Relationship Between Mannose-binding Lectin Gene Polymorphism And Infections In Children

Background and Objective: Mannose-binding lectin (MBL;also referred to as mannan-binding lectin or mannose-binding protein) is a calcium-dependent plasma coUagenous lectin and plays an important role in innate immune defence against infectious agents, especially in children whose adaptive immune system is not immature. MBL has various oligomeric structures ranging from dimers to hexamers which are based on subunits comprising three identical peptide chains (structural unit). Each chain is characterised by a carbohydrat-recognition domain( CRD) in carboxy-terminal region and a coUagenous region in N-terminal region, which are related closely to it's physiology function The CRD is able to make co-ordination bonds with the 3- and 4-hydroxyl groups of appropriate sugars decorating microbial surfaces such as N-acetyl-D-glucosamine, mannose and fucose. When MBL bind to pathogenic microbial surfaces , the complement system are activated, which are mediated by the MBL-associated serine proteases (MASP) binding covalently to the coUagenous region of MBL, this activation mechanisms of the complement system are called lectin pathywayor MBL pathyway. MBL has another functions , the promotion of complement-independent opsonophagocytosis and the modulation of inflammation besides the activation mechanisms of the complement system. Function of MBL are greatly related to MBL levels and mutimeric structure in circulation, low plasma concentrations cause a failure of the structural unit to oligomerize , which are fuction -defective and related to unexplained recurrent or prolonged infections in children. MBL is encoded by MBL gene (mbl2). Six single nucleotide polymorphisms (SNPs) sites have been described of the MBL gene, including D, B and C variants at +223, +230 and +239 sites in exon 1, while A indicating wild-type;H/L and X/Y loci at positions -550, -221 in promoter;P/Q loci at +4 site in 5' untranslated region. The 6 polymorphism sites are closely linked and comprised several haplotypes , some of which are associated with decreased level of MBL in circulation. The SNP of the 6 polymorphism sites and the frequencies of haplotypes are various in different nations, which cause very large variation in MBL plasma concentration , thus ,the susceptibility of disease is different from nation to nation. The respiratory tract infection , is the most common disease in children, and repeated respiratory tract infection (RRTI ) is one of the most common disease which cause children to be in hospital and affects the physical and mental health in children greatly. Up till now, no study on MBL gene polymorphism covering all six sites was published in Chinese population . Low MBL level may be a risk factor for RRTI, thus, investiation on the correlation between RRTI and MBL genetic deficiency are surely needed. The objective of the study is to analyze the SNPs and haplotypes of MBL gene, the correlation among haplotypes, MBL levels in circulation, and repeated infections , besides, to find new mutation in MBL gene.Part one SNP at-550,-221, +4, +223,+230 and+239 sites in promoter, 5'UT and exon 1 of MBL gene and haplotypes in Hans childrenObjective To study the frequencies of SNPs and haplotypes in Hans children in Zhejiang province, and compared the results with the analogous studies in different nations.Methods (1) Research population: Two hundred and six Hans children, including 105 healthy children and 101 infected children hospitalized in the children's hospital, were included in the study. The infection group consists of RRTI ( 57 cases), acute respiratory tract infection ( ARTI, 21 cases ) , localized abscess ( 13 cases ) and otitis media group ( 10 cases ). (2) Collection of blood samlpes and extraction of genomic DNA: About 1.5ml of blood samlpes from 206 children were collected into clean tubes with dry EDTA, and genomic DNA was extracted by QIAamp blood mini kit after plasma was aliquoted. (3) Analysis of SNPs and haplotypes of MBL gene: Both PCR-SSP and sequencing methods were used in determining the SNPs of the 6 polymorphism sites, and a further method, molecular clone was used when identified the