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

Abstract and Introduction


Background and Aims: Non-alcoholic fatty liver disease (NAFLD) is a global health burden. Risk factors for disease severity include older age, increased body mass index (BMI), diabetes, genetic variants, dietary factors and gut microbiota alterations. However, the interdependence of these factors and their individual impact on disease severity remain unknown.

Methods: In this cross-sectional study, we performed 16S gene sequencing using fecal samples, collected dietary intake, PNPLA3 gene variants and clinical and liver histology parameters in a well-described cohort of 180 NAFLD patients. Principal component analyses were used for dimensionality reduction of dietary and microbiota data. Simple and multiple stepwise ordinal regression analyses were performed.

Results: Complete data were available for 57 NAFLD patients. In the simple regression analysis, features associated with the metabolic syndrome had the highest importance regarding liver disease severity. In the multiple regression analysis, BMI was the most important factor associated with the fibrosis stage (OR per kg/m2: 1.23, 95% CI 1.10–1.37, P < .001). The PNPLA3 risk allele had the strongest association with the histological grade of steatosis (OR 5.32, 95% CI 1.56–18.11, P = .007), followed by specific dietary patterns. Low abundances of Faecalibacterium, Bacteroides and Prevotella and high abundances of Gemmiger were associated with the degree of inflammation, ballooning and stages of fibrosis, even after taking other cofactors into account.

Conclusions: BMI had the strongest association with histological fibrosis, but PNPLA3 gene variants, gut bacterial features and dietary factors were all associated with different histology features, which underscore the multifactorial pathogenesis of NAFLD.


Non-alcoholic fatty liver disease (NAFLD) has evolved as the most common chronic liver disease in the world affecting approximately 25%-30% of the population in Western countries.[1] Whereas nonalcoholic fatty liver, the most common type, has a relatively benign prognosis, the risk for disease progression including the development of liver fibrosis, cirrhosis and the development of hepatocellular cancer is increased in individuals with non-alcoholic steatohepatitis (NASH) and progressive fibrosis.[2] As approximately 20% of patients with NAFLD will develop progressive disease[3] and no treatment options except for lifestyle modifications are available, the early identification of patients at risk is important.

NAFLD represents a multifactorial disease, and several risk factors for disease progression in the setting of NAFLD have been identified. These include older age, increased body weight, the presence of type 2 diabetes, genetic variants such as the common PNPLA3 (encoding patatin like phospholipase domain-containing protein 3) p.I148 M polymorphism and dietary factors.[4] In addition, changes in the gut bacterial microbiota composition with an overgrowth of potentially detrimental bacterial species and a deficit of potentially beneficial gut bacteria, as well as a reduced bacterial diversity, have been identified as additional cofactors that might contribute to the deterioration of NAFLD.[5–8] Several mechanisms could explain how specific changes in the microbiota composition might modulate NAFLD. Gut barrier dysfunction, which has been described in patients with NAFLD, can lead to the translocation of microbes or microbial metabolites to the liver, where they can induce an inflammatory response and activate profibrotic pathways. In addition, gut bacteria are responsible for the deconjugation of bile acids that are produced by the liver and for the conversion of primary into secondary bile acids. Bile acids act as signalling molecules that bind to host nuclear and G-protein-coupled receptors, which have an impact on several host metabolic functions. Synthesis of short-chain fatty acids by gut bacteria, an increased energy harvest by gut bacteria and endogenous ethanol production represent other potential mechanisms on how the gut bacterial microbiome might affect NAFLD.[9]

Because most studies investigated associations between these factors and disease severity individually, it is unclear whether the observed gut microbiota changes in more advanced NAFLD are independently associated with the disease or if they are rather secondary to the frequently observed metabolic comorbidities such as type 2 diabetes, obesity and dyslipidemia. Further, the importance of the gut microbiota in relation to clinical factors, diet and the PNPLA3 polymorphism remains unclear.

In this study, we analysed gut microbiota, dietary data, PNPLA3 p.I148M genotypes and various clinical parameters from a very well-characterized NAFLD cohort. We aimed to investigate, which of the individual gut microbial, dietary, genetic and clinical factors have the most important association with NAFLD severity when taking all factors into consideration. For this purpose, we performed simple and multiple stepwise ordinal regression analyses using the liver histology features steatosis, inflammation, ballooning, the NAFLD activity score and the stage of fibrosis, as outcome parameters.