Pathogenicity Analysis Of Rare Heterozygous Missense Mutations In RBM20 And MURC Genes

BackgroundWe reported a patient in clinic presented with dilated right ventricle and ventricular arrhythmia carrying two rare missense mutations of RBM20(RNA Binding Motif Protein 20)and MURC(Muscle-Restricted Coiled-Coil Protein)gene.Both of them were related with structure of myofibrils.The pathogenetic variants in some specific sites could cause the certain phenotypes,such as ventricular arrhythmias and dilation,which highly matched the patient’s symptoms.Whereas,the pathogenicity of the two variant sites has never been investigated yet.This study will analyze the pathogenicity of these two mutation sites under instruction of Standards and Guidelines for the Interpretation of Sequence Variants:a Joint Consensus Recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology.Methods1.The patient’s 21,522 genes(including cardiomyopathy and arrhythmia-related genes)were sequenced by using peripheral blood next-generation sequencing technology.And the pathogenic phenotypes of RBM20 and MURC genes were matched with the patient’ s clinical manifestations.2.We used bioinformatics tools to predict pathogenicity of this two specific mutation sites of RBM20 and MURC gene by investigating population frequency and pathogenic records through related database,analyzing the evolutionary conservation of specific mutation sites through multiple sequence alignment,predicting the hydrophobicity and structure of the protein before and after mutation with the Kyte Doolittle algorithm and I-TASSER(Iterative Threading Assembly Refinement)modeling respectively.And we also used certain algorithms and tools to predict their pathogenicity.3.In vitro we cloned human RBM20 and MURC cDNA into pHB vectors and introduced the two specific mutations by site directed mutagenesis and transferred them into human cardiomyocytes by liposome.The difference in mRNA of RBM20 and MURC genes was detected by qPCR before and after the mutation and the difference in their protein expression was determined by Western Blot.Results1.The patient carried two rare heterozygous missense mutations:RBM20 gene c.DNA 224th cytosine mutated into thymine,resulting in serine residue to leucine respectively(c.224C>T/p.Ser75Leu het);MURC gene c.DNA 71th adenlne mutated into thymine,causing Aspartic acid residue into Valine(c.71A>T/p.Asp24Val het).2.The population frequency of the RBM20 variant fluctuated from 0.0000 to 0.0002.The region of 73-76th residuals of RBM20 protein was highly conserved in Mammals.The Kyte Doolittle hydrophobicity score of the peptide at 72-79th residuals was increased by 0.511～0.911.The variants caused the disappearance of predicted hydrogen bond with a distance of 2.2A.SIFT predicted the pathogenicity of the mutation as "Damaging" and PolyPhen-2 predicted it as "Probably Damaging".The population frequency of the MURC variant fluctuated from 0.0000 to 0.0001.The region of 22-38th residuals of MURC protein was highly conserved in Mammals.The Kyte Doolittle hydrophobicity score of the peptide at 20-27th residuals increased by 0.855～0.856.The variants caused the disappearance of predicted two hydrogen bonds with distance of 1.8A and 1.9 A respectively.SIFT predicted the pathogenicity of the mutation as "Damaging " and PolyPhen-2 predicted it as " Possibly Damaging.3.Compared with the Wild Type group,the Mutation groups of both RBM20 and MURC gene had significantly lower mRNA expression(P<0.01).Compared with the Wild Type group,the Mutation groups of both RBM20 and MURC gene had significantly lower protein expression(P<0.01).ConclusionThese two rare heterozygous missense mutations RBM20 c.224C>T/p.Ser75Leu and MURC c.71A>T/p.Asp24 Val can cause the decreased expression of their mRNA and protein and were defined as Likely Pathogenic.