Fig 1: Protein abundance scores for 137 SLCO1B1 variants. Variants having abundance scores less than or equal to 0.5728 SLCO1B1 (1296C>A) were classified as “severely damaging” variants, whereas variants having abundance scores equal to or above 0.5768 (SLCO1B1*1B, 388A>G, rs2306283) but less than 0.6015 (SLCO1B1*27, 1200C>G, rs59113707) were classified as “damaging.” Variants having abundance scores higher than 0.6015 were classified as “tolerated.” The results shown are averages abundance scores for four replicates. S.D. values are listed in Supplemental Table 1.
Fig 2: Variant frequencies by bin for the six newly identified “severely” damaging variants (1462G>A, 1246G>A, 215G>A, 1508A>G 1828C>T, and 1296C>A) for SLCO1B1, their distribution into each of the four bins, and similar data for the common SLCO1B1*5 allele.
Fig 3: Validation of SLCO1B1 variants identified as containing severely damaging variants. (A) Western blot validation of SLCO1B1 variants identified as containing severely damaging variants. The protein expression of SLCO1B1 in BFP-/mCherry+ cells integrating severely damaging variants were validated by Western blot analysis. mCherry was used as a loading control. A control lane contained WT SLCO1B1. (B) Concentration-dependent uptake of estradiol 17-ß-d-glucuronide by SLCO1B1 WT BFP-/mCherry+ cells and the six newly identified severely damaging SLCO1B1 variant BFP-/mCherry+ cells after 1-minute incubations. The quantity of radioactively labeled estradiol 17-ß-d-glucuronide that accumulated within the cells was determined by liquid scintillation counting. The data are expressed in CPM normalized by the amount of protein content in milligrams. Data are presented as mean uptake for three replicate experiments. (C) The bar graph shows the uptake of estradiol 17-ß-d-glucuronide (24 nM) for variants in SLCO1B1 TM4 in BFP-/mCherry+ cells after 1-minute incubations. The uptake activities of variants in severely damaging variants against WT were tested by one-way ANOVA; ****P < 0.0001. (D) Concentration-dependent uptake of estradiol 17-ß-d-glucuronide for variants in SLCO1B1 TM4 in BFP-/mCherry+ cells after 1-minute incubations. Data are presented as means ± S.D. of CPM per mg protein for three replicated experiments. (E) The bar graph shows the uptake of estradiol 17- ß-d-glucuronide (24 nM) for variants in SLCO1B1 TM4 in BFP-/mCherry+ cells after 1-minute incubations. The uptake activities of variants in TM4 against WT were tested by one-way ANOVA; *P < 0.05; ****P < 0.0001.
Fig 4: Flow cytometry of SLCO1B1 constructs with known variants and FACS of pooled SLCO1B1 variant libraries. (A) The SLCO1B1 expression cassette is depicted diagrammatically. When this vector is integrated into a “landing pad” in HEK293 cells, it results in the expression of recombinant protein that is labeled with GFP-labeled SLCO1B1, whereas the cell itself will express mCherry, so the ratio of GFP to mCherry serves as an indication of the stability of the expressed protein, i.e., the higher that ratio, the more stable the protein encoded by the expressed variants. The SLCO1B1 expression cassette was integrated into landing pad through attB and attP recombination. (B and C) Flow cytometry analysis of BFP-/mCherrry+ cells that had integrated wild-type or known damaging variant such as SLCO1B1*2. Note that for the WT protein, most of the cells eluted toward higher GFP/mCherry ratios, whereas cells containing damaging variants eluted at significantly lower GFP/mCherry ratios than did cells expressing the WT. Mean GFP/mCherry ratios for those variants were consistent with Western blot results obtained during our previous study. (D) Cells integrating SLCO1B1 pooled variant libraries were sorted into four bins based on their GFP/mCherry ratios. The variants were categorized into three groups: severely damaging variants fell into bin 1, damaging variants fell into bin 2 and bin 3, and tolerated variants fell into bin 4. Gates were set based on WT SLCO1B1 and SLCO1B1*2. Pools of sorted cells in each bin were collected and used as input material for subsequent amplicon DNA sequencing. HA-L, left homologous arm; HA-R, right homologous arm; IRES, internal ribosome entry site.
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