Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

Abstract

Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease. To elucidate the molecular basis of NAFLD, we performed an exome-wide association study of liver fat content. Three variants were associated with higher liver fat levels at the exome-wide significance level of 3.6 × 10(-7): two in PNPLA3, an established locus for NAFLD, and one (encoding p.Glu167Lys) in TM6SF2, a gene of unknown function. The TM6SF2 variant encoding p.Glu167Lys was also associated with higher circulating levels of alanine transaminase, a marker of liver injury, and with lower levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides and alkaline phosphatase in 3 independent populations (n > 80,000). When recombinant protein was expressed in cultured hepatocytes, 50% less Glu167Lys TM6SF2 protein was produced relative to wild-type TM6SF2. Adeno-associated virus-mediated short hairpin RNA knockdown of Tm6sf2 in mice increased liver triglyceride content by threefold and decreased very-low-density lipoprotein (VLDL) secretion by 50%. Taken together, these data indicate that TM6SF2 activity is required for normal VLDL secretion and that impaired TM6SF2 function causally contributes to NAFLD.

Figure 1

Exome-wide association with hepatic triglyceride content in the Dallas Heart Study (DHS). (a) Manhattan plot showing the association of 138,374 sequence variants on the HumanExome Array (BeadChip, Illumina) with hepatic triglyceride content in the DHS (n=2,736). The dashed line denotes the Bonferroni corrected significance threshold. (b) Quantile-quantile plot of −log₁₀ P-values. (c) Evolutionary conservation of TM6SF2. Bolded letters denote residues that are conserved among all species shown. Residue 167 is shown in red. (d). Mean hepatic triglyceride content (± s.e.m.) by TM6SF2 genotype (rs58542926) in the DHS. The association was tested using linear regression with adjustment for age, gender, ancestry and BMI. (e) Levels of TM6SF2 mRNA in human tissues. Quantitative Real-time PCR was performed on mRNA extracted from human tissues (Clontech). Each bar represents the average of a triplicate measurement expressed as a fraction of the Ct value obtained from the tissue expressing the highest level (small intestine). The values were normalized to the levels of the 36B4 transcript.

Figure 2

Expression of TM6SF2 in cultured hepatocytes. (a) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. (b) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.

Figure 3

Knockdown of Tm6sf2 expression in the liver of mice is associated with increased hepatic triglyceride content. (a) Adeno-associated virus vectors (AAV) expressing two different anti-Tm6sf2 shRNAs or vector alone were administered intravenously into the tail veins of 8-week old chow-fed C47BL/6J male mice (n=8/group). After two weeks, the livers were harvested and the levels of TM6SF2 mRNA were measured using Real-Time PCR (left). Lipids were extracted from the livers of the AAV-treated mice and triglyceride levels were quantified using enzymatic assays (middle). The plasma levels of cholesterol and triglyceride were measured in ad-lib fed AAV-treated mice at the end of the dark cycle (right). The experiment was performed three times and the results were similar. (b) Plasma lipoproteins from chow-fed C47BL/6J male mice (n=6/group) after a 4-h fast were fractionated using FPLC as described in the Methods. Cholesterol and triglyceride were measured enzymatically in each column fraction. The experiment was performed twice and the results were similar. (c) Hepatic VLDL secretion in Tm6sf2 KD mice. Plasma triglyceride levels after 4 hour fasting (left), and triglyceride accumulation after Triton WR 1339 injection (right) were measured in chow-fed male mice (8 week old, 4/group) treated with the AAV Tm6sf2-shRNA8 or control mice as described in the Methods. Two weeks after infection, mice were fasted for 4 h and injected via the tail vein with Triton WR1339 (500 μg/g body weight). Blood was sampled at the indicated times and plasma triglyceride levels were measured. Mean triglyceride levels at each time point are shown. The slopes of the lines were calculated by least squares regression and compared using a t-test. The experiment was performed twice and the results were similar. Error bars indicate standard deviations.

Figure 4

Sucrose feeding in TM6SF2-KD mice. AAV expressing anti-Tm6sf2 shRNAs or vector alone were administered intravenously into the tail veins of 8-week old C47BL/6J male mice (n=6/group) and the mice were fed a high-sucrose diet for 4 weeks. (a) Lipids were extracted from the livers of the AAV-treated mice and hepatic triglyceride levels were quantified using enzymatic assays. (b) Plasma levels of cholesterol and triglyceride in TM6SF2-KD mice were measured enzymatically. The differences in mean hepatic triglyceride concentrations were compared using two-sample t-tests. (c) Sections from liver tissue of the same mice stained with Oil Red O, as described in Methods. Magnification: main image ×20, inset ×64. Bar represents 40 μm. The experiment was performed twice and the results were similar. Error bars show standard deviations.