Identification of the Bile Acid Transporter Slco1a6 as a Candidate Gene That Broadly Affects Gene Expression in Mouse Pancreatic Islets

Abstract

We surveyed gene expression in six tissues in an F2 intercross between mouse strains C57BL/6J (abbreviated B6) and BTBR T(+) tf/J (abbreviated BTBR) made genetically obese with the Leptin(ob) mutation. We identified a number of expression quantitative trait loci (eQTL) affecting the expression of numerous genes distal to the locus, called trans-eQTL hotspots. Some of these trans-eQTL hotspots showed effects in multiple tissues, whereas some were specific to a single tissue. An unusually large number of transcripts (∼8% of genes) mapped in trans to a hotspot on chromosome 6, specifically in pancreatic islets. By considering the first two principal components of the expression of genes mapping to this region, we were able to convert the multivariate phenotype into a simple Mendelian trait. Fine mapping the locus by traditional methods reduced the QTL interval to a 298-kb region containing only three genes, including Slco1a6, one member of a large family of organic anion transporters. Direct genomic sequencing of all Slco1a6 exons identified a nonsynonymous coding SNP that converts a highly conserved proline residue at amino acid position 564 to serine. Molecular modeling suggests that Pro564 faces an aqueous pore within this 12-transmembrane domain-spanning protein. When transiently overexpressed in HEK293 cells, BTBR organic anion transporting polypeptide (OATP)1A6-mediated cellular uptake of the bile acid taurocholic acid (TCA) was enhanced compared to B6 OATP1A6. Our results suggest that genetic variation in Slco1a6 leads to altered transport of TCA (and potentially other bile acids) by pancreatic islets, resulting in broad gene regulation.

Keywords: diabetes; eQTL; fine mapping; obesity; positional cloning.

Figure 1

Inferred eQTL with LOD ≥5, by tissue. Points correspond to peak LOD scores from single-QTL genome scans with each microarray probe with known genomic position. The y-axis is the position of the probe and the x-axis is the inferred QTL position. Points are shaded according to the corresponding LOD score, though we threshold at 100: all points with LOD ≥100 are black.

Figure 2

Inferred chromosome 6 eQTL with LOD ≥5, by tissue. Each point corresponds to a microarray probe and indicates the maximum LOD score on chromosome 6 for that probe and the position of the peak LOD score. Brown points correspond to probes whose genomic location is on chromosome 6; blue points are for probes on other chromosomes.

Figure 3

The first two principal components for the islet gene expression data for the 181 microarray probes that map to the chromosome 6 trans-eQTL hotspot with LOD ≥100 but do not reside on chromosome 6. Each point is a mouse; points for mice without a recombination event in the 10-cM interval centered at the peak marker are colored by their genotype in the region. Yellow points correspond to the 74 mice with recombination events in the interval.

Figure 4

Fine mapping of the islet chromosome 6 eQTL. (A) Initial SNP genotypes of the 64 mice with recombination events in the 10-Mbp (7 cM) region around the QTL, along with their inferred QTL genotypes (shown at the center of the inferred interval). The highlighted box indicates 29 mice with recombination events in the QTL interval. (B) Additional genotypes on five markers in the QTL interval, for 28 of the 29 mice with recombination event flanking the QTL. The highlighted box indicates 8 mice with recombination event flanking the QTL. (C) Additional genotypes in the reduced QTL interval, for 8 mice with recombination events flanking the QTL. (D) The QTL interval is further reduced to 298 kbp (141.979–142.277 Mbp), a region containing three genes.

Figure 5

Functional characterization of mouse OATP1A6. (A) Pymol views of a homology model of mouse OATP1A6 with the proline at position 564 highlighted. (B) Mouse OATP1A6-mediated uptake of some common OATP substrates was determined in HEK293 cells transiently transfected with empty vector (EV, gray points), C57BL/6-OATP1A6 (blue points), or BTBR OATP1A6 (green points). Uptake of 10 µM taurocholic acid (TCA), cholic acid (CA), estrone-3-sulfate (E3S), methotrexate (MTX), (D-Pen², D-Pen⁵)-enkephalin (DPDPE), estradiol-17-β-glucuronide (E17βG), and bromosulfophthalein (BSP) was measured at 37 °C for 5 min. (C) Uptake of TCA was measured at various concentrations from 1 to 100 µM at 37 °C for 1 min with empty vector and B6 or BTBR OATP1A6-expressing HEK293 cells. Net uptake was calculated by subtracting the values of empty vector-transfected cells from either B6 or BTBR OATP1A6 transfected cells. Resulting data were fitted to the Michaelis–Menten equation to obtain K_m and V_max values. (D) Uptake of 10 µM TCA was measured at 1 and 5 min with normal B6 OATP1A6-Pro⁵⁶⁴ or mutant Ser⁵⁶⁴ and with normal BTBR OATP1A6-Ser⁵⁶⁴ or mutant Pro⁵⁶⁴. For the uptake experiments, each point shown is derived from an individual experiment performed with triplicate determinations.