Using an independent sample of 1,092 well-phenotyped morbidly obese subjects who underwent bariatric surgery and in whom liver histology was determined, we found that despite similar BMI, subjects with NAFLD had a higher prevalence of diabetes, hyperlipidemia and greater waist circumference than those without NAFLD. These data support that NAFLD is associated with insulin resistance and other features of the metabolic syndrome largely independent of obesity. Association analyses of five NAFLD-associated genetic variants identified by the GOLD Consortium GWAS meta-analysis in our bariatric cohort revealed a nonsynonymous variant in PLPLA3
to be associated not only with the presence of steatosis but also with NASH, a more progressive and clinically ominous manifestation of NAFLD. These finding extend a previously reported studies to patients with extreme obesity. These findings are consistent with other studies in which the G allele of PLPLA3
rs738409 was not only associated with simple fat accumulation, but also with the degree of liver steatosis as evaluated by liver biopsy (23
). In other studies, the same allele was associated with steatosis, portal inflammation, lobular inflammation, Mallory-Denk bodies, NAFLD activity score, and fibrosis (8
Furthermore, we replicated the GOLD Consortium’s association of the T allele of rs2228603 in NCAN with degree of liver steatosis as well as with signs of lobular inflammation and perivenular fibrosis, which are consistent with NASH, a more advanced stage of NAFLD. For GCKR rs780094 and PPP1R3B rs4240624, although not statistically significantly associated with NAFLD, the direction of effect and the effect size showed similar trends to the GOLD Consortium results. It is likely that the inability to achieve statistical significance is due to lack of power to discern the effect of variants of modest effect size. The absence of even a trend for association of LYPLAL1 rs1213785 with NAFLD may be due to differences among populations, a false negative finding, or a false positive finding in the initial GOLD Consortium study. Assuming a 24% population prevalence of NAFLD, our sample had 80% power to detect an OR of 1.48 for presence of steatosis for a variant with allele frequency of at least 0.05 at p<0.05. Using a higher population prevalence of 50% which may be more suitable for a bariatric population, our sample had 80% power to detect an OR of 1.29 for a variant with an allele frequency of at least 0.05 at p<0.05.
Our data revealed that the PPP1R3B
SNP rs4240624 was not significantly associated with liver steatosis, lobular inflammation, or hepatocyte ballooning. However, this variant was the only SNP in our study that tended to be associated with cirrhosis (p=0.07). In the GOLD Consortium meta-analysis, PPP1R3B
was associated with fatty liver identified on CT scan, but it was not associated with histologic features of NAFLD. As hypothesized by Speliotes et al, this may suggest that PP1R3B
is related to liver steatosis but not hepatocyte inflammation and fibrosis (11
). Alternatively, the SNP may be associated with intrahepatic accumulation of a substance that resembles steatosis on abdominal radiographic imaging and causes cirrhosis (i.e., glycogen storage disease type IV, which is characterized by glycogen precipitation in the liver that results in cirrhosis).
Our data also replicate distinct patterns of serum lipids for some NAFLD-associated variants. The GOLD Consortium meta-analysis found no association between lipid levels and PNPLA3
rs738409[G]. Our data suggests a relationship between the NAFLD-associated G allele and lower total- and HDL-cholesterol levels in the full cohort (p=0.03). This may be a type I error or alternatively due to a difference in ascertainment; all subjects in our study were selected for extreme obesity. With regard to serum triglycerides, there was no association between this SNP and serum triglyceride levels in the overall cohort, but there was a trend toward an association with lower triglyceride levels in patients with NAFLD (p=0.08). In support of this finding, the relationship between this SNP and serum triglyceride levels has been reported by others (26
As in the GOLD Consortium, we found association of the NAFLD-associated NCAN rs2228603[T] allele with lower serum LDL-cholesterol, total cholesterol and triglyceride levels, particularly in those with NAFLD. Interestingly, this association was not found in equally obese patients without NAFLD suggesting that NAFLD and lower serum lipids may be mechanistically linked in rs22286603[T] carriers. Since NAFLD is clinically associated with higher serum lipids, this seemingly paradoxical finding suggests that more than one NAFLD subtype may exist. Elucidation of the causative gene/variant(s) at this locus may uncover a novel disease mechanism in which sequestration of triglycerides in the liver, either as a result of increased triglyceride uptake and/or decreased lipolysis result in lower serum lipid levels.
