Association Of Mannose-binding Lectin Gene Polymorphism And Its Plasma Level With Neonatal Infections

Infection is one of the most common causes of newborn diseases, especially inpremature and low birth weight infants, and even leads to dead, which bring theeconomic and social problems. There have been reported that neonatal infectionrelates with genetic and congenital immune system abnormalities, and easily occurredin child with innate immunity deficiency. Recent studies showed that mannosebinding lectin (mannose binding lectin, MBL) deficiency is the most common causeof immunodeficiency, and may be the major cause of neonatal infection in our country.MBL is one of the important molecules of the innate immune system. MBL combinesthe sugar chain of pathogenic microorganism’s surface, activates MBL-associatedserine protease by collagen like region, and then activates the complement system. SoMBL Plays an important role in preventing infants, whose adaptive immune systemsare premature, from infection, especially in premature. The plasma MBLconcentration is mainly affected by its gene polymorphism. The point mutation ofMBL structure gene exon1on52、54and57codon that have affects on proteinfunction. These point mutations lead to the destruction of collagen like regionstructure of MBL which could not be oligometric and polypeptide chains easily bedegraded in the plasma, and eventually lead to become nonfunctional proteins. TheSNP(single nucleotide polymorphisms, SNPs) of promoter region of MBL geneinfluences its plasma concentration on transcriptional level. It is sure that there havethree polymorphism sites: H/L, X/Y, P/Q, locates in-550,-221and5’UTR+4respectively. Previous researches all focused on these six loci polymorphism ofpromoter region and exon1of MBL gene.In this experiment, the SNP of MBL gene was analyzed. The relationship amongthe SNP of MBL gene, MBL plasma levels and neonatal infections were discussed. Objective1. Study on the6SNP sites which include promoter region (-550,-221and5’UTR+4loci) and exon1regions (+223,+230and+239loci), and haplotypes which becomposed of multiple SNP sites of MBL on Fujian Han newborns (including term andpreterm infants). Analyze the difference of these6key sites and haplotypes of MBLgene polymorphism between the term and the preterm groups. The aim of the presentstudy is to evaluate the frequency of a common SNP of MBL gene.2. Analyze polymorphism of MBL gene and plasma concentration of MBL, and to explore therelationship between gene polymorphisms and neonatal infections such as pneumonia and sepsis3. Try to find new sites of MBL gene polymorphism in Fujian Han newborns.Methods1. The subject of study were inpatients of Neonatal intensive care unit of Fujianmaternal child health hospital from2013January to June, including82preterminfants,100term infants. The95cases of infection group included60cases ofpneumonia and35cases of sepsis. The control group was with87cases. DNA wasextracted from blood samples and the products of PCR were purified and thenanalyzed sequences using dideoxy chain termination sequencing.2. MBL concentrations were detected using ELISA method, and the clinical andlaboratory data were collected.3. The level of MBL between the two groups was compared by Wilcoxonnonparametric test method. Kruskal-Wallis nonparametric test method was applied inthe comparison of multiple groups. The comparison of the allele frequency, genotypicfrequency and the ratio of constituent used χ2Test, and the test level was set to0.05Results1. In these cases of Han children, genotypic frequencies in-550loci of promoter were0.236for G/G,0.489for G/C and0.275for C/C. Allele frequencies were0.481and0.519for G and C respectively. Genotypic frequencies in-221loci were0.055forC/C,0.308for C/G and0.637for G/G. Allele frequencies in-221loci were0.209and0.791for C and G respectively. Genotypic frequencies in5’UTR+4loci were0.764 for C/C,0.214for C/T and0.022for T/T. Allele frequencies were0.87and0.13for Cand T respectively. By χ2Test, Allele frequencies of3sites in the promoter regionaccorded with the Hardy-Weinberg equilibrium. Genotypic frequencies in+230lociwere0.731for G/G,0.247for G/A and0.022for A/A. Allele frequencies in+230siteswere0.854and0.146for G and A respectively, and the results accorded with theHardy-Weinberg equilibrium by χ2test.2. The analysis results of-550,-221and+230site by SHEsis software showed thatthere existed high linkage disequilibrium. In this study, there are6haplotypes of thepromoter and exon1: HYPA, LYPB, LYPA, LXPB, LXPA and LYQA. HYPA is themost common, and allele frequency was0.463, the others were0.137,0.062,0.009,0.2and0.111respectively. Besides LYQA, the others were consistent with the Hanchildren in Zhejiang area, but the frequencies of HYPA and LYPA were differencebetween these two areas. LXPB and LYQA had not been reported in foreign data.3. Among three key sites in the promoter region, the