Sequence Variation Of EBV-Encoded Gene In Leukemia And Myelodysplastic Syndrome

Background and Objective:Epstein–Barr virus(EBV)is recognized as a tumor virus that is closely related to the development of multiple tumors,such as nasopharyngeal carcinoma(NPC),gastric cancer,Hodgkin’s lymphoma,natural killer T-cell lymphoma,and others.In recent years,many studies have focused on the relationship between leukemia and EBV.The unique pattern of EBV expression can be considered a marker of the particular differentiation window of the B-cell that can result in chronic leukemia(CL).Recent studies have showed EBV infection in acute leukemia(AL).However,the potential role of EBV in hematic tumors still remains unclear.We have detected EBER and LMP1 gene variations in various samples including samples from donors with gastric carcinomas,nasopharyngeal carcinomas,and lymphomas,as well as samples from healthy donors.However,the variations of these EBV-coding genes in hematological diseases have not been detected.To find EBV gene variations in more EBV-associated diseases in Northern China and try to give clues to the link between these mutations and EBV-related tumors,we decided to investigate sequence variations in the EBER and LMP1,and also compare these results with those from previous reports.The traditional EBNA2 variants(EBV type1/type2)and F/f type were also analyzed.Methods: Peripheral blood specimens of 313 leukemia patients and 99 myelodysplastic syndrome(MDS)patients were obtained from the Affiliated Hospital of Qingdao University,which located in Shandong Province,Northern China.Four milliliters of the peripheral blood samples were collected and centrifuged at 1200 rpm for 10 min.EDTA was added to anticoagulate.The intermediate leukocyte layer was extracted and the DNA was extracted by protease K digestion and phenol-chloroform method.Nested polymerase chain reaction(PCR)was performed to analyze the EBV genotypes 1/2 according to the sequence divergence within EBNA-2.Restriction fragment length polymorphism(RFLP)was performed to analyze genotypes F/f.EBER and LMP1 subtypes were analyzed by the Nested polymerase chain reaction technology and DNA sequencing,and the nucleotide and amino acid sequences of EBER and LMP1 genes were compared with the standard strain B95-8 by DNAstar.Results:(1)Type 1 EBV was found in 59(73.8%,59/80)leukemia,30(88.2%,30/34)MDS and 57(78.9%,45/57)healthy donors,while type 2 was found in 7(8.7%,7/80)leukemia,30(88.2%,30/34)MDS and 10(17.6%,10/57)healthy donors as well as type1+2 was found in 14(17.5 %,14/80)leukemia,1(3%,1/34)MDS and 2(3.5%,2/57)healthy donors.(2)Type F was detected in 98.8%(82/83)leukemia,93.3%(28/30)MDS samples and 98(94.2%,98/104)healthy donors while Type f was detected in 1.2%(1/83)leukemia,6.7%(2/30)MDS samples and 6(5.8%,6/104)healthy donors.The distribution of EBV genotypes 1/2 was not significantly different among the leukemia,myelodysplastic syndrome and healthy donors groups,neither was the distribution of EBV genotypes F/f.(3)The dominate subtype of EBER was the EB-6m prototype in leukemia and MDS(73.2 %(30/41),83.3%(10/12)respectively).The detection rate was lower than that of healthy people(96.7%,89/92),and the difference was significant(p<0.05).(4)The China 1,XhoⅠ(-)and del-LMP1 were the dominate subtype of LMP1 in leukemia,MDS and healthy donors.(5)The China 1 subtype was found in 70%(28/40)leukemia,90%(9/10)MDS and 69.8%(30/43),while XhoⅠ(-)was found in 75%(30/40)leukemia,90%(9/10)MDS and 74.4%(32/43)healthy donors.The del-LMP1 was found in 77.5 %(31/40)leukemia,90 %(9/10)MDS and 69.8%(30/43)healthy donors.The distribution of LMP1 subtypes(China 1,XhoⅠ(-)and del-LMP1)were not significantly different among the leukemia,myelodysplastic syndrome and healthy donors.Conclusion:(1)The genotypes 1 and F were the predominant EBV genotypes in leukemia and myelodysplastic syndrome in Qingdao,Shandong province.(2)The predominant genotype of EBER is EB-6m in leukemia and MDS.(3)The China 1,XhoⅠ(-)and del-LMP1 were the dominate subtype of LMP1 in leukemia and MDS.The distribution of the three variants were not significantly different among the leukemia,myelodysplastic syndrome and healthy donors groups,suggesting that the EBV strains with three variants might be not associated with leukemia and MDS.