Genetic Variants And Functional Analyses Of The CTSL Gene Promoter In Dilated Cardiomyopathy

Objective: In this study,we investigated the role of DNA sequence variants（DSVs）in the promoter region of the cathepsin L（CTSL）gene in the development of dilated cardiomyopathy（DCM）.Biological function of identified DSVs in the promoter region of the CTSL gene and related molecular mechanisms were also explored.Methods:1 Blood specimens were collected from DCM group（135 cases）and control group（186 cases）,and genomic DNA was extracted.2 PCR primers were designed to amplify the promoter region of CTSL gene,which were then directly sequenced.DSVs were identified by comparison between DCM group and control group.3 p GL3-CTSL reporter gene vector was constructed with ligation of p MD19-T vector,transformation of competent cells,screening of recombinant plasmids,plasmid extraction and double digestion experiments.Transient transfections were conducted with p GL3-CTSL reporter vector and an internal control vector（p RL-TK）in HEK-293 cells and NRCMs cells.Relative effects of DSVs on CTSL gene promoter transcriptional activity were calculated.4 DSV-affected binding of transcription factors was predicted with TRANSFAC database（https://portal.genexplain.com/）.Synthetic biotin-labeled probes were used for gel mobility shift assays（EMSA）to detect transcription factor-DNA interactions in vitro.Results:1 By DNA sequencing,a total of 4 variations were identified in this study population.One DSV（g.87725317G>A）and one SNP[g.87726114G>A（rs1163419261）] were only present in one DCM patient.Two SNPs [g.87725838G>C（rs187982055）] and [ g.87725948C>A（rs3118869）] were present in both DCM group and control group with similar frequencies.2 In HEK-293 cells and NRCMs cells,transfection experiments revealed that p GL3-87725317A+87726114A significantly altered the CTSL gene promoter transcriptional activity.3 With TRANSFAC database,it was predicted that DSV g.87725317G>A may create a binding site for TCF-7L1 and eliminate binding sites for RFX5,RFX1,ETV5,RFX2 and ESR1.SNP[g.87726114G>A（rs1163419261）] may eliminate binding sites for NHLH1 and MYC.4 EMSA results showed that DSV（g.87725317G>A）created a binding site for an unknown transcription factor in both HEK-293 cells and NRCMs cells.Conclusion: DSVs in CTSL gene promoter may contributed to the development of DCM by altering the CTSL promoter transcriptional activity.