Screening And Identification Of Serum Biomarkers For Pulmonary Tuberculosis And Analysis Of FCN2SNPs With Susceptibility To Pulmonary Tuberculosis

Part I Discovery and Identification of Serum Biomarkers for Pulmonary Tuberculosis using iTRAQ2D LC-MS/MSBackground:Tuberculosis (TB) is a chronic respiratory infectious diseases which cause serious damage to the human health by Mycobacterium tuberculosis (Mtb) and pulmonary TB is the most common type of TB. Pathogenic role of Mtb is related with inflammation caused by proliferation of bacteria, as well as the body impairment produced the delayed type hypersensitivity. Despite expansion of the directly observed treatment short course strategy and substantial investment to improve treatment completion rates, accurate diagnosis of disease remains one of the biggest obstacles to global TB control. The infection of TB remains a significant cause of mortality and morbidity worldwide. Early diagnosis and prompt treatment of disease is essential for effective control of TB. Delayed diagnosis might accelerate disease exacerbation, increase the risk of death and promote TB spread. In recent decades, there has not been a significant advance in the diagnostic techniques of TB, making early diagnosis of TB outdated and inappropriate. The organisms utilize innate and vacquired immunity to defend against pathogens infection. There are some pathophysiological changes in the interactions process of host-pathogens. Detection of these changes provides novel method for diagnosis of pulmonary TB. Serum is an ideal source of diagnostic biomarkers because of easy accessibility, noninvasiveness, and rapid response to disease. In this study, differential expressed serum proteins were screened in patients with pulmonary TB, healthy controls and other lung diseases using iTRAQ labeling coupled with2D LC-MS/MS technique and then the candidate protein biomarkers were validated using ELISA. Further analysis of receiver operating characteristic (ROC) curve revealed the sensitivity, specificity, and accuracy of the potential protein biomarkers, and provided experimental data for the diagnosis of pulmonary TB.Methods:A total of160subjects were collected, including40cases of healthy controls group,40cases of pulmonary TB group, and80cases of differential diagnosis group (40cases of lung cancer group and40cases of pneumonia group. The differential expressed serum proteins were screened in patients with pulmonary TB, healthy controls and other lung diseases using iTRAQ labeling coupled with2D LC-MS/MS technique. The Proteinpilot software was used to identify and quantify proteins. The bioinformatics analysis was used to analyze cell composition, molecular function and biological processes of differentially expressed proteins. ELISA method were applied to analyze the differentially expressed serum protein. The receiver operation characteristic curve was used to analyze the sensitivity and specificity of the each protein for diagnosis, respectively.Results:We applied iTRAQ2D LC-MS/MS technique to investigate protein profiles in patients with pulmonary TB, healthy controls and other lung diseases. A total of434proteins were identified.34differentially expressed proteins (24up-regulated proteins and10down-regulated proteins) were screened in the serum of pulmonary TB patients. Significant differences in protein S100-A9(S100A9), extracellular superoxide dismutase [Cu-Zn](SOD3), and matrix metalloproteinase9(MMP9) were found between pulmonary TB and other lung diseases by ELISA. Correlations analysis revealed that the serum concentration of MMP9in the pulmonary TB was in moderate correlation with SOD3(r-0.581) and S100A9(r=0.471), while SOD3was in weak correlation with S100A9(r=0.287). The combination of serum S100A9, SOD3, and MMP9levels could achieve92.5%sensitivity and95%specificity to discriminate between pulmonary TB and healthy controls,90%sensitivity and87.5%specificity to discriminate between pulmonary TB and pneumonia, and85%sensitivity and92.5%specificity to discriminate between pulmonary TB and lung cancer, respectively.Conclusions:(l)The iTRAQ2D LC-MS/MS combined with ELISA technology platform was established to detect pulmonary TB serum protein;(2) The panel of differentially expressed proteins S100A9, SOD3, and MMP9discriminate between pulmonary TB and other lung diseases with higher sensitivity and specificity, and they might be potential biomarkers. The results provided experimental basis for the diagnosis of pulmonary TB.