Stefan Stender

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

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.

Reduced atherogenesis in cholesterol-fed diabetic rabbits. Giant lipoproteins do not enter the arterial wall.

In cholesterol-fed rabbits, alloxan-dlabetes has an antt-atherogenlc effect, which is associated with severe elevation of plasma triglyceride concentrations. To study this effect, we measured llpoprotein sizes and aortic permeability coefficients for chotesteryl ester and for albumin In hypertriglycerldemlc diabetic cholesterol-fed rabbits and In normotriglyceridemlc cholesterol-fed rabbits. With the same high cholesterol concentration in plasma, hypertrlglyceridemlc diabetic rabbits had 70% of plasma cholesterol In very large llpoprotelns (diameter >75 nm), whereas normotrlglycerldemlc rabbits had only about 10% of plasma cholesterol in these giant llpoprotelns. The aortic permeability coefficients for cholesteryl ester In hypertriglycerldemlc diabetic cholesterol-fed rabbits was only 10% to 50% of that In normotriglyceridemlc cholesterol-fed rabbits. Aortic permeability coefficients for albumin did not differ significantly between the hypertrlglyceridemlc and normotriglyceridemlc rabbits. The results suggest that the large size of a major fraction of plasma llpoprotelns in the hypertrlglyceridemlc diabetic cholesterol-fed rabbits Is responsible for the relatively low aortic permeability coefficient for cholesteryl ester from plasma and hence for reduced atherogenesis In these animals.

Low-Density Lipoprotein Cholesterol and the Risk of Cancer: A Mendelian Randomization Study

BACKGROUND Low plasma levels of low-density lipoprotein (LDL) cholesterol are associated with an increased risk of cancer, but whether this association is causal is unclear. METHODS We studied 10 613 participants in the Copenhagen City Heart Study (CCHS) and 59 566 participants in the Copenhagen General Population Study, 6816 of whom had developed cancer by May 2009. Individuals were genotyped for PCSK9 R46L (rs11591147), ABCG8 D19H (rs11887534), and APOE R112C (rs429358) and R158C (rs7412) polymorphisms, all of which are associated with lifelong reduced plasma LDL cholesterol levels. Plasma LDL cholesterol was calculated using the Friedewald equation in samples in which the triglyceride level was less than 354 mg/dL and measured directly by colorimetry for samples with higher triglyceride levels. Risk of cancer was estimated prospectively using Cox proportional hazards regression analyses and cross-sectionally by logistic regression analyses. Causality was studied using instrumental variable analysis. All statistical tests were two-sided. RESULTS In the CCHS, compared with plasma LDL cholesterol levels greater than the 66th percentile (>158 mg/dL), those lower than the 10th percentile (< 87 mg/dL) were associated with a 43% increase (95% confidence interval [CI] = 15% to 79% increase) in the risk of cancer. The polymorphisms were associated with up to a 38% reduction (95% CI = 36% to 41% reduction) in LDL cholesterol levels but not with increased risk of cancer. The causal odds ratio for cancer for a 50% reduction in plasma LDL cholesterol level due to all the genotypes in both studies combined was 0.96 (95% CI = 0.87 to 1.05), whereas the hazard ratio of cancer for a 50% reduction in plasma LDL cholesterol level in the CCHS was 1.10 (95% CI = 1.01 to 1.21) (P for causal odds ratio vs observed hazard ratio = .03). CONCLUSION Low plasma LDL cholesterol levels were robustly associated with an increased risk of cancer, but genetically decreased LDL cholesterol was not. This finding suggests that low LDL cholesterol levels per se do not cause cancer.

No effect of nifedipine on atherogenesis in cholesterol-fed rabbits.

It was recently reported that the calcium antagonist nifedipine suppresses aortic cholesterol accumulation in cholesterol-fed rabbits without reducing hypercholesterolemia. We extended this study on plasma lipoprotein levels and aortic influx of cholesteryl ester. We gave 40 mg per day of nifedipine orally to 17 rabbits fed a 2% cholesterol diet for 8 weeks. For the same period of time 15 control rabbits received placebo capsules and the same diet. During the study, total cholesterol, HDL, LDL, and VLDL cholesterol concentrations in plasma were not significantly different in the experimental and control animals. At the end of the study we found no difference in the two groups in accumulation of cholesterol in the intima media of the proximal thoracic aorta, the distal thoracic aorta, and the corresponding media layers. Furthermore, aortic influx of free cholesterol, cholesteryl ester, and albumin from plasma measured by radioactive tracers was not significantly affected by nifedipine.