variation frequency in-550locuswas no significant difference between full-term and premature group (χ2=2.04,P>0.05). Allele frequencies of C and G in-221locus in premature group were0.268and0.732respectively, while the control group was0.160and0.840respectively, sofrequency of variation between the two group was significant difference (χ2=5.9,P<0.05). There was no significant difference in allele frequencies of C and T of+4site between the preterm and term group (χ2=0.467, P>0.05). Only the+230locuspolymorphism change was found of three key sites in the exon1region, which allelefrequency of G and A showed no significant difference between term and preterminfants group (χ2=0.739, P>0.05). There were5haplotypes LXPA, LYPB, LYPA,LYQA and HYPA of6key sites of promoter and exon region, in which LXPA of thepremature group was significantly higher than that of full-term group, OR=1.794(95%CI1.06～3.017, P<0.05), and LYPB of the preterm group was significantlyhigher than that of preterm infants group, OR=0.533(95%CI0.28～0.99, P<0.05).The other three haplotypes had no significant difference between two groups.4. The deletion of6bases AGAGAA in the promoter region from-327to the-332sitewas found, in which including homozygous deletion in4cases. At the same time, polymorphism of-435G/A,-427A/C,-349A/G,-336A/G and-70C/T change werefound upstream and downstream of the site. There had strong linkage amongc.4665-4670delAGAGAA,-435G/A,-427A/C,-349A/G,-336A/G and-70C/T bylinkage disequilibrium test.5. The variation frequencies in-550and+4locus of promoter region of MBL genehad no significant difference between the infection group and the control. Thevariation frequency of-221locus in infection group was higher than in control group(0.722vs0.644, χ2=7.14, P<0.05). The variation frequency in+230site of infectiongroup was0.141, while the control group was0.061, the infection group was higherthan that of the control group (χ2=6.4, P<0.05). Comparison of MBL monomergenotipic frequency between infection group and control groups showed that LYPBand LYQA were higher than the control group (0.129vs0.021,0.152vs0.001,χ2=14.74、27.9, P<0.005), and the LYPA lower than the control group (0.072vs0.238,χ2=20.2, P<0.05), the others were no significant difference.6. Lower plasma concentration of MBL is one of the risk factors of neonatal infection.The median concentration of MBL was1560ng/ml in healthy group, which washigher than that in infection group(452ng/ml), the difference is significant (z=-4.566,P<0.05). At the same time, the infection group was separated into neonatal pneumoniagroup and sepsis group, the median concentration of MBL was445ng/ml and770ng/ml respectively, and there was no significant difference between these two diseasegroups.7. Research on the relationship between the genotype and MBL concentration. Geneexon is a key factor for the plasma levels of MBL. For different exon: A/A>A/B>B/B,the median plasma concentration of MBL was2409ng/ml,1069ng/ml and33.3ng/ml respectively, and there had significant difference (P<0.001). For the same exon,the plasma concentration of MBL was the highest in HYP/HYP type (3400ng/ml) andthe lowest in LYP/LYQ (310ng/ml).Conclusions1. Genotypic frequencies of-550,-221,+4and+230loci of MBL gene in Han children were0.519,0.791,0.13and0.146respectively. There were no polymorphicchanges in+223and+239loci.2. Han children in XX were with MBL gene promoter haplotype HYP, LYP, LXP andLYQ4, with monomer genotypes HYPA, LYPB, LYPA, LXPB, LXPA and LYQA, inwhich HYPA was the most common. The new monomer genotypes LYQA was found.3. The distribution frequencies of haplotypes were vary in term and preterm group,which LXPA in preterm group was significantly higher than that of full-term group,while LYPB of full-term group was significantly higher than that of preterm group.4. C.4665-4670delAGAGAA polymorphisms were detected. The polymorphism of-435G/A,-427A/C,-349A/G,-336A/G and-70C/T change were found and had stronglinkage among these loci.5．Relationship between the genotype of MBL and neonatal infection (neonatalpneumonia, sepsis): The variation frequency of-221and+230locus were higher ininfection group than in normal group. LYPB and LYQA in the infection group wassignificantly higher than that in normal group, but LYPA in the control group wassignificantly higher than that in infection group.6. Lower plasma concentration of MBL is one of the risk factors of neonatal infection.The concentration of MBL was significant difference between infection group andhealthy group, but no significant difference between these two disease groups(neonatal pneumonia and sepsis).7. Relationship between the genotype and MBL concentration: Exon is a key factorfor the plasma levels of MBL. To different exon: A/A>A/B>B/B, there had significantdifference. To the same exon, the plasma concentration of MBL was the highest inHYP/HYP type and the lowest in LYP/LYQ type.