## 3 Discussion  
Herein, we provide evidence that hepatic _SLC10A1_/NTCP expression is subject to a considerable inter-individual variability caused by various factors. In agreement with previous data which we had described for the hepatic uptake transporters _SLC22A1_/OCT1 [67] and _SLC01B1_/OATP1B1 [56] cholestasis, alcohol consumption, and inflammation determined by C-reactive protein altered _SLC10A1_/NTCP expression whereas age and gender are of minor importance. Moreover, it cannot be excluded that protein expression/function may be altered by disease status as previously described [57; 68; 69]. Our comprehensive analyses of genetic variants in the promoter, the coding as well as 3'UTR/5'UTR of _SLC10A1_ indicated that especially missense variants seem to be uncommon in Caucasians, which is consistent with a recent study [50] and was confirmed when considering population frequencies from gnomAD (Supplementary Figure S8). Cluster analysis based on genome-wide genetic data indicates that all individuals of our cohort are of European ancestry (Supplementary Figure S9). Using deep sequencing of all exons and intronic flanking regions, only two non-synonymous variants (rs200149939, p.R185C; rs200746820, p.S213R) were identified heterozygosity in single individuals of our population. Population frequencies from gnomAD indicate that both missense variants are also only rarely detected in other ethnic populations, e.g., in the East Asian (rs200149939, MAF = 0.01%), African (rs200149939, MAF = 0.004%; rs200746820, MAF = 0.004%) or South American population (rs200149939, MAF = 0.003%; rs200746820, MAF = 0.006%) [70]. Since we did not observe a  
Figure 7: _SLC10A1_/NTCP expression and hepatic metabolism. Spearman correlations between mRNA expression and protein levels and endogenous metabolites in the 143 liver samples. _SLC10A1_ mRNA expression was measured by qPCR and protein abundance was analyzed through LC-MS/MS. Metabolite levels were quantified using LC-MS technology. Correlations higher than 0.25 or lower than \(-0.2\) are labeled, whereas correlations not significant after adjustment for multiple testing using the Benjamini-Hochberg procedure are only weakly colored. remarkable alteration in hepatic bile acid levels as endogenous NTCP substrate in individuals carrying these variants, a substantial functional effect for bile acid transport appears to be unlikely. However, the bile acid levels determined in our liver tissues might not only consist of intracellular bile acids but might additionally include canalicular or ductal bile acids [71]. We, therefore, generated HEK cells recombinantly expressing the variants p.R185C or p.S213R. Using these cells, we confirmed that both variants do not affect the bile acid transport function of NTCP. In summary, none of the identified genetic variants in the _SLC10A1_ gene region could explain the observed inter-individual variability in hepatic _SLC10A1_/NTCP expression. This could be confirmed using _SLC10A1_ gene expression and germline genetic data from adjacent non-tumor liver tissue from the hepatocellular cohort of TCGA. In addition, our GWAS did not reveal any genetic variants in other genomic loci that are significantly associated with either mRNA expression or protein levels in our liver cohort to explain the inter-individual variability. Thus, epigenetic factors (e.g., DNA methylation), identified in our study might be more important for the regulation of hepatic _SLC10A1_/NTCP expression. We demonstrated that DNA methylation indeed had an impact on the regulation and expression of NTCP in vitro and in vivo. _SLC10A1_ promoter activity was significantly influenced by DNA methylation and treatment of cells with the demethylating agent AZA led to an increase in _SLC10A1_ mRNA expression. In the liver cohort, DNA methylation levels of certain CpG sites located in the proximal promoter region and in exon 1 in front of the translational start site were significantly associated also with NTCP protein levels and only to a lesser extent with _SLC10A1_ mRNA.