title: Single nucleotide polymorphisms in the human reduced folate carrier: characterization of a high-frequency G/A variant at position 80 and transport properties of the His(27) and Arg(27) carriers.
abstract: The presence of sequence variants in the human reduced folate carrier (hRFC) was assessed in leukemia blasts from children with acute lymphoblastic leukemia (ALL) and in normal peripheral blood specimens. A CATG frame shift insertion at position 191 was detected in 10-60% of hRFC transcripts from 10 of 16 ALL specimens, by RFLP analysis and direct sequencing of hRFC cDNAs. In genomic DNAs prepared from 105 leukemia (n = 54) and non-leukemia (n = 51) specimens, PCR amplifications and direct sequencing of exon 3 identified a high-frequency G to A single nucleotide polymorphism at position 80 that resulted in a change of arginine-27 to histidine-27. The allelic frequencies of G/A80 were nearly identical for the non-leukemia (42.2% CGC and 57.8% CAC) and leukemia (40.7% CGC and 59.3% CAC) genomic DNAs. In cDNAs prepared from 10 of these ALL patients, identical allelic frequencies (40 and 60%, respectively) were recorded. In up to 62 genomic DNAs, hRFC-coding exons 4-7 were PCR-amplified and sequenced. A high-abundance C/T696 polymorphism was detected with nearly identical frequencies for both alleles, and a heterozygous C/A1242 sequence variant was identified in two ALL specimens. Both C/T696 and C/A1242 were phenotypically silent. In transport assays with [(3)H]methotrexate and [(3)H]5-formyl tetrahydrofolate, nearly identical uptake rates were measured for the arginine-27- and histidine-27-hRFC proteins expressed in transport-impaired K562 cells. Although there were no significant differences between the kinetic parameters for methotrexate transport for the hRFC forms, minor (approximately 2-fold) differences were measured in the K(i)s for other substrates including Tomudex, 5,10-dideazatetrahydrofolate, GW1843U89, and 10-ethyl-10-deazaaminopterin and for 5-formyl tetrahydrofolate.
authors.affiliation: Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 East Warren Avenue, Detroit, MI 48201, USA.
authors.forename: J R
authors.initials: JR
authors.lastname: Whetstine
authors.forename: A J
authors.initials: AJ
authors.lastname: Gifford
authors.forename: T
authors.initials: T
authors.lastname: Witt
authors.forename: X Y
authors.initials: XY
authors.lastname: Liu
authors.forename: R M
authors.initials: RM
authors.lastname: Flatley
authors.forename: M
authors.initials: M
authors.lastname: Norris
authors.forename: M
authors.initials: M
authors.lastname: Haber
authors.forename: J W
authors.initials: JW
authors.lastname: Taub
authors.forename: Y
authors.initials: Y
authors.lastname: Ravindranath
authors.forename: L H
authors.initials: LH
authors.lastname: Matherly
mesh_terms: D019943:Amino Acid Substitution; D001402:B-Lymphocytes / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics*; D002648:Child; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004273:DNA, Neoplasm / Q000737:chemistry / Q000235:genetics; D005787:Gene Frequency; D006801:Humans; D020014:K562 Cells; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000493:pharmacokinetics; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics / Q000473:pathology*; D012334:RNA, Neoplasm / Q000235:genetics / Q000378:metabolism; D058978:Reduced Folate Carrier Protein; D013234:Stem Cells / Q000378:metabolism; D014162:Transfection
chemical_list: D002352:Carrier Proteins; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D026901:Membrane Transport Proteins; D012334:RNA, Neoplasm; D058978:Reduced Folate Carrier Protein; C548699:SLC19A1 protein, human; D008727:Methotrexate