Improved age estimation from semen using sperm-specific age-related CpG markers.

Accurate age estimation from semen has the potential to greatly narrow the pool of unidentified suspects in sexual assault investigations. However, previous efforts utilizing semen age-related CpG (AR-CpG) markers have shown lower accuracy compared to blood AR-CpG-based methods. This discrepancy may be attributed to DNA methylation (DNAm) interferences from "round cells" such as leukocytes and immature sperm cells in semen. This study aimed to develop age calculators based on sperm-specific AR-CpG markers and to achieve performance-improved age estimates from sperm DNA. Through an analysis of publicly available MethylationEPIC microarray data from 90 sperm samples of healthy males aged 22-51 years, we identified 31 sperm-specific AR-CpG markers with absolute Pearson's R values > 0.5 and Benjamini-Hochberg adjusted p values < 0.013. The top 19 AR-CpG markers with the largest absolute R values and beta ranges > 0.10, along with 3 reported semen AR-CpG markers (cg06304190, cg06979108, and cg12837463), were integrated into two methylation SNaPshot panels (Ⅰ and Ⅱ), each containing 11 markers. The 21 qualified AR-CpG markers showed absolute R values ≥ 0.427 in an independent validation cohort of 253 sperm DNA samples (22-67 years), with cg21843517 exhibiting the strongest age correlation (R = 0.853). The optimal models, constructed using sperm DNAm data of the training set (n = 214, 22-67 years) and markers from panel Ⅰ (n = 11), panel Ⅱ (n = 10), or both panels, achieved mean absolute errors (MAEs) of 2.526-4.746, 3.890-5.715, and > 9.800 years on the test sets of sperm (n = 39, 23-64 years), semen (same donors as the sperm test set), and whole blood (n = 40, 22-65 years), respectively. The simplified models incorporating 3, 5, 9, or 14 AR-CpG markers (MAE = 2.918-4.139 years for sperm) still outperformed the Lee et al. original model (MAE = 6.444 years for semen) and the reconstructed panel Lee model (MAE = 6.011 years for sperm). The final models, utilizing all sperm DNAm data (n = 253) and markers from panel Ⅰ, panel Ⅱ, or both panels, yielded mean MAEs of 2.587, 2.766, and 2.200 years, respectively, on the 50 test sets generated by 5 repeats of 10-fold cross-validations. Additionally, multiple markers in both panels demonstrated the ability to discern sperm or semen from blood with 100% accuracy. In summary, our study substantiates the potential of sperm-specific AR-CpG markers for precise age estimation from sperm DNA, providing an improved toolset for forensic investigations.

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.

Exploring the forensic effectiveness and population genetic differentiation by self-constructed 41 multi-InDel panel in Yunnan Zhuang group.

Yunnan is one of the main residences of the Zhuang group which is one of the 55 ethnic minorities in China. At present, there are relatively few researches on population genetics and forensic science of the Yunnan Zhuang group. Therefore, this study used a self-constructed panel containing 41 multi-InDel markers to analyze the genetic polymorphisms of 173 individuals from Yunnan Zhuang group. The results indicated that these 41 multi-InDels in Yunnan Zhuang group were highly polymorphic markers expect for three markers. The cumulative match probability and combined exclusion probability values of the 40 multi-InDels (MI38 marker was excluded) were 8.0671E-26 and 0.9999995959, respectively. In addition, population genetic analyses were performed on genotyping data of 41 multi-InDel markers among the Yunnan Zhuang and 26 reference populations, revealing that the Yunnan Zhuang group was genetically close to the five populations in East Asia. According to the STRUCTURE analysis, the Yunnan Zhuang group presented similar ancestral compositions to the five populations from East Asia, and when the K value was three, the five intercontinental populations showed their different genetic structures. In conclusion, the 41 multi-InDel markers could be used as an effective tool for individual identification and paternity testing of the Zhuang group in Yunnan province, as well as for their ancestry information inference studies.

Synthesis of molecularly-imprinted polymers towards a group of amphetamine-type stimulants by reflux precipitation polymerization with a pseudo template.