Aortic permeability to LDL as a predictor of aortic cholesterol accumulation in cholesterol-fed rabbits.

The aim of this study was to investigate the possibility that the permeability characteristics of the arterial wall are related to the development of atherosclerosis. The in vivo regional variation of aortic permeability to iodinated human low density lipoprotein (LDL) in normal rabbits was compared with the regional variation in aortic cholesterol accumulation in cholesterol-fed rabbits. Aortas were divided into the aortic arch, thoracic aorta, and abdominal aorta, and each of these three parts was further subdivided into four segments of similar size. The permeability to LDL was 40 +/- 7 nl.cm-2.hr-1 (mean +/- SEM, n = 11) in the most proximal segment of the aortic arch and decreased throughout the length of the aorta to 3 +/- 1 nl.cm-2.hr-1 in the most caudal segment of the abdominal aorta. In such normal rabbits the aortic cholesterol content was similar in all 12 arterial segments at 0.08 +/- 0.005 mumol/cm2 (mean +/- SEM, n = 3 x 12). Aortic cholesterol accumulation was determined in other rabbits with an average plasma cholesterol level of 32 +/- 1 mmol/l for 96 days; the cholesterol content in the most proximal segment of the aortic arch was 2.7 +/- 0.5 mumol/cm2 (mean +/- SEM, n = 11) and decreased with increasing distance from the heart to 0.17 +/- 0.03 mumol/cm2 in the most caudal segment of the abdominal aorta. Linear regression analysis showed a close positive association between the permeability to LDL of a given aortic segment and the cholesterol accumulation in that same aortic segment after cholesterol feeding (r2 = 0.96, p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Genetically elevated bilirubin and risk of ischaemic heart disease: three Mendelian randomization studies and a meta-analysis.

Elevated plasma levels of bilirubin, an endogenous antioxidant, have been associated with reduced risk of ischaemic heart disease (IHD) and myocardial infarction (MI). Whether this is a causal relationship remains unclear.

Elevated body mass index as a causal risk factor for symptomatic gallstone disease: a Mendelian randomization study.

Elevated body mass index (BMI) is associated with an increased risk of gallstone disease. Whether this reflects a causal association is unknown. Using a Mendelian randomization approach, we studied 77,679 individuals from the general population. Of these, 4,106 developed symptomatic gallstone disease during up to 34 years of follow‐up. Subjects were genotyped for three common variants known to associate with BMI: FTO(rs9939609); MC4R(rs17782313); and TMEM18(rs6548238). The number of BMI‐increasing alleles was calculated for each participant. In observational analyses, mean baseline BMI was 55% (11.6 kg/m2) increased in individuals in the fifth quintile versus the first quintile, similar in women and men. The corresponding multifactorially adjusted hazard ratio (HR) for symptomatic gallstone disease was 2.84 (95% confidence interval [CI]: 2.32‐3.46) overall, 3.36 (95% CI: 2.62‐4.31) in women, and 1.51 (95% CI: 1.09‐2.11) in men (P trend: 0.001 to <0.001; P interaction: BMI*sex on risk = 0.01). In genetic analyses, carrying 6 versus 0‐1 BMI‐increasing alleles was associated with a 5.2% (1.3 kg/m2) increase in BMI overall and with increases of 4.3% in women and 6.1% in men (all P trend: <0.001). Corresponding HRs for symptomatic gallstone disease were 1.43 (95% CI: 0.99‐2.05) overall, 1.54 (95% CI: 1.00‐2.35) in women, and 1.19 (95% CI: 0.60‐2.38) in men (P trend = 0.007, 0.02, and 0.26, respectively; P interaction allele score*sex on risk = 0.49). The estimated causal odds ratio (OR) for symptomatic gallstone disease, by instrumental variable analysis for a 1 kg/m2 increase in genetically determined BMI, was 1.17 (95% CI: 0.99‐1.37) overall and 1.20 (95% CI: 1.00‐1.44) and 1.02 (95% CI: 0.90‐1.16) in women and men, respectively. Corresponding observational HRs were 1.07 (95% CI: 1.06‐1.08), 1.08 (95% CI: 1.07‐1.10), and 1.04 (95% CI: 1.02‐1.07), respectively. Conclusion: These results are compatible with a causal association between elevated BMI and increased risk of symptomatic gallstone disease, which is most pronounced in women. (Hepatology 2013; 58:2133–2141)

