Polymorphism Determination of Mutations Found in the Human Reduced Folate Carrier Gene

Abstract

The reduced folate carrier (RFC) is important for cancer patients because it is responsible for the uptake of antifolate drugs like methotrexate (MTX) which causes cell and tumor death. It is known that defective antifolate uptake is caused by alterations in the RFC gene, and two mutations have been found: a glutatmate to lysine substitution at amino acid position 45 (Glu-Lys45) and an arginine to histidine substitution at amino acid position 27 (Arg-His27). The Arg-His27 mutation causes the appearance of a restriction site for the enzyme DraIII, and the Glu-Lys45 mutation causes the disappearance of a BanI restriction enzyme cutting site. The region encompassing the RFC gene of human genomic DNA of a random population were amplified by polymerase chain reaction (PCR). These amplifications were then subjected to restriction digests, one by DrallI and another by BanI. A polymorphism can be defined as a difference in DNA sequence among individuals. Genetic variations occurring in more than 1% of a population are considered to be polymorphisms. From the 35 patients screened with the Dram digestions, an allele frequency of 60% for the wildtype allele and 40% for the mutant allele was determined, and therefore, the Arg-His27 mutation can be considered to be a polymorphism. The BanI digestions for the Glu-Lys45 mutation provided inconclusive evidence. However, through the sequencing of certain patients it was discovered that another BanI cutting site alteration exists on the RFC gene. An amino acid substitution seems to occur in one of the patients at position 3 in which a leucine substitutes a proline (Pro-Leu3). Because proline is commonly known as a helix breaker, the discovery of this polymorphism may be a lead into changes in structure and function of the RFC protein. Via the determined polymorphism of the Arg-His27 and the discovery of other possible polymorphisms, insight can be drawn as to why certain cancer patients respond to and uptake MTX more successfully.