## 2 Results
### Impact of SLC10A1 Missense Variants on SLC10A1/NTCP Function  
In order to examine the functional influence of the only two missense variants (rs200149939, p.R185C; rs200746820, p.S213R), that were identified in our human liver cohort, we first determined the location of these variants in the recently reported NTCP models. Both variants are predicted to lie outside the bile acid-binding region (Figure 2A-C) and are classified as tolerated by algorithm SIFT, CADD, REVEL, and MetaLR while PolyPhen classified p.R185C as tolerated and p.S213R as possibly damaging and MutationAssessor classified both variants as possibly damaging (Figure 2D). Discrepancies in in silico predictions of NTCP variant function were also observed by [PERSON] et al. [50] corroborating our findings that algorithms are limited to predicting NTCP function and in vitro functional assays are required. We next measured bile acid concentrations of unconjugated, glycine-conjugated, and taurine-conjugated bile acids in the liver cytosol of the patients using mass spectrometry. As shown in Figure 3A, bile acid content does not appear to be altered in heterozygous carriers of the two missense variants compared to non-carriers. However, both variants were found only once heterozygosity in the 143 liver samples limiting statistical analyses. Figure 2: In silico analysis of NTCP missense variants. (**A–C**) Location of p.R185 and p.S213 in the three recently reported human NTCP structures. In all three structures, p.R185 is located at the start of the transmembrane segment (TM) 6. Amino acid S213 is located extracellularly in the topological models reported by (**A**) [PERSON] et al. [47] and (**B**) [PERSON] et al. [46]. In the model reported by (**C**) [PERSON] et al. [48], this amino acid is located at the end of TM6. Putative conserved residues in the Na\({}^{+}\)-binding sites are located in TM2 (p.Q8), TM3b (p.S105/p.N106), TM4 (p.T123), and TM8a (p.E257, p.Q261); key residues for bile acid-binding are in TM1 (p.L27, p.L31, p.L35) and in TM5 (p.K157, p.G158) [48] and their approximate locations are indicated by white circles. TM1, TM5, and TM6 form the “panel domain” whereas the other TMs form the “core domain”. (**D**) Predicted functional consequences of p.R185C and p.S213R as obtained from Ensembl Release 106 ([[https://www.ensembl.org](https://www.ensembl.org)]([https://www.ensembl.org](https://www.ensembl.org)) accessed on 6 June 2022). Green: tolerated; orange: possibly damaging. We therefore stably expressed NTCP carrying either of the two missense variants in human embryonic kidney (HEK) cells. NTCP and the variant proteins were fully glycosylated (Figure 3B) and showed correct immunolocalization in the plasma membrane (Figure 3C). The prototypic substrate tautocholate was taken up by NTCP, p.R185C and p.S213R with comparable K\({}_{\rm m}\) values (Figure 3D,E). Figure 3: Functional characterization of NTCP missense variants in human liver and cell lines. (**A**) Box scatter plots show bile acid concentrations measured in liver cytosol using Q-TOF LC/MS. Individual levels are shown using gray colored circles. Heterozygous carriers of the two missense variants compared  
to non-carriers are illustrated by colored points: magenta, rs200149939, p.R185C; purple, rs200746820, p.S213R. The measured bile acids are CA: cholic acid, CDCA: chenodeoxycholic acid, DCA: deoxycholic acid, GCA: glycochenodeoxycholic acid, GDCA: glycodeoxycholic acid, GLCA: glycolithocholic acid, GUDCA: glycoursedeoxycholic acid, LCA: lithocholic acid, TCA: taurocholic acid, TCDCA: taurochenodeoxycholic acid, TIDCA: taurodeoxycholic acid, TLCA: taurochloride acid TUDCA: tauroursodeoxycholic acid, UDCA: tursodeoxycholic acid. The lines indicate the medians. The whiskers correspond to the 25 th and 75 th percentiles. (**B**) Immunoblot analysis of protein lysates from HEK cells expressing NTCP reference sequence, p.R185C or p.S213R using affinity-purified NTCP-specific GNG antibody [58].