Thymidylate Synthetase Gene Polymorphism：Relationship With The Risk Of Childhood Acute Leukemia And Effects On High Dose Methotrexate Related Toxicity

Objective：To investigate polymorphisms of open reading frame (ORF)and3’-untranslated region (3’-UTR) in Thymidylate Synthase gene (TYMS).Then, to analyze correlation between these polymorphisms andsusceptibility of childhood acute leukemia (AL) in the Chinese Hanpopulation. Thirdly, to evaluate the influence of polymorphisms for TYMSon toxicities related to high dose methotrexate (HD-MTX) in children withacute lymphoblastic leukemia (ALL).Methods: Reverse transcriptase (RT)-PCR-denaturing gradient gelelectrophoresis (DGGE) combined with direct sequencing were carried outto conform SNPs of ORF and the polymorphisms of3’-UTR in TYMS in108children with AL and121healthy controls. Allele frequency andgenotype distribution of TYMS polymorphisms were analyzed in the twogroups. A case-control study was performed to evaluate the association between these polymorphisms and susceptibility of AL. Linkagedisequilibrium of polymorphisms in TYMS was also analyzed. There were85children with ALL in the108children with AL.52children with ALLwere treated with HD-MTX. The clinical manifestations of the patientswere evaluated retrospectively, such as general situation, mucocutaneousdamage, myelosuppression, and blood and urine routine, hepatic and renalfunction, ECG, myocardial enzymes, and so on. Then effects of TYMS genepolymorphism on toxicities of HD-MTX were investigated.Results:1. Only two SNPs, A381G and T349C were found in the ORF ofTYMS. The allele frequencies of TYMS A381G (Glu127Glu) of AL patientsand normal controls were13.0%versus12.4%, and no significantdifference was observed in the two groups (P>0.05), which suggested thatthere was no correlation between TYMS A381G and susceptibility to AL. Inaddition, TYMS T349C (Phe117Leu) was firstly identified in Chinesechildren and its allele frequency was1.2%.2. Two SNPs (C1100T, A1170G) and1494del6/ins6were conformedin3’-UTR of TYMS. Of all108AL patients, the proportions of CC, CT andTT genotypes of C1100T were10.2%,40.7%and49.1%, C allelefrequency was30.6%; the proportions of AA, AG, and GG genotypes ofA1170G were47.2%,38.0%and14.8%, G allele frequency was33.8%; theproportions of+6bp/+6bp,+6bp/-6bp and-6bp/-6bp genotypes of 1494del6/ins6were8.3%,44.5%,47.2%,+6bp allele frequency was30.6%.Distribution of the3polymorphisms in TYMS had no significant differencebetween AL group and control group (P>0.05).3. Linkage disequilibrium among TYMS A381G, C1100T, A1170G,and1494del6/ins6was observed paired (P<10-4), especially betweenC1100T and1494del6/ins6. But all haplotype frequencies showed nosignificant difference between AL group and control group (P>0.05).4. In75children with ALL, the relationships between each TYMSpolymorphism and clinical characteristics such as age, sex, risk wereanalyzed respectively. Compared with TYMS1170AA genotype, number ofboys carrying AG and GG were about three fold that of girls (OR=3.05,95%CI=1.14~8.19, P=0.023). No correlation was observed betweenTYMS A381G, C1100T,1494del6/ins6and the clinical features of childrenwith ALL (P>0.05).5. Of52children with ALL received HD-MTX chemotherapy,92.3%had bone marrow suppression;40.4%liver dysfunction;32.7%gastrointestinal reactions;25.0%oral mucositis and11.5%cardiac toxicity.Most of the toxicities were so light that the patients could tolerate.6. TYMS A1170G was related with the risk of neutropenia. TYMS1170GG genotype played a protective role, which reduced the risk ofneutropenia (OR=0.07,95%CI=0.01-0.64, P=0.026). TYMS A381G,C1100T and1494del6/ins6polymorphisms were not correlated with HD-MTX related toxicities (P>0.05).Conclusions：1. In our study，only two SNPs, A381G and T349C are found in theORF of TYMS. The allele frequency and genotype distribution of themwere identified in Chinese AL patients and normal controls. There is nocorrelation between TYMS A381G and susceptibility of AL. TYMS T349Cand its allele frequency are firstly identified in the Chinese population.Association of TYMS T349C with susceptibility of AL and and its functionneed further study.2. To our knowledge, this is the first report about C1100T, A1170Gand1494del6/ins6of3’-UTR of TYMS in Chinese children with AL.Linkage disequilibrium between C1100T and1494del6/ins6is identified.These polymorphisms are not associated with the incidence of Chinesechildhood AL.3. TYMS A1170G may be correlated with gender of children withALL.4. Side effects are common in children with ALL treated by HD-MTXchemotherapy, but most are not severe. The most common of side effectsare bone marrow suppression and abnormal liver function, followed bygastrointestinal reaction and oral mucositis.5. TYMS A1170G may be associated with HD-MTX related toxicity inchildhood ALL. TYMS1170GG genotype may reduce risk of MTX-related neutropenia.