败血病患儿血浆甘露糖结合凝集素表达水平的变化及其基因分型

Mannose-binding lectin plasma concentration variation and gene polymorphism in children with septicemia

目的了解败血病患儿血浆甘露糖结合凝集素（MBL）表达水平的变化及其基因启动子和外显子Ⅰ区的多态性。方法用ELISA法检测血浆MBL浓度，用序列特异性引物（SSP）PCR法和序列分析法对MBL基因进行分型。统计分析采用非参数检验，遗传学分析采用SHEsis软件。结果31例败血病患儿急性期和恢复期血浆MBL浓度中位数分别为2978ng／mL和1254ng／mL，急性期明显高于恢复期（P〈0．01），且两者间呈线性相关（r=0．781）。PCR—SSP和序列分析显示，启动子-550位点H／H、H／L和L／L型分别占45．2％、29．0％和25．8％，等位基因突变频率为0．290，各型等位基因分布不符合Hardy-Weinberg平衡（Χ^2=4．90，P〈0．05）；-221位点Y／Y和X／Y型分别占83．9％和16．1％，无X／X型，等位基因突变频率为0．098；各型等位基因分布符合Hardy-Weinberg平衡（Χ^2=0．008，P〉0．05）。外显子Ⅰ A型、A／B型和B／B型分别占71．0％、25．8％和3．2％，＋54密码子等位基因突变频率为0．161，各型外显子的分布符合Hardy-Weinberg平衡（Χ^2=0．066，P〉0．05）。所有样本5’端非转录区＋4位点均为P型，无C或D型外显子发现。A／A型外显子急性期和恢复期MBL浓度中位数分别为3782ng／mL和1436ng／mL，显著高于A／B型外显子的565ng／mL（P〈0．01）和233ng／mL（P〈0．01）以及B／B型外显子的16ng／mL和9ng／mL，但A／A型急性期MBL浓度的增加倍数为1．76±1．10，其他型为1．11±0．82，差异无统计学意义（t=1．577，P=0．126）。结论败血病患儿急性期血浆MBL浓度显著高于恢复期，且两者呈线性相关，血浆MBL浓度高低与MBL结构基因关系最密切。

Objective To investigate the mannose-binding lectin（MBL） plasma concentration in both acute and convalescent stages in children with septicemia and to study the polymorphism in promoter and exon 1 region in MBL gene. Methods MBL plasma concentrations were measured using enzyme-linked immunosorbent assay（ELISA） method with human MBL ELISA kit, while the MBL gene polymorphisms were determined with both sequence-specific primer polymerase chain reaction （PCR） method and sequence analysis method by using BigDye Mix 3730 genetic analyzer. Genetic analysis software, SHEsis, and nonparametric test, t test and Χ^2 test with SPSS 11.0 software were used. Results The MBL plasma concentrations in acute stage（median value= 2 978 ng/mL） were significantly higher than that（median value= 1 254 ng/mL） in convalescent period（P〈0.01） in all 31 septicemic children（r= 0. 781 ）. The results of PCR-SSP and sequence analysis showed that the variant allele frequency of the codon 54 of MBL gene was 0. 161, 71.0% of 31 cases were A/A exon type, while 25.8%o were A/B type and 3.2% were B/B type instead. Neither C nor D exon type was found. The frequency of point mutation in the promoter region at -550 （H/L variants） was 0. 290 and at 221（X/Y variants） position was 0. 098, the percentage of H/H type, H/L type and L/L type at -550 position was 45.2%, 29.0% and 25.8% respectively, while that of Y/Y type, X/Y type and X/X type was 83. 9%, 16. 1% and 0 instead. No polymorphism was found in the 5＇ untranslated region at ＋4（P/Q variants） position. The allele distribution for -221 site and ＋230 site in MBL gene of 31 children was found to be consistent with Hardy-Weinberg equilibrium. Analyzing the correlation between MBL structural genotype and plasma concentration, found that the structural variants had a dominant effect on the concentration of MBL. The A/A wild type was associated with the highest plasma concentration with median values as high as 3 782 ng/mL in acute stage and 1 436 ng/mL in convalescent period, the A/B type was associated with lower MBL levels（median values 565 ng/mL vs 233 ng/mL） and the B/B type with the lowest levels, the difference was significant between A/A and non A/A type（P〈0.01） in both acute stage and convalescent period. No significant difference was found about the increased levels of MBL in acute stage between A/A and non A/A types （t = 1. 577,P = 0. 126）. Conclusions The MBL plasma concentration is significantly higher in acute stage compared with that in convalescent period in children with septicemia, and the structural variants have a dominant effect on the plasma concentration of MBL.