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.
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.
Whetstine
J R
JR
Department of Pharmacology, Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 East Warren Avenue, Detroit, MI 48201, USA.
Gifford
A J
AJ
Witt
T
T
Liu
X Y
XY
Flatley
R M
RM
Norris
M
M
Haber
M
M
Taub
J W
JW
Ravindranath
Y
Y
Matherly
L H
LH
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
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