Experimental long-distance haplotyping of OCA2-HERC2 variants

The regulatory HERC2 SNP, rs12913832, is strongly associated with blue and brown eye colour. However, eye colour in heterozygous rs12913832 individuals is observed to vary greatly. Missense mutations in OCA2, such as rs1800407 and rs74653330, are associated with lighter eye colour in some but not all heterozygous rs12913832 individuals. Determining the physical linkage of these variants might help to further explain eye colour variation. So far, experimental haplotyping of these variants has been challenging because the genomic distance between them (∼ 135 kb) exceeds the fragment lengths produced by commonly used DNA isolation kits. The aim for this study was to explore novel methods for long distance haplotyping to assess associations between OCA2-HERC2 haplotypes and eye colour. DNA was isolated from frozen blood samples collected from Norwegians that are known to be heterozygous for both HERC2 rs12913832 and OCA2 SNPs, either rs1800407 (n = 23) or rs74653330 (n = 17), using the newly commercially available Monarch® HMW (heigh molecular weight) DNA Extraction Kit (New England BioLabs_inc). We successfully isolated DNA fragments up to 210 kb, which were long enough to haplotype OCA2-HERC2 loci by droplet digital PCR (ddPCR). Three haplotypes were observed in the study population: rs12913832:A-rs1800407:T in 22/23 individuals, rs12913832:A-rs1800407:C in 1/23 individuals and rs12913832:A-rs74653330:T in 16/16 individuals. As expected, all individuals with the rs12913832:A-rs74653330:T haplotype had intermediate to blue eye colour. However, the rs12913832:A-rs1800407:T haplotype was observed in both blue and brown-eyed individuals, suggesting more research is needed.     

Human eye colour and HERC2, OCA2 and MATP

Prediction of human eye colour by forensic genetic methods is of great value in certain crime investigations. Strong associations between blue/brown eye colour and the SNP loci rs1129038 and rs12913832 in the HERC2 gene were recently described. Weaker associations between eye colour and other genetic markers also exist. In 395 randomly selected Danes, we investigated the predictive values of various combinations of SNP alleles in the HERC2, OCA2 and MATP (SLC45A2) genes and compared the results to the eye colours as they were described by the individuals themselves. The highest predictive value of typing either the HERC2 SNPs rs1129038 and/or rs12913832 that are in strong linkage disequilibrium was observed when eye colour was divided into two groups, (1) blue, grey and green (light) and (2) brown and hazel (dark). Sequence variations in rs11636232 and rs7170852 in HERC2, rs1800407 in OCA2 and rs16891982 in MATP showed additional association with eye colours in addition to the effect of HERC2 rs1129038. Diplotype analysis of three sequence variations in HERC2 and one sequence variation in OCA2 showed the best discrimination between light and dark eye colours with a likelihood ratio of 29.3.     

The genetics of eye colours in an Italian population measured with an objective method for eye colour quantification

Brown and blue eye colours are primarily explained by the single nucleotide polymorphism (SNP) HERC2 rs12913832. However, the genetics of eye colours that appear to be neither blue nor brown are not well understood. In this study, 230 unrelated Italian individuals were typed for 32 SNP loci in pigmentary genes. High resolution digital images of the participants’ eyes were taken and the iris region was successfully extracted with the use of the custom designed software Digital Iris Analysis Tool (DIAT) from 218 of the 230 (95%) images. The software counted the numbers of blue and brown pixels in the iris region and calculated a Pixel Index of the Eye (PIE-score) that described the eye colours quantitatively. The PIE-score ranged from −1 to 1 (brown to blue). We investigated the association of the PIE-scores extracted from the eye images with the genotypes of the 32 pigmentary SNPs. We observed a statistically significant association between the PIE-scores and the SNP loci rs12913832, rs4778241, rs7495174 in the HERC2/OCA2 region and the locus rs16891982 in SLC45A2.     

Eye colour single nucleotide polymorphisms (SNPs) variants in Thai population

Human eye colour is recognized as one of the visible characterized features that would be valuable for forensic identification by predicting these phenotypes with particular genotypes. Recent gene association study had figured the remarkable variants mainly in OCA2 and HERC2 genes. In this study, eight human eye colour single nucleotide polymorphisms (SNPs) in five pigmentation genes, OCA2 (rs7495174, rs4778241 and rs4778138), HERC2 (rs12913832 and rs1667394), SLC24A4 (rs12896399), SLC45A2 (rs16891982) and TYR (rs1393350) were investigated in 374 Thai samples. The homozygote genotypes were predominantly observed in rs16891982 (GG/0.9920), rs1393350 (GG/0.9973) and rs12913832 (AA/0.9866). The allele frequency comparison with other populations in HapMap had identified the major allele of East Asia and Thai. Furthermore, the analysis of haplotype block of HERC2-OCA2 comprised five SNPs (rs12913832, rs1667394, rs7495174, rs4778241 and rs4778138) presented the most haplotype was AG-GAG (0.5199), which was rarely observed in European population.     

Analysis of genetic polymorphisms associated with the presence of freckles for phenotypic prediction

The prediction of externally visible characteristics (EVCs) is a commonly used practice by the forensic sciences as an important resource in the investigation of criminal cases in which the identity of perpetrators or victims is unknown or even to recognize decomposed cadavers. With this purpose, genetic markers associated with pigmentation traits have been widely studied by forensic scientists and, nowadays, it is possible to predict phenotypic characteristics such as hair, eyes and skin colour, as well as the presence of skin freckles by analysing single nucleotide polymorphisms (SNPs). In this study, we analysed the association of six SNPs located in pigmentation genes to the presence of freckles in individuals from the Brazilian population for forensic DNA phenotyping. The study was based within the context of a larger project on a population sample of 534 adult Brazilians of