haplotypes. Results (1) The sensitivity and specificity of PCR-SSP method in determining SNPs in promoter region of MBL gene: Defining sequencying as gold standard, the sensitivity were 90.9%, 94.4% and 95.0%, the specificity were 97.4%, 93.0% and 100.0% , respectively in detecting H/H, H/L,and L/L types with PCR-SSP method at position -550 , while the sensitivity were 84.8% and 100.0%, the specificity were 100.0% and 85.1% respectively in testing the Y/Y and Y/X types at position -221 loci instead. Low sensitivity and specificity were found in analysing the SNP at +4 site in 5'UTR. (2) In all 206 Hans children, 57.3%, 18.9% and 23.8% were H/H, H/L and L/L types at position -550 , the frequencies of Hwas 0.667and L was 0.333;About position -221, 85.4% were Y/Y type and 14.6% were Y/X type, the frequencies of Y and X type were 0.927and 0.073 respectively;The percentages of A/A, A/B and B/B exon 1 were70.9%^ 25.2% and 3.9%, respectively, the variants (B) frequency was 0.165, while the wild , A exon 1 was 0.835 instead. The allele distribution for -221 site and +230 site in MBLgene of 206 nans was in good agreement with Hardy-Weinberg equilibrium. No mutation was found at +4site in 5'UT, +223 and +239 site in exon 1 (3) High linkage disequilibrium were found between -550 site and -221, +230 sites.Three were only 3 promoter types, HYP, LYP and LXP be found in the study , with allele frequencies as 0.667, 0.260 and 0.073, respectively. Six haplotypes , HYPA, LYPA, LXPA, LYPB, LXPB and HYPB, were identified, and the HYPA haplotype was the most common ( frequency= 0.648), followed by LYPA(frequency=0.165);The new haplotypes, HYPB and LXPB identified in the study, were never been reported in references.Conclusion (1) The variant frequencies at-550, -221 and +230 sites of MBL gene in Zhejiang Hans children were 0.333 ^ 0.073 and 0.165 respectively;No mutation was found at 5'UT+4site, +223 (52 codon) site, and+239 site (57codon) in MBLgene in the study. (2) Six haplotypes of MBLgene, HYPA, LYPA, LXPA, HYPB, LYPB, and LXPB were found and the HYPAwas the most common haplotype;Two new haplotypes, HYPB and LXPB were identified in the study.Part Two Correlations among the polymorphisms of MBL gene, plasmaMBL levels and infections in children Objective To study the correlations among the polymorphisms of MBL gene,plasma MBL levels and infections (especially the repeated respiratory tract infection) in children.Methods (1) Gene frequencies were calculated by a simple gene-counting method, and yï¿¡ test was used with SPSS 11.0 (statistics package for social science) when compared the SNPs and allele frequencies between healthy and infection groups. (2) Plasma levels of MBL were determined by ELISA method with human MBL ELISA kit (has a minimum detection level of 0.41ng/ml) , the Netherlands, HyCult biotechnology b.v. (3) Comparisons about plasma levels of MBL among different groups were analyzed with nonparametric test with SPSS 11.0.Results (1) Compared with the healthy group, the infection group covering all disease groups, had more higher variant frequency at position -550 (0.391 vs 0.276, /=6.12, i><0.05) and no difference was found at position -221 and +230 (P>0.05). (2) Compared with the healthy group, the variant frequencies at -550 site (0.482 vs 0.276, /=5.37, P<0.05) and +230 site (0.254 vs 0.143, /=6.17, P<0.05) were significantly higher in RRTI group, while in the ARTI, the localized abscess or the tympanitis group, no significant variant frequency was found. No difference of mutation frequency was found between each infection group and healthy group. The haplotype of MBL gene, due to the SNPs of the 6 polymorphism sites , HYPA occurred more common in healthy group (frequency=0.695) than in RRTI group (frequency=0.509, /=11.02, P<0.005), while LYPB was more common in RRTI group (frequency=0.158) than in healthy group instead (frequency=0.076, /=5.25, i><0.05)0 (3) The median values of MBL plasma concentrations in healthy, RRTI, ARTI , localized abscess and otitis media group were 1065 ng/ml, 798ng/ml, 1477 ng/ml, 1707 ng/ml and 1779 ng/ml respectively .Compared with the healthy group, higher levels of MBL plasma concentrations was found in both the ARTI group (P=0.030) and the