Objective: To explore the characteristics and significances of gene mutations in pulmonary adenocarcinoma, and to provide evidence for targeted medication. Methods: High throughput sequencing based target-capture sequencing was performed in 104 patients with pulmonary adenocarcinoma to detect the mutational status of 56 cancer-related genes. All patients were diagnosed in the First People's Hospital of Kunshan from May 2017 to August 2018. The mutational characteristics of pulmonary adenocarcinoma was analyzed and compared with European and American pulmonary adenocarcinoma populations. The correlations between mutational characteristics and clinical features were analyzed, and the mutation sites for targeted medication were screened. Results: Among 104 patients with pulmonary adenocarcinoma, totally 34 mutational genes were detected in 84 patients (81%, 84/104). Highly frequent mutations included epidermal growth factor receptor (EGFR) (49%, 51/104), TP53 (21%, 22/104), KRAS (13%, 14/104), and BRAF (6%, 6/104). Among all the 187 variants, 76% (142/187) were non-synonymous missense mutations, 13% (24/187) were small fragment deletions, 6% (12/187) were copy number variants, 3% (5/187) were small fragment insertions, and 2% (4/187) were nonsense site mutations. Among 104 patients with pulmonary adenocarcinoma, 34 targeted drug-associated mutations of 13 genes were detected in 68 patients (65%), and 19 (18%) patients harbored ≥ 2 targeted drug-associated mutations. EGFR mutations were more common in female patients than in male patients [62% (34/55)vs. 35% (17/49), χ²= 7.629, P= 0.006], while KRAS mutations were more frequent in male patients than in female patients [22% (11/49) vs. 5% (3/55), χ²= 6.424, P= 0.011]. The mutation frequencies of gene EGFR, TP53, KRAS, and CDKN2A in Chinese single-center (the First People's Hospital of Kunshan) and European and American adenocarcinoma populations were significantly different (all P < 0.05). Conclusions The molecular mutational characteristics of pulmonary adenocarcinoma are complex, and vary greatly among different populations. High throughput sequencing-based multiple-gene detection can reveal its mutational features comprehensively, and that has important roles in personal targeted medication guidance, drug-resistance monitoring and prognosis evaluation.

Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum. Based on our primary study on tegument surface proteins of S. japonicun, a cDNA encoding a 66 kDa calcium-binding protein of S. japonicum (Chinese strain) was cloned, sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S. japonicum. The expression of SjIrV1 were detected by Real-time PCR, using cDNA templates isolated from 7, 14, 21, 28, 35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages, and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms. The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a (+) vector and transformed into competent Escherichia coli BL21 for expression. The recombinant protein was purified using a Ni-NTA purification system, and confirmed by high performance liquid chromatography (RP-HPLC) and tandem mass spectrometry (MS/MS). Western blotting analysis showed that recombinant SjIrV1 (rSjIrV1) could be recognized by the S. japonicum infected mouse serum and the mouse serum specific to rSjIrV1, respectively. Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms. ELISA test revealed that IgG, IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice. The study suggested that rSjIrV1 might play an important role in the development of S. japonicum.
Animals ; Antibodies, Helminth ; blood ; Calcium-Binding Proteins ; genetics ; metabolism ; Cloning, Molecular ; Escherichia coli ; metabolism ; Female ; Genetic Vectors ; Helminth Proteins ; genetics ; metabolism ; Male ; Mice ; Recombinant Proteins ; genetics ; metabolism ; Schistosoma japonicum ; genetics ; metabolism