By contrast, the NAFLD-associated GCKR rs780094[T] allele, was associated with higher serum triglycerides. These findings are similar to the GOLD Consortium analysis as well as other studies (20
). Finally, we did not detect any association between the LYPLAL1
SNPs and serum lipids. It is possible that the lack of association may be due to inadequate sample size and power, especially for PPP1R3
in which the GOLD Consortium reported increased LDL- and HDL-cholesterol.
locus contains at least 20 genes in a 500 kb region on chromosome 19p13 (28
). None of these genes are particularly compelling positional candidates for NAFLD or lipid homeostasis. Additional studies are needed to fine map this locus and perform functional studies. Neurocan, the protein product of NCAN
, is a chondroitin sulfate proteoglycan primarily expressed in the nervous system that is thought to be involved in cell adhesion and migration (31
). Rs2228603 is located in exon 3 of NCAN
and encodes a non-conservative nonsynonymous mutation (Pro92Ser), which is predicted by the software tool PolyPhen-2 to alter protein structure and function (35
). While neurocan itself has not yet been shown to be expressed in the liver or to directly affect lipid metabolism or hepatic steatosis, it is becoming increasingly recognized that the central nervous system (CNS) is an important regulator of peripheral glucose and triglyceride metabolism (36
). The liver is highly innervated by both sympathetic and parasympathetic nerves (39
). Bruinstroop et al have shown that post-prandial serum triglyceride levels were significantly elevated in parasympathetic or sympathetic denervated rats compared to sham-operated animals (40
). Robertson et al demonstrated that vagal stimulation increases hepatic secretion of very low-density lipoprotein (VLDL) triglyceride (41
). Since NCAN
rs2228603 has been associated with increased liver fat and decreased serum triglyceride (11
), it is plausible that this variant is associated with a brain-liver axis that, when deregulated, increases the risk for NAFLD. Furthermore, this axis may be modulated by increased dietary fat intake, as suggested by a 50% increase in hepatic VLDL synthesis after fat intake (43
Further evidence for central control of liver lipid metabolism comes from studies of neuropeptide Y (NPY), a 36-amino acid peptide neurotransmitter secreted by the hypothalamus that has glucose and lipid regulatory effects in the liver. Fasting rats treated with intracerebroventricular injection of NPY had increased VLDL secretion into the bloodstream by 2.5-fold (44
). Intracerebroventricular administration of an NPY-Y5 receptor agonist reproduced this effect, while an NPY-Y1 receptor antagonist decreased VLDL secretion. These findings demonstrate that the CNS can control VLDL secretion.
In summary, in light of the increased risk for NAFLD conferred by obesity, we have extended findings of the GOLD Consortium to include a large population of bariatric surgery patients in whom liver steatosis, inflammation, and fibrosis were documented histologically. Specifically, we have shown that variants in PNPLA3 and NCAN are associated not only with liver steatosis, but also NASH, a more progressive and clinically ominous manifestation of NAFLD. NCAN is a gene expressed in brain and thus may identify an untapped potential role for the CNS in the mechanism of fatty liver. Alternatively variants in other genes at this locus may be responsible for the NAFLD phenotype. Fine mapping and functional studies will be required to identify the causative genes/variants, which may provide insight into a novel pathway for NAFLD development, leading to new strategies for prevention and treatment of this disease. In addition, follow up analysis of the effects of gastric bypass surgery on liver steatosis and inflammation and dyslipidemia in our bariatric cohort may provide additional insight into the potential role of these loci as determinants of metabolic responses to weight loss.