Study On Codon-54 Mutation Of The MBL Gene In Population Of Hans And Uigurs In China

Mannan-binding lectin(MBL) is an important innate immune defence molecule of the collectin family in the superfamily of C-type lectins. By recognizing and binding to certain carbohydrate moieties on various pathogens,it can promote the killing of microbes either by acting directly as an opsonin or by activating the lectin complement pathway. However,the function of MBL depends on a certain of levels of serum MBL. MBL deficiency and low levels of serum MBL are the basis for a common opsonic deficiency and are associated with recurrent infections and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis.Now,it has been shown that MBL deficiency and low levels of serum MBL are strongly associated with the presence of three variant alleles B,C,D in MBL structural gene,which codons 54,57 and 52 amino acid of MBL polypeptide respectively. The normal allele is known as A. Each variant is the result of a single point mutation in exon 1 that disrupts the collagen-like structure of the MBL polypeptide. This leads to a reduction of functional MBL in individuals. It was surprised that the frequencies of mutation of the exon 1 of MBL allele reached to 0.29 in all and were much more different in population of different races. In Asian,the low serum concentrations were primarily due to an extremely high allele frequency of the codon 54(B) variant.China is a populous country with many nationalities,it is necessary to study the genotypes and frequencies of alleles of the exon 1 of MBL gene in Chinese so as to provide decision-making evidence for prevention and treatment of MBL deficiency. Therefore,the genotypes and frequencies ofthe codon 54 of the MBL structural gene of 2 Chinese nationalities(Hans andUigurs) have been studied.Objectives:1. To establish a simple,inexpensive,rapid technology to detect point mutation or SNP of MBL gene with high sensitivity and specificity,which should be of utility in the most of medical laboratories in our country.2. To establish the frequency of codon 54 mutant allele and compare the difference of genotypes of MBL structural gene in the population of Hans and Uigurs.Methods:1. Blood samples of the population of Hans and Uigurs were collected,genome DNA extracted,and basis PCR conditions of amplifying target sequence established.2. By using single strand conformation polymorphism analysis of polymerase chain reaction (PCR-SSCP),the frequency of codon 54 mutant allele of MBL structural gene in the population of Hans was investigated.3. The molecular beacon (MB) hybridization technique with visual monitoring was established. By means of MB,the codon 54 mutation of MBL structural gene in the population of Hans and Uigurs was detected. RESULTS:1. Genome DNA of all blood samples were extracted by using erythrocyte lysis solution and PCR buffer/nonionic detergent/proteinase K. A 109bp fragment was amplified from the human genome DNA.2. In 166 Han samples,19(11.45%) heterozygous genotypes of codon 54 mutant allele were found by PCR-SSCP. No homozygote of mutant allele was detected.3. The technique of molecular beacon hybridization with visual monitoring was established successfully. In 138 Han samples,homozygous and heterozygous genotypes of codon 54 allele in MBL structural gene were 2.17% and 21.01% respectively,while in 120 Uigurs samples,those were 0.83% and 18.34%.CONCLUSIONS:1. With strong specificity and easy to manipulation,this simple,inexpensive,rapid molecular beacon hybridization technique permits visual monitoring of gene point mutation and SNP,which shows better advantage than PCR-SSCP.2. The frequency of codon 54 mutant allele in exon 1 of MBL structural gene is 0.13 in the population of Hans,while that is 0.10 in Uigurs. There are no obvious difference for the frequencies and genotypes distribution for the MBL gene codon 54 mutant allele between Hans and Uigurs.