Methylation-Based Age Prediction Using Pyrosequencing Platform from Seminal Stains in Han Chinese Males

Various methods have been performed to predict an unknown individual's age from biological traces in forensic investigations. A considerably accurate age prediction for the semen donor can help narrow down the search in a sexual assault case. The aim of this study was to develop an assay for age prediction from seminal stains in Han Chinese males. We built a sperm-specific linear regression model using bisulfite pyrosequencing. Validations were conducted with a Mean Absolute Deviation from the chronological age (MAD) of 4.219 years in liquid semen, 4.158 years in fresh seminal stains, 4.393 years in aged seminal stains, and 3.880 years in mixed stains, respectively. Furthermore, our strategy enables accurate age prediction using a forensic casework sample. The strategy indicated that we produced an accurate and reliable age prediction tool for the semen donors in Han Chinese males for forensic purposes.     

Whole Plastome Sequences of Two Drug-Type Cannabis: Insights Into the Use of Plastid in Forensic Analyses

DNA is one of the fastest growing tools in forensic sciences, increasing reliability in forensic reports and judgments. The use of DNA has increased in different areas of the forensic sciences, such as investigation of plant species, where plastid DNA has been used to elucidate and generate evidence in cases of traceability of genetically modified and controlled plants. Even with several advances and the practice of using DNA in forensic investigations, there are just few studies related to the identification of genetic tools for the characterization of drug and nondrug-types of Cannabis. Herein, the whole plastomes of two drug-type Cannabis are presented and have their structures compared with other Cannabis plastomes deposited in the GenBank, focusing in the forensic use of plastome sequences. The plastomes of Cannabis sativa “Brazuka” and of the hybrid Cannabis AK Royal Automatic presented general structure that does not differs from the reported for other C. sativa cultivars. A phylogenomic analyses grouped C. sativa “Brazuka” with the nondrug C. sativa cultivars, while the hybrid Cannabis AK Royal Automatic placed isolated, basal to this group. This suggests that the analysis of plastomes is useful toward genetic identification of hybrids in relation to C. sativa.     

Bone Mineral Density Adult Age Estimation in Forensic Anthropology: A Test of the DXAGE Application

Estimating age‐at‐death of individuals represented only by skeletonized human remains is a fundamental aspect of forensic anthropological casework. Recently, several researchers have proposed that bone mineral density (BMD) is a useful predictor of age‐at‐death in forensic contexts. Navega et al. (JFS 63(2):497–503) developed an online application called DXAGE for calculating age‐at‐death from BMD parameters. This study tests the utility of DXAGE by utilizing data from the National Health and Nutrition Examination Survey (NHANES). BMD data from a female subsample (n = 470) of the NHANES 2007–2008 dataset were analyzed, and the relationship between predicted age and real age was examined. Inaccuracy was 14.25 years, and bias was ?7.20 years. Results show that there is a weak correlation between predicted and actual age (r = 0.47) using the DXAGE application. While BMD data are potentially useful for predicting age age‐at‐death, the DXAGE application should be used cautiously in forensic anthropological contexts.     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.     

Morphoscopic Trait Expression in “Hispanic” Populations

This study evaluates population variation of eight cranial morphoscopic traits using samples of known southwest Hispanics (n = 72), Guatemalans (n = 106), American Blacks (n = 146), and American Whites (n = 218). We applied the support vector machine (SVM) method to build a prediction model based on a subsample (20%) of the data; the remainder of the data was used as a test sample. The SVM approach effectively differentiated between the four groups with correct classification rates between 72% (Guatemalan group) and 94% (American Black group). However, when the Guatemalan and southwest Hispanic samples were pooled, the same model correctly classified all groups with a higher degree of accuracy (American Black = 96%; American White = 77%; and the pooled Hispanic sample = 91%). This study also identified significant differences between the two Hispanic groups in six of the eight traits using univariate statistical tests. These results speak to the unique population histories of these samples and the current use of the term “Hispanic” within forensic anthropology. Finally, we argue that the SVM can be used as a classification model for ancestry estimation in a forensic context and as a diagnostic tool may broaden the application of morphoscopic trait data for the assessment of ancestry.     

