| Cytomegalovirus infection (CMV-I) and CMV related disease occurred after hematopoietic stem cell transplantation have long been suspected of having some relationship with the graft-versus-host-disease(GVHD) and some degree of myelosuppression in Allogeneic hematopoietic stem cell transplantation(Allo-HSCT). They are the major causes of transplantation related mortality. CMV is the biggest human DNA herpes virus. Previous studies suggest that CMV glycoprotein B (gB) maybe the target for antibodies production and may play a role in viral entry into cells. Related analysis of gene encoding envelope gB showed that clinical isolates adopted one of four CMV sequence configurations, allowing all isolates to be assigned a gB genotype of 1-4.Objective To establish the convenient and specific molecular method for CMV surveillance in recipients of Allo-HSCT and analyze the correlation between CMVgB genotypes and transplant-related complications through restricted endoenzyme digestion.Patients A total of 79 patients undergoing Allo-HSCT in our hospital were included in this study from Apri.2001~Apri.2003. In these patients:27 cases with acute myeloid leukemia ,15 with acute lymphoblastic leukemia, 17 with chronic myeloid leukemia and 20 cases with other hematological diseases. Of these, 49 patients underwent Allo-PBSCT(including 1 with selected CD34+ cell transplantation(Allo-PBCD34+T), 10 with NST, 4 with HLA mismatched transplantation). 17 underwent Allo-BMT, 9 underwent Allo-PBSCT pluse BMT, 4 underwent CBSCT. The median age is 32(range from 11 to 60) and the ratio of male and female was 1.63:1.Methods Nested PCR was carried out using primers 1292, 1676 and 1613, which amplify the region in the coding sequence of the CMV gB DNA described as Beverly Torok-Storb et al, 1997. Then, the PCR product was simultaneous digested with Ras I and Hinf I in two separate reactions. The digested DNA was resolved into informative banding patterns on 10% polyacrylamid gels. Such methods have never been reported domestically before.Results Among the 135 specimens, close correlation between the result of CMV-pp65 immunohistochemistry assay and PCR array has been evidenced(Table 1).Of these, 42 (53.2%)cases[66 specimen(48.9%)]were positive. The gB genotype from 42 cases(45 samples) were identified as gB 1,21(46.7%); gB2,14(31.1%); gB3,7(15.6%); gB4,3(6.7%), respectively, by restriction enzyme analysis. Of these, three patients were simultaneously or successively infected with CMV gBl and gB2. The incidence of GVHD hi groups of CMV(+) and CMV(-) was 80.9% vs. 32.4%,respectively(P<0.01). Incidence of aGVHD H-IV /cGVHD in different CMVgB types is as follows: gB1, 81.0%; gB2,50.0%; gB3,42.9% as well as gB4,0; respectively.(Table 2-4).Table 1. The result of CMV-pp65 immunohistochemistry assay and PCR CMVgBTable 2. Correlation between CMV infection and GVHDTable 3. Correlation between CMV gB genotypes and GVHDTable 4. Correlation between CMV gB genotype and GVHD degree.Conclusion The results indicated:l.Nested-PCR for CMVgB gene detection is a sensitive and specific molecular analysis method. 2. CMV infection is closely correlated with GVHD after Allo-HSCT, CMVgB 1 and gB 2 are the main types in our hospital and particularly correlated with II° - IV°aGVHD or cGVHD( PO.05 ). 3. Some evidence shows that myelosuppression after Allo-HSCT maybe correlated with CMVgB3 infection,but no statistics significance has been tested. 4. All the samples from biopsy colon tissues have proved CMV negative by this method. 5. Sequencing analysis of CMV gB genotypse have shown some genetic variations in gB types 1 and 3 compared with GeneBank standard sequences (M60931). It perhaps has some epidemiological significance and may influence viral tropism or pathogenicity. |
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