| ObjectiveCytomegalovirus (CMV) is one of the common source of opportunistic infections after hematopoietic stem cell transplantation (HSCT). The mortality of CMV disease is over 80% early in HSCT. Prevention and early diagnosis were the key measures to reduce the occurrence of CMV disease. CMV infection and disease are influenced by the host genes, the virus genotypes and immune system. In this study, we explore the interaction between CMV infection and immune reconstruction,HLA and non-HLA genes polymorphisms,KIR genes and CMV gB genotypes,which would help us to optimize dornor selection and manage CMV infection.Methods[Section 1] CMV gB genotyping was determined with Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and Clone Sequencing in 115 patients with CMV infection. The viral load were detected by Real-time Quantitative PCR.[Section 2] PCR with sequence specific olignuleotide probe ( PCR /SSO) were used for low resolution HLA genotyping in 433 pairs of donors and recipients before HSCT. HLA-A,B,C DR,DQ polymorphism and HLA-E genotype were analysed in CMV positive and negative patients.[Section 3] The KIR genotype was determined by Sequence-specific primer polymerase chain reaction (PCR-SSP) in 138 pairs of donors and recipients before HSCT. Posttransplant monitoring for CMV infection was performed by indirect immune histochemically assays.[Section 4] Non-HLA gene (ACE,CD14,MPO,MBL) single nucleotide polymorphism were determined by sequence-specific primer polymerase chain reaction (PCR-SSP) and sequencing in 64 pairs of donors and recipients before HSCT and the difference of these genes SNPs were analysed in CMV positive and negative patients.[Section 5] A total of 92 consecutive patients receiving HSCT through 2008 to 2010 were analyzed. Posttransplant monitoring for CMV infection was performed by indirect immune histochemically assays. T cell subsets immune reconstitution, NK cell receptors expression and SOCS1,SOCS3 transcription were detected by flow cytometry (FCM ) or Real-time quantitative PCR at 30d,60d,90d after HSCT in CMV positive and negative patients.Results[Section 1] The distribution of CMV gB genotypes in HSCT recipients were as following: gB1, 42/115 (36.52%); gB2, 3/115 (2.61%); gB3, 43/115(37.39%); gB4, 2/115 (1.74%). There were 20 patients (17.39%) who had a combination of 2 different CMV genotypes.There were 5 patients (4.35%) with a CMV genotype that lacked an RsaI digestion site, herein named gB5. The median viral loads of gB1 were 2.7×103, gB3 were 4.0×103 and mixed-gB were 1.2×104. There was no difference of viral loads between gB1 and gB3 group(P>0.050). The viral loads were higher in mixed-gB group than in single-gB group (P<0.050). the median therapy time was 17 days in mixed-gB patients and 14 days in single-gB patients. There was statistically significant difference between two group. With multivariate analysis, gB3 was associated with high risk for CMV pneumonitis.[Section 2] There are 10 gene allele of HLA-A, 23 gene allele of HLA-B, 13 gene allele of HLA-DR, 8 gene allele of HLA-C and 5 gene allel of HLA-DQ. The frequency of HLA-Bw4 was 46.40% and HLA-Bw6 was 53.60%. The frequency of HLA-C1 was 81.22% and HLA-C2 was 18.78%. The allele frequency of HLA-A24 and HLA-DR15 were increased and HLA-Cw6 was decreased in CMV positive group following HLA-matched HSCT. The CMV infection rate was higher in HLA-mismatched group than in HLA-matched group(P<0.050). The allele frequency of HLA-A33,HLA-B46,HLA-B58,HLA-DR3,HLA-Cw1 were increased and HLA-Cw6 was decreased in CMV disease group. CMV infection rate was higher in HLA-E*0101/0101 group than in other HLA-E*0103 group (P<0.050).[Section 3] There was no difference in KIR gene allele and haplotype between donors and recipients. The allele frequency of 2DS2 and 2DS4*003-007 of donors were decreased in CMV positive group. The CMV infection rate were higher in patients received KIR haplotype A/A donor than in the others received KIR haplotype B/B donor (P<0.050). With multivariate analysis, 2DS4*003-007 and KIR haplotype B/B of donor reduce CMV infection adjusting for confounding variables.[Section 4] The distribution of ACE gene single nucleotide polymorphism were DD 14/128(10.94%), ID72/128(56.25%), II 42/128(38.81%). The distribution of CD14 -159 allele gene single nucleotide polymorphism were CC 18/128(14.06%), CT 81/128 (63.28%),TT 29/128 (22.66%). The distribution of MPO- 463 allele gene single nucleotide polymorphism were G 100/128( 78.13%), A 2/128(1.56%), GA 26/128 (20.31%). The distribution of MBL gene single nucleotide polymorphism were H 28/128 (21.88%), HL 73/128 (57.03%), L 27/128 (21.09%), Y 87/128(67.97%), YX 38/128(29.69%), X 3/128 (2.34%) , A 94/128(73.44%), AB 32/128(25.00%), B 2/128(1.56%). The allele frequency of ACE, CD14, MPO were no differcence between CMV positive and negative patients. The gene frequency of MBL-HL was increased in CMV positive group .[Section 5] The hematopoetic reconstitution were observed in all 92 patients.The median time of granulocyte count exceeding 0.5×10E9/L was 13d (9~22d) and the median time of platelet count exceeding 20×10E9/L was 23d (11~45d). There were 40 patients with CMVpp65 postive within 90 d after HSCT and the median time of CMVpp65 postive was 54d (28~84d). Compared with CMV negative group,CD3+ cell and CD3+CD4+ cell percentage were lower in CMV positive group at 30d (P<0.05). CD3-CD16+CD56+ cell percentage was higher in CMV positive group at 60d (P<0.050). the expression of CD3-HLA-DR+ was lower in CMV positive group at 90d after HSCT (P<0.050). The expression of NKG2C were higher in CMV positive group at 60d and 90d after HSCT. Moreover, SOCS1 and SOCS3 transcripts were higher in patients after HSCT than that in normal control ,while SOCS3 transcript was higher in CMV positive group than in CMV negative group.Conclusion1) CMVgB genetypes were associated with viral load, treatment time and CMV disease. CMV gB3 infection was a risky indicator for CMV pneumoitis.2) HLA mismatch was CMV infection risk factors. In HLA-matched transplantation, HLA-A24, HLA-DR15, HLA-Cw6 and HLA–E*0101 were associated with CMV infection .3) Active KIRs genes were associated with CMV infection after HSCT and 2DS4*003-007, haplotype BB of donor were were in favor to reducing CMV infection.4) MBL gene single nucleotide polymorphisms were associated with CMV infection after HSCT.5) Delayed T lymphoctyes reconstruction, high proportion of NK cells, NKG2C expression and high level of SOCS3 transcript were associated with CMV infection after HSCT.In summary: CMV disease after HSCT was associated with CMVgB genetypes. Analysis of donor-recipient HLA genotype, donor KIR genotyping and MBL single nucleotide polymorphism before HSCT and monitoring of T cell reconstitution, NK cell reconstitution, NKG2C and SOCS3 expression after HSCT contribute to CMV infection prevention and treatment. |
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