In this study, using a large cohort of patients with NAFLD and well-defined histological information, we confirm the association of the minor alleles of rs738409 and other SNPs in the PNPLA3 gene region with liver steatosis and liver enzymes. More importantly, we show a strong association of PNPLA3 with histological parameters of disease activity (inflammation and Mallory-Denk bodies) and with fibrosis severity, associations that are independent of the effect on steatosis. Thus, the presence of the minor allele of rs738409 is a strong predictor of NAFLD histological severity.
Interestingly, the rs738409 G allele was present in the same frequency in patients with NASH or patients with steatosis alone, despite its marked enrichment in both groups compared to population controls, and despite its association with disease severity. This is probably related to the lack of association between rs738409 and hepatocyte ballooning, a histological feature that heavily favors a diagnosis of NASH by the pathologist (27
). Another explanation may result from a potential selection bias. In order to be included, patients had to have been referred to a hepatologist and have an indication for a liver biopsy. Thus, it is possible that the patients with steatosis only are not fully representative of simple steatosis and have very mild features of NASH (perhaps obscured by sampling error).
One study to date has investigated the association of rs738409 with histological parameters of NAFLD (29
) by comparing 172 patients with NAFLD (103 of whom had liver biopsies) to 94 normal controls. In that study, the minor allele was associated with histological steatosis and overall severity, similar to our findings. Our larger sample size enabled us to study in greater detail the associations with specific components of histological severity and to demonstrate their independence of steatosis grade. In contrast to our findings, Sookoian et al (29
) observed a significant difference in rs738409 genotypes between patients with NASH and steatosis. The two studies differ in the population prevalence of the minor allele, in the definition of NASH and in the recruitment strategy of patients. Further studies are necessary to resolve this discordant result.
NAFLD is becoming increasingly prevalent in pediatric patients (30
). We did not identify an association between the PNPLA3 SNPs and severity in the pediatric cohort, probably because of a milder disease at an early stage of the NASH natural course, as evident by the smaller proportion of pediatric patients with a definite NASH diagnosis. However, the presence of the rs738409 G allele was associated with a younger age at the time of liver biopsy, suggesting a younger age of disease presentation.
There are important racial and ethnic differences in the prevalence of fatty liver disease and the predisposition to NASH; patients of Hispanic ethnicity are more likely to have NASH and NAFLD, while African-Americans seem to be relatively protected (31
). The racial distribution in our adult cohort did not allow us to determine whether the effects of genetic variation explain the ethnic-racial differences or are independent of them.
PNPLA3 encodes for adiponutrin, a membrane-bound protein expressed primarily in adipose tissue (32
). The expression level of adiponutrin mRNA is tightly regulated by food intake (33
) through changes in insulin levels (35
). The function of adiponutrin is not fully understood. It has close homology with adipose triglyceride lipase (37
) and seems to have both triglyceride lipase and acyl-CoA-independent transacylase activity (38
). rs738409C->G is a nonsynonymous coding SNP, creating an I148M change in the adiponutrin protein. Recently, this mutation was suggested to impair triglyceride hydrolysis in hepatocytes, a potential explanation for increased triglyceride accumulation (26
). Interestingly, despite the association with liver steatosis, this SNP does not seem to be associated with insulin resistance (10
) or with obesity (39
). It has been reported to be associated with lower cholesterol and LDL-cholesterol levels (14
), but whether this is related to the liver disease is unclear. It is possible that another genetic variant, strongly linked to rs738409, may be responsible for the difference in phenotypes.
We have also found an association of NASH fibrosis with several SNPs on chromosome 10, in the vicinity of the CHUK1 gene, encoding IKKα which is involved in the NFκB signaling and CPN1 which encodes carboxypeptidase N, a metalloproteinase that regulates the activity of kinins and anaphylotoxins. Despite the association with increased fibrosis, the minor alleles at this locus are associated with lower ALT in our cohort as well as in other studies (11
). This apparently paradoxical association is explained by the non-monotonous relationship between ALT and fibrosis (increasing fibrosis stage associated initially with higher ALT and then with lower ALT) in patients with NAFLD. These SNPs are not associated with any other histological aspect of NAFLD, and thus, it is unclear whether they are associated with NASH fibrosis specifically, or with fibrosis in other liver diseases as well. Further studies are needed to confirm their role in NAFLD and other liver diseases.
Our findings of association with histological parameters reach conventional, but not GWAS-level significance. Although this is often advocated in genetic studies, we believe it may not be applicable in this case for several reasons. First, we have performed a candidate-gene analysis with only 6 SNPs, as compared to hundreds of thousands in GWA studies. Second, as we tested associations with several phenotypes, we corrected for multiple comparisons using the FDR procedure (25
). Since the histological attributes we tested are not independent of each other, the use of the Bonferroni correction would have been too conservative and would greatly reduce the power to detect an association. The FDR procedure is much more suitable for this situation (41
). Third, the separate and independent association of rs738409 with different histological aspects of NAFLD, all in the same direction of effect, strongly supports a true association. Finally, the histological scoring scheme for NAFLD is semi-quantitative and has a limited dynamic range. This factor markedly reduces the ability to achieve high significance values that can be observed with discrete traits such as liver enzymes or liver fat quantification by MRS.
In summary, we confirm the association of the rs738409 G allele in the PNPLA3 (adiponutrin) gene with steatosis in the largest cohort of histologically-proven NAFLD reported to date. Furthermore, this is the first study to describe an association of this SNP with histological parameters of disease severity, including inflammation, Mallory-Denk bodies and fibrosis. In pediatric patients, the high-risk allele seems to be associated with earlier presentation of disease. Our findings suggest that the rs738409 G allele may predispose patients to fat accumulation in the liver, but that other factors, environmental or hereditary, may be required for the development of inflammation, cellular injury and fibrosis. However, once patients develop NASH, the rs738409 G allele predisposes them to worse injury. In addition, we report an association of SNPs in the PNPLA3 locus and in the CPN1-CHUK locus on chromosome 10 with severity of NASH fibrosis. The mechanism underlying these associations requires further investigation.