Identification of Mixtures of Two Types of Body Fluids Using the Multiplex Methylation System and Random Forest Models.

OBJECTIVE: Body fluid mixtures are complex biological samples that frequently occur in crime scenes, and can provide important clues for criminal case analysis. DNA methylation assay has been applied in the identification of human body fluids, and has exhibited excellent performance in predicting single-source body fluids. The present study aims to develop a methylation SNaPshot multiplex system for body fluid identification, and accurately predict the mixture samples. In addition, the value of DNA methylation in the prediction of body fluid mixtures was further explored. METHODS: In the present study, 420 samples of body fluid mixtures and 250 samples of single body fluids were tested using an optimized multiplex methylation system. Each kind of body fluid sample presented the specific methylation profiles of the 10 markers. RESULTS: Significant differences in methylation levels were observed between the mixtures and single body fluids. For all kinds of mixtures, the Spearman's correlation analysis revealed a significantly strong correlation between the methylation levels and component proportions (1:20, 1:10, 1:5, 1:1, 5:1, 10:1 and 20:1). Two random forest classification models were trained for the prediction of mixture types and the prediction of the mixture proportion of 2 components, based on the methylation levels of 10 markers. For the mixture prediction, Model-1 presented outstanding prediction accuracy, which reached up to 99.3% in 427 training samples, and had a remarkable accuracy of 100% in 243 independent test samples. For the mixture proportion prediction, Model-2 demonstrated an excellent accuracy of 98.8% in 252 training samples, and 98.2% in 168 independent test samples. The total prediction accuracy reached 99.3% for body fluid mixtures and 98.6% for the mixture proportions. CONCLUSION: These results indicate the excellent capability and powerful value of the multiplex methylation system in the identification of forensic body fluid mixtures.

General Formulas for Calculating Commonly Used Kinship Index.

OBJECTIVES: To derive general formulas for calculating commonly used kinship index (KI). METHODS: By introducing the Kronecker symbol, the formulas used to calculate the same KI under different genotype combinations were summarized into a unified expression. RESULTS: The general formulas were successfully derived for KI in various case situations, including the paternity index, full sibling index, half sibling index, avuncular index, grandpaternity index, first-cousin index, and second-cousin index between two individuals without or with the mother being involved; grandpaternity index between grandparents and a grandchild without or with the mother being involved; half sibling index between two children with two mothers being involved; full sibling index among three children; and half sibling index among three children with no, one, or two mothers being involved. CONCLUSIONS: The general formulas given in this study simplify the calculation of KIs and facilitate fast and accurate calculation through programming.

Analysis of the Yp11.2 Deletion Region of Phenotypically Normal Males with an AMELY-Null Allele in the Chinese Han Population.

Aim: The amelogenin gene is a widely used gender marker for forensic DNA profiling. Males who have the amelogenin Y (AMELY) allele deletion can be mistakenly identified as females if genotyping is performed only on the amelogenin gene. The aim of this study was to investigate the frequency of the AMELY allele deletion in the Chinese Han population and analyze the possible genetic variation on the Y chromosome. Materials and Methods: The amelogenin gene of 12,735 unrelated males from the Chinese Han population were genotyped using common forensic short tandem repeat (STR) kits. The AMELY allele deletion was verified by redesigned primers and sequencing. Eighteen Y-specific sequence tagged sites (STSs) on the Yp11.2 region were selected to delineate the deletion breakpoints on the Y chromosome. Results: Three males were confirmed to have no AMELY allele. The frequency rate of the AMELY-null allele was 0.236% (3/12,735) in the Chinese Han population of Central China; 2.73 Mb of sequence on the Y chromosome were absent in all the AMELY-negative samples. The deleted region was mapped using SRY, AMELY, 5 Y-STRs, and 18 STSs, which belong to the class I deleted pattern. The three unrelated males shared the same Y-STR haplotype with four males from other Chinese populations, all of whom have the AMELY-null allele. The haplogroup of these males was identified as the O3 haplogroup. Conclusion: The AMELY allele deletion in the Chinese population was accompanied by the deletion of the Y-STR loci on the Yp11.2 region. Therefore, another Y-specific marker should be tested simultaneously when unknown samples are examined as part of a criminal investigation.

Multistep microsatellite mutation leading to father-child mismatch of FGA locus in a case of non-exclusion parentage.

A non-exclusion paternity case with a mismatch in the autosomal short tandem repeats (STR) locus FGA is reported. The genotypes of the suspected father, the mother and the questioned child in FGA locus were 18/25, 20/26 and 20/22, respectively. Examination of 38 autosomal STR loci revealed no mismatches, and the paternity index is up to 1.3618×10(6). The haplotype of 16 Y chromosomal STR in the child matched completely with that of the father. These results suggested that the suspected father is the biological father of the child and that a rare three- or four-step microsatellite mutation had occurred in the paternal allele of FGA.

