Successful nuclear DNA profiling of rootless hair shafts: a novel approach.

Historically, rootless hair shaft samples submitted to a forensic laboratory for DNA analysis are reserved for mitochondrial DNA (mtDNA) analysis due to the presence of highly degraded as well as insufficient amounts of nuclear DNA. Although mtDNA has been very successful in obtaining results from rootless hair, this system has its limitations, namely, it is a lineage marker that cannot differentiate between maternally related genotypes. Given the high incidence of hairs as forensic evidence, there is a need for the use of a nuclear DNA test system capable of producing reliable results for hair shaft forensic evidence. This study reports the utilization of an enhanced DNA extraction methodology for hairs, in combination with a recently developed novel, nuclear DNA typing assay, InnoTyper® 21, to improve the success rate for obtaining informative results from highly compromised, degraded, and trace forensic samples such as rootless hair shafts. The InnoTyper 21 kit is a small amplicon retrotransposon marker typing system compatible with currently used capillary electrophoresis platforms. This system contains 20 Alu element markers, ranging in size from 60 to 125 bp, making the assay highly sensitive for extremely degraded forensic samples and thus enabling recovery of nuclear DNA profiles from samples that would otherwise require mtDNA sequencing. A subset of samples was also tested with the GlobalFiler kit with less success due to the larger amplicon sizes in comparison with InnoTyper 21. Results were variable but very promising, with approximately 40% of the total number of hairs tested producing interpretable nuclear DNA profiles with InnoTyper 21. These results demonstrate the ability of the utilized methodologies to produce nuclear DNA results with high statistical power from rootless hair shafts.

A human identification system for hair shaft using RNA polymorphism.

Hair is one of the common pieces of evidence at crime scenes, with abundant mitochondrial DNA but limited nuclear DNA in its shaft. It also helps to narrow the investigation scope to maternal lineage but fails to provide unique individual information. We assumed that RNA in hair shafts would be an alternative resource used to perform human identification based on the facts that (1) RNA retains the polymorphic information; (2) the multi-copy of RNA in a cell resists degradation as compared to the one-copy of nuclear DNA. In this study, we explored the potential of RNA polymorphism in hair shafts for forensic individual identification. A SNaPshot typing system was constructed using 18 SNPs located on 11 genes (ABCA13, AHNAK, EXPH5, KMT2D, KRT35, PPP1R15A, RBM33, S100A5, TBC1D4, TMC5, TRPV2). The RNA typing system was evaluated for sensitivity, species specificity, and feasibility for aged hair samples. Hair samples from a Shanxi population in China were used for the population study of the system. The detection limit of the assay was 2 ng RNA. The CDP of these 11 genes was 0.999969 in the Shanxi population. We also identified the concordance of the RNA and DNA typing results. In summary, we developed an RNA typing method to perform human identification from hair shafts, which performed as accurately as nuclear DNA typing. Our method provides a potential basis for solving the human identification problem from hair shafts, as well as other biological materials that lack nuclear DNA.

Validation of the InnoXtract™ DNA extraction method for bone, teeth, and rootless hair.

Forensic casework samples often include human hairs, teeth, and bones. Hairs with roots are routinely processed for DNA analysis, while rootless hairs are either not tested or processed using mitochondrial DNA. Bones and teeth are submitted for human remains identifications for missing persons and mass disaster cases. DNA extraction from these low templates and degraded samples is challenging. The new InnoXtract DNA extraction method utilizes magnetic beads that are optimized to bind small DNA fragments, as small as 100 base pairs, to purify high-yield DNA from compromised samples. This validation study evaluates InnoXtract's ability to obtain amplifiable DNA from samples such as rootless hairs and skeletal remains. Studies performed include sensitivity, stability, repeatability, reproducibility, non-probative samples, and comparison to standard organic extractions. Sensitivity studies demonstrate average yield recoveries ranging from 53% to 100% and 73% to 85% for the InnoXtract hair and bone methods, respectively. Studies demonstrate consistent results across a range of sample types, such as insulted and un-insulted bone and teeth, as well as hair shafts from donors of various ages, gender, race, and hair characteristics. The InnoXtract bone method outperformed organic extraction. The method was successfully automated on a MagMAX™ Express-96, with recoveries over 70% relative to the manual version. InnoXtract has the potential as an automated high-throughput, high-yield bone extraction method with 6 h of total extraction time for up to 96 samples. The validation study results demonstrate that the InnoXtract kits produce high-yield and high-quality DNA from compromised bone, teeth, and hair shaft samples.

Optimal processing for proteomic genotyping of single human hairs.

The use of hair evidence for human identification is undergoing considerable improvement through the adoption of proteomic genotyping. Unlike traditional microscopic comparisons, protein sequencing provides quantitative and empirically based estimates for random match probability. Non-synonymous SNPs are translated as single amino acid polymorphisms and result in genetically variant peptides. Using high resolution mass spectrometry, these peptides can be detected in hair shaft proteins and used to infer the genotypes of corresponding SNP alleles. We describe experiments to optimize the proteomic genotyping approach to individual identification from a single human scalp hair 2 cm in length (∼100 µg). This is a necessary step to develop a protocol that will be useful to forensic investigators. To increase peptide yield from hair, and to maximize genetically variant peptide and ancestral information, we examined the conditions for reduction, alkylation, and protein digestion that specifically address the distinctive chemistry of the hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 h of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 h of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times increased the average number of genetically variant peptides from 20 ±â€¯5 to 73 ±â€¯5 (p = 1 × 10-13), excluding intractable hair samples. Random match probabilities reached up to 1 in 620 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original hair protein extraction method. Ancestral information was also present in the data. While the number of genetically variant peptides detected were equivalent for both European and African subjects, the estimated random match probabilities for inferred genotypes of European subjects were considerably smaller in African reference populations and vice versa, resulting in a difference in likelihood ratios of 6.8 orders of magnitude. This research will assure uniformity in results across different biogeographic backgrounds and enhance the use of novel peptide analysis in forensic science by helping to optimize genetically variant peptide yields and discovery. This work also introduces two algorithms, GVP Finder and GVP Scout, which facilitate searches, calculate random match probabilities, and aid in discovery of genetically variant peptides.