Sterol transporter adenosine triphosphate–binding cassette transporter G8, gallstones, and biliary cancer in 62,000 individuals from the general population

Gallstone disease, a risk factor for biliary cancer, has a strong heritable component, but the underlying genes are largely unknown. To test the hypothesis that ABCG8 (adenosine triphosphate–binding cassette transporter G8) Asp19His (D19H) genotype predicted risk of gallstones and biliary cancer in the general population, we studied 62,279 white individuals from The Copenhagen City Heart Study and The Copenhagen General Population Study, randomly selected to reflect the adult Danish population aged 20 to 80+ years. Endpoints were recorded from January 1976 through May 2009. During a mean follow‐up of, respectively, 31 and 4.4 years, 3124 participants developed symptomatic gallstone disease and 30 developed biliary cancer. The multifactorially adjusted hazard ratio for symptomatic gallstone disease was 1.9 (95% confidence interval, 1.7‐2.1) in DH heterozygotes (prevalence, 12%), and 3.3 (2.3‐4.6) in HH homozygotes (0.4%) versus noncarriers (P for trend <0.001). Mean age at onset of symptomatic gallstone disease was 56 years for noncarriers, 54 for DH heterozygotes, and 52 for HH homozygotes (P for trend <0.001). The fraction of all gallstones attributed to D19H was 11%. The multifactorially adjusted hazard ratio for biliary cancer was 4.0 (1.9‐8.4) in DH heterozygotes and HH homozygotes combined versus noncarriers (P < 0.001). The fraction of all biliary cancers attributed to the D19H genotype was 27%. Finally, D19H genotype associated with stepwise increases in plasma levels of alanine aminotransferase and gamma glutamyltransferase of up to 14% and 25% in HH homozygotes, and with corresponding stepwise reductions in plasma levels of total and low‐density lipoprotein cholesterol of up to 5% versus noncarriers (all comparisons, P for trend <0.001). Conclusion: In this general population cohort, ABCG8 D19H genotype was an important predictor of both symptomatic gallstone disease and biliary cancer. (HEPATOLOGY 2011:53:640‐648)

Different efflux pathways for high and low density lipoproteins from porcine aortic intima.

To study the efflux of high (HDL) and low (LDL) density lipoproteins from the arterial wall in vivo, a surgical model in pigs was used. An isolated segment of the lesion-free thoracic aorta was pulse labeled from the lumen of the artery with 3H-cholesteryl ester labeled HDL and 14C-cholesteryl ester labeled LDL. Subsequently, the labeled aortic segment was exposed to cold chase in vivo. The transfer of HDL cholesteryl ester from plasma into intima expressed as intimal clearance was three to seven times greater than that of LDL cholesteryl ester. At least 50%, but possibly as much as 95%, of the HDL cholesteryl ester that entered the arterial intima during a period of 4 hours penetrated the arterial wall beyond the internal elastic lamina. In contrast, less than 15% of the LDL cholesteryl ester that entered the arterial intima in the same period penetrated beyond the luminal layer. After 24 hours of cold chase in vivo, more than 80% of both labeled HDL esterified cholesterol and labeled LDL esterified cholesterol had disappeared from the arterial wall. Transmural profiles after 9 hours of cold chase showed that labeled HDL was present throughout the entire arterial wall, whereas labeled LDL in quantitative amounts was present only in the luminal layer. The results suggest that the most important efflux route for HDL esterified cholesterol is through the vasa vasorum and lymphatics in the outer media and adventitia, whereas LDL esterified cholesterol predominantly leaves intima via the lumen of the artery.

Adiposity amplifies the genetic risk of fatty liver disease conferred by multiple loci

Complex traits arise from the interplay between genetic and environmental factors. The actions of these factors usually appear to be additive, and few compelling examples of gene–environment synergy have been documented. Here we show that adiposity significantly amplifies the effect of three sequence variants (encoding PNPLA3 p.I148M, TM6SF2 p.E167K, and GCKR p.P446L) associated with nonalcoholic fatty liver disease (NAFLD). Synergy between adiposity and genotype promoted the full spectrum of NAFLD, from steatosis to hepatic inflammation to cirrhosis. We found no evidence of strong interaction between adiposity and sequence variants influencing other adiposity-associated traits. These results indicate that adiposity augments genetic risk of NAFLD at multiple loci that confer susceptibility to hepatic steatosis through diverse metabolic mechanisms.