Duchenne and Becker muscular dystrophies (DMD/BMD) are the most commonly inherited neuromuscular disease. However, accurate and convenient molecular diagnosis cannot be achieved easily because of the enormous size of the dystrophin gene and complex causative mutation spectrum. Such traditional methods as multiplex ligation-dependent probe amplification plus Sanger sequencing require multiple steps to fulfill the diagnosis of DMD/BMD. Here, we introduce a new single-step method for the genetic analysis of DMD patients and female carriers in real clinical settings and demonstrate the validation of its accuracy. A total of 89 patients, 18 female carriers and 245 non-DMD patients were evaluated using our targeted NGS approaches. Compared with traditional methods, our new method yielded 99.99% specificity and 98.96% sensitivity for copy number variations detection and 100% accuracy for the identification of single-nucleotide variation mutations. Additionally, this method is able to detect partial deletions/duplications, thus offering precise personal DMD gene information for gene therapy. We detected novel partial deletions of exons in nine samples for which the breakpoints were located within exonic regions. The results proved that our new method is suitable for routine clinical practice, with shorter turnaround time, higher accuracy, and better insight into comprehensive genetic information (detailed breakpoints) for ensuing gene therapy.
Pulmonary inflammation can contribute to the development of lung cancer in humans. We investigated whether pulmonary inflammation alters the genotoxicity of polycyclic aromatic hydrocarbons (PAHs) in the lungs of mice and what mechanisms are involved. To model nonallergic acute inflammation, mice were exposed intranasally to lipopolysaccharide (LPS; 20 µg/mouse) and then instilled intratracheally with benzo[a]pyrene (BaP; 0.5 mg/mouse). BaP-DNA adduct levels, measured by 32P-postlabeling analysis, were approximately 3-fold higher in the lungs of LPS/BaP-treated mice than in mice treated with BaP alone. Pulmonary Cyp1a1 enzyme activity was decreased in LPS/BaP-treated mice relative to BaP-treated mice suggesting that pulmonary inflammation impacted on BaP-induced Cyp1a1 activity in the lung. Our results showed that Cyp1a1 appears to be important for BaP detoxification in vivo and that the decrease of pulmonary Cyp1a1 activity in LPS/BaP-treated mice results in a decrease of pulmonary BaP detoxification, thereby enhancing BaP genotoxicity (ie, DNA adduct formation) in the lung. Because less BaP was detoxified by Cyp1a1 in the lungs of LPS/BaP-treated mice, more BaP circulated via the blood to extrapulmonary tissues relative to mice treated with BaP only. Indeed, we observed higher BaP-DNA adduct levels in livers of LPS/BaP-treated mice compared with BaP-treated mice. Our results indicate that pulmonary inflammation could be a critical determinant in the induction of genotoxicity in the lung by PAHs like BaP. Cyp1a1 appears to be involved in both BaP bioactivation and detoxification although the contribution of other enzymes to BaP-DNA adduct formation in lung and liver under inflammatory conditions remains to be explored.
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