Haplotype combinations of the Met408Val and Met420del polymorphisms in the OCT1 gene in humans and their functional activity. (A-B) Combined representation of the Met408Val and Met420-deletion genotypes (A) and haplotypes (B) of Caucasians from our previous clinical studies and from selected (CEU and GRB) or all populations from the 1000 Genomes Project (the individual genotype data that was used in the calculations was obtained from http://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/).⁵  Individual haplotypes were inferred using PHASE version 2.1.⁶ The genotype/haplotype frequencies are given as number (and %) of all individuals in the corresponding population. CEU, population of Utah residents (CEPH) with Northern and Western European ancestry; GRB, population of British in England and Scotland. (C) Strategy (upper 2 panels) and an example of the results (lower panel) of haplotype sequencing analyses using semiconductor-based massively parallel sequencing. A 155-bp region from the chromosome 6 was amplified via PCR. The region contained both the Met408Val and Met420-deletion loci. This haplotype-specific single-molecule DNA analysis enabled us to experimentally verify the presence of different haplotype combinations in individuals heterozygous for the Met408Val and Met420-deletion polymorphisms. The PCR products were purified, pooled together, subjected to template preparation using Ion OneTouch, and sequenced using Ion PGM (both from Life Technologies, Darmstadt, Germany). We obtained on average 19 000 sequencing reads per chromosome that covered both loci. The results were visualized using Integrative Genomics Viewer version 2.3 (Broad Institute; http://www.broadinstitute.org/igv/). Each gray line represents a single sequencing read. The reads containing the Val408 allele are denoted with a G indicating a disagreement with the Met408 allele that is present in the human reference sequence (that is given in the bottom of the figure). The presence of the Met420 deletion is designated with a black dash indicating a disagreement with the presence of ATG in the human reference sequence. This is a representative example of part of the reads of a single sample demonstrating that individuals heterozygous for both Met408 and Met420 deletion did not carry the 2 alleles on the same chromosome. (D) Comparison of the uptake activity of hOCT1 carrying all theoretically possible combinations of the Met420del and Met408Val polymorphisms. The uptake was measured in human embryonic kidney 293 cells stably transfected with the hOCT1 variants or with an empty control plasmid pcDNA5. The stably transfected cells were generated using the FlpIn system for targeted chromosomal integration (Life Technologies) following a procedure described before.⁷ The uptake was measured as the total cellular accumulation after a 2-minute incubation at 37°C with 1 µM MPP⁺, 5 µM TEA⁺, or 5 µM ASP⁺. The concentrations used were below (MPP⁺ and TEA⁺) or close to (ASP⁺) the known K_M values for the substrates. The concentrations of TEA⁺ and MPP⁺ used in this study were identical to the one used by Giannoudis et al¹  and Shu et al,⁸  respectively. Two minutes of incubation were within the linear range for time dependence of the OCT1-mediated uptake. Shown are means and standard error of the means of at least 3 independent experiments, each performed in duplicate. Met420 deletion caused a significant 70% decrease in TEA⁺ uptake. The decrease was less strong (18.6%) when ASP⁺ was used and missing when MPP⁺ was used as a substrate. In comparison, the exchange of Met408 to Val408 caused only a 12% increase in TEA⁺ uptake. The effects of Met408Val substitution were similar in size both with the Met420 and Del420 background and vice versa. This suggests independent additive effects and a lack of epistatic interaction between the 2 polymorphisms.