localized abscess group (P=0.021) , while no significant lower MBL level was found in ARTI group (P=0.052);Compared with the RRTI group, the ARTI group (P=0.011) , the localized abscess group (P=0.005) and the otitis media group (P=0.041) had higher MBL plasma levels instead. (4 ) The MBL plasma concentrations in acute stage (median value =2978 ng/ml) was significantly higher than that (median value =1254 ng/ml) in convalescent period (P^ 0.000) in all 31 septicemia children with linear correlation( r =0.781, P<0.01). (5) Analyzing the correlation between genotype and plasma concentration, we 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 1189 ng/ml , the A/B type was associated with lower MBL levels (median values was 744ng/ml) and the B/B type with the lowest levels ( median values was as low as 3ng/ml), the difference among A/A, A/B and B/B genotype was significant (P ^0.000). Compared the plasma MBL level between different promoter types with the same structural gene, we found that the HYP/HYP( wild type ) had a higher level of MBL (median=1579 ng/ml) and the promoter variants had lower MBL concentration (median: 818-1109 ng/ml) in A/A exon 1, and the same (median=981 ng/ml in wild type promoter and median= 264-875 ng/ml in promoter variants) in A/B exonl, too.Conclusion (1) The variants frequencies at -550 and +230(54 codon ) sites were significantly higher in RRTI group than those in healthy group, which cause the difference of HYPA and LYPA frequencies between the RRTI group and the healthy group. Therewas relation between MBL gene polymorphism . (2) Low plasma MBL concentration was a risk factor associated with RRTI. (3) The MBL plasma concentration was significant higher in acute stage than that in convalescent period with linear correlation in children with septicemia. (4) The levels of MBL due to genotype, and the structural variants had a dominant effect on the plasma concentration of MBL, A/A>A/B>B/B;HYP/HYP modulate higher MBL plasma concentration than promoter variants with the same exon 1 type.Part Three Molecular clone and sequencing analysis of promoter region and exon 1 in MBL geneObjective To identify the heterozygote variants performed with sequencing method and determine the haplotypes in variants with more than 2 heterozygote mutation site, and to find new mutation in MBL gene.Methods (1) After the selection of DNA template , the promoter and exon 1 of human MBL gene was amplified by PCR and purified with DNA gel extraction kit made by V-gene. (2) Molecular clone: The purified DNA fragment was inserted into vector plasmid pMD18-T, and then transformed the Escherichia coli TGI. The positive clones were selected on ampicillin plate and plasmid DNA was extracted. Transformants were screened by digesting the DNA with restriction endonucleases EcoRV. (3) Sequencying: The plasmid DNA was sequenced using a forward M13R(-48) and a reverse M13R(-47) primer in an DNA BigDye Mix 3730 sequencer. (4) Dnaman software was used in comparing the sequence in the study with the whole sequence of MBL gene in GeneBank.Results (1) The heterozygote mutation was identified with clone technique in those with double peaks at polymorphism site by sequencying PCR product. (2) Thehaplotypes of variants with more than 2 heterozygote mutations at polymorphism site were identified. (3) Four new mutations were found in promoter region of MBL gene: heterozygote or homozygote mutation at - 436 loci: G (gualine) was substituted by A (adenine) in 5 RRTI children and one in healthy children;heterozygote mutation at -548 site, adenine was substituted by gualine in a RRTI child (D28) with very low MBL level which did not accord with genotype;The remained two other mutation were as follows: adenine was substituted by gualine at -119 site in a RRTI child (L71);Gualine was substituted by adenine at -112 site in a RRTI child (L36).Conclusion (1) Heterozygote variants were identifed with clone technique in individual with double peaks at polymorphism site directly sequenced from PCR products. (2) Four new mutation was found at position-548, -436, -119 and -112 in promoter region of MBL gene, and further study are needed on the probable relationship between the new mutations and infection.