Identification and Functional Characterization of Novel Genetic Variations in theOCTN1Promoter

Abstract

Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter.

Association study betweenOCTN1functional haplotypes and Crohn's disease in a Korean population

“…Therefore, we hypothesized that patients with decreased OCTN1 activity by genetic variants would be susceptible to intestinal inflammation, and would demonstrate more severe CD phenotypes than patients with normal OCTN1 activity. In our previous study, we determined that the OCTN1 haplotypes that contain two variants, g.-1145A>G and g.-248C>G, showed reduced promoter activity [8]. Among the six OCTN1 promoter haplotypes in the present study, five haplotypes (H2 to H6) contained these variants.…”

“…Here, we hypothesized that the OCTN1 functional haplotypes that demonstrated decreased promoter activity could be associated with a severe clinical course of CD, as the well-known OCTN1 variant L503F demonstrated a reduction in transport activity in a previous study [4]. In our population, five OCTN1 promoter haplotypes, (H2 to H6) were predicted to have decreased promoter activity according to the results of our previous study [8]. Therefore, we divided the 194 patients into two groups according to the five OCTN1 haplotypes: a variant group (n=86) and a control group (n=108).…”

“…In the present study, we evaluated the clinical usefulness of OCTN1 functional haplotypes identified in our previous study [8]. First, we investigated the association between genetic variants of the OCTN1 promoter and CD.…”

“…In the present study, four OCTN1 promoter variants, g.-1875T>A, g.-1745A>G, g.-1145A>g, and g.-248C>G, which showed significant changes in promoter activity in our previous study [8], were genotyped by a SNaPshot assay using an automated DNA analyzer (DNA Link, Inc., Seoul, Korea).…”

“…We compared the frequencies of OCTN1 functional variants or haplotypes reported in our previous study [8] between patients with CD and healthy control groups. Table 1 shows the frequency of the four variants between the two groups.…”