Table3_Exploring TSPAN4 promoter methylation as a diagnostic biomarker for tuberculosis.DOC

Background:Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a persistent infectious disease threatening human health. The existing diagnostic methods still have significant shortcomings, including a low positivity rate in pathogen-based diagnoses and the inability of immunological diagnostics to detect active TB. Hence, it is urgent to develop new techniques to detect TB more accurate and earlier. This research aims to scrutinize and authenticate DNA methylation markers suitable for tuberculosis diagnosis. Concurrently, Providing a new approach for tuberculosis diagnosis.Methods:Blood samples from patients with newly diagnosed tuberculosis and healthy controls (HC) were utilized in this study. Examining methylation microarray data from 40 whole blood samples (22TB + 18HC), we employed two procedures: signature gene methylated position analysis and signature region methylated position analysis to pinpoint distinctive methylated positions. Based on the screening results, diagnostic classifiers are constructed through machine learning, and validation was conducted through pyrosequencing in a separate queue (22TB + 18HC). Culminating in the development of a new tuberculosis diagnostic method via quantitative real-time methylation specific PCR (qMSP).Results:The combination of the two procedures revealed a total of 10 methylated positions, all of which were located in the promoter region. These 10 signature methylated positions facilitated the construction of a diagnostic classifier, exhibiting robust diagnostic accuracy in both cross-validation and external test sets. The LDA model demonstrated the best classification performance, achieving an AUC of 0.83, specificity of 0.8, and sensitivity of 0.86 on the external test set. Furthermore, the validation of signature methylated positions through pyrosequencing demonstrated high agreement with screening outcomes. Additionally, qMSP detection of 2 potential hypomethylated positions (cg04552852 and cg12464638) exhibited promising results, yielding an AUC of 0.794, specificity of 0.720, and sensitivity of 0.816.Conclusion:Our study demonstrates that the validated signature methylated positions through pyrosequencing emerge as plausible biomarkers for tuberculosis diagnosis. The specific methylation markers in the TSPAN4 gene, identified in whole blood samples, hold promise for improving tuberculosis diagnosis. This approach could significantly enhance diagnostic accuracy and speed, offering a new avenue for early detection and treatment.

背景：结核病（TB），由分枝杆菌属的结核分枝杆菌（Mtb）引起，是一种持续存在的传染病，对人类健康构成严重威胁。现有的诊断方法仍存在显著不足，包括基于病原体的诊断中阳性率低，以及免疫学诊断无法检测活动性TB。因此，迫切需要开发新的技术以更准确、更早地检测TB。本研究旨在审慎鉴定和验证适用于结核病诊断的DNA甲基化标记。同时，提供一种新的结核病诊断方法。方法：本研究利用了新诊断的结核病患者和健康对照者（HC）的血液样本。通过分析40份全血样本（22TB + 18HC）的甲基化微阵列数据，我们采用了两种程序：特征基因甲基化位置分析和特征区域甲基化位置分析，以确定独特的甲基化位置。基于筛选结果，通过机器学习构建诊断分类器，并通过单独队列（22TB + 18HC）中的焦磷酸测序进行验证。最终，通过定量实时甲基化特异性PCR（qMSP）开发了一种新的结核病诊断方法。结果：两种程序的组合揭示了总共10个甲基化位置，这些位置均位于启动子区域。这10个特征甲基化位置有助于构建诊断分类器，在交叉验证和外部测试集中均表现出稳健的诊断准确性。LDA模型在外部测试集中展现了最佳的分类性能，实现了AUC为0.83，特异性为0.8，敏感性为0.86。此外，通过焦磷酸测序验证的特征甲基化位置与筛选结果高度一致。此外，对2个潜在的低甲基化位置（cg04552852和cg12464638）的qMSP检测显示出有希望的结果，AUC为0.794，特异性为0.720，敏感性为0.816。结论：本研究表明，通过焦磷酸测序验证的特征甲基化位置可作为结核病诊断的潜在生物标志物。在整血样本中确定的TSPAN4基因的特定甲基化标记有望改善结核病诊断。这种方法可以显著提高诊断准确性和速度，为早期检测和治疗开辟了新的途径。