Detection Of D120G Mutation In MASP2 By HRM Assay And Preliminary Study On Significance Of MASP2 In Children With URTI

Human health and quality of life are endangered by infection diseases, such as upper respiratory tract infection (URTI) and hepatitis B. URTI, which mostly occurs in winter and spring, is the most common pediatric disease. URTI accounts for the largest proportion in patients in pediatric outpatient services and wards. Hepatitis B is an epidemic infectious disease in our country. About 0.3 million people died of severe hepatitis, liver cirrhosis, liver cancer or other liver diseases caused by HBV infection each year. The pathogen susceptibility depends on the body’s line resistance, and innate immunity is the first-line of resistance to defense infection.Complement system, which played an important role in microbial resistance to variety pathogenic microorganisms, mediated immune defense functions such as release of inflammatory mediators, recruitment of inflammatory cells, destruction of pathogens, elimination of immune complex deposition and apoptotic cells, is an important component of the innate immune defense. The lectin pathway, as the third pathway of complement activation, is an important component of the innate immunity system. In lectin pathway, mannan-binding lectin(MBL) and ficolins circulate in complexes with MBL-associated serine protease 1/2, MASP1/MASP2 (MASP1/MASP2) homologs of Clr and Cls. The former recognised and combined with the mannose, N-acetyl glucose and other carbohydrate structures which on the surface of pathogenic microorganism through the carbohydrate-recognition domain(CRD), thus the latter was activated, the activated MASP2 cleaves complement factors C4 and C2 to form the C3 convertase C4b2a, thus initiating the activation of the complement system.MASP2 is composed of 686 amino acids with a total length of 20717 bp, and it contains of 12 exons and 12 introns. It is coding genes in the chromosome 1p.36.22. The single peptide chain of MASP2 is composed of six modules and a linker region: an N-terminal complement subcomponent Clr/Cls-like domain(CUB1), followed by an epidermal growth factor(EGF)-like domain of the Ca2+-binding type, a second CUB domain(CUB2), two contiguous complement control protein modules(CCP1 and CCP2), a short linker and finally a chymotrypsin-like serine protease(SP) domain. The present study indicates that the three domains of MASP2 interacted with MBL-CLR via the N-terminal three modules (CUB1-EGF-CUB2) depending on the Ca2+, and the other three domains of MASP2 C-terminal is serine protease activity center, complement activation mainly relys on the SP domain to play the role of serine protease, cleave both complement factors C4 and C2, hence forming the C3 convertase. Therefore, MASP2 plays a crucial role in the lectin pathway because both MBL and other ficolins(H, M and L) need it to activate complement. MASP2 deficiency was first described in a patient with multiple infections and autoimmune manifestations due to an exon 3 mutation. Thus the isoelectric point changed because of the exchange of acidic aspartic acid(Asp, D) with neutral glycine(Gly, G) at position 120 (p.D120G), which leads to significant low levels of serum MASP2. Moreover, the mutant MASP2 could not associate with MBL and thus could not form the active mannan-binding lectin-MASPs complexes, so as to cause or aggravate pathogen infection.Thus, MASP2 deficiency may be one of the reasons of recurrent infectious with a variety of pathogenic microorganisms. Rapid genotyping MASP2 using high-resolution melting assay(HRM) might predict the risk of infectious disease. At present, plasma MASP2 concentration about relationship with URTI have not been reported, therefore, MASP2 in children with URTI has been preliminarily studied which has a significant influence.Part 1 Genotyping of MASP2 at position 120 (p.D120G) by high-resolution melting assayHRM analytical technologies has the advantages of low-cost, high throughput, no need to design expensive probe and immediate conductment of a high-resolution melting analysis after polymerase chain reaction(PCR) with all reaction in a closed tube to reduce pollution. PCR products after HRM analysis can be used for DNA sequencing as well. This technology has been adopted in clinical chemistry, food security, and human pathology considering the advantages listed. In this study, we have developed a HRM assay which is capable of detecting the mutation in MASP2 at position 120 (D120G).Samples and Methods:1. Samples:153 patients were recruited and peripheral blood samples were obtained after informed consent to validate the accuracy of the HRM assay. Genomic DNA was extracted from 1 ml EDTA-anticoagulated peripheral blood by the phenol/chloroform method.2. Molecular analysis:A PCR composed of separate amplicons was designed to generate sequences containing the polymorphic site of interest in the MASP2 genes. Design primes of MASP2 genes at position 120 (D120G) by HRM assay. Homozygous mutant type of MASP2 genes is obtained