| Part Ⅰ Screening and Identification of Biomarkers for Drug-sensitive TuberculosisBackground:Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that most often affects the lungs. Clinically, TB is diagnosed by bacteriological examination of sputum. However, bacteriological examination is often time consuming (4-8 weeks), resulting in increased morbidity and mortality. So, there is a lack of efficient biomarkers or methods for TB diagnosis.Methods:Serum samples were collected from 431 subjects, including 150 healthy controls,131 untreated drug-sensitive tuberculosis (DS-TB) patients,912-month treated DS-TB patients and 59 6-month treated DS-TB (cured DS-TB) patients. We screened proteomic and transcriptomic differences between the untreated DS-TB group, cured DS-TB group and healthy controls group (10 cases/group, including biological replicates) by using iTRAQ-2D LC-MS/MS and Solexa sequencing. Furthermore, enzyme-linked immunosorbent assay (ELISA) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to validate proteins and miRNAs expression. Receiver operating characteristic (ROC) curve and logistic regression analysis were used to evaluate the potential efficacy of the DS-TB diagnostic model.Results:The iTRAQ-2D LC-MS/MS and Solexa sequencing data showed 35 differentially expressed proteins and 64 differentially expressed miRNAs in DS-TB patients. Compared to the healthy controls group, difference in Gene Ontology categories was found between cured and untreated DS-TB patients:metabolic pathway (13); organelles (13) and catalytic activity (7). KEGG analysis revealed differences in the complement and coagulation pathway, and phagosome pathway. Significant differences in ALB, ARHGDIB, FCN2, miR-92a-3p, miR-125a-5p, and miR-148b-3p were found among untreated DS-TB patients,2-month treated DS-TB patients, cured DS-TB patients and healthy controls by ELISA and qRT-PCR validation. We established the diagnostic model with an accuracy of 94.37% and evaluation model with an accuracy of 85.33%. ALB, ARHGDIB, FCN2, miR-92a-3p, miR-125a-5p, and miR-148b-3p may serve as potential biomarkers for DS-TB diagnosis.Conclusions:(1) Screening and identification of biomarkers for DS-TB by using iTRAQ-2D LC-MS/MS combined Solexa sequencing, and ELISA and SYBR green qRT-PCR validation; (2) Identification of new biomarkers such as ALB, ARHGDIB, FCN2, miR-92a-3p, miR-125a-5p and miR-148b-3p for DS-TB; (3) Construction of a diagnostic model with 94.12% sensitivity and 94.59% specificity, and a evaluation model with 79.41% sensitivity and 90.24% specificity. Our study provided potential biomarkers for DS-TB diagnosis.Part Ⅱ Screening and Identification of Biomarkers for Multidrug-resistance TuberculosisBackground:Pulmonary tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Based on the response of Mtb infection to drug, TB can be divided into drug-sensitive tuberculosis (DS-TB) and drug-resistance tuberculosis; the one resistant to both isoniazid and rifampicin is called multidrug-resistant tuberculosis (MDR-TB). However, the clinical methods to diagnose MDR-TB are time-consuming and/or sensitivity lacking, resulting in the increased morbidity and mortality. So, there is an urgent need to develop rapid, sensitive and efficient methods for MDR-TB diagnosis in order to prevent pulmonary TB spread.Methods:Serum samples were collected from 323 subjects, including 150 healthy controls,131 DS-TB patients, and 42 MDR-TB patients. We screened proteomic and transcriptomic differences between the MDR-TB group, DS-TB group, and healthy controls group (10 cases/group, including biological replicates) by using iTRAQ-2D LC-MS/MS and Solexa sequencing. Furthermore, enzyme-linked immunosorbent assay (ELISA) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to validate proteins and miRNAs expression. Receiver operating characteristic (ROC) curve and decision tree model were used to establish the MDR-TB diagnostic model.Results:The iTRAQ-2D LC-MS/MS and Solexa sequencing data showed 50 differentially expressed proteins and 43 differentially expressed miRNAs in the MDR-TB group, compared with the DS-TB group and healthy controls. Among them, 11 proteins and 14 miRNAs were