人类反转座核元件甲基化研究进展

人类反转座核元件LINE-1序列和Alu序列甲基化水平的变化与多种疾病的发生、发展有密切的联系,因此是相关疾病,尤其肿瘤和癌症预测、诊断、治疗及预后的新标记。近年来,二者在法医学方面的研究也逐渐展开,主要集中在同卵双生子的鉴别、年龄的鉴定、组织的鉴定等方面,有望成为法医DNA分析领域新一代的遗传标记。本文综述LINE-1序列和Alu序列甲基化在医学、法医学等领域的研究进展,希望能为相关研究和应用提供参考。     

DNA in the Criminal Justice System: The DNA Success Story in Perspective,

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high‐volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high‐volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high‐volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high‐volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool.     

Age Estimation in Living Egyptians Using Signal Joint T‐cell Receptor Excision Circle Rearrangement

Age estimation is one of the challenges in forensic sciences. There are many techniques to estimate the age. Molecular biology approach is one of these techniques. Signal joint T‐cell receptor excision circles gene (sjTRECs), is one of this approach. We aimed to use sjTRECs as a suitable marker for age estimation among Egyptian population. TaqMan qPCR approach was used to quantify sjTREC levels in blood samples obtained from 153 healthy Egyptian individuals ranging from a few weeks to 70 years. Our results showed a significant negative correlation between sjTREC levels and age with p ≤ 0.05. Moreover, the individual's age can be determined through this formula Age = ?30.671+ (?5.998Y) (Y is dCtTBP ? sjTREC) with standard error ±7.35 years. Within the forensic context, sjTREC' levels can be used to estimate the Egyptian individual's age accurately.     

Direct PCR Improves the Recovery of DNA from Various Substrates

This study reports on the comparison of a standard extraction process with the direct PCR approach of processing low-level DNA swabs typical in forensic investigations. Varying concentrations of control DNA were deposited onto three commonly encountered substrates, brass, plastic, and glass, left to dry, and swabbed using premoistened DNA-free nylon FLOQswabs^™. Swabs (n = 90) were either processed using the DNA IQ^™ kit or, for direct PCR, swab fibers (~2 mm²) were added directly to the PCR with no prior extraction. A significant increase in the height of the alleles (p < 0.005) was observed when using the direct PCR approach over the extraction methodology when controlling for surface type and mass of DNA deposited. The findings indicate the potential use of direct PCR for increasing the PCR product obtained from low-template DNA samples in addition to minimizing contamination and saving resources.     

Forensic Dental Age Estimation by Measuring Root Dentin Translucency Area Using a New Digital Technique

Dentin translucency measurement is an easy yet relatively accurate approach to postmortem age estimation. Translucency area represents a two‐dimensional change and may reflect age variations better than length. Manually measuring area is challenging and this paper proposes a new digital method using commercially available computer hardware and software. Area and length were measured on 100 tooth sections (age range, 19–82 years) of 250 μm thickness. Regression analysis revealed lower standard error of estimate and higher correlation with age for length than for area (R = 0.62 vs. 0.60). However, test of regression formulae on a control sample (n = 33, 21–85 years) showed smaller mean absolute difference (8.3 vs. 8.8 years) and greater frequency of smaller errors (73% vs. 67% age estimates ≤ ±10 years) for area than for length. These suggest that digital area measurements of root translucency may be used as an alternative to length in forensic age estimation.     

Assessing Carbon and Hydrogen Isotopic Fractionation of Diesel Fuel n-alkanes During Progressive Evaporation

Compound-specific isotope analysis offers potential for fingerprinting of diesel fuels, however, possible confounding effects of isotopic fractionation due to evaporation need to be assessed. This study measured the fractionation of the stable carbon and hydrogen isotopes in n-alkane compounds in neat diesel fuel during evaporation. Isotope ratios were measured using a continuous flow gas chromatograph/isotope ratio mass spectrometer. Diesel samples were progressively evaporated at 24 ± 2°C for 21 days. Increasing depletion of deuterium in nC₁₂–nC₁₇ alkanes in the remaining liquid with increasing carbon chain length was observed. Negligible carbon isotope fractionation was observed. Preferential vaporization was measured for the shorter chain n-alkanes and the trend decreased with increasing chain length. The decrease in δ²H values indicates the preferential vaporization of the isotopically heavier species consistent with available quantitative data for hydrocarbons. These results are most important in the application of stable isotope technology to forensic analysis of diesel.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