by PCR mutagenesis, homozygous wild type of MASP2 genes was fabricated and conserved in our laboratory. Mutant heterozygote type of MASP2 genes was gained by mixure of homozygous mutant type of MASP2 genes and homozygous wild type of MASP2 genes in equal volume and equal concentration. Different kinds of genotype samples of MASP2 were used as standards to establish HRM assay conditions. Wild type samples of MASP2 can be got from DNA sequencing and used to optimize HRM assay conditions.153 samples were randomly selected for direct DNA sequencing validation, and different genotype MASP2 plasmid were used in within-run and between-run experiments ten times for reproducibility and stability assessment. In addition, ten-fold serial dilutions of mutation MASP2 plasmid were used to assess the detection limit. At the same time, mixing homozygous mutant type of MASP2 genes and homozygous wild type of MASP2 genes in different proportions to obtained mutant heterozygote type of MASP2 genes in different concentration gradient which were used to assess the detection limit of proportation mutant heterozygote type of MASP2 genes.3. Statistical analysis:To validate the accuracy of the method, a number of samples from each of the genotype groups detected by HRM assay were selected to perform DNA sequencing. To validate the stability of the method, different MASP2 genotypes were analyzed in within-run and between-run experiment ten times. Statistical analysis was conducted with SPSS 16.0 software.4. Comprehensive analysis of the experimental results.Results:After adjustment and optimization of the PCR system, the MASP2 genes were efficiently amplified and easily genotyped by HRM analysis. A total of 153 genomic DNA samples were tested to validate this assay. The precision of HRM genotyping was assessed by comparing the retention times and calculating the coefficients of variation (CV). Statistical analysis indicated good reproducibility and stability with a CV of melting temperatures less than 0.1%. To assess robustness, MASP2 homozygous mutation plasmid were ten-fold serially diluted to 10 ng,1 ng, 100 pg,10 pg,1 pg, and analyzed. All concentrations could be detected.Discussion:In this study, the method we present is proved to be high-throughput, high reproducibility and reliable which are suitable for rapid genotyping of MASP’2 gene. We only find one heterozygote mutation type of MASP2 genes in 153 sample. Hence, we predict the mutation genotype of MASP2 gene at position 120 (D120G) in southern China is very rare. This study established a simple, low-cost, high-throughput, rapid, and highly sensitive method for genotyping MASP2 gene, which provides an alternative method in genetic counseling.Part 2 Significance of mannan binding lectin-associated serine proteaes 2 in children with upper respiratory tract infectionAs reported, MASP2 is in tight relation with many diseases. But we don’t know its relationship with URTI clearly. To explore the the significance of serum levels of mannan binding lectin-associated serine protease 2 (MASP2) in children with upper respiratory tract infection (URTI).Method:103 children with URTI and 35 healthy children were respectively selected as subjects investigated. Plasma levels of MASP2 and C reactive protein (CRP) were detected and the routine blood test was performed in all the children. According to the clinical parameter of CRP and white blood cell (WBC) and the different stage of infection with or without treatment, the children with URTI were respectively divided into the elevated CRP group (n=48) and the normal CRP group (n=54), the elevated WBC group (n=61) and the normal WBC group (n=40), the early stage of infection without treatment group (n=68) and the mid-late stage of infection with treatment group (n=35). All data were statistically analysized by SPSS 16.0 software. Application of bioinformatics analysis whether there is a common stress components in MASP2 gene fragments, acute phase protein the MBL gene fragments, atypical acute phase reaction protein CRP gene fragment.Results:The plasma MASP2 concentration in the URTI group was significantly higher than that in the healthy control group (P<0.001) and strikingly correlated with age (r=0.302, P＜0.01). The plasma MASP2 concentration in the elevated CRP group was correlated to the CRP value (r=0.310, P<0.05), but not in the normal CRP group (P>0.05); The plasma MASP2 level in the elevated WBC group was significantly correlated to the WBC value (r=0.392, P＜0.01), but not in the normal WBC group (P>0.05); The plasma MASP2 concentration in the early stage infection without treatment group was significantly higher than that in the mid-late stage of infection with treatment (P＜0.01); We find that MASP2 gene, acute phase protein the MBL gene, atypical acute phase reaction protein CRP gene have an common stress components, hepatic nuclear factors-4a motif domains.Conclusion:MASP2 may be an acute-phase protein, and the plasma MASP2 level might be deserved as a new reference index in diagnosis of URTI in children.