| Objective: To investigate the distribution of WT1 rs16754 in patients with acute myeloid leukemia(AML)and its relationship with the risk of disease.To compare the clinical features,the abnormal rate of karyotype and the mutation rate of FLT3-ITD in AML patients who were homozygous major alleles(GG type)and at least one minor allele(GA/AA type)of WT1 rs16754.To compare the complete remission and overall survival time in patients with GG type and GA/AA type group and clarify the relationship between the polymorphism of WT1 rs16754 and chemotherapy sensitivity and prognosis in AML.To analyze the relationship between WT1 gene rs16754 polymorphism and prognosis of AML by meta analysis.Methods:1.The genotype distribution of WT1 rs16754 and its relationship with the risk of diseaseBone marrow or peripheral blood samples were collected from 115 newly diagnosed AML patients and 177 healthy volunteers,extracted their DNA,a pair of primers were designed on both sides of the rs16754 polymorphism loci of WT1 gene,then distinguished their genotypes by using high resolution melting curve method(HRM).5 samples of each genotype were randomly selected and seventh exon(151bp)were amplified by polymerase chain reaction(PCR).The accuracy of classification results was verified by sequencing.The difference of genotype distribution between patients group and healthy control group were compared.2.Clinical features between different genotypes of WT1 rs16754 in AML patientsThe clinical indexes of AML patients were collected,including age,gender,peripheral white blood cell count,erythrocyte count,hemoglobin concentration,platelet count,neutrophil count,lactate dehydrogenase,percentage of bone marrow blast cells,WHO 2008 type.R banding technique was used for karyotype analysis;polymerase chain reaction was used to amplify the FLT3-ITD gene.The product was photographed and read by 10% polyacrylamide gel electrophoresis.The above indicators of AML patients with homozygous major alleles or at least one minor allele were statistically described and compared,respectively.3.Comparison of chemotherapy response and survival status of AML patients with different genetype of WT1 rs16754The chemotherapy regimen of AML patients was obtained by consulting the case records and the chemotherapy regimen between the homozygous major allele and at least one minor allele AML patients group.Access to medical records and telephone follow-up to follow up,Overall survival(OS)was defined as the time of diagnosis to death or last follow-up,and the complete remission rate and overall survival time of the two groups were compared.4.Meta analysis of WT1 rs16754 polymorphism with prognosis in AML patientsStudies published in Pub Med,CNKI,Wan Fang,CBM database were searched using the search terms ‘WT1',‘Polymorphism' and ‘Leukemia,Myeloid,Acute' respectively.Deadline was December 1,2015.Results:1.In 115 patients with primary AML,the genetype frequency for GG was 59(51.3%),GA was 43(37.4%),and AA in 13 cases(11.3%).In healthy control group,the genetype for GG was 94 cases(53.1%),GA was72(40.7%),AA was 11(6.2%).Among them,1 cases were failed by HRM method repeatedly,and the PCR product was sequenced.The direct sequencing results of 15 samples were completely consistent with the HRM method.There was no significant difference in genotype distribution between the two groups(P=0.296),and there was no significant difference in age(P=0.472)and gender(P=0.596)between the two groups.The genotype distribution in the control group was subject to the law of Hardy-Weinberg genetic equilibrium(P=0.938).For the study population,G is the major allele of rs16754,which is not related to the risk of AML.2.There were statistically significant differences in the percentage of bone marrow blast cells(P=0.028)in 56(48.7%)GA/AA and 59(51.3%)GG patients.There were no significant differences in the age(P=0.825)and sex(P=0.483)distribution between the in the patients with genotype GA/AA and GG of WT1 rs16754.The white blood cell count(P=0.823),red blood cell count(P=0.479),hemoglobin(P=0.356),platelet count(P=0.455),neutrophil count(P=0.737),lactate dehydrogenase(P=0.804),WHO 2008 type(P=0.439),chromosome karyotype analysis(P=0.357)and the rate of FLT3-ITD mutation(P=0.480)had no statistically significant differences.3.After chemotherapy,51 GG patients(86.4%)of 59 patients(56)had CR,47 patients with type GA/AA(83.9%).The difference of CR rate was not statistically significant(P=0.704).Of the 115 patients,16 patients were lost to follow-up,99 patients received survival data,including 45 cases in GA/AA group and 54 cases in GG group? A total of 5 patients underwent transplantation,in which 2 cases of allogeneic hematopoietic stem cell transplantation,2 cases of autologous hematopoietic stem cell transplantation,1 cases of micro transplantation.Differences were not observed in the transplantation rate between the two groups(P=1.000).Kaplan-Meier method was used to estimate the rate of OS in patients.After Log-rank test,there was significant difference in the rates of OS between the two groups(P=0.035).The rate of OS in group GG was higher than that in group GA/AA,suggesting that allele A might be a risk factor for prognosis.Using COX proportional hazard model to adjust age(over 60 years old and <60 years old)and gender this two factors,the results showed that A allele of rs16754 was an independent prognostic factor for survival of AML patients.The mortality risk of patients with type GA/AA was higher than that in patients with type GG(HR=1.681,95% CI 1.046~2.700,P=0.032).4.A total of 11 English articles and the results of the first part of this study are included in this meta analysis,with a total of 2904 patients accrued.Meta analysis showed that there was no association between rs16754 and CR(RR=1.02,95% CI:0.99-1.06,P=0.23)in AML patients,and the same to overall survival(OS)(HR=0.77,95% CI:0.52-1.15,P= 0.21),5-year OS(RR=1.10,95% CI:0.90-1.34,P=0.37)and relapse-free survival(RFS)(HR=0.80,95% CI: 0.54-1.19,P=0.27).Conclusions:1.The polymorphism locus of WT1 rs16754 was not associated with the risk of AML.The patients were divided into GA/AA group and GG group,there were no significant differences in age and sex distribution between the two groups.2.There were significant differences in WBC,RBC,HGB,PLT,neutrophil count,LDH level,WHO(2008)and karyotype between the GA/AA group and GG group.FLT3-ITD mutation is an indicator of poor prognosis for AML,the mutation rate of FLT3-ITD between the two groups had no difference,but the percentage of bone marrow blast cells of GA/AA group is higher.3.The polymorphism locus of WT1 rs16754 was not associated with sensitivity to chemotherapy.Analysis the survival time of 99 AML patients,the rate of OS in group GG was higher than that in group GA/AA.After adjusted age(over 60 years old and <60 years old)and gender,the results showed that A allele of rs16754 was an independent prognostic factor for survival of AML patients,suggesting that allele A might be a risk factor for prognosis.4.No correlation was found between WT1 gene rs16754 polymorphism and AML prognosis. |
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