We extend previous work by showing that the G allele of rs738409 in PNPLA3
is associated with histologic steatosis as well as NASH, fibrosis and cirrhosis. Particularly striking is the association of the G allele of rs738409 with decreased likelihood of having a zone 3 centered distribution of steatosis. Zone 3 centered steatosis is more often observed in early stages of NAFLD and is less likely to be associated with ballooned hepatocytes, Mallory-Denk bodies or advanced fibrosis than panacinar or azonal distributions of steatosis.23
Zone 3 hepatocytes are characterized by higher levels of glycolysis, liponeogenesis and ketogenesis than periportal zone 1 hepatocytes which in turn have a higher level of gluconeogenesis, urea synthesis, and bile acid and cholesterol synthesis.24-27
Although zone 3 hepatocytes may be metabolically well-suited for lipogenesis in the normal liver, in advanced disease the ability of this zone to buffer the energy overload may be overwhelmed and fat deposition throughout the liver may predominate. When the normal mechanisms that protect hepatocytes from fatty acid damage get overwhelmed, lipotoxicity, cell death and triggering of stellate cell activation ensue.28
These processes can lead to the recruitment of inflammatory cells to the liver and to the deposition of extracellular matrix, resulting in fibrosis and cirrhosis.28
Consistent with this hypothesis, the G allele of rs738409 in PNPLA3
is associated more frequently with diffuse fat deposition (not just limited to zone 3) it may promote NASH, fibrosis and cirrhosis throughout the liver.
One of the strengths of this work is that we were able to match cases of NAFLD with controls to enable us to find variants that associate with NAFLD. An alternative approach would have been to examine severity of phenotypes within the NAFLD sample alone. The case-control approach provided greater power: with fewer than 600 cases of NAFLD, we were able to show genetic associations of rs738409 with P values ranging from 1×10−38 to 1×10−47, whereas in a case-only analysis, our lowest P value was 5 × 10−11. This increased power by adding MIGen is due to the increased overall sample size as well as increased phenotypic breadth of the samples since few or no individuals in the NASH CRN just have just steatosis or no steatosis, respectively. The addition of these ancestry-matched controls did not appear to generate large numbers of false positives in that only variants near PNPLA3 strongly associated with NAFLD, whereas the other tested variants did not.
Limitations of this work include the ascertainment of the NASH CRN on the basis of NAFLD at one timepoint and the analysis of individuals of European ancestry; thus, results may not be directly translatable to differently ascertained samples of other ancestries or be the same over time. Although we have tried to assess the effect of the G allele of rs738409 in PNPLA3 in large meta-analyses, a small effect on metabolic syndrome components cannot be ruled out. Further the results may be affected by unmeasured confounders but the results are undiminished when controlling for measured confounders. The presence of liver disease in MIGen would cause misclassification and would bias our results towards the null, slightly reducing power compared with an equivalently sized sample known to be free of liver disease. However, this misclassification would not cause spurious associations, so the strong association between PNPLA3 and NAFLD/ NASH remains valid even in this setting. Finally, because many of the histologic subphenotypes of NAFLD are highly intercorrelated in the NASH CRN sample, further work will be needed to better characterize and possibly distinguish the specific effects of rs738409 on these phenotypes in patients with histologic NAFLD.
This is to our knowledge the first well-powered assessment of the effects on histology-based NAFLD of genetic variants previously associated with liver enzyme levels. Our results suggest that variation at PNPLA3 specifically and strongly influences the development of advanced NAFLD including NASH, fibrosis and cirrhosis. Given that PNPLA3 appears to be part of a family of enzymes that affect lipid metabolism, this suggests that altering lipid metabolism, particularly within the liver, can affect accumulation of fat and subsequent development of NAFLD. These results therefore suggest that certain inherited variations in lipid metabolism precede and could lead to the development of liver disease. Because this variant appears to not have a strong population effect on diabetes, obesity or serum lipid levels, the development of fatty liver disease can be at least partially dissociated from epidemiologically correlated variables. Thus, through genetic analyses, we may be able to delineate the causal pathways that lead to specific disease complications of metabolic risk factors such as NAFLD and, in the future, selectively target them for therapeutic intervention.