| Background:Atrial fibrillation(AF)is a common and harmful clinical tachyarrhythmia.The etiology of atrial fibrillation has shown that the occurrence of atrial fibrillation is more common in patients with cardiac organic disease.However,some patients with atrial fibrillation have neither the traditional risk factors,nor have determine cause.And this unexplained atrial fibrillation is called idiopathic atrial fibrillation(IAF).The researchers found that patients with IAF usually showed a familial aggregation,therefore thy speculated that IAF may be associated with genetic factors.KCNA 5 is a member of KCNA family which is responsible for encoding Kv1(Shaker)potassium ion channel,and Kv1.5 potassium ion channel encoded by KCNA5is an important subtypes of Kv1 family.In human heart,Kv1.5 is specifically expressed in atrial myocytes.KCNA5 is a susceptibility gene for atrial fibrillation,and the gene is located on human chromosome 12p13,encodingαsubunit of Kv1.5 ion channe.As one of the most extensive,the most basic form of the gene mutation,single nucleotide polymorphism(SNP)is always a hot topic for genetic research.Screening for atrial fibrillation-related susceptibility gene SNP sites can provide a theoretical basis searching for biomarkers,determining high-risk groups and developing personalized medicine programs for atrial fibrillation.However,there is no definitive study with KCNA5 gene.Based on the above background and early studies of KCNA5 genes,this study selected rs3741930(C/T)and rs1056468(A/T)sites on KCNA5 gene,and then conducted a case-control study.The distribution of KCNA5 SNP(rs3741930 and rs1056468)in the two groups was detected and compared,to explore the association between KCNA5 SNP(rs3741930 and rs1056468)and idiopathic atrial fibrillation(IAF)in molecular biology level and provide new potential sites for the diagnosis and personalized treatment of atrial fibrillation.Part One Allele frequencies distribution of KCNA5 SNP loci in IAF patientsObjective:To investigate the relationship between allele frequencies of two KCNA5 SNP loci and idiopathic atrial fibrillation.Methods:In our study,282 patients with IAF admitted were collected,and 300 healthy and age-matched healthy people were used as the control group.In both case and control groups,5 mL peripheral blood were drawn.Genomic DNA from the whole blood cells was extracted.The target fragment was amplified by PCR using genomic DNA as template,and then identified,purified,recovered and sequenced.Statistical analysis of allele frequencies of KCNA5 SNP loci in patients with atrial fibrillation and healthy people.Results:The distribution of rs3741930 locus both in IAF group and control group were in line with H-WE.C,T allele frequency of rs3741930 in IAF group was(235)41.7%,(329)58.3%;while the frequency distribution in healthy control group was(208)34.7%,(392)65.3%.C allele frequency in IAF group were significantly higher compared with the control group(P=0.014).The distribution of rs1056468 locus both in IAF group and control group were in line with H-WE.A,T allele frequency of rs1056468 in IAF group was(387)68.6%,(177)31.4%;while the frequency distribution in healthy control group was(384)64%,(216)36%.A allele frequencies in IAF group and control group had no significant difference(P=0.095).Summary:Rs3741930 may be a new risk locus for idiopathic atrial fibrillation.That people who carrying rs3741930-C allele are more likely to suffer from IAF.Rs1056468 had no relation with idiopathic atrial fibrillation disease.Part Two Genotype frequency distribution of KCNA5 rs3741930 and rs1056468 in IAF patientsObjective:To investigate the association between genotype frequency distribution of two KCNA5 SNP loci and idiopathic atrial fibrillation.Methods:We investigated the genotype frequency distribution of the SNPs of rs3741930 and rs1056468 on KCNA5 gene by DNA sequencing,and comparative differences between the different genotypes distribution of KPNA5 SNP loci and the clinical parameters in IAF patients.Results:The CC,CT and TT genotype frequencies in the IAF group were(47)16.7%,(141)50.0%and(94)33.3%respectively;while in the control group,those were(30)10%,(148)49.3%and(122)40.7%respectively;rs3741930-CC genotype frequency in IAF group were significantly higher compared with the control group(P=0.019).The AA,AT and TT genotype frequencies in the IAF group were(125)44.3%,(137)48.6%and(20)7.1%respectively;while in the control group,those were(116)38.7%,(152)50.7%and(32)10.6%respectively;All the three genotype frequencies in IAF group had no significant difference compared with the control group.Among different genotypes,there was no significant correlation among age,systolic blood pressure(SBP),diastolic blood pressure(DBP),heart rate(HR),left atrium diameter(LAD),left ventricular ejection fraction(LVEF)and other clinical indicators(P>0.05).While the BMI in CC and CC+TT groups of rs3741930 locus were 26.9±2.3 Kg/m~2 and 24.8±2.5 Kg/m~2 respectively.There were statistical differences between the two groups(p=0.019).BMI in AA and AA+TT groups of rs1056468 locus were 24.9±2.7 Kg/m~2 and26.4±2.4 Kg/m~2 respectively;There was statistically significant significance between the two groups(P=0.014).Summary:Rs3741930 may be a new risk locus for idiopathic atrial fibrillation.Genotype frequency distribution of rs1056468 had no relation with IAF.KCNA5 SNP loci rs3741930 and rs1056468 may effect on the BMI of IAF.Part Three Effect of C allele and T allele of rs3741930 locus on Kv1.5 currentObjective:To further verify the relationship between KCNA5 poly-morphism rs3741930 and idiopathic atrial fibrillation.Methods:Transfect KCNA5 gene with rs3741930 SNP into HEK293cells to make heterologous expression systems.The Ikur of heterologous expression systems of rs3741930 were recorded by whole-cell patch clamp.Results:Whole-cell patch clamp results showed a difference in Ikur between those two groups.The current density of Ikur at 60 mV in the rs3741930 T genotype was 45.36±7.79 pA/pF(n=13),while the current was reduced to 39.52±4.74 pA/pF(n=11)in C genotype,(P=0.0416).Norma-lization with a current of+60 mV as 100%,observing the morphology of the current-voltage curve,it was found that the morphology of the current-voltage curves of the two HEK293 cells was highly coincident.Summary:The reason why people carrying rs3741930-CC genotype were more likely to suffer from IAF may be due to a reduction in the number of KCNA5 protein expression on the cell membrane.Conclusion:Rs3741930 may be a new risk locus for idiopathic atrial fibrillation.The reason why people carrying rs3741930-C allele and rs-3741930-CC genotype were more likely to suffer from IAF may be due to a reduction in the number of KCNA5 protein expression on the cell membrane.KCNA5 SNP loci rs3741930 and rs1056468 may effect on the BMI of IAF. |
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