| Background:Hypertrophic cardiomyopathy(HCM)is a hereditary cardiovascular disease characterized by left ventricular hypertrophy.The main hazards of HCM include sudden cardiac death(SCD),premature heart failure and stroke,which is not only the main cause of sudden death in young people,but also an important cause of heart transplantation.Molecular genetic studies had confirmed that the most common cause of this disease is the mutation of sarcomere gene,which encodes the element of cardiac contraction mechanism.Previous studies have found that HCM patients have large clinical heterogeneity,and the relationship between HCM genotypes and phenotypes is unclear,so it is still difficult to predict disease progression,guide related treatment and judge prognosis by HCM patients’genotypes.At present,all kinds of clinical treatments for HCM are symptomatic treatment.However,as a single gene genetic disease,the main cause of HCM lies in the gene mutation of cardiomyocytes,and the current treatment methods do not address its etiology,so it can’t fundamentally cure HCM.Base editor is a new type of gene editing technology,which breaks through the limitations of previous gene therapy methods,gene correction effect will not disappear with the passage of time,and can achieve long-term inhibition of mutation phenotype.In this study,we analyzed the clinical phenotypic and prognostic characteristics of MYH7 gene Arg453Cys mutation and TNNT2 gene Arg102Trp mutation HCM,which are relatively common in HCM,and discussed whether base editors can correct HCM pathogenic mutation in vitro and effectively treat HCM.This study is expected to provide new ideas and scientific basis for the treatment of HCM with clear pathogenic mutations.Methods:In order to explore the correlation between genotype and phenotype of HCM patients,we included 986 unrelated HCM patients and 761 non-HCM controls HCM patients were examined by medical history,physical examination,blood test and echocardiography,and followed up regularly.At the same time,we sequenced the study population by whole exon sequencing.Then we collected and analyzed the clinical phenotypic characteristics of HCM patients with Arg453Cys mutation in MYH7 gene and Arg102Trp mutation in TNNT2 gene,and preliminarily discussed the relationship between the above two mutations and HCM clinical phenotype.In order to explore the gene therapy methods based on the above genetic background,we chose the base editor technology as the gene therapy method for HCM.Firstly,we used CRISPR/Cas9 technology to construct a HCM mouse model carrying the above two mutations relative to humans,and extracted mutant mouse embryonic fibroblasts(MEF).The designed base editor and sgRNA plasmid vector were simultaneously transfected into MEF cells by cell electroporation transfection technique,and the successfully transfected cells were enriched by flow sorting.The DNA of the cells was extracted for highthroughput sequencing.Finally,the editing efficiency and bystander editing were calculated by analyzing the sequencing data,in order to evaluate whether the base editor technique can correct the pathogenic mutation of HCM,safety and other side effects of treating HCM.Results:We found that 6 cases(0.6%)of HCM patients carried the Arg453Cys mutation located in exon 14 of MYH7 gene.The 1357th base of CDS sequence was mutated from C to T,resulting in the conversion of amino acid 453rd from arginine to cysteine.The followup results showed that the average onset age of the 6 patients was young,the condition was easy to deteriorate,and even cardiovascular death occurred.3 cases(0.3%)of the 986 patients were found carrying the Arg102Trp mutation located in exon 10 of the TNNT2 gene,and the 304th base mutation in the CDS sequence changed from C to T,resulting in the conversion of the 102nd amino acid from arginine to tryptophan.All the 3 patients showed mild hypertrophy(maximum wall thickness≤20mm),but they were still prone to disease progression and cardiovascular death.In addition,neither of these two mutations was detected in 761 non-HCM controls.We explored the optimal transfection conditions of MEF cells by electroporation,and finally found that the electroporation parameters of 1350V pulse voltage,10ms pulse time and 1 pulse had higher transfection efficiency on MEF cells.Then we detected the DNA sequence of MEF cells which had successfully transfected,through Sanger sequencing and high-throughput sequencing.The results showed that the highest editing efficiency of 12.33%was achieved for the Arg454Cys mutation in Myh6,with no obvious bystander editing.For the Arg127Trp mutation in Tnnt2,we achieved 0.53%～15.58%editing efficiency,but at the same time produced 0.02%～0.92%of bystander editingConclusion:Our study found that the Arg453Cys mutation in MYH7 gene and the Arg102Trp mutation in TNNT2 gene are relatively common among Chinese HCM patients.Although the patients with these mutations have high clinical heterogeneity,they have a higher risk of cardiovascular death.Our study explored the possibility of base editor applied to HCM pathogenic mutation correction,and achieved certain editing efficiency in correcting Arg454Cys mutation in Myh6 gene and Arg127Trp mutation in Tnnt2 gene,while producing less bystander editing. |
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