Age-related Study Of 7 AR-CpGs In Blood Samples From Han Population

Background Age is the phenotypic information of individuals.Infering the individual age of the biomedical materials at the forensic field can greatly narrow the scope of investigation in the case,it is of great significance for criminal investigation.Traditional forensic science mainly adopts an anthropological method to infer the age of individuals according to the age-related characteristics of histomorphological indicators such as bones and teeth.Although this method is relatively mature,there are still problems such as large errors,empirical dependence,and limited application when the bone,tooth and other tissues are missing.Generally,the biological samples left at the forensic field are mostly various types of body fluid stains,exfoliated cells,tissue fragments,etc.How to effectively predict the age of an individual through these biological samples is a key issue of forensic science.With the development of molecular biology and the deepening of understanding of development and aging processes,a series of aging-related biomarkers have been discovered,including mitochondrial DNA oxidative damage and fragment deletion,AGEs content,telomere length,and protein D-Aspartic ratio,sjTRECs abundance,mRNA expression levels,microRNA and DNA methylation levels.In the field of forensic science,the application value of these aging-related biomarkers in individual age inference has also been studied in depth.In general,compared with other age-related biomarkers,the use of DNA methylation markers to infer individual age has the advantage of wide application range and high inference accuracy.At present,there are relatively few studies in this area in China,and there is a significant difference in the correlation between DNA methylation markers and age in different geographical populations.Therefore,it is necessary to further analyze and verify the currently reported age-related DNA methylation markers in Chinese population and evaluate their applicability to Chinese population.Objective The aims of this study were analyzed the age correlations of 7 reported AR-CpGs in blood samples from Chinese population,and exploredt their application value in Chinese individual age estimation.Methods ? Review the relevant literature and screen out 7 AR-CpGs sites.? Search the sequence and design the PCR primers and SBE primers of 7 AR-CpGs sites using MethPrimer software.? Examine the reliability of the designed primers by polyacrylamide gel electrophoresis and single locus SNaPshot reaction.? According to the different direction of the extended primers,the seven markers were divided into two groups,two composite PCR amplification systems and two composite SBE single base extension systems were constructed by adjusting the primer concentration and the amplification annealing temperature.? Blood samples from 230 healthy Han unrelated individuals were collected as training samples,after DNA was extracted,quantified,and converted by bisulfite,the constructed composite detection system was used for detection.? Analyze the age-related and gender differences of each AR-CpG locus in the training samples,and use the stepwise regression model to construct individual age-prediction models for males,females,and regardless of gender individuals.? A blood sample of 80 healthy Han Chinese independent individuals was randomly collected as testing samples.The age was inferred by our composite detection system and age-prediction model we established in this paper,and compared with the real age to evaluate the accuracy of individual age estimation.Results ? According to the literature,seven AR-CpGs sites were screened for this study,cg02228185?ASPA₁?,cg09809672?EDARADD?,cg19283806?CCDC102B?,cg04208403?ZNF423?,chr17: 44390358?GRCh38,ITGA2B₁?,cg14361627?KLF14?and cg06639320?FHL2?.? We successfully constructed two sets of composite PCR amplification system and two sets of composite SBE single base extension system,combination 1 includes four CpG sites of cg02228185,cg09809672,cg19283806 and cg04208403,and combination 2 includes three CpG sites of chr17: 44390358?GRCh38?,cg14361627 and Cg06639320.? In the training samples,the age correlation of cg19283806 in male,female and total samples was the highest,the relevant systems were 0.9001,0.8151 and 0.8706 respectively.The age correlation of cg04208403 in male,female and total sample groups was the lowest,the relevant coefficients are 0.5686,0.4498 and 0.5303 respectively.?4?The age-inferred model constructed by stepwise regression model for the training samples is: 72.61045-39.17263×chr17: 44390358?GRCh38?+131.78740×cg14361627+43.74092×cg06639320 + 9.93087 × cg04208403-15.51575 × cg02228185-69.32841 ×cg19283806,the MAD values of the training samples and the verification samples are 4.20 and 4.83 respectively.?5?For the analysis of the three age groups,the prediction deviation of training samples in the 20-40 years old is the lowest,MAD is 3.82 years,and the prediction accuracy within 5 years is the highest,it can reach 73.2%.The predicted deviation of the testing samples in the age from 0 to 20 years old is the lowest,MAD is 4.24 years,and the prediction accuracy in the age range from 20 to 40 years old is the highest within 5 years,which can reach 74.1%.Conclusion ? In the blood samples of the Han population,except for the age correlation of cg04208403,the other six AR-CpGs had a good age correlation;? The individual age-inferred model we constructed in this paper has a high accuracy in the Chinese Han population.