final multiple regression model. Therefore, failure to enter the final model rather indicates that other colinear factors are of higher importance when considering all variables. Overall, longitudinal studies are needed to investigate which of the studied factors are the most relevant in terms of liver-related and overall outcomes.

Focusing only on patients with all data completely available allowed us to analyse a very well-characterized cohort and limited our sample size. In relation to the high number of explanatory variables, this might have led to model instability, and the results need to be confirmed in larger cohorts.

Collecting diet over 14 days provides very detailed information about food choices and has some advantages over other collection methods such as food frequency questionnaires.[38,39] Dietary assessment however is generally very vulnerable to distortion and even if patients were instructed on how to use the dietary report in as much detail as possible, high motivation is needed to follow the requirements, and the analysis relies on honesty and accuracy of the patients' disclosures. Even if we included energy misreporting in our analyses, miscalculation or underreporting of food portions might have occurred and could have biassed the results. In addition, focusing more than usual on the diet due to the dietary recording can create more awareness and therefore may change dietary habits, even though patients were instructed not to change usual dietary habits over the recording period.

In conclusion, specific gut microbial, dietary, the PNPLA3 polymorphism and clinical features are significantly associated with different histology features, which underscore the multifactorial pathogenesis of NAFLD. A higher BMI, the PNPLA3 risk variant, lower relative abundances of Faecalibacterium or Prevotella, higher relative abundances of Gemmiger and dietary patterns low in fibre and specific vitamins as well as enriched in amino acids, uric acid and purine seem to represent factors of crucial importance for NAFLD disease severity. A better understanding of synergistic effects driving simple steatosis to more advanced disease stages might help to develop improved strategies for the prevention of disease progression and could result in better individualized treatment strategies such as personalized nutrition or microbe-based therapeutics resulting in effective prevention of disease progression and improved treatment of the global burden of NAFLD.