Shells and Bones: A Forensic Medicine Study of the Association of Terrestrial Snail Allopeas micra with Buried Human Remains in Brazil

Little is known regarding the scavenger fauna associated with buried human corpses, particularly in clandestine burials. We report the presence of 20 shells of the terrestrial snail Allopeas micra, within hollow bones of human remains buried for 5 years, during the process of collecting DNA material. The fact that a large number of shells of A. micra had been found in the corpse and in the crime scene supports the assumption that there was no attempt to remove the corpse from the area where the crime occurred. Despite this, our observations cannot be used to estimate the postmortem interval because there is no precise knowledge about the development of this species. This is the first record of a terrestrial snail associated with a human corpse and its role in this forensic medicine case.     

Forensic Autopsies can Determine Latent Prostate Cancer Prevalence

To evaluate the usefulness of forensic autopsies in determining latent prostate cancer (PC) prevalence, we examined latent PC prevalence from autopsies and compared our findings between decedents with and without cancer. Data from forensic autopsies performed in Japan from 2004 to 2014 were obtained. For each prostate, histopathological examinations were performed in both the base and the apex sections. Three hundred and seventeen Japanese decedents were selected for analysis. The mean age of decedents was 56.4 ± 17.8 years (range, 14–94 years). Among this population, 39.4% died suddenly of disease and 60.6% died of external causes. Latent PC was identified in 45 (14.2%) decedents, who ranged from 27 to 93 years old (mean, 71.1 ± 12.9 years). The prevalence of clinically significant PC with a Gleason score of 7 or more was 8.8%, and the rate increased with age. Fifteen males had cancers other than PC. The prevalence of overall latent PC was significantly higher for those with cancer compared with those without (40.0% vs. 12.9%; P = 0.003). In this study, the use of forensic autopsy materials provided the opportunity to obtain a more accurate natural history of PC, as the decedents in this situation would have been more likely to have died suddenly while behaving as normal prior to death, and less likely to have been impacted by long-term medical interventions.     

A Comparative Assessment of the Chen et al. and Suchey‐Brooks Pubic Aging Methods on a North American Sample, ,

Accurately estimating the age‐at‐death of adult human skeletons is fundamental in forensic anthropology. This study evaluates the accuracy of two pubic bone age estimation methods—Chen et al. and Suchey‐Brooks. Specimens were obtained from a known collection of modern pubic bones curated at the Maricopa County Forensic Science Center in Phoenix, Arizona. A sample of 296 left male pubic bones of European ancestry was statistically evaluated via bias, absolute mean error, and intra‐ and inter‐observer error. Results indicate that the two methods are similar; the Suchey‐Brooks method is the most accurate for aging young adults (error c. 7 years), while the Revised Chen et al. method is most accurate for aging middle‐age adults (error c. 6 years). Thus, the Chen et al. method is an important contribution to forensic anthropology for aging older adult skeletal remains. There are, however, some limitations such as subjectivity and the intricate scoring system of Chen et al. method.     

A Test of Sex Estimation in Subadults Using the Elevation of the Auricular Surface from Four Samples of Known Age and Sex

Biological sex is foundational to the work of forensic anthropologists and bioarcheologists. The lack of reliable biological sex estimation methods for subadults has, thus, greatly limited forensic and bioarcheological analyses. Auricular surface elevation showed promise as a subadult sex estimation method in previous studies. This study examined two auricular surface elevation evaluation methods on four subadult samples of known age, sex, and ancestry. Samples were scored as “male,” “female,” or “indeterminate” and results were examined with chi‐square analysis. No consistent sex estimation pattern, accuracy, or predictive value was produced between samples. Only one test was significant using Fisher's exact test analysis (FET = 7.501, p < 0.022): the composite approach on the Hamann‐Todd sample. While age, sample size, or developmental factors may play a role in these results, clearly sample variation does as well. This study found auricular surface elevation was not a useful subadult sex estimation method.     