Proteomic genotyping: Using mass spectrometry to infer SNP genotypes in pigmented and non-pigmented hair.

Proteomic genotyping uses genetically variant peptides that contain single amino acid polymorphisms to infer the genotype of corresponding non-synonymous SNP alleles. We have focused on hair proteins as a source of protein-based genetic information in a forensic context. An optimized sample processing protocol for hair shafts has been developed for use on a single hair that allows us to conduct validation protocols on real world samples. This includes whether the inferred SNP genotypes are robust and not systematically affected by biological or chemical variation in hair proteomes that might be obtained from a crime scene. To this end we analyzed the hair of 4 mature individuals with a mixture of pigmented and non-pigmented hair. We demonstrate significant changes in the proteomes of grey versus pigmented hair. Vesicle specific proteins and lipid catabolism proteins were enriched in pigmented hair, and housekeeping proteins and lipid anabolic enzymes were enriched in grey, non-pigmented hair. The resulting profiles of genetically variant peptides, however, were more correlated with profiles from the same individuals regardless of pigmentation status. Together with other published evidence, this finding indicates that profiles of genetically variant peptides are robust and more correlated with other genetically variant peptide profiles from the same individual irrespective of changes occurring in the hair protein profile. Based on this small sample, investigators using profiles of genetically variant peptides to infer random match probabilities should not expect to observe differences based on the pigmentation of the hair shaft.

Comparison of protein expression levels and proteomically-inferred genotypes using human hair from different body sites.

The microanatomy of human hair differs as a function of the site of origin on the body. This was a major consideration when anatomical features of hair were used as a means of comparison and human identification. Recent advances have demonstrated that proteomics of the hair shaft can be used to develop profiles of protein abundance and genetically variant peptides, the latter in turn being used to infer genotypes of SNP alleles. Because the profile of proteins would be expected to change as hair anatomy changes, it is an open question if the profile of genetically variant peptides will also change. While some sample to sample variation is expected, a potential drawback of using genetically variant peptides to infer an individual genotype is that the proteomic profile might change as a function of body site origin as well as an individual's genotype. The hypothesis in this study is that the profile of hair shaft genetically variant peptides depends more on an individual's genotype than on the site of hair shaft origin. To test this an analysis of both protein expression levels and genetically variant peptides was conducted on 4 body sites (scalp, axillary, beard and pubic hair) from 5 individuals with 4 biological replicates. Levels of protein expression were estimated using label-free quantification on resulting proteomic mass spectrometry datasets. The same datasets were then also analyzed for the presence of genetically variant peptides. This study demonstrates that the protein profiles of hair shafts varied as a function of somatic origin. By contrast the profile of genetically variant peptides, and resulting inferred genotype of SNP alleles, were more dependent on the individual. In this study random match probabilities ranged up to 1 in 196. Individual identification based on genetically variant peptides therefore can be obtained from human hair without regard to the site of origin. If the site of hair shaft origin was legally relevant then microscopic analysis is still necessary. This study demonstrates the utility of proteomic analysis for extracting forensic information from hair shaft evidence.

Hair detection and lesion segmentation in dermoscopic images using domain knowledge.

Automated segmentation and dermoscopic hair detection are one of the significant challenges in computer-aided diagnosis (CAD) of melanocytic lesions. Additionally, due to the presence of artifacts and variation in skin texture and smooth lesion boundaries, the accuracy of such methods gets hampered. The objective of this research is to develop an automated hair detection and lesion segmentation algorithm using lesion-specific properties to improve the accuracy. The aforementioned objective is achieved in two ways. Firstly, a novel hair detection algorithm is designed by considering the properties of dermoscopic hair. Second, a novel chroma-based geometric deformable model is used to effectively differentiate the lesion from the surrounding skin. The speed function incorporates the chrominance properties of the lesion to stop evolution at the lesion boundary. Automatic initialization of the initial contour and chrominance-based speed function aids in providing robust and flexible segmentation. The proposed approach is tested on 200 images from PH2 and 900 images from ISBI 2016 datasets. Average accuracy, sensitivity, specificity, and overlap scores of 93.4, 87.6, 95.3, and 11.52% respectively are obtained for the PH2 dataset. Similarly, the proposed method resulted in average accuracy, sensitivity, specificity, and overlap scores of 94.6, 82.4, 97.2, and 7.20% respectively for the ISBI 2016 dataset. Statistical and quantitative analyses prove the reliability of the algorithm for incorporation in CAD systems. Graphical Abstract Overview of proposed system.

A Case of Epidermal Cyst containing Hair Shafts and Trichilemmal Cyst occurring on the Knees / 대한피부과학회지

We report a case of 4-year-old girl with erythematous papule and nodule on the right knee and left knee, respectively. Histopathologically, the findings of walls of the each cystic lesions were those of the epidermal cyst containing hair shafts of lanugo size on the right knee and the trichilemmal cyst on the left knee. The observation of hair shafts in a cystic cavity represents an important clue for identifying some unusual varieties of cystic lesions. We report a interesting case of epidermal cyst containing hair shafts and trichilemmal cyst simultaneously and independently occurred on the right knee and left knee, respectively.