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 le ukemia (ALL) and in normal peripheral blood specimens. A CATG frame shift i nsertion 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 identifie d a high-frequency G to A single. nucleotide polymorphism. at position 80 t hat resulted in a change of arginine-27 to histidine-27. The allelic freque ncies 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 prep ared from 10 of these ALL patients, identical allelic frequencies (40 and 6 0%, respectively) were recorded. In up to 62, genomic DNAs, hRFC-coding exo ns 4-7 were PCR-amplified and sequenced. A high-abundance C/T696 polymorphi sm was detected with nearly identical frequencies for both alleles, and a h eterozygous C/A1242 sequence variant was identified in two ALL specimens. B oth C/T696 and CYA1242 were phenotypically silent. In transport assays with [H-3]methotrexate and [H-3]5-formyl tetrahydrofolate, nearly identical upt ake rates were measured for the arginine-27- and histidine-27-hRFC proteins expressed in transport-impaired K562 cells. Although there were no signifi cant differences between the kinetic parameters for methotrexate transport for the hRFC forms, minor (similar to2-fold) differences were measured in t he K(i)s for other substrates including Tomudex, 5,10-dideazatetrahydrofola te, GW1843U89, and 10-ethyl-10-deazaaminopterin and for 5-formyl tetrahydro folate.