Study On The Function And Mechanism Of A Novel Mutant Of KIT Gene In Familial Hereditary Gastric Multiple Stromal Tumor Disease

Objective: Gastrointestinal stromal tumors(GIST)are the most common mesenchymal tumors of the digestive tract,most of which contain mutations that activate the c-kit or platelet-derived growth factor receptor-α(PDGFRα)gene.The main clinical manifestations of GIST are abdominal distension,abdominal pain and abdominal mass.The gold standard for clinical diagnosis of GIST is pathological diagnosis,including morphological examination of tumor cells,immunohistochemistry and molecular diagnosis.Familial GIST is a rare autosomal dominant inherited disease that has only been reported in a few families.Similar to GIST caused by somatic mutations,KIT and PDGFRA are the most commonly mutated genes in familial GIST.The clinical manifestations of hereditary GIST are earlier onset and multiple tumor lesions,with a few patients accompanied by skin pigmentation and mast cell growth.Based on a rare familial GIST case,this study used whole exon sequencing and targeted Sanger sequencing to screen pathogenic genes,combined with basic tumor research methods,from clinical diagnosis to basic molecular mechanism,to provide a theoretical basis for subsequent accurate management of this case of disease.Methods: Clinical diagnosis of GIST was carried out based on the imaging features of endoscopic ultrasonography combined with positive pathological indicators.Whole exome sequencing was performed using patients’ tumor tissues and peripheral blood of family members to search for potential driver mutations.Sanger targeted sequencing was performed using paraffin-embedded samples and oral epithelial cells of family members for germ line mutation verification.Mutation Taster,SIFT,and Polyphen2 were used to predict the mutation function.Gene mutants and overexpressed plasmids were constructed by molecular cloning technology,and the effects of candidate mutations on cells were studied in vitro cell experiments.The effects of gene mutations on cell proliferation were detected by CCK8 assay,and potential therapeutic schemes were screened by combining with tyrosine kinase inhibitor(TKI)assay.The expressions of PI3K/m TOR,RAS/RAF/ERK and JAK/STAT path-related proteins were detected by Western blotting experiments to explain the relevant mechanisms downstream of the mutation.Results:1.A novel KIT primitive mutation was detected in this genetic gastrointestinal stromal tumor(c.G2485 C,p.A829P);2.The mutation is predicted to be pathogenic and alter the structure and function of KIT protein;3.The mutation can promote the proliferation of 293 T cells,and TKI related experiments showed that the mutation was resistant to imatinib,the first-line chemotherapy drug of GIST,and sensitive to ripretinib,a multi-target tyrosine kinase inhibitor.4.The mutation can lead to ligand-independent activation of KIT protein,and activate the downstream PI3K/m TOR,RAS/RAF/ERK and JAK/STAT pathways to varying degrees.5.The mutation resulted in ligand-independent proliferation of Ba/F3 cells and sustained KIT protein activation in the cell line as well as significant activation of the downstream JAK/STAT pathway.Conclusion:1.KIT-A829 P mutation is the driving factor of multiple gastric stromal tumors inherited in this family.2.KIT-A829 P promotes cell proliferation through continuous activation of KIT gene and JAK/STAT3 pathway;3.Ripretinib is an effective targeting drug for GIST with KIT-A829 P mutation.