Development of a 9-locus X-STR multiplex PCR system for genetic analysis of three ethnic populations in China.

X-chromosome short tandem repeats (X-STR) analysis has been confirmed to be effective for kinship testing such as in deficiency paternity cases. The aim of this study was to develop a new multiplex polymerase chain reaction (PCR) system that can simultaneously amplify 9 X-STR loci (GATA172D05, DXS10159, DXS6797, HPRTB, DXS10079, DXS6789, DXS9895, DXS10146 and GATA31E08) in the same PCR reaction, and to obtain the database of the 9 X-STR loci in three ethnic populations in China. The genetic data of 815 (404 females and 411 males) unrelated Han Chinese from Hubei province, and Yi and Zhuang Chinese from Yunnan province were analyzed by using this multiplex system. The results showed that a total of 93 alleles for all these loci were found, and 7 to 20 alleles for each locus were observed. All of the analyzed loci were in agreement with Hardy-Weinberg equilibrium after Bonferroni correction in the three studied populations. The polymorphism information content (PIC) and power of discrimination (PD) in females were 0.6566-0.8531 and 0.8639-0.9684, respectively. Pairwise comparisons of allele frequency distribution showed significant differences in the most of these loci between different populations. The results indicate that this multiplex system is very useful for forensic analysis of different ethnic populations in China.

Multistep microsatellite mutation in a case of non-exclusion parentage.

A non-exclusion paternity with multistep mutation in the locus D5S818 was reported. Examination of 39 autosomal short tandem repeats (STR) loci revealed a mismatch of the maternally or paternally transmitted allele in the locus D5S818 in the questioned child. The composition of the alleles of this locus in the mother, the questioned child and the alleged father are 11/13, 7/13 and 13, respectively. The sequence analysis of the regions flanking the locus D5S818 of the mother, the questioned child and the alleged father excluded the possibility of null allele as a cause of the allelic mismatch in the child. The combined paternity index of 39 autosomal STRs is up to 2.461×10(9). Genotyping of sixteen Y-STR loci in the questioned child matched completely with the alleged father. The results prove that the alleged father is the biological father of the questioned child with four-step or six-step microsatellite mutation in the locus D5S818.

Age-related DNA methylation changes for forensic age-prediction.

There is no available method of age-prediction for biological samples. The accumulating evidences indicate that DNA methylation patterns change with age. Aging resembles a developmentally regulated process that is tightly controlled by specific epigenetic modifications and age-associated methylation changes exist in human genome. In this study, three age-related methylation fragments were isolated and identified in blood of 40 donors. Age-related methylation changes with each fragment was validated and replicated in a general population sample of 65 donors over a wide age range (11-72 years). Methylation of these fragments is linearly correlated with age over a range of six decades (r = 0.80-0.88). Using average methylation of CpG sites of three fragments, a regression model that explained 95 % of the variance in age was built and is able to predict an individual's age with great accuracy (R (2 )= 0.93). The predicted value is highly correlated with the observed age in the sample (r = 0.96) and has great accuracy of average 4 years difference between predicted age and true age. This study implicates that DNA methylation can be an available biological marker of age-prediction. Further measurement of relevant markers in the genome could be a tool in routine screening to predict age of forensic biological samples.

Isolation and identification of age-related DNA methylation markers for forensic age-prediction.

Age-prediction is an important part of forensic science. There is no available method of individual age-prediction for general forensic biological samples at crime scenes. Accumulating evidence indicates that aging resembles a developmentally regulated process tightly controlled by specific age-associated methylation exists in human genome. This study isolated and identified eight gene fragments in which the degree of cytosine methylation is significantly correlated with age in blood of 40 donors. Furthermore, we validated two CpG sites of each gene fragment and replicated our results in a general population sample of 40 males and 25 females with a wide age-range (11-72 years). The methylation of these fragments is linear with age over a range of six decades (Fragment P1 (r=-0.64), P2 (r=-0.58), P3 (r=-0.79), R1 (r=0.82), R2 (r=0.63), R3 (r=0.59), R4 (r=0.63) and R5 (r=0.62)). Using average methylation of two CpG sites from each fragment, we built a regression model that explained 95% of the variance in age and is able to predict the age of an individual with great accuracy (R(2)=0.918). The predicted values are highly correlated with the observed age in the sample (r=0.91). This study implicates that DNA methylation will be an available biological marker of age-prediction. Furthermore, measurement of relevant sites in the genome could be a tool in routine forensic screening to predict age of biological samples.

[Research progress on the phenotype informative SNP in forensic science].

Single nucleotide polymorphism (SNP) refers to the single base sequence variation in specific location of the human genome. Phenotype informative SNP has gradually become one of the research hot spots in forensic science. In this paper, the forensic research situation and application prospect of phenotype informative SNP in the characteristics of hair, eye and skin color, height, and facial feature are reviewed.

[Development of SNP multiplexes utilizing universal reporter primers for forensic purposes].

OBJECTIVE: To establish a simple, fast and economical technique for multiplex-typing SNPs and to explore its potential forensic application. METHODS: Five Y-SNP loci (IMS-JST164520, IMS-JST021354, IMS-JST003305, M119 and M134) were selected and the allele specific primers of each locus were designed with the universal reporter primers tailed at their 5' end. Alleles of these loci were amplified first by allele specific primers, then amplified by universal reporter primers tagged by fluorescent dye. RESULTS: A fluorescent-multiplex PCR system of the five Y-SNP loci was established. The typing results showed that two different colors of product peaks denoted two different alleles of a SNP locus, and the fragment sizes of alleles among different SNP loci were different. The haplotype diversity of these five loci was estimated to be 0.8655 in Wuhan Han population. CONCLUSION: The multiplex-typing SNPs based on the universal reporter primers is a simple, fast, and economical technique, and may have good application value in forensic medicine.

[Haplotype and heteroplasmy in the HV II region of mtDNA in human blood detected by PCR-DGGE].

OBJECTIVE: To establish a simple and effective technique for detecting haplotype and heteroplasmy of mtDNA, and investigate their frequencies in Chinese Han population. METHODS: The fragments from 29-290 nt of mtDNA HV II from peripheral leukocytes of 200 unrelated Wuhan Han individuals were analyzed by using PCR-DGGE technique. RESULTS: Seventeen haplotypes were found in the range of 29-290 nt, and the haplotype diversity (HD) was 0.8844. The heterogeneity was observed from 4 individuals, and its frequency was 2%. CONCLUSION: PCR-DGGE is a simple, sensitive and effective technique in analyzing polymorphism and heteroplasmy of mtDNA, and can be used in forensic practice.

[The application of PCR-SSCP in forensic mtDNA typing].

OBJECTIVE: To study the application of PCR-SSCP in forensic mtDNA typing. METHODS: Primers flanking the mtDNA HV-I and HV-II regions were designed. By PCR-SSCP techniques, 70 family trios and 140 unrelated Wuhan Han individuals were investigated and analyzed. RESULTS: In 70 family trios, the SSCP profiles in region HV-I and HV-II of children were not same to that of their fathers in 98.57% and 97.13% respectively but were identical with their mothers. In 140 unrelated Wuhan Han individuals, 21 haplotypes were found in HVI, GD = 0.9556; 16 haplotypes were found in HVII, GD = 0.9356. CONCLUSION: PCR-SSCP technique may be useful in forensic mtDNA typing, especially for screening the suspects.

[Detecting the haplotype of three Y-STR loci by fluorescent multiplex amplification].

OBJECTIVE: To establish a fluorescent multiplex PCR system for typing Y-STR loci Y-GATA-A7.1, DYS456 and DYS443, and investigate their haplotype frequencies in Chinese Han population. METHODS: 203 unrelated males of Han population living in Zhengzhou were typed by fluorescent multiplex amplification system and ABI 3100 genetic analyzer. RESULTS: In Zhengzhou Han population, 5,6 and 6 different alleles were observed for Y-GATA-A7.1, DYS456 and DYS443 loci, and their gene diversity (GD) were 0.669 2, 0.583 9 and 0.705 3 respectively. A total of 44 different haplotypes formed by these three loci was identified and the haplotype diversity (HD) reached 0.952 3. CONCLUSION: The fluorescent multiplex system for these three Y-STR loci will be very powerful for forensic individual identification and paternity testing in Chinese Han population.

[Genetic polymorphism of 23 Y chromosome biallelic markers in Wuhan Han population].

To search polymorphic Y chromosome biallelic markers in Chinese Han population, and obtain their population genetic data. Genotyping of 23 biallelic markers on human Y chromosome (M7, M9, M50, M88, M89, M95, M111, M117, M119, M121, M122, M134, M159, M164, M175, M214, LINE1, MSY2, RPS4Y711, SRY465, IMS-JST164520, IMS-JST021354 and IMS-JST003305) were carried out in a sample of 160 unrelated Chinese male individuals living in Wuhan using fragment length discrepant allele specific PCR (FLDAS-PCR) and PAGE technique. In all 23 biallelic markers, genetic polymorphism were identified for 20 loci in Wuhan Han population except for M50, M159 and M164, and the ranges of gene diversity (GD) were 0.0126-0.4855. A total of 35 different haplogroups (Hg1-35) were observed and the haplogroup diversity (HD) was 0.9471. The haplogroups formed by 20 biallelic markers are highly polymorphic, and can be used in forensic science and population evolution studies.

[A simple and rapid modified--new method for SNP typing by fragment length discrepant allele specific PCR].

OBJECTIVE: To establish a new method for single nucleotide polymorphism (SNP) typing based on allele specific PCR: fragment length discrepant allele specific PCR (FLDAS-PCR), and study the influence on specific extension by introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers. METHODS: For SNP loci rs759117 and rs760887, two allele specific forward primers, with different length and a mismatch introduced at the third or fourth 3'-terminal base, and a public reverse primer were designed for SNP typing. The genotyping of SNP was determined by the two allele specific fragments different in size after polyacrylamide gel and silver staining. RESULTS: The different homozygote genotypes comprised a single band with different size respectively, and the heterozygote genotypes comprised two bands. Typing results were completely consistent with those by direct sequencing. Non-specific primer extension was decreased remarkably after introducing a mismatch at the third or fourth 3'-terminal base of allele specific primers, and the stringency of PCR reaction was cut down. CONCLUSION: FLDAS-PCR is a simple, rapid and efficient new method for SNP typing. During FLDAS-PCR, specific primers with a mismatch at the third or fourth 3'-terminal base have more power to identify two alleles.

[The multiplex analysis of epigenetic markers and genetic markers by post-digestion mutagenically separated PCR].

OBJECTIVE: To establish a novel method for the multiplex analysis of the methylation and single nucleotide polymorphism (SNP). METHODS: The imprinted SNP rs220028 was chosen as a model. Genomic DNA, after being digested with methylation sensitive restriction enzyme, were typed by mutagenically separated PCR (MS-PCR). The polymorphism of restriction site was excluded by PCR-RFLP. RESULTS: By post-digestion MS-PCR, the methylated allele was detected selectively, the maternal origin of which was confirmed by pedigree analysis; A=0.5085, G=0.4915,PIC=0.3749. CONCLUSION: The multiplex analysis of methylation markers and SNP can be achieved by post-digestion MS-PCR. The imprinted SNP locus rs220028 is a potentially useful marker in screening Prader-Willi/Angelman syndrome.

[The expression of bFGF and its receptor FGFR1 in rat after fluid-percussive brain injury].

OBJECTIVE: Study on the pattern of changes of bFGF and FGFR1 immunoreactivity occurred in the experimental brain injury model for the purpose of providing the scientific basis for molecular pathological diagnosis, forensic identification, clinical treatment as well as further ascertaining the molecular mechanism of brain injury. METHODS: Male SD rats were divided into normal control, sham operation control and injury groups. The rats of injury groups were subjected to moderate lateral fluid percussion brain injury (0.2 mPa). The injury groups were then subdivided into 30 min, 1, 3, 6, 12 h, 1, 3, 7 d groups according to the time elapsed after injury. The SP immunohistochemistry method was used to examine the expression of both bFGF and FGFR1 factors in rat brain. RESULTS: In the brain of normal control and sham operation control groups, the low expression levels of bFGF and FGFR1 were observed. The increase of bFGF and FGFR1 immunoreactivity could be observed 6 h after injury in cortex and brain stem, reached to the peak at 1 d and remained at the high level up to 3 d, then partly declined at 7 d. In hippocampus, however, the increase occur as early as 3 h after injury, reached to the peak at 1 d and then decreased progressively, and returned to basal level at 7 d. CONCLUSION: The results suggested that brain injury induced the gene expressions of bFGF and FGFR1. The bFGF may contribute to maintenance of nerve cell survival and the repair of damaged neural tissues after CNS injury and the patterns of their level change were quite regular and can be used for timing of injury in forensic medicine aspect.

[Application of chi-square test and exact test in Hardy-Weinberg equilibrium testing].

This article review the application of chi-square test of various data handling methods and exact test in Hardy-Weinberg equilibrium testing of human genetic marker in population genetics. The importance of HWE-exact test in multiallelic system was emphasized, especially in the study of forensic VNTR and STR typing.

[Genetic polymorphism of the STR DXS9898 locus in Han population].

OBJECTIVE: Studying the genetic polymorphism of X-STR locus DXS9898 in Han population. METHODS: 296 unrelated Chinese individuals (199 females and 97 males) living in Chengdu were investigated using PCR and PAG electrophoresis followed by silver staining. RESULTS: 6 alleles were observed and the range of fragment size was 189-214 bp. The genotype distribution of DXS9898 locus was in accordance with Hardy-Weinberg equilibrium. Family survey confirmed Mendelian inheritance of alleles. The observed heterozygosity in females was 0.5930, the discriminating power (Dp) were 0.5667 and 0.9420 for males and females respectively. The power of exclusion were 0.5862 and 0.4392 for trio and duo respectively. CONCLUSION: The results demonstrated that the locus is highly polymorphic and can be used in forensic identification and parentage testing.