Polymorphisms of SLC22A9 (hOAT7) in Korean Females with Osteoporosis

Abstract

Among solute carrier proteins, the organic anion transporters (OATs) play an important role for the elimination or reabsorption of endogenous and exogenous negatively charged anionic compounds. Among OATs, SLC22A9 (hOAT7) transports estrone sulfate with high affinity. The net decrease of estrogen, especially in post-menopausal women induces rapid bone loss. The present study was performed to search the SNP within exon regions of SLC22A9 in Korean females with osteoporosis. Fifty healthy controls and 50 osteoporosis patients were screened for the genetic polymorphism in the coding region of SLC22A9 using GC-clamped PCR and denaturing gradient gel electrophoresis (DGGE). Six SNPs were found on the SLC22A9 gene from Korean women with/without osteoporosis. The SNPs were located as follows: two SNPs in the osteoporosis group (A645G and T1277C), three SNPs in the control group (G1449T, C1467T and C1487T) and one SNP in both the osteoporosis and control groups (G767A). The G767A, T1277C and C1487T SNPs result in an amino acid substitution, from synonymous vs nonsynonymous substitution arginine to glutamine (R256Q), phenylalanine to serine (F426S) and proline to leucine (P496L), respectively. The Km values and Vmax of the wild type, R256Q, P496L and F426S were 8.84, 8.87, 9.83 and 12.74 µM, and 1.97, 1.96, 2.06 and 1.55 pmol/oocyte/h, respectively. The present study demonstrates that the SLC22A9 variant F426S is causing inter-individual variation that is leading to the differences in transport of the steroid sulfate conjugate (estrone sulfate) and, therefore this could be used as a marker for certain disease including osteoporosis.

Keywords: Denaturing gradient gel electrophoresis; Estrone sulfate; GC-clamp; Osteoporosis; Polymorphism; SLC22A9.

Fig. 1. The exon-intron organization of the SLC22A9 genome and its relationship to the SLC22A9 cDNA. Gray boxes represent the protein-coding regions of the exons.

Fig. 2. The typical band patterns of agarose or acrylamide gel electrophoresis. (A) Normal individual genomic DNA amplified using DGGE primer sets for the respective mutation found in exons by duplex PCR. (B) The duplex PCR products from the same genomic DNAs were loaded on a polyacrylamide gel. (C) The aberrant band patterns and sequencing electropherograms of observed SLC22A9 SNPs in osteoporosis and normal subjects. The synonymous A645G, G1449T and C1467T were found in exon 3 and 9, respectively. The G767A, T1277C and C1487T were found in exon 4, 7 and 9, respectively.

Fig. 3. Transport activity of the SLC22A9 and its variants. (A) Concentration dependence of estrone sulfate mediated by wild type SLC22A9 and variants in Xenopus laevis oocytes. Defolliculated stage VI and V oocytes were injected with 25 ng/oocyte of wild-type and variants cRNAs, and incubated for 3 days in Barth's solution at 18℃. After three days, [³H] estrone sulfate uptake experiment was performed using various concentrations (25 nM~50 µM). Each point employs 8~10 oocytes and represented mean±standard error of three independent experiments. (B) Eadie-Hofstee plot analysis for determination of Km and Vmax values.