Investigation Of Single Nucleotide Polymorphisms, Haplotypes And Genotypes Of Mannan-binding Lectin Gene In Four Chinese Nationalities

Mannan-binding lectin (MBL) is one of members of the collectin family in the superfamily of C-type lectins and is considered as a key component of innate immunity. MBL recognizes mannose and N-acetylglucosamine moieties on a great of variety of pathogens, leading to the destruction of them and the cells infected by them by opsonization and phagocytosis and(or) activation of the complement system.MBL deficiency had been reported to be an important element in an ever increasing number of diseases, such as recurrent infections, autoimmune diseases, cystic fibrosis, etc. Six important single nucleotide polymorphisms (SNPs) of MBL gene strongly influenced the concentration of MBL in serum, which are located in the 5' regulatory region at positions -550, named alleles H/L, at positions -221, alleles X/Y, and at position 4, named alleles P/Q, and in the coding sequence of exon 1 at positions 223 (codon 52), 230 (codon 54), and 239 (codon 57), named alleles D, B and C respectively, wild-type named A. Since each of the three point mutations in exon 1 is in linkage disequilibrium with a different promoter haplotype, only seven common haplotypes have been described: HYPA, HYPD, LYPA, LYPB, LYQA, LYQC and LXPA in 64 possible haplotypes. Combination of various promoter genotypes regulating transcription of the MBL gene and the different structural gene variants are responsible for the large interracial and interindividual variations in MBL serum levels.China is a populous country with many nationalities. It is necessary to study the genetic polymorphisms of MBL gene in Chinese so as to provide decision-making evidence for prevention and treatment of MBL deficiency. Therefore, the main SNPs, haplotypes and genotypes of MBL gene in the populations of 4 minority nationalities (Wa, Bai, Yi and Hani) have been investigated in the present study. The investigation of SNPs of MBL gene is important to the researches and clinical practices, and may set up the foundation for the new trend of medicine—individual therapy.Chapter One Construction of Seven Standard Plasmids of CommonHaplotypes of MBL Gene Objective:To construct the standard plasmids of 7 common haplotypes of MBL gene. Methods:1. Using the DNA samples whose the haplotypes and genotypes of MBL gene had been clear as PCR templates, the fragments of haplotypes involved the promoter region and exon 1 of MBL gene were amplified by SSP-PCR and the standard plasmids of haplotypes were constructed by cloning the fragments into T vector.2. Using the standard plasmids of HYPA and LYQA haplotypes as templates, the imitative standard plasmids were constructed by site-directed mutagenesis to chang the bases located at codon 52 and codon 57 of exon 1 of MBL gene respectively. Results:The standard plasmids of haplotypes HYPA, LXPA, LYQA, LYPA, LYPB, HYPD and LYQC of MBL gene were obtained successfully. Conclusions:The standard plasmids of 7 common haplotypes of MBL gene will provide the standard control in detecting the SNPs, haplotypes and genotypes of MBL gene with SSP-PCR and Real-time PCR.Chapter Two Using SSP-PCR to Study the SNPs, Haplotypes and Genotypes ofMBL Gene in Wa and Bai Nationalities Objective:For those samples from Wa and Bai nationalities whose the haplotypes andgenotypes of promoter region of MBL gene had been clear, the point mutations at codon 52, 54 and 57 of exonl in MBL gene were detected by using SSP-PCR and the distribution of alleles, haplotypes and genotypes of the gene were analyzed. Methods:1. The three point mutations at codon 52, 54 and 57 of exonl in MBL gene were detected by SSP-PCR.2. Combining the data of haplotypes and genotypes of promoter region and the three point mutations at codon 52, 54 and 57 of exonl in MBL gene, the frequencies of the main SNPs, haplotypes and genotypes of MBL gene and the significance of their distribution in the two populations were analyzed.Results:1. The SSP-PCR system for detecting three point mutations of exon 1 of MBL gene was established, and the haplotypes and genotypes of MBL gene were analyzed.2. The distribution of alleles, haplotypes and genotypes of main SNPs of MBL gene in these two populations were defined. 54 point mutation of MBL gene was found in Bai and Wa populations, but no 52 and 57 point mutations. The haplotypes are maintly LYQA and HYPA. The genotypes are mainly LXPA/LYQA, HYPA/ LYQA, LYPA/LYQA, LYQA/LYQA and HYPA/HYPA in Bai population, and LXPA/LYQA, HYPA/LYQA, LYPA/LYQA, LYQA/LYQA and HYPA/HYPA in Wa population.Conclusions:1. The established SSP-PCR system is sensitive and reliable, and can be used to detect the point mutation and SNPs.2. Significance of difference was found in the distributions of alleles, haplotypes and genotypes of MBL gene between Wa and Bai populations.Chapter Three Combination of Real-time PCR-MB and SSP-PCR to Study the Main SNPs, Haplotypes and Genotypes of MBL Gene in Yi and HaniNationalitiesObjectives:1. To establish a simple, inexpensive and rapid method to detect the six main SNPs of MBL gene with high