A State Census of Unsubmitted Sexual Assault Kits: Comparing Forensic DNA Testing Outcomes by Geographic and Population Density Characteristics*

A growing number of U.S. cities and states have large numbers of unsubmitted sexual assault kits (SAKs) in police property facilities. Prior research conducted in large urban cities has found that testing these kits yields a sizable number of DNA profiles that meet FBI eligibility for upload to the national criminal DNA database CODIS (Combined DNA Index System) and uploaded profiles return a substantial number of matches to existing criminal profiles in CODIS. It is unknown whether these findings are unique to large urban cities with high crime rates. The purpose of current study was to document forensic testing outcomes from a state census of previously unsubmitted SAKs, which included large urban–suburban centers, as well as smaller cities and rural counties. We inventoried all previously unsubmitted SAKs in Michigan (N = 3422 SAKs) and submitted all kits for forensic DNA testing. A total of n = 1239 SAKs had a DNA profile that met eligibility for upload into CODIS (36.2% unconditional, 56.5% conditional CODIS eligible rate) and n = 585 SAKs yielded a CODIS Hit (17.1% unconditional, 47.2% conditional CODIS hit rate). These rates are consistent with studies from urban areas suggesting approximately half of SAKs tested yield a CODIS profile and approximately half of those uploaded profiles yield a hit. We compared SAK forensic testing outcomes by geographic and population density characteristics, and although rates were often higher in larger metropolitan areas, the obtained rates in micropolitan and rural areas suggest testing is warranted in smaller jurisdictions as well.     

A Test of the Passalacqua Age at Death Estimation Method Using the Sacrum

A test of the accuracy of the Passalacqua (J Forensic Sci, 5, 2009, 255) sacrum method in a forensic context was performed on a sample of 153 individuals from the J.C.B. Grant Skeletal Collection. The Passalacqua (J Forensic Sci, 5, 2009, 255) method assesses seven traits of the sacrum using a 7‐digit coding system. An accuracy of 97.3% was achieved using the Passalacqua (J Forensic Sci, 5, 2009, 255) method to estimate adult skeletal age. On average each age estimate differed by 12.87 years from the known age. The method underestimated the age of individuals by an average of 4.3 years. An intra‐observer error of 6.6% suggests that the method can be performed with precision. Correlation and regression analysis found that the sacral traits used in the Passalacqua (J Forensic Sci, 5, 2009, 255) method did not have a strong relationship with age or an ability to strongly predict age. Overall, the method was not practical for use in a forensic context due to the broad age ranges, despite the high accuracy and low intra‐observer error.     

A New Robust Epigenetic Model for Forensic Age Prediction

Forensic DNA phenotyping refers to an emerging field of forensic sciences aimed at the prediction of externally visible characteristics of unknown sample donors directly from biological materials. The aging process significantly affects most of the above characteristics making the development of a reliable method of age prediction very important. Today, the so-called “epigenetic clocks” represent the most accurate models for age prediction. Since they are technically not achievable in a typical forensic laboratory, forensic DNA technology has triggered efforts toward the simplification of these models. The present study aimed to build an epigenetic clock using a set of methylation markers of five different genes in a sample of the Italian population of different ages covering the whole span of adult life. In a sample of 330 subjects, 42 selected markers were analyzed with a machine learning approach for building a prediction model for age prediction. A ridge linear regression model including eight of the proposed markers was identified as the best performing model across a plethora of candidates. This model was tested on an independent sample of 83 subjects providing a median error of 4.5 years. In the present study, an epigenetic model for age prediction was validated in a sample of the Italian population. However, its applicability to advanced ages still represents the main limitation in forensic caseworks.     

Analytical Thresholds and Sensitivity: Establishing RFU Thresholds for Forensic DNA Analysis,

Determining appropriate analytical thresholds (ATs) for forensic DNA analysis is critical to maximize allele detection. In this study, six methods to determine ATs for forensic DNA purposes were examined and compared. Four of the methods rely on analysis of the baseline noise of a number of negatives, while two utilize the relationship between relative fluorescence unit signal and DNA input in the polymerase chain reaction (PCR) derived from a dilution series ranging from 1 to 0.06 ng. Results showed that when a substantial mass of DNA (i.e., >1 ng) was amplified, the baseline noise increased, suggesting the application of an AT derived from negatives should only be applied to samples with low levels of DNA. Further, the number and intensity of these noise peaks increased with increasing injection times, indicating that to maximize the ability to detect alleles, ATs should be validated for each post‐PCR procedure employed.