Determination of amphetamine-type drugs (ATSs) in urine and wastewater is a simplified approach for the widespread monitoring of ATSs abuse. To improve the sensitivity of the analytical methods, molecularly imprinted polymers (MIPs) based solid-phase extraction (SPE) pretreatment attracted great attention in this field. Generally, smaller particle sizes and more uniform morphology of the MIPs could provide higher detection sensitivity. Our previous works showed reflux precipitation polymerization (RPP) is a method for synthesizing monodispersed MIPs with small particle size. However, synthesis of uniform spherical MIPs towards a group of targets has never been reported. Therefore, in the present work, MIPs towards a group of ATSs were synthesized via RPP with a pseudo template for the first time. After screening potential pseudo-templates, N-methylphenylethylamine (MPEA) was selected as the optimal pseudo-template. MPEA-MIPs were characterized by scanning electron microscope (SEM), FT-IR spectroscopy and X-ray photoelectron spectroscopy (XPS) spectra. Adsorption isotherms, adsorption kinetics and selectivity were evaluated, and the experimental results indicated that the MPEA-MIPs possessed good selectivity and adsorption property towards ATSs. After optimization of the MIP-SPE procedure, the MIP-SPE cartridges were then coupled with liquid chromatography and tandem mass spectrometry (LC-MS/MS) for determination of ATSs. The evaluation results showed that MIP-SPE-LC-MS/MS displayed good linearity (R2 >0.991) in the linear range (1.0-50.0 µg/L for urine and 0.5-50.0 µg/L for wastewater), and low matrix effect (85-112%). The limit of detection (LOD) was 0.05 -0.29 µg/L, and the accuracy (85-115%) and repeatability (RSD ≤ 15%) were satisfactory at low, medium and high concentrations. To the best of our knowledge, this is the first time that dummy MIPs towards a group of ATSs were synthesized by RPP polymerization, which showed great potential for the detection of ATSs in urine and wastewater.

Accurate age estimation from blood samples of Han Chinese individuals using eight high-performance age-related CpG sites.

Age-related CpG sites (AR-CpGs) are currently the most promising biomarkers for forensic age estimation. In our previous studies, we first validated the age correlation of seven reported AR-CpGs in blood samples of Chinese Han population. Subsequently, we screened some good age predictors from blood samples of Chinese Han population, and built pyrosequencing-based age prediction models. However, it is still important to select a set of high-performance AR-CpGs in a specific racial group and establish a simple and efficient method for accurate age estimation for forensic purpose. In this study, eight AR-CpGs, namely chr6: 11,044,628 (ELOVL2), cg06639320 (FHL2), chr1: 207,823,723 (C1orf132), cg19283806 (CCDC102B), cg14361627 (KLF14), cg17740900 (SYNE2), cg07553761 (TRIM59), and cg26947034, were selected based on our previous studies, and a multiplex methylation SNaPshot assay was developed to investigate DNA methylation levels at these AR-CpGs in 529 blood samples (aged 2-82 years) from Han Chinese population. All selected CpG sites showed strong age correlation with the correlation coefficient (r) from 0.8363 to 0.9251. Multiple linear regression (MLR) and support vector regression (SVR) age prediction models were simultaneously established to fit change characteristics of DNA methylation levels of eight AR-CpGs with the age in 374 donors' blood samples. The MLR model enabled age prediction with R2 = 0.923, mean absolute error (MAE) = 3.52, while the SVR model enabled age prediction with R2 = 0.935, MAE = 2.88. One hundred fifty-five independent samples were used as a validation set to test the two models' performance, and the prediction MAE for the validation set was 3.71 and 3.34 for the MLR and SVR models, respectively. For the MLR and SVR models, the correct prediction rate at ± 5 years reached a high level of 79.35% and 83.23%, respectively. In general, these statistical parameters indicated that the SVR model outperformed the MLR model in age prediction of the Han Chinese population. In addition, our method provides sufficient sensitivity in forensic applications and allows for 100% efficiency when examining bloodstains kept in room conditions for up to 43 days. These results indicate that our multiplex methylation SNaPshot assay is a reliable, effective, and accurate method for age prediction in blood samples from the Chinese Han population.

Assessment of Multiple Annealing and Looping-Based Amplification Cycle-Based Whole-Genome Amplification for Short Tandem Repeat Genotyping of Low Copy Number-DNA.

