High-dose
methotrexate is a standard component of
therapy for high-grade
osteosarcoma. Its effectiveness may be limited by intrinsic and acquired resistance. Decreased
reduced folate carrier (RFC) expression has been shown in approximately half of
osteosarcomas at diagnosis. Mutations and polymorphisms in the RFC gene have been reported in various cell lines. The purpose of this study was to investigate sequence alterations in the RFC gene in
osteosarcoma tumor samples. The entire coding region of the RFC gene in samples from 162
osteosarcoma patients was screened by
DNA single-stranded conformational polymorphism, followed by direct sequencing of any region with altered mobility. A previously identified polymorphism at
cDNA position number 174 of RFC exon 2 was observed. Sixty-one samples (37.6%) were heterozygous with both A/G at this position (His(27)/Arg(27)), 52 samples (32.2%) were homozygous with G (Arg(27)), and 49 samples (30.2%) were homozygous with A (His(27)). Fifteen (9.2%) samples were identified with other RFC sequence variants in exon 2, none of which have been reported. The sequence variants in exon 2 included a G to A substitution at
cDNA position 231, a G to A substitution at
cDNA position 155, a C to T substitution at
cDNA position 114, and a T to C substitution at
cDNA position 104, resulting in a
serine to
asparagine substitution at
amino acid 46, a
glutamate to
lysine substitution at
amino acid 21, an
alanine to
valine substitution at
amino acid 7, and a
serine to
proline substitution at
amino acid 4, respectively. A deletion of A at
cDNA position 126 resulting in a frameshift was also observed. Some of these variants were observed in multiple samples. Eight samples had altered single-stranded conformational polymorphism patterns in exon 3 that were associated with
nucleotide changes that altered the amino acid sequence. All of these RFC sequence variants appeared to be heterozygous. Heterozygous C/T and homozygous C also were observed at RFC
cDNA position 790 in exon 3, which does not alter the
amino acid coding sequence. This study shows that RFC sequence alterations are frequent in samples from
osteosarcoma patients. Additional studies are under way to determine the clinical significance of these sequence alterations and their effect on
methotrexate transport and resistance.