Study On Genetic Structure Variation Of DCC Gene In Pathogenesis Of APC(-) Familial Adenomatous Polyposis

Objective:Familial adenomatous polyposis（FAP）is a type of hereditary colorectal cancer with poor prognosis.It is primarily caused by mutations in the APC gene, though the etiology of some cases of FAP remains unknown.In this study,we screened an APC（-）/FAP family and found that an inherited structural variant of the DCC gene may be the cause of the disease.The relationship between the structural variant and the development of FAP was investigated in this study at both in vitro and in vivo levels.Methods:A total of 7 samples were collected from a family of clinically diagnosed patients with classic FAP seen at the Department of Oncology,The First Affiliated Hospital of Kunming Medical University in August 2019.Of these,one sample was from the proband,three were from patients clearly diagnosed with FAP by colonoscopic screening,1 of whom underwent total colectomy and 2 others are under surveillance,and the remaining samples from the family serving as study controls.An inherited structural variant of the DCC gene was found in all four patients with FAP in this family,which caused a deletion in exons 2-3 of the DCC gene(named DCC^ΔE2-3).In addition,chromosomal translocations were discovered,but no pathogenic mutations in the APC gene were found.To investigate whether the inherited structural variant of the DCC gene is a pathogenic mutation in this family,we designed knockout plasmids for exons 2-3 of the DCC gene using CRISPR/Cas9,and then transfected them into the normal human intestinal epithelial cell line NCM460 and the human intestinal cancer cell line SW480,successfully constructing stable clonal homozygous cell lines.Next,proliferation,migration,invasion,apoptosis,and cell cycle assays were performed before and after transfection to analyze the effects of DCC-SV on cell functions in vitro.The effects of DCC-SV on malignant transformation of normal cells,tumor cell proliferation,and tumorigenicity in vivo were investigated by subcutaneous tumorigenicity assays in nude mice.The gene clusters and signaling pathways affected by DCC-SV were analyzed by transcriptomics.Results:In this family,WES sequencing results suggested that there were no APC gene mutation but a truncated exon 2-3 in the DCC gene,and it was hypothesized that the inherited structural variant of the DCC gene was a potential pathogenic factor in this family.In vitro functional assays after deletion of exons 2-3 of the DCC gene indicated no significant differences in proliferation,cell cycle,migration,and invasion in NCM460^MUTcells compared to NCM460^WTcells（p>0.05）.In contrast,SW480^MUTcells exhibited stem cell characteristics and positive results on the 3D sphere-formation assay.Among intestinal cancer stem cell markers（CD29,CD24,Lgr5,CD166,CD133,SOX2）,the expression of Lgr5 was increased and the expression of CD29 was decreased.In subcutaneous tumorigenicity assays in nude mice,NCM460^MUTwas also not tumorigenic compared to NCM460^WT.Nude mice injected with SW480^MUTcells exhibited shorter time to tumor formation and larger tumor volume and weight than SW480^WT（p<0.05）.Transcriptomics results suggested that deletion of the DCC gene in SW480^MUTcells primarily affected the MAPK signaling pathway,the TGF-βsignaling pathway,signaling pathways regulating pluripotency of stem cells,colorectal cancer,and ferroptosis signaling pathways.Conclusions:The inherited structural variant of the DCC gene(DCC^ΔE2-3)identified in this study was the causative factor in this APC（-）/FAP family.Screening for SV,in addition to SNP and CNV,for FAP of unknown etiology is an additional screening strategy for pathogenic genes in FAP.