Eb Virus Related Tumor Virus Gene Polymorphism

Background and objective: Epstein-Barr virus (EBV) is associated with human malignancies such as Burkitt's lymphoma (BL), Hodgkin's lymphoma (HD), nasopharyngeal carcinoma (NPC) and gastric carcinoma. The fact that EBV infection is ubiquitous in the world but the incidence distribution of EBV-associated malignancies differs in geographic regions raises the possibility that particular EBV strains contribute to the development of specific EBV-associated malignancies. However, whether certain EBV subtypes represent geographical polymorphism or are preferentially associated with particular malignancies remains controversial. In China, most studies of the sequence variations of EBV genome have been limited to the populations in Southern China, the endemic area of NPC. The populations in the non-endemic area for NPC have not been extensively determined. To characterize the sequence variations in multiple loci of EBV genome in NPC non-endemic area of China and to explore the association of EBV variants with EBV-associated tumors, the gene polymorphism of EBV genome in isolates from NPC, EBV-associated gastric carcinoma (EBVaGC) and healthy donors in Shandong Province were analyzed.Method In situ hybridization for EBV-encoded small RNA1 (EBER1) was used to screen 175 NPC cases and 1678 gastric carcinoma cases to select EBV positive tumor cases, and polymerase chain reaction (PCR) and southern hybridization for BamHI W fragment were used to screen 268 throat washing (TW) samples of healthy donors to select EBV-positive controls. All the controls and tumor cases were inhabitants of Han nationality in Shangdong Province. PCR and restriction fragment length polymorphism (RFLP) were used to determine the genotypes in EBV-positive samples, including EBV types 1 and 2 at EBNA3C locus, variations of F to f at BamHI F and C to D at BamHI W1/I1 boundary region. The gene polymorphisms of EBER, nuclear antigen 1 (EBNA1), latent membrane 1 (LMP1) and BamHI-A rightward frame 0 (BARF0) were analyzed by PCR and sequencing. The variants of each gene were classified according to the signature changes. The distribution of virus genotypes and sequence variants were compared among the groups and compared with the previous data in NPC endemic area as well as other areas. Moreover, the potential influences on functional domains of the genes were also analyzed.Results①Totally,141 of 175 (80.6%) NPCs and 101 of 1656 (6.1%) GCs were screened for EBER1 positive in tumor cells, and 108 of 268 (40.3%) TW samples were positive for EBV BamHI W fragment, some of which with high quality of DNA were used for gene polymorphism analysis. ②The frequencies of type 1, type F and type C were 82.1% (215/262),91.8% (236/257) and 63.6% (147/247) in the cases detected, respectively. The type D was detected with higher rate in EBVaGC (52.4%,33/63) than in NPC (32.2%,39/121) and TW of healthy donors (28.6%,18/63) (EBVaGC vs NPC:P=0.010; EBVaGC vs TW: P=0.020), while the distributions of the type 1/2 and type F/f among EBVaGC, NPC and healthy donors were not significantly different.③Three main distinct variants of EBER genes, designated as EB-6m, EB-8m and EB-10m, were identified in 154 samples. EB-6m had a previously identified EBER sequence identical to P3HR-1 and was the most common variant. EB-8m and EB-10m were new EBER variants with six common mutations (nucleotide positions in EBER2 transcript:44,46,57,61,93 and 167) in EBER2 genes. They were found in 13/47 (27.7%) NPC cases, whereas in only 1/50 (2.0%) EBVaGC cases and not found in TW cases. The distributions were significantly different (P<0.05). Four (nucleotides 44,46,57 and 61) of the six common changes identified in EBER2 genes are located in the stem-loops of EBER2 secondary structure, which have been shown to bind to several cellular proteins.④Four patterns of the EBNA1 variations, V-val, P-thrV, V-leuV and P-alaV, were observed in 137 samples, and V-val was the most common subtype in all the three groups. The distribution of the EBNA1 subtypes among EBVaGC, NPC and healthy donors was similar. In addition, preferential linkages between EBNA1 subtypes and EBNA3C variants were found to exist.⑤Three main variants of LMP1 gene, China 1, Med- and China 2, were observed in 130 samples, and China 1 was the most common subtype in all the three groups. The frequencies of China 1, XhoI(-) (loss of an XhoI site in N-terminus of LMP1) and del-LMP1 (30 bp deletion in C-terminus of LMP1) in EBVaGC were significantly higher than those in NPC or TW (P<0.01). Preferential linkages between LMP1 subtypes and EBNA3C variants were found to exist. The distribution of LMP1 subtypes in type 1 isolates among EBVaGC, NPC and TW was not significant.⑥BARFO genes carried 9 common AA changes in most of the isolates compared with B95-8. According to the signature changes, two groups and four subtypes were identified in 127 cases, among which 118 isolates showed similar mutation pattern and were classified as one group. Other 9 isolates were arranged into another group. The distribution of BARFO subtypes among EBVaGC, NPC and TW was not significant. One known functional CTL epitope (amino acids 356-364) and the interaction domain with Notch (amino acids 351-371) were conserved in all isolates except for isolates from three samples.⑦The similarities and differences with the previous data in Southern China are as follows:1) Similar to Southern China, the type 1 strain, V-val subtype of EBNA1 and China 1 subtype of LMP1 were the most common subtypes in our area.2) The frequency of type D strains in our area was higher than Southern China.3)In contrast to a higher frequency of type f strains in NPC than that in host populations in Southern China, a lower frequency of type f strains in NPC in our area was found and it similarly distributed among NPC, EBVaGC and healthy donors.4) Several EBNAl subtypes (V-val, P-thrV and V-leuV) were found in NPC biopsies with the same prevalence in the host population in our area, while V-val is the only subtype in NPC biopsies of Southern China with several subtypes in the host populations.5) The frequency of amino acid 335 G→D mutation of LMP1 was lower in our area than Southern China, whereas a higher frequency of amino acid 326 E→Q mutation was found in our area.6) Med- but not B95-8 subtype of LMPlwas found in our area in contrast to Southern China.Conclusions①Type 1 and Type F are the predominant genotypes in Shandong Province. Type D strain is associated with EBVaGC.②The higher frequency of new EBER variants in NPC than EBVaGC and healthy donors suggests EBER locus can be useful to identify different EBV isolates, and it would be interesting to evaluate the association of EBER polymorphisms with EBV-associated tumors.③V-val is the dominant subtype in our area and EBNA1 gene variations are geographically restricted rather than tumor-specific polymorphisms.④China 1 is the major LMP1 subtype in our area. LMP1 variations represent geographically restricted polymorphism, whereas the higher frequency of China 1 strain with amino acid 326 E→Q mutation in EBVaGC than in NPC and TW may also suggests its association with EBVaGC.⑤The conserved mutations in BARFO and the similar distribution of BARFO variants among EBVaGC, NPC and TW may suggests no association between BARFO variants and EBV-associated tumors.⑥The differences in gene polymorphisms of EBV genome between the populations of the same ethnic background in different regions may contributes to the geographical distribution of EBV-associated malignancies.