sensitivity and specificity, which can also be applied toanalyze the haplotypes and genotypes of the gene.2. To define the distribution of the alleles, haplotypes and genotypes of main SNPs of MBL gene in Yi and Hani nationalities by investigating the main SNPs with the method above. Methods:1. Blood samples form Yi and Hani nationalities were collected and genomic DNA were extracted form the leukocytes.2. Alleles H/L and P/Q located in MBL gene promoter region were typed by SSP-PCR, the fragments containing alleles X/Y amplified by PCR and the alleles X/Y located in MBL gene promoter region were analyzed by real-time PCR-molecular beacons(MB) fluorescence detection method.3. Based on the haplotypes and genotypes of promoter region of MBL gene, the haplotype fragments of exon 1 of MBL gene were amplified by SSP-PCR using different sequence specific primers and universal antisence primers. Three point mutations at codons 52, 54 and 57 of exonl of MBL gene were detected by real-time PCR-MB using the haplotypes fragment as templates.4. Combination of the result of SSP-PCR and real-time PCR-MB, the alleles, haplotypes and genotypes of the six main SNPs of MBL gene were determined.5. The differences of intertribal distribution of alleles, haplotypes and genotypes of MBL gene of the four nationalities were analyzed.Results:1. SSP-PCR for detecting the alleles H/L and P/Q in the promoter region of MBL gene and Real-time PCR-MB for detecting the alleles X/Y in the promoter region and three point mutations at codons 52, 54 and 57 of exonl of MBL gene were established.2. In Yi and Hani populations, the frequency of alleles L and H were similar in Yi population, but allele L was higher than H in Hani population, and allele Y was higher than X, allele P was consumedly higher than Q. The frequency of allele B was 0.082 in Yi population and 0.119 in Hani population, alleles D and C were not found in these two populations. The haplotypes were mainly HYPA and LXPA in Yi population and HYPA and LYQA in Hani population. The genotypes are mainly HYPA/HYPA, LXPA/LXPA and HYPA/LYQA in Yi population and HYPA/HYPA,HYPA/LYQA and HYPA/LYPB in Hani population.3. The distribution of alleles, haplotypes and genotypes of 6 main SNPs of MBL gene in 4 populations, Wa, Bai, Yi and Hani, were defined:(1) In these 4 populations, the distribution of alleles H/L and P/Q was statistically significant (p<0.01 and PO.01 respectively).The frequencies of allele L were higher than those of allele H in all of the 4 populations and higher in Wa and Bai than those in Yi and Hani populations. The frequencies of allele P were higher than those of allele Q in all of these 4 populations. The distribution of allele X/Y was not statistically signigficant in these 4 populations, but the frequencies of allele Y were higher than allele X in all of these 4 populations, whose general frequency was about 80%. Point mutations at codons 52 and 57 of exonl weren't found in these 4 populations. The distribution of the point mutation at codon 54 of exonl was statistically significant (P<0.01) among 4 populations and higher in Hani population.(2) The distribution of haplotypes of MBL gene in these 4 populations was statistically significant (p<0.01). The frequencies of haplotypes HYPA and LYQA wee higher in Bai and Wa populations and haplotypes HYPA and LXPA in Yi population and HYPA and LYQA in Hani population, general frequency of HYPA was more than 40% in Yi and Hani populations.(3) The distribution of genotypes of MBL gene in the 4 populations were statistically significant (p<0.01). Consequently, the frequencies of genotypes LYPA/LYQA, LYQA/LYQA, LXPA/LYQA, HYPA/LYQA and HYPA/HYPA in Wa population, genotypes LYPA/LYQA, LYQA/LYQA, LXPA/LYQA and HYPA/HYPA in Bai population, genotypes HYPA/HYPA, LXPA/LXPA, HYPA/LYQA and HYPA/LYPA in Yi population, genotypes HYPA/HYPA, HYPA/LYQA and HYPA/LYPB in Hani population, were higher, with the highest frequency of HYPA of 31.9% in Yi population and 24.8%in Hani population. Conclusions:1. With high specificity and easy manipulation, this simple, inexpensive and rapid real time PCR-MB method permits automatic high-throughput real time monitoring of gene point mutations or SNPs, which shows advantages over the traditional approaches for identifying point mutations and SNPs. The combination of SSP-PCR and real time PCR-MB method facilitates the investigation of haplotypesand genotypes for functional genes.2. Among 4 populations, Wa, Bai, Yi and Hani, the distribution of alleles L/H and P/Q of promoter region and 54 point mutation of exonl are statistically significant and no point mutations at codons 52 and 57 of exonl have been found. The distribution of allele X/Y is not statistically significant. The distribution of haplotypes and genotypes of MBL gene are statistically significant. The frequencies of haplotype HYPA are higher than those of others, the frequencies of genotype HYPA/HYPA are higher than those of others and the frequencies of genotypes LYPA/LYQA and LYQA/LYQA are higher in Bai and Wa populations than those in Yi and Hani populations, and HYPA/LYQA is higher in Yi and Hani populations.