Screening For Genetic Mutations Of Sudden Unexplained Death In Yunnan

Objective:Sudden unexpected death(SUD)is a hot research field in forensic science.Yunnan sudden unexplained death(YNSUD),happening in some remote mountainous and semi-mountainous areas in Yunnan province,are characterized as familial aggregation,spatial aggregation and time aggregation.In addition,there are still some SUD without these aggregative characteristics in Yunnan province.The SUD in Yunnan has become a serious public health problem,because of unknown etiology,without effective prevention and control measures.In this study,the mutations of related genes were screened in SUD in Yunnan,in order to explore the causes of death and the mechanism of death.Methods:1.Sample:95 SUD cases(90 sporadic SUD cases and 5 YNSUD cases)were selected in Forensic Medicine Institute of Kunming Medical University from 1984 to 2015,after forensic autopsy,histological examination and routine drug test were carefully performed.2.Screening of gene mutations associated with sudden death(SD):According to recent reports,the mutation hotspots of SD related genes were selected,including KCNQ1-Exon3 572-592 base,KCNQ1-Exon6 base 805-905,KCNH2-Exon7 1681-1902 base,KCNH2-Exon9 base 2398+5,SCN5A-Exon3 283-362 base and SCN5A-Exon28 base 5291-6016.Primers were designed by Primer Premier 5 software.Using polymerase chain reaction and direct DNA sequencing,the mutation hotspots areas were detected,then the comprehensive mutation was analyzed by NCBI-Blast and the protein function of missense mutations was predicted.3.Whole genome sequencing for screening YNSUD related genes:A total of 2 YNSUD cases and 1 pedigree sample were selected for the whole genome sequencing.Pedigree analysis was performed to explore YNSUD related genes.Results:1.Screening results of SD related mutations:(1)30 genes mutations related with sudden death were detected from 90 sporadic SUD cases.1 case of missense mutation was found at the KCNQ1 gene exon3 c.576G>T(p.R192L),the pspe=0.982.1 case of heterozygous mutation was found at the KCNH2 gene exon7 c.1789T>A(p.Y597N),the pspe=0.994.1 case of heterozygous mutation was found at the KCNH2 gene exon7 c.1769G>A(p.G590D),the pspe=0.559.19 cases of heterozygous mutations were found at the KCNH2 gene exon7 c.1801 G>A(p.G601S),the pspe=0.125.10 cases of heterozygous mutations were found at the KCNH2 gene exon7 c.1800C>A(p.S600R),the pspe=0.03.3 cases of synonymous mutations were found at the KCNH2 gene exon7 c.1765C>T,SCN5A gene exon28 c.5295T>C and SCN5A gene exon28 c.5447C>T.(2)1 case of heterozygous mutation was found at the KCNH2 gene exon7 c.1801G>A(p.G601S)in YNSUD,the pspe=0.125.2.Whole genome sequencing for screening YNSUD related genes:5 mutations of OBSCN and TTN genes were found in 2 YNSUD cases,but not in the family sample.3 mutations of OBSCN gene in the 1 chromosome are c.228548197G>A(codon changes:cGt/cAt),c.228550426C>T(codon changes:Ccc/Tcc)and c.228564884G>A(codon changes:cGc/cAc);2 mutations of TTN gene in the 2 chromosome are c.179558366A>G(codon changes:Att/Gtt)and c.179582537C>G(codon changes:gCg/gAg).Conclusion:1.SUD and YNSUD cases,five novel mutations were screened out,which were in KCNQ1 gene 576 position and KCNH2 gene 1789,1769,1800,1801 position.2.Through the screening of missense mutations and the predicting of protein function,we found that the mutations of KCNQ1 gene exon3 c.576G>T(p.R192L)and KCNH2 gene exon7 c.1789T>A(p.Y597N)were more likely to lead to the function change of ion channel proteins associated with SUD.3.With whole genome sequencing,5 mutations of OBSCN and TTN genes(OBSCN gene c.228548197G>A,c.228550426C>T and c.228564884G>A;TTN c.179558366A>G and c.179582537C>G)were found.4.SUD and YNSUD cases may be associated with the mutations of related pathogenic genes.