related by integrative analysis and the network was established mainly in complement and coagulation cascades. According to the relevance between miRNAs and proteins (predicted genes), we validated three miRNA-protein (predicted gene) pairs:miR-4433b-5p-CD44, miR-424-5p-F11, and miR-199b-5p-KNG1. A negative correlation was observed between miR-199b-5p and KNG1. Furthermore, the decision tree we established to identify MDR-TB patients possess a sensitivity of 100.00% and a specificity of 88.33%. And 10-fold cross-validation showed that the accuracy of the model was 83.33%.Conclusions:(1) We screened and identified MDR-TB biomarkers by using iTRAQ-2D LC-MS/MS combined Solexa sequencing and found 11 differentially expressed proteins and 14 differentially expressed miRNAs; (2) By ELISA and SYBR green qRT-PCR validation, we established the diagnostic model for MDR-TB with CD44, F11, KNG1, miR-4433b-5p, miR-424-5p and miR-199b-5p biomarkers; (3) The diagnostic model for MDR-TB possess a sensitivity of 100.00% and a specificity of 88.33%. Our study provided potential biomarkers for MDR-TB diagnosis.Part Ⅲ TB Association Study of CTLA4 Single Nucleotide Polymorphisms with Susceptibility to Pulmonary TuberculosisBackground:According to the World Health Organization report, although about one third of the world’s population is believed to be infected with Mycobacterium tuberculosis, but only 5-15% of people develop active tuberculosis (TB), suggesting that individual differences may play an important role in susceptibility to TB. Our previous study showed that single nucleotide polymorphism (SNP) is associated with susceptibility to TB. The cytotoxic T lymphocyte antigen-4 (CTLA4) gene is a key negative regulator of the T lymphocyte immune response. It has been found that CTLA4 +49>G (rs231775),+6230G>A (rs3087243), and 1143G>A (rs11571319) polymorphisms are associated with susceptibility to many autoimmune diseases, and can down-regulate the inhibition of cellular immune response of CTLA4.Methods:Three SNPs (rs231775, rs3087243, and rs11571319) in CTLA4 were genotyped by using the PCR and DNA sequencing methods in order to reveal the susceptibility and pathology correlation to pulmonary TB in a Chinese Han population (274 TB patients and 266 healthy controls). Linkage disequilibrium and haplotype analysis were performed by Haploview 4.2 software. According to the National Tuberculosis Association (NTA) classification, we divided the TB patients into minimal, moderate and advanced groups. Based on the radiological findings (CT scan and Chest X-ray), we divided TB patients into the single lung lesion group and the double lung lesion group.Results:There were no significant differences in allele frequencies in 3 SNPs between the TB group and the healthy controls group (.P>0.05). We found that the frequency of CTLA4+49AG genotype in the pulmonary TB patients (38.42%) was significantly lower than that of the healthy controls (49.77%), (P=0.038,95%CI=0.436-0.978). However, no associations were found between the other 2 SNPs (+6230>A,11430G>A) and TB (P>0.05). There was a strong linkage disequilibrium (D> 0.075, r2> 0.3) between CTLA4+49 and +6230 site. Haplotype analysis showed that the frequency of haplotype AGG in the healthy controls group (0.069) was significantly higher than the pulmonary TB patients group (0.014), (P<0.0001,95%CI=0.072-0.468). In addition, haplotype GGA was found to be significantly related to TB with double lung lesion rather than single lung lesion (,P=0.018). However, no significant differences were found after NTA classification (P>0.05).Conclusions:(1) This study is the first to report that +49,+6230,11430 SNPs in the CTLA4 gene can be associated with pulmonary TB; (2)+6230 and 11430 CTLA4 SNPs had no relationship with susceptibility to TB in the Chinese Han population. However, +49AG genotype may reduce the risk of being infected with TB; (3) +49A/+6230G/11430G may have a protective function in healthy controls, and +49G/+6230G/11430A was associated with the double lung lesion. The present study provides a new experimental basis to understand the pathogenesis of TB. |
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