(3)Several proteins including Sushi, nidogen and EGF-like domain-containing protein1(SNED1), L-lactate dehydrogenase A chain (LDHA), Filamin-A (FLNA) and Costars family protein C6orfll5(ABRAC1) had more significant fold changes. Although the roles of these unselected proteins are largely unknown in TB disease, and the commercial ELISA kits for these proteins are unavailable, further studies on these unselected proteins may help us to find more effective diagnostic biomarkers for pulmonary TB. Part II The FCN2Gene Polymorphisms are Associated with Susceptibility to Pulmonary TuberculosisBackground:One-third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), of which5-10%will eventually develop active tuberculosis (TB). In addition, previous studies in our laboratory revealed the correlation of gene polymorphisms and susceptibility to TB suggested genetic factors always affected individual’s susceptibility to TB. Lectin pathway is an important way of complement activation. It plays an important role in the process of the host defense against pathogens infection. Ficolin-2is pattern recognition innate immune molecule. A variety of pathogens can be recognized and bound by Ficolin-2through MBL-associated serine proteases such as MASP2activate complement pathway. The pathogen surface is marked with C3b that can induce macrophage phagocytosis of pathogens, the formation of the membrane attack complex and the destruction of their cell membranes, results in direct elimination of the pathogens. However, to date, FCN2polymorphisms associated with susceptibility to pulmonary TB have not yet been reported. The present study aimed to investigate the associations of7single nucleotide polymorphisms (SNPs) in the FCN2gene promoter region and exon8region with pulmonary TB in the Chinese Han populations. The study provided novel ideas for prevention and control of pulmonary TB.Methods:Seven SNPs (+6359C＞T and+6424G>T) in exon8and (-986G>A,-602G>A,-557A>G,-64A>C and-4A>G) in promoter region of the FCN2gene were genotyped using the PCR and DNA sequencing technology in254cases of healthy control group and282cases of pulmonary TB group. Allele and genotype frequency were analized in the study. The correlation between SNPs and TB was analyzed in five different inheritance patterns (codominant, dominant, recessive, overdominant, and log-additive) using logistic regression method. Linkage disequilibrium analysis was performed between7SNPs sites by SNPstats and Haploview4.2software.Results:No significant differences were observed in the distribution of allelic frequencies of seven SNPs between the pulmonary TB group and the healthy control group. However, the frequency of homozygous variation genotype (P=0.037,-557A>G; P=0.038,-64A>C; P=0.024,+6424G>T) in tuberculosis group was lower than the healthy group and there were significant differences between the two groups. After adjustment for age and gender,-557A>G (P=0.019; OR=0.24;95%CI,0.07-0.89),-64A>C (P=0.019; OR=0.24;95%CI,0.07-0.89) and+6424G>T (P=0.012; OR=0.23;95%CI,0.06-0.82) were found to be recessive models in association with pulmonary TB. In addition,-64A>C (P=0.047) and+6424G>T (P=0.03) were found to be codominant models in association with pulmonary TB. There were strong linkage disequilibrium (D’>0.75, P<0.0001) between6SNPs of FCN2gene except -602G>A. By calculating linkage disequilibrium, the frequency of haplotype showed no significant correlation with pulmonary TB.Conclusions:(1) This study is the first to report that seven SNPs in the FCN2gene promoter region and exon8region can be associated with pulmonary TB by a case-control study, combined with statistical analysis.(2) The homozygous genotype of-557A>G,-64A>C and+6424G>T in FCN2gene may be protective factors for TB, and be recessive models in association with pulmonary TB. In addition,-64A>C and+6424G>T were found to be codominant models in association with pulmonary TB.(3)-986G>A,-602G>A,-4A>G and+6359C＞T SNPs in the FCN2gene were not associated with pulmonary TB in the Chinese Southern Han populations.