儿童巨细胞病毒感染与甘露聚糖结合凝集素表达相关性研究

The expression of mannan-binding lectin in children with human cytomegalovirus infection

目的探讨甘露聚糖结合凝集素(MBL)基因多态性及血浆蛋白水平与儿童巨细胞病毒(HCMV)感染的相关性。方法收集2007年3～11月在浙江大学医学院附属儿童医院诊断为HCMV感染的患儿作为HCMV感染组,选择同期行健康体检的儿童作为对照组。对两组行MBL基因检测和分型,并对HCMV感染组进行随访,分别测定其急性期和恢复期血浆MBL蛋白水平。分别比较两组基因变异频率和血浆MBL蛋白水平。结果HCMV感染组纳入104例,对照组纳入105例;HCMV感染组有50例患儿进行随访,HCMV感染组MBL基因启动子区-550位点的L型变异频率明显高于对照组(56.7%vs34.3%,P=0.001),野生单体基因型HYPA的频率明显低于对照组(47.6%vs62.8%,P=0.002),完整基因型中高水平表达基因型(YA/YA)的频率低于对照组(41.3%vs60.0%,P=0.007),而低水平表达基因型(YA/XA,YA/YB,XA/XA)的频率高于对照组(52.9%vs29.5%,P=0.001)。HCMV感染组急性期和恢复期血浆MBL蛋白水平均低于对照组(P=0.019和0.000)。HCMV感染组急性期血浆MBL蛋白水平高于恢复期(P=0.000)。结论MBL基因多态性导致的血浆MBL蛋白水平低下与儿童HCMV感染相关,提示MBL可能对儿童HCMV感染具有保护作用。

Objective Human cytomegalovirus （HCMV） is a main pathogen responsible for a variety of congenital defects and pediatric infections. Mannan-binding lectin （MBL） is a serum lectin that mediates phagocytosis and activates complement. Its deficiency has been associated with increased susceptibility to infectious diseases, mainly in childhood. The aim of this study was to explore the relationship between genetic polymorphism and protein level of MBL and HCMV infection in children. Methods 104 children diagnosed as HCMV infection （64 males and 40 females ranging from one to twenty-four months old with a mean age of 4.29 months）, were investigated. All patients were seropositive for HCMV-IgM antibody tested by ELISA, and with HCMV DNA varied from 4 x 103 copies/ml to 7 ×10^6 copies/ml detected by quantitative-PCR in urine. All patients had clinical symptoms and single or multiple organ damages （ including hepatitis, pneumonitis, colitis, thrombocytopenia and involvement of central nervous system）. 50 patients were followed-up until they were recovery （seronegative for HCMV-IgM antibody）. 105 healthy, symptom- free children from the same area were assessed as control. Genomic DNA of total 209 cases including both patients and controls were extracted from blood according to standard phenol-chloroform procedure. Six SNPs in the MBL2 gene （ -550G/C, -221C/G and ＋4C/T of the promoter, CGT52TGT, GGC54GAC and GGA57GAA of the exon 1 ） were analyzed by a sequencing-based typing method. A DNA fragment including promoter region and exon 1 （953 bp） was obtained by polymerase chain reaction （PCR） amplification using the sense primer 5＇ -CCT GCC AGA AAG TAG AGA GG-3＇ and anti-sense primer 5＇-CCA GGC AGT TFC CTC TGG AAG G-3＇. PCR products were observed by agarose gel electrophoresis and subjected to DNA sequencing. The concentrations of plasma MBL were measured in 105 healthy controls and 50 HCMV-infectious patients at both acute and convalescent phases by a sandwich enzyme immunoassay with a human MBL ELISA kit. Results Mutations at the ＋ 4 site of the promoter and the codon 52 or codon 57 of the exon 1 were not found in this study. There was no significant difference between the frequency of type B mutation at the codon 54 of the exon 1 in HCMV-infectious patients and that of the healthy controls. Compared to the controls, the frequency of L variant at the promoter -550 site was significantly higher in patients （56.7% vs 34.3%, P =0. 001 ）, while the frequency of wild haplotype HYPA was lower （47.6% vs 62.8% , P=0. 002）. The frequency of complete genotype of high expression group （YA/YA） was lower （41.3% vs 60.0%, P =0. 007）, and that of low expression group （YA/XA, YA/YB, XA/XA） was higher （52.9% vs 29.5%, P = 0. 001 ） in patients compared with the controls. The MBL protein levels of patients in both acute and convalescent phases significantly decreased when comparing to the controls （P = 0. 019 and P = 0. 000, respectively ）. Conclusions Low MBL protein level as a result of genetic polymorphism seems to be correlative with HCMV infection in children. It can be speculated that as an acute response protein and a pattern-recognition molecule of the innate immune system, MBL may play a role in protecting children from HCMV infection.