| Background and Objective: Epstein–Barr virus(EBV)infection is ubiquitous worldwide,but certain EBV-associated tumors are endemic and exhibit biased geographic distribution worldwide.The relation between EBV gene polymophism and tumors has attracted much attention,but whether there is tumor related EBV gene variation is still controversial.Most previous studies have focused on EBV encoding genes,but less attention has been paid to its non-coding genes.EBV-encoded BART miRNAs play important roles in viral infection and tumor development.The aim of this study is to clarify the regional distribution of BART cluster 1 gene variation and its relation to EBV-associated tumors by comprehensively analyzing the sequences of.BART cluster 1 gene in EBV-associated tumors and healthy donors(HDs)from NPC-endemic China and NPC-non-endemic China combined with the sequences of EBV genomes derived from different people in different parts of the world.Another aim of the study is to investigate the effect of BART cluster 1 gene variation on the expression of mature miRNAs.Methods:(1)Nested PCR and DNA sequencing were used to detect the sequences of BART cluster 1 gene in 354 EBV positive specimens from Guangdong and Shandong.BART cluster 1 sequences were cut from 906 EBV genomes with clear geographic and disease origin in Gen Bank.The sequences were analyzed by Lasergene and MEGA software and classified according to the signature changes and the phylogenetic tree.(2)The distributions of BART cluster 1 variants and single nucleotide polymorphisms(SNPs)were compared among different populations in the same region,as well as among same populations in different regions.Specific variants and SNPs and the risk for diseases were analyzed.(3)Linkage analysis of BART cluster 1 gene variation in EBV genome with SNPs T162476 C and C163364 T of BALF2 gene,which previously reported to be associated with NPC risk,was performed.(4)Plasmids containing different BART cluster1 gene variants were constructed and transfected into EBV negative NPC cells HONE1.The expression level of mature miRNAs produced by BART cluster 1 was detected by q RT-PCR.Results:(1)BART cluster 1 gene sequences from specimens and genomes were classified into six variants(BART-A,-B,-C,-D,-E and-P),and the first five variants were further divided into two subtypes respectively.Four variants,BART-A,-B,-C and–D,were detected in the specimens from Guangdong and Shandong.Among which,BART-A and BART-C were found in a few specimens.The most common variant in Guangdong was BART-D2,followed by BART-B,and BART-D2 was the main subtype of NPC.The most common variant in Shandong was BART-D1,followed by BART-B.Six variants(BART-A,-B,-C,-D,-E and-P)were found in 906 EBV genomes.BART-E and-P were only found in a few genomes.BART-A was distributed in most of the regions,but its detection rate was low.BART-B and BART-C were found mainly in Asian,and BART-C was more common in Southeast Asia.BART-D1 was the mainly variant in Africa,Europe&Australia&USA and NPC-non-endemic East Asia,while a relatively higher proportion of BART-B was also found in NPC-non-endemic East Asia.BART-D2 was only found in Asia,and was only common in NPC-endemic China.In addition,BART-D2 was the main subtypes of NPC in NPC-endemic China.(2)The detection rates of BART-D2 and SNP C139946 T were 87.0% and 91.5%respectively in 283 NPCs from NPC-endemic China,which were higher than 26non-NPC malignancies(19.2% and 69.2%)and 242 HDs(43.8% and 74.8%)in the same area,and were also higher than those of 118 NPCs from NPC-non-endemic East Asia(31.3% and 40.7%)and 20 NPCs from Southeast Asia(5.0% and 10.0%).The detection rates of BART-D2 and C139946 T in non-NPC malignancies and HDs from NPC-endemic China were higher than non-NPC malignancies(The number of cases was147,95 and 37,respectively)and HDs&Non-malignancies(The number of cases was170,34 and 40,respectively)from NPC-non-endemic East Asia,Africa and Europe&Australia&USA,respectively(The detection rates of BART-D2 and C139946 T were 0.0%～1.8% and 0.0%～7.5%,respectively).In addition,the detection rates of BART-D2 and C139946 T in NPCs from NPC-non-endemic East Asia were higher than non-NPC malignancies and HDs&Non-malignancies in the same area,and were also higher than NPCs from Southeast Asia.All the above differences were significant(P=0.008～P<0.001).(3)BART cluster 1 gene variant and SNP C139946 T were significantly correlated with SNPs T162476 C and C163364T(P<0.001).The high-risk variants of SNPs 162476 C and 163364 T mainly corresponded to the BART-D2 and 139946 T.(4)Compared with HD,BART-D2 and 139946 T increased the risk of NPC in NPC-endemic China(BART-D2:OR=9.692,95CI=6.158-15.254;139946T:OR=4.579,95CI=2.642-7.934).BART-D2 and 139946 T also increased the risk of NPC in NPC-non-endemic China(BART-D2 :OR=8.222,95CI=2.413-28.015;139946T:OR=9.086,95CI=3.084-26.771),but they were not associated with the risk of non-NPC tumor.(5)The expression level of EBV-miR-BART4-5p、1-5p、15、5-3p and 6-3p increased,while that of 3-5p and 6-5p decreased in HONE1 cells transfected with BART cluster 1gene of BART-D2 compared with EBV standard stain.The differences were significant(P<0.05).Conclusions:(1)EBV BART cluster 1 gene variation exhibits regional distribution,and unique variation pattern is found in NPC-endemic China.(2)The BART-D2 and SNP C139946 T of EBV BART cluster 1 are associated with NPC,especially in the NPC-endemic area,and increase the risk of NPC.(3)The BART-D2 subtype of EBV BART cluster 1 gene may affect the expression of mature BART miRNAs. |
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