Gene And Functional Verification Of Familial Sick Sinus Syndrome

Background:Sick sinus syndrome?SSS?is a common cardiovascular disease in the elderly.It can be characterized by pathological sinus bradycardia and sinus arrest,resulting in dizziness and syncope,and implantable electronic pacemakers are the only effective treatment currently.The symptom in the offspring of familial sinus syndrome?FSSS?occurs earlier than the previous generation,and often manifests as genetic continuity.At the molecular level,the occurrence and development of sick sinus syndrome can be attributed to genetic factors.For example,sodium voltage-gated channel alpha subunit 5?SCN5A?,ankyrin 2?ANK2?,hyperpolarization activated cyclic nucleotide gated potassium channel 4?HCN4?,and myosin heavy chain 6?MYH6?have all been identified as contributing to the risk of SSS.However,some family members of the FSSS reported no functional mutations or abnormalities of the above genes,suggesting it may be due to other mutation or dysfunction.In this study,we explored the pathogenesis and genetic regulatory networks of FSSS by explor:ing the key genes.To provide important support for early detection,prevention and treatment of SSS.At the same time,it provides an important theoretical basis for the development of biological pacemakers for SSS.Materials and methods:1.Family history was carefully reviewed,medical and personal history was obtained from each participant,drawning the family map,and analyzing the dominant/recessive characteristics of the family.2.Collection and preservation of blood samples:Peripheral blood samples were collected from all probands using 5 mL tubes with ethylene diamine tetraacetic acid?EDTA?and stored in dry ice.3.Genomic DNA extraction:A TIANamp Blood DNA Kit?TIANGEN,Beijing?was used for genomic DNA extraction.4.Whole-genome sequencing and bioinformatics analysis:Genome sequencing was performed for this family to identify the disease-causative mutation for this family.Whole-genome sequencing?WGS?with 150 bp paired-end reads was performed on an Illumina Hiseq 4000 sequencing platform?Novogene,Beijing?.After sequencing,the original sequence data was filtered,the quality of the sequencing data was evaluated,the depth,the coverage,the number of single nucleotide variants?SNV?and indel mutations were calculated,and the mutation site screening and the harmful classification of the mutation sites were performed.5.Small family genetic screening and verification disease-causing gene mutations:The variants were selected based on disease clinical symptoms using the Phenolyzer online software.potassium voltage-gated channel modifier subfamily G member 2?KCNG2?were further validated in members of the Chinese family using the polymerase chain reaction?PCR?.Evolutionary conservation of the mutated amino acids was assessed via Clustal Omega online software through alignment of the KCNG2 orthologous protein sequences of the following species.The effect of point mutations for the candidate risk gene was predicted by PredictProteinonline software.PredictProtein and SWISS-MODEL online software were used to predict the protein structures of the candidate risk gene,including the wild-type and mutated forms.Potential alterations to the three-dimensional?3D?protein structure of the candidate risk gene,including the wild-type and mutated versions,were determined using PyMol software?version 1.5?.Results:1.Clinical data:The symptoms,electrocardiogram and dynamic electrocardiogram of the proband and the proband mother were consistent with the diagnostic criteria of sick sinus syndrome,and the children of the proband were sinus bradycardia compared with the normal.2.Sequencing results:The average overall sequencing depth was more than 39× and coverage was 99.45%in the six sequenced genomes.A total of 82 variants passed the initial filtration process and were confirmed to be carried by all four patients.These post-filtration variants were related to 64 genes.The Phenolyzer software was used to link the above-mentioned 64 genes to sick sinus syndrome and identified 58 genes.Among the 58 genes,a protein-coding gene which is markedly expressed in human heart,named KCNG2,was found.The result also was shown that KCNG2 gene was closely related to sick sinus syndrome.3.KCNG2 verification:In the Chinese family mentioned above,the recurrent heterozygous mutation?NM 012283:exonl:c.401₄12del:?in KCNG2 gene was identified in all four affected individuals.The mutation could cause several amino acid deletions?NP₀36415.1:p.134Arg_Thr138delinsPro?.Residues Arg134 to 138Thr of KCNG2 protein were predicted to be conserved among multiple species.The position of the mutation may be have a great effect on KCNG2 protein.The secondary structure between wild type and the mutated form of the KCNG2 protein found the mutant KCNG2 gene?NM₀12283:exonl:c.401₄12de1:?,the location of protein-binding sites within the KCNG2 protein would change?Fig.6?.In addition,the potential effect of the mutation on protein 3D conformation examined that the number of helixes within the KCNG2 protein was predicted to decrease for the KCNG2 gene mutantConclusions:1.KCNG2?NM₀12283:exonl:c.401₄12del:?mutation was first discovered in FSSS.2.The KCNG2 gene mutation can cause several amino acid deletions?NP₀36415.1:p.134Arg Thrl38delinsPro?,affecting the secondary structure and three-dimensional structure of the protein..