| Objective:The purpose of this study was to investigate coding single nucleotide polymorphisms (cSNPs) in reduced folate carrier gene (RFC) in Chinese children with acute leukemia (AL) and normal control children, and to describe the allele frequency distributions in the cases and controls. These informations could provide the basis for connection between the polymorphism of RFC and methotrexate (MTX) resistance in patients with AL, and provide clue for association between the SNP of RFC and the occurrence of ALMethods:The cDNAs from 95 childhood AL patients bone marrow and from 135 control peripheral blood were prepared by reverse transcriptase (RT)-polymerase chain reaction (PCR). The open reading frames (ORFs) of human reduced folate carrier (hRFC) were analyzed for cSNPs by denaturing gradient gel electrophoresis (DGGE), constant denaturant gel electrophoresis (CDGE) and DNA sequencing. The primes for first PCR and nested PCR were designed by the analyzed softwares such as WinMeltrM2.0, Oligol.l and Prime5.0. The frequencies of allele and genotype of each SNP were analyzed by software SPSS 13.0 in AL group and control group.Results:Four novel cSNPs in the hRFC gene were found first time in this study. These novel cSNPs were named as variation 1,2,3 and 4, and three of them were missense mutation. Variation 1 caused a C to T substitution, produced Arg>Trp (R>W) substitution; Variation 2 caused a T to C substitution, produced Leu>Pro (L>P); Variation 4 caused a T to A substitution, Leu>Gln (L>Q). Variation 3 was a synonymous mutation, causes a G to A substitution, Thr>Thr (T>T). The allelic frequencies of these variation 1,2,3 and 4 were 0.22%,0.22%,0.22%,0.87% in 230 children, and 0.5%,0,0.5%,0 in 95 AL patients, and 0,0.4%,0,1.5% in 135 normal children, respectively. Moreover, four known variations were determined in these samples, including 80G>A,696C>T,972G>A and 1242C>A. The allelic frequencies of these variations were 45.7%,44.0%,9.6%,0.22% in 230 children; and 51.6%,40.0%,9.5%,0.5% in 95 AL patients; and 41.5%,47.0%,9.6%,0 in 135 normal children, respectively. For three higher frequency of variation at 80G>A (45.7 %),696C>T (44.0%) and 972 G>A (9.6%), their allele frequencies and genotype frequencies were assessed by a chi-square test. The results showed that 80G>A had statistically significant difference between the two groups (p<0.05). Compared with the genotype of G, the genotype of A increased the risk of acute leukemia by 1.500 times. RFC 80GA and 80AA also increased the risk of acute leukemia, which made the incidence of leukemia increasing 2.370-fold. The 95% confidence interval was 1.034～2.183 and 1.256～4.130, respectively. However, the difference was not statistically significant at 696C>T and 972 G>A (p>0.05). The study did not detect any other known mutations in 230 samples, which published in the International SNP Bank (Number:rsl37432739). These undiscovered cSNPs included a total of twenty-six, that were:10T>A,20C>T, 61G>A,137G>A,167G>C,246G>C,423T>C,665T>C,761T>C,775G>T,784C>T, 786A>G,872T>C,921A>G,1014C>T,1022A>G,1146C>T,1250T>C,1367A>G, 1372G>T,1392A>G,1396C>T,1406T>C,1434A>G,1564G>A and 1673T>C.Conclusion:In methodology, it was first time to use the advance technology of PCR-DGGE/CDGE with DNA sequencing for screening and assessment of RFC cSNPs. Four novel cSNPs (Variation 1,2,3 and 4) were identified in the hRFC gene and displayed allelic frequencies of 0.22%,0.22%,0.22%,0.87%, respectively. Variation 1,2 and 4 were missense mutations, which could had potential influence on RFC transportation and were worth further to study their function. In addition,, three known mutations (696C>T, 972G>A,1242G>A) were first found in Chinese children, the allelic frequencies were 44.0 %,9.6%,0.22% respectively. Meanwhile, one known mutation (80G>A) was first found in Chinese children with AL, the allelic frequencies in 230 samples were 45.7%, and 51.6% in AL patients,41.5% in normal children. Given the results of this study, we first found that RFC 80G>A (R27H) variant may contribute to increasing the susceptibility to AL, while no association in 696C>T and 972G>A. Following cSNPs reported abroad were not found in this study:10T>A,20C>T,61G>A,137G>A,167G>C,246G>C,423T>C, 665T>C,761T>C,775G>T,784C>T,786A>G,872T>C,921A>G,1014C>T,1022A>G, 1146C>T,1250T>C,1367A>G,1372G>T,1392A>G,1396C>T,1406T>C,1434A>G, 1564G>A and 1673T>C (refer to the International SNP Bank rsl37432739 and cSNPs from other republished articles). |
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