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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 His²⁷ and Arg²⁷ Carriers¹

Departments of Pharmacology [J. R. W., L. H. M.] and Pediatrics [Y. R., J. W. T.], and the Experimental and Clinical Therapeutics Program [J. R. W., T. W., X. Y. L., R. M. F., J. W. T., Y. R., L. H. M.], Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, Children’s Hospital of Michigan, Detroit, Michigan 48201 [Y. R., J. W. T.], and Children’s Cancer Institute Australia for Medical Research, Randwick, Sydney, New South Wales, Australia [A. J. G., M. N., M. H.]

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 [³H]methotrexate and [³H]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 (2-fold) differences were measured in the K_is for other substrates including Tomudex, 5,10-dideazatetrahydrofolate, GW1843U89, and 10-ethyl-10-deazaaminopterin and for 5-formyl tetrahydrofolate.

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