Detection of the nt230[del4] MDR1 mutation in dogs by a fluorogenic 5' nuclease TaqMan allelic discrimination method.

For detection of the nt230[del4] MDR1 mutation, a 4-bp deletion in the canine MDR1 (ABCB1) gene, a TaqMan allelic discrimination assay was designed that allows for MDR1 genotyping without post-PCR processing. Directly after completion of the PCR amplification, the MDR1 genotype can be assigned based on selective fluorescence measurement. For primer selection the locus of a potential 265A>G single nucleotide polymorphism was omitted; this locus is covered by the oligonucleotide PCR primers from most of the hitherto established MDR1 genotyping methods. Dogs homozygous for the nt230[del4] MDR1 mutation show highly increased susceptibility to many drugs commonly used in veterinary medicine including ivermectin. As more than 10 dog breeds are predisposed to this mutation, reliable genotyping methods are necessary to identify affected dogs before drug treatment. This study provides a new allelic discrimination method that detects the MDR1 mutation with high specificity and reliability and is useful for routine diagnostics.

Cloning and functional characterization of human sodium-dependent organic anion transporter (SLC10A6).

We have cloned human sodium-dependent organic anion transporter (SOAT) cDNA, which consists of 1502 bp and encodes a 377-amino acid protein. SOAT shows 42% sequence identity to the ileal apical sodium-dependent bile acid transporter ASBT and 33% sequence identity to the hepatic Na(+)/taurocholate-cotransporting polypeptide NTCP. Immunoprecipitation of a SOAT-FLAG-tagged protein revealed a glycosylated form at 46 kDa that decreased to 42 kDa after PNGase F treatment. SOAT exhibits a seven-transmembrane domain topology with an outside-to-inside orientation of the N-terminal and C-terminal ends. SOAT mRNA is most highly expressed in testis. Relatively high SOAT expression was also detected in placenta and pancreas. We established a stable SOAT-HEK293 cell line that showed sodium-dependent transport of dehydroepiandrosterone sulfate, estrone-3-sulfate, and pregnenolone sulfate with apparent K(m) values of 28.7, 12.0, and 11.3 microm, respectively. Although bile acids, such as taurocholic acid, cholic acid, and chenodeoxycholic acid, were not substrates of SOAT, the sulfoconjugated bile acid taurolithocholic acid-3-sulfate was transported by SOAT-HEK293 cells in a sodium-dependent manner and showed competitive inhibition of SOAT transport with an apparent K(i) value of 0.24 mum. Several nonsteroidal organosulfates also strongly inhibited SOAT, including 1-(omega-sulfooxyethyl)pyrene, bromosulfophthalein, 2- and 4-sulfooxymethylpyrene, and alpha-naphthylsulfate. Among these inhibitors, 2- and 4-sulfooxymethylpyrene were competitive inhibitors of SOAT, with apparent K(i) values of 4.3 and 5.5 microm, respectively, and they were also transported by SOAT-HEK293 cells.