Study On The Association Of MtDNA Copy Number And Heterogeneity,as Well As Variations Of Captured Genes In The Target Region Of With Human Longevity

Objective:1.This study aimed to investigate the relationship between mitochondrial DNA(mtDNA)copy number,heterogeneity and human longevity through next-generation sequencing.2.This study aimed to test the relationship between genetic variations in target regions and human longevity through next generation sequencing.Material and Methods:1.The subjects were 100 long-lived individuals aged over 95 years and 100 control individuals aged 60-80 years from Lingao county,Hainan province.2.mtDNAs from longevity group and control group were analyzed by using high-throughput sequencing technique.BWA,SAMtools and Sambamba software were used to compare high-throughput sequencing sequences with human reference mitochondrial genome,and to analyze quantitatively mtDNA copy number and variations.To reveal the association between mtDNA copy number and heterogenetity with longevity,statistics and annotations of SNV and In Del were performed using ANNOVAR which includes db SNP database,1000 genome project and other existing databases?3.Targeted sequencing of 180 target genes.Three genes are selected from Longevity Map genes associated with longevity,eighteen genes from genes involved in DNA damage repair,thirty eight genes from lysosomal autophagy related genes and one hundred and twenty one genes from the upstream and downstream regions of 10 kb of the above selected genes.The taget regions of the longevity and control groups were sequenced by next-generation sequencing.The target sequences were compared to the human reference genome(GRCH37/hg19),and variation analysis,annotation and statistical analysis of SNV and In Del were performed using ANNOVAR which includes db SNP database,1000 genome project and other existing databases)to screen the genetic variations associated with longevity.Results:1.Comparison of mtDNA copy number between longevity group and control group showed no significant difference between the two groups(P=0.098).2.The overall heterogeneity of mtDNA in the longevity group was higher than that of the control group with significant difference(P=2.78×10-4)by comparing the heterogeneity between the longevity group and the control group.3.It was found that the mitochondrial heterogeneity in the longevity group was significantly higher than that of the control population(P = 1.5×10-5)by comparing the mitochondrial heterogeneity at the interval of(27)5% mutation rate.The result suggested that the heterogeneity within this interval may increase with age.4.It was found that there was no statistically mitochondrial heterogeneity difference between the longevity group and control group by comparing the mitochondrial heterogeneity at the intervals of 5-10% mutation rate(P=0.415)and(29)20%mutation rate(P = 0.796).The results suggested that mitochondrial heterogeneity at the intervals of 5-10% and(29)20% mutation rates did not increase with age.5.There was no significant difference between synonymous and non-synonymous mutations in the mitochondrial heterogeneity at interval of(29)5% mutation rate between the longevity and control groups(P=0.224).6.Based on the association analysis of genotype and allele in the target region,it was found that there was significant difference in the SNPs of frequency(27)1%(rare variation)and frequency ?1%(low frequency variation)from1000g--ALL database between the longevity group and the control group(P=0.029),suggesting some low-frequency and rare mutations may be associated with longevity.7.By comparing the allelles frequency in the target region between the longevity group and the control groups,the genetic variations related to longevity was found,which were located in the exon regions of GLB1,ERCC6,ACP2,CTSH and AP1B1;the intron regions of GLB1,ATR,SCARB,FAM47 E,GDI1,AP1B1,APOE,CTSH,NR1H3,ACP2,DDB2,LIPA,PGBD3,ERCC6,AP1S1,GNE,ARSB,SLC36A3,VKORC1L1 and GUSB;the 5'-UTR regions of GUSB,ERCC6,and ENC1;the 3'-UTR regions of SLC36A3,VKORC1L1,AP1S1,NAT16,ERCC6 and CTSH;the splicing regions of EIF4A1;the nc RNA_exonic regions of MIR4653;the nc RNA_intronic regions of BLOC1S1-RDH5 and SENP3-EIF4A1.8.By comparing the genotypes between the longevity and the control groups in the target region,the genetic variations associated with longevity was found,which were located in the exon regions of GLB1,GUSB,ERCC6,ACP2,RDH5 and CTSH,respectively,the Intron regions of GLB1,ATR,SCARB2,FAM47 E,ENC1,HEXB,ARSB,ATG10,VKORC1L1,GUSB,AP1S1,CLTA,GNE,ERCC6,PGBD3,LIPA,RRP8,ILK,DDB2,ACP2,ACP2,NR1H3,CTSC,ANKRD24,APOE,AP1B1 and XRCC6;the 3'-UTR regions of VKORC1L1,AP1S1,NAT16,ERCC6 and CTSH;.the nc RNA_exonic regions of BLOC1S1-RDH5 and SENP3-EIF4A1;the splicing regions of EIF4A1 and ERCC2;the 5'-UTR regions of GUSB and ERCC6.Conclusions:1.It was found that there was no significant difference in the copy number of mtDNA between the longevity and the control groups(P=0.098),suggesting that the copy number of mtDNA was not significantly reduced in the longevity population.2.The overall heterogeneity of mtDNA in the longevity group was significantly higher than that in the control population,but only in the mutations rate(27)5%,and not in the mutation rate(29)5%.Therefore,the relationship between heterogeneity of mtDNA and longevity is needed to enlarge sample size to further confirm the results.3.Based on the association analysis of genotype and allele by comparing with the1000g--ALL database,it was found that there were significant differences in SNPs with frequency(27)1%(rare variation)and ?1%(low frequency variation)(P= 0.029),suggesting that some low frequency mutations and rare mutations may be associated with longevity.4.By capture target sequencing,it was revealed that 36 genetic variations may be associated with longevity in Hainan population,These genetic variants were located in these genes,which as follows:GLB1,ERCC6,ACP2,CTSH,AP1B1,ATR,SCARB,FAM47 E,GDI1,APOE,NR1H3,DDB2,AP1S1,GNE,SLC36A3,VKORC1L1,GUSB,ENC1,NAT16,EIF4A1,MIR4653,BLOC1S1-RDH5,SENP3-EIF4A1,RDH5,SCARB2,HEXB,ARSB,ATG10,CLTA,PGBD3,LIPA,RRP8,ILK,ANKRD24,XRCC6 and ERCC2.These genes are involved in lysosomal autophagy pathways and DNA damage repair pathways,suggesting that genetic variation in the pathways of lysosomal autophagy and DNA damage repair may be associated with longevity.