Aim: A common problem in forensic practice is the lack of sufficient amounts of good quality genomic DNA. A possible solution is the amplification of the available genomic DNA before locus-specific polymerase chain reaction (PCR) analysis. The aim of this study was to evaluate multiple annealing and looping-based amplification cycle (MALBAC)-based whole-genome amplification (WGA) for short tandem repeat (STR) genotyping of low copy number DNA (LCN-DNA). Materials and Methods: DNA isolated from five blood samples was quantified and diluted to 250, 150, 100, 50, 25, and 5 pg/µL. After preamplification with MALBAC, WGA products were quantified. PCR-STR genotyping was performed in triplicate using dilution or purification-treated WGA products for each level of DNA. STR profiles were analyzed and compared with that from non-WGA DNA. Results: The purification treatment performed better than dilution of the MALBAC-based WGA products. Compared with the non-WGA DNA, both the average number and peak heights of correct alleles were significantly improved after preamplification with the MALBAC-based WGA at DNA inputs of ≤50 pg. Like other WGA methods, allele dropout and allele drop-in were observed in the profiling results for many samples. Conclusions: MALBAC shows great potential in LCN-DNA analysis and could find broader application in the fields of forensics and genetics.

A novel multiplex assay system based on 10 methylation markers for forensic identification of body fluids.

Identifying the source of body fluids found at a crime scene is an essential forensic step. Some methods based on DNA methylation played significant role in body fluids identification. Since DNA methylation is related to multiple factors, such as race, age, and diseases, it is necessary to know the methylation profile of a given population. In this study, we tested 19 body fluid-specific methylation markers in a Chinese Han population. A novel multiplex assay system based on the selected markers with smaller variation in methylation and stronger tissue-specific methylation were developed for the identification of body fluids. The multiplex assay were tested in 265 body fluid samples. A random forest model was established to predict the tissue source based on the methylation data of the 10 markers. The multiplex assay was evaluated by testing the sensitivity, the mixtures, and old samples. For the result, the novel multiplex assay based on 10 selected methylation markers presented good methylation profiles in all tested samples. The random forest model worked extremely well in predicting the source of body fluids, with an accuracy of 100% and 97.5% in training data and test data, respectively. The multiplex assay could accurately predict the tissue source from 0.5 ng genomic DNA, six-months-old samples and distinguish the minor component from a mixture of two components. Our results indicated that the methylation multiplex assay and the random forest model could provide a convenient tool for forensic practitioners in body fluid identification.

Mutation analysis of 28 autosomal short tandem repeats in the Chinese Han population.

Short tandem repeats (STRs) have been extensively used in forensic genetics. However, according to previous studies, the mutation rates of STRs are relatively high and are affected by many factors. Therefore, it is important to analyze STR mutations and determine the influence of underlying factors on STR mutation rates. Mutation rates of 28 autosomal STRs were determined from 8708 paternity testing cases in the Chinese Han population, and the relationships between STR mutation rates and population, sex, age, allele length and heterozygosity were investigated. A total of 279 mutations were observed at 27 loci in a total of 233,530 meiosis cases, including 273 (97.8%) one-step, 5 (1.8%) two-step and 1 (0.4%) three-step mutations. The overall average mutation rate was 1.19 × 10-3 (95% CI 1.06 × 10-3 - 1.34 × 10-3) ranging from 0 (TPOX) to 2.79 × 10-3 (D13S325). Mutation rate comparisons revealed statistically significant differences at several STRs among populations. Paternal mutations occurred more frequently than maternal mutations, at a ratio of 6.04:1, and the mutation rate tended to increase with paternal age. Moreover, our study revealed a bias towards contraction mutations for long alleles and expansion mutations for short alleles. No obvious bias was observed in the overall mutation direction. In addition, STR loci with higher expected heterozygosity (Hexp) tended to have higher mutation rates. This work revealed the relationships between STR mutation rates and several influencing factors, providing useful data and information for further research on STR mutations in forensic genetics.

Genome-wide identification of age-related CpG sites for age estimation from blood DNA of Han Chinese individuals.

Age-related CpG (AR-CpG) sites are currently the most promising molecular markers for forensic age estimation. However, the AR-CpG sites of Han Chinese population remains to be systematically characterized. In this study, we performed genome-wide methylation analyses on 42 whole blood DNA from healthy Han Chinese volunteers (aged from 18 to 62 years) using the Illumina MethylationEPIC BeadChip microarray. As expected, both known and novel AR-CpG sites were identified. Considering the sex difference in aging rate, we then separately selected AR-CpG candidates and built pyrosequencing-based multiple linear regression models for age estimation of males and females. The model constructed from the male sample group (n = 167, aged from 1.50 to 85.71 years) explained 95.22% of variation in age using five AR-CpG sites (chr6:11044864 ELOVL2, chr1:207997068 C1orf132, cg19283806 CCDC102B, cg17740900, and chr10:73740306 CHST3) and yielded a mean absolute error (MAE) of 2.79 years. The model constructed from the female sample group (n = 141, aged from 3.33 to 80.38 years) explained 94.90% of variation in age with six AR-CpG sites (chr6:11044867 ELOVL2, chr1:207997060 C1orf132, chr2:106015757 FHL2, cg26947034, chr16: 67184108 B3GNT9, and chr20:44658203 SLC12A5) and yielded an MAE of 2.53 years. Besides, the estimated age was highly correlated with the actual age (R > 0.97). The robustness of these AR-CpG markers was demonstrated by 10-fold cross-validations. In conclusion, we updated the AR-CpG sites of Han Chinese population and provided two sets of AR-CpG sites for accurate age estimation.

Genetic analysis of 32 InDels in four ethnic minorities from Chinese Xinjiang.

The present study used the previously constructed 32-plex InDels panel to investigated the genetic diversity of four ethnic minorities (Hui, Mongol, Uygur and Kazakh) from Xinjiang, and analyzed the genetic relationships between the four populations and 27 reference populations. No significant deviations were observed from the Hardy-Weinberg equilibrium (HWE) at the 32 InDels for each population. The average observed heterozygosity (Hexp), average polymorphic information content (PIC), combined power of discrimination (CPD) and cumulative probability of exclusion (CPE) for the 32 InDels were all higher than the Qiagen Investigator DIPplex kit in the four populations from Xinjiang. The CPD ranged from 0.999999999999903 (Kazakh) to 0.999999999999952 (Hui) and CPE ranged from 0.9971 (Uygur) to 0.9985 (Hui), which indicated that the 32 InDels were capable for individual identification and could be a supplementary tool in paternity test for these populations. Population genetic analysis by the method of analysis of molecular variance (AMOVA), FST, phylogenetic tree, TreeMix-based topology, multi-dimensional scale analysis (MDS), principal components analysis (PCA) and STRUCTURE analysis showed that Xinjiang Hui population has a close relationship with East Asians (EAS), especially Chinese Han, and the populations of Xinjiang Mongol, Uygur and Kazakh showed mixed ancestral components related to EAS and Europeans (EUR).

Lighting up forensic science by aggregation-induced emission: A review.

Forensic science requires a fast, sensitive, and anti-interfering imaging tool for on-site investigation and bio-analysis. The aggregation-induced emission (AIE) phenomenon exhibits remarkable luminescence properties (large Stokes shift, diverse molecular structures, and high photo-stability), which can provide a viable solution for on-site analysis, while at the same time overcoming the problem of aggregation-caused quenching (ACQ). Based on the outstanding performance in chemical analysis and bio-sensing, AIE materials have great prospects in the field of forensic science. Therefore, the application of AIE in forensic science has been summarized for the first time in this article. After a brief introduction to the concept and development of AIE, its applications in the determination of toxic or hazardous substances, based on data on poisoning deaths, has been summarized. Subsequently, besides the bio-imaging function, other applications of AIE in analyzing markers related to forensic genetics, forensic pathology, (focusing on the corpse) and clinical forensics (focusing on the living) have been discussed. In addition, applications of AIE molecules in criminal investigations, including recognition of fingerprints and blood stains, detection of explosives and chemical warfare agents, and anti-counterfeiting have also been presented. It is hoped that this review will light up the future of forensic science by stimulating more research work on the suitability of AIE materials in advancing forensic science.

The evaluation of seven age-related CpGs for forensic purpose in blood from Chinese Han population.

Age prediction of biological samples is one of the important tasks in forensic DNA phenotyping, and DNA methylation is regarded as the most promising biomarker for forensic age prediction. To date, numerous CpG sites have been reported to be age-related based on the changes in methylation. In this study, seven age-related CpG (AR-CpG) sites, cg02228185 (ASPA), cg09809672 (EDARADD), cg19283806 (CCDC102B), cg04208403 (ZNF423), chr17: 44,390,358 of GRCh38/hg38 (ITGA2B), cg14361627 (KLF14), and cg06639320 (FHL2), were selected and analyzed in 310 blood samples using a multiplex methylation SNaPshot assay to evaluate the value of selected AR-CpGs in age prediction in blood from Chinese Han population. The study confirmed the correlation of all the investigated markers with human age, and the correlation of cg19283806 with age is the highest while cg04208403 is the lowest in the Chinese Han population. Two different age prediction models, stepwise regression and support vector regression (SVR), were established based on the methylation SNaPshot data using 230 blood samples from donors aged 2-86 years old. The stepwise regression model included six AR-CpGs (except cg09809672) and enabled age prediction with R2 = 0.85, mean absolute deviation (MAD) = 4.22, while the SVR model enabled age prediction with R2 = 0.86, MAD = 4.01. An independent set of 80 samples was used to test the two models' performance and the prediction MAD for the validation set was 4.71 and 4.56 for the stepwise regression and SVR models, respectively. The number of correct predictions for ±5 years achieved a high level of 67.50 % and 73.75 %, respectively for the stepwise regression and SVR models. In general, the SVR model was superior to the linear regression model in age prediction. These results suggest that these seven CpG sites would be useful for age prediction in blood samples from the Chinese Han population.

Circulating tumor DNA methylation profiles enable early diagnosis, prognosis prediction, and screening for colorectal cancer.

Circulating tumor DNA (ctDNA) has emerged as a useful diagnostic and prognostic biomarker in many cancers. Here, we conducted a study to investigate the potential use of ctDNA methylation markers for the diagnosis and prognostication of colorectal cancer (CRC) and used a prospective cohort to validate their effectiveness in screening patients at high risk of CRC. We first identified CRC-specific methylation signatures by comparing CRC tissues to normal blood leukocytes. Then, we applied a machine learning algorithm to develop a predictive diagnostic and a prognostic model using cell-free DNA (cfDNA) samples from a cohort of 801 patients with CRC and 1021 normal controls. The obtained diagnostic prediction model discriminated patients with CRC from normal controls with high accuracy (area under curve = 0.96). The prognostic prediction model also effectively predicted the prognosis and survival of patients with CRC (P < 0.001). In addition, we generated a ctDNA-based molecular classification of CRC using an unsupervised clustering method and obtained two subgroups of patients with CRC with significantly different overall survival (P = 0.011 in validation cohort). Last, we found that a single ctDNA methylation marker, cg10673833, could yield high sensitivity (89.7%) and specificity (86.8%) for detection of CRC and precancerous lesions in a high-risk population of 1493 participants in a prospective cohort study. Together, our findings showed the value of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of CRC.

Characterisation, verification and genetic basis of anomalous STR patterns: a report of four cases of X-chromosome STR biallelic patterns in human males.

Analysis of the characteristics and genetic basis of the anomalous short tandem repeat (STR) pattern encountered in forensic cases has been shown to be useful for analysing STR profiles in routine forensic casework. Here, we report biallelic patterns at several X-chromosome STR (X-STR) loci in human males revealed by forensic parameters investigation using the commercial AGCU X19 Kit. The presence of these patterns was verified by reanalysis using new samples and bidirectional Sanger sequencing of the singleplex polymerase chain reaction (PCR) products. And the genetic basis for their production was inferred based on the relative peak heights at the amelogenin locus and the affected locus (DXS10159, DXS10134 and DXS10079) and the normalised peak height ratios between the affected locus and adjacent loci relative to the control sample 9947A. The inference results suggested that two cases of biallelic pattern at the DXS10159 locus would be caused by local duplications, while in the other two cases, both the biallelic patterns at loci DXS10134 and DXS10079 would be due to somatic mutations. One case where the male showed a biallelic pattern at the DXS10159 locus (Xp11.21) was further analysed. Quantitative PCR (qPCR) revealed a microduplication (< 0.2 Mb) spanning at least 13.9 kb in Xp11.21 encompassing the DXS10159 locus. Finally, a workflow for analysing anomalous STR patterns was summarised. In conclusion, this study is a detailed report of X-STR biallelic patterns in human males, which serves as an effective complement to the database and provides an example for the analysis of anomalous STR patterns.

Development of a new 32-plex InDels panel for forensic purpose.

Insertion/deletion polymorphisms (InDels) have attracted more and more attention of forensic researchers because of their low mutation rate and small amplicons. In the face of challenging forensic cases and degraded DNA, InDels break through the limitations of traditional STRs and provided a new direction for forensic identification. In this study, a multiplex panel consisting of 32 InDels and amelogenin was established and the InDels were selected with minimum allele frequencies (MAF) ≥ 0.4 in Chinese Han population based on the 1000 Genomes Project and published articles. The 32-plex InDels assay was evaluated using the southern Chinese Han population from Hubei province. All markers were proved to be highly polymorphic with an average observed heterozygosity (Hobs) of 0.4885. The combined power of discrimination (CPD) and the combined probability of paternity exclusion (CPE) were 0.999999999999966 and 0.9982, respectively. Further validation studies indicated that the 32-plex InDels assay was highly sensitive to DNA template amounts as low as 31.25 pg and was effective for degraded DNA. The obtained allele frequencies from this study were compared with published data of other populations.

Differences of microRNA expression profiles between monozygotic twins' blood samples.

Monozygotic (MZ) twins are widely regarded as genetically identical, and traditional DNA typing methods are insufficient in identifying MZ twins. So the discrimination of MZ twins become a forensic problem. MicroRNAs (miRNAs) are a class of small, endogenous, non-protein-coding RNA molecules of approximately 22 nucleotides in length, and exist extensively in a variety of eukaryotic cells. MiRNAs regulate gene expression and play fundamental roles in multiple biological processes, including cell differentiation, proliferation and apoptosis as well as aging and disease processes. The goal of this study is to explore the differential expression of miRNAs within MZ twin pairs, and aimed to find new biomarkers for distinguishing MZ twins. Thus, the miRNA expression profiles of seven pairs of healthy MZ twins of different sex and age were analyzed by miRNA microarray. A total of 545 miRNAs were found to be differentially expressed in these MZ twin pairs, and 2, 5, 22, 53 and 132 differentially expressed miRNAs were shared across six, five, four, three and two pairs of MZ twins respectively. These findings had been confirmed by real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays on select miRNAs, including miR-151a-3p, miR-3653-3p, miR-142-3p, miR-4325, miR-16-5p, let-7i-5p, miR-222-3p, miR-550b-3p, miR-4791 and miR-27a-3p. The results demonstrated that there are differences in the expression of miRNAs within MZ twin pairs, suggesting a role of miRNAs in identifying MZ twins.

Microdeletion at 8q24.13 rather than multistep microsatellite mutation resulting in the genetic inconsistency at the D8S1179 locus in a true trio.

When using microsatellite loci for DNA paternity testing, genetic inconsistencies sometimes occur in true trios and duos and may be erroneously attributed to germline mutations of microsatellite alleles. Here, we reported a typical case and discussed the issue of how to find out the cause of a genetic inconsistency. In our case, a genetic inconsistency in a true trio was observed at the D8S1179 locus, where the father has only allele 10 as compared to only allele 16 of his son. A set of tests were then performed. The results showed that the inconsistency was not result from the germline mutation of allele 10 to allele 16, or from the presence of null alleles due to primer binding site mutations, but from the microdeletion at 8q24.13, about 2.99 to 49.76 kb, detected in both the father and his son, which revealed by deletion mapping using short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs). In conclusion, genetic inconsistencies observed in true trios or duos cannot be rashly attributed to germline STR mutations, especially multistep mutations, in the absence of verification or specification; otherwise, the reliability of the genetic proofs established will be challenged.

Comparison of different methods for repairing damaged DNA from buffered and unbuffered formalin-fixed tissues.

Formalin fixation is considered an important process for preservation of human tissue samples for long periods. However, this process not only results in cross-linking complicating isolation of nucleic acid but also introduces polymerase "blocks" during polymerase chain reaction (PCR). At present, many protocols have already been developed aiming at extracting high amounts of amplifiable DNA from formalin-fixed tissues (FFTs). However, there are few methods for repairing formalin-damaged DNA. In this study, we compared the effectiveness of several post-extraction enzymatic repair techniques, including Taq DNA polymerase, DNA polymerase I and T4 DNA ligase, the PreCR™ Repair Mix and Restorase® DNA Polymerase, in restoring STR profiles from formalin-damaged DNA. Our results indicated that formalin-damaged DNA may be repaired partly with Taq DNA polymerase and the Restorase® DNA Polymerase, and lost alleles may be restored and STR peak heights may increase upon repair with them. Moreover, the repair ability of the protocol 2 with Taq DNA polymerase surpasses the Restorase® DNA Polymerase.

An optimized rapid bisulfite conversion method with high recovery of cell-free DNA.

BACKGROUND: Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA. RESULTS: We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods. CONCLUSIONS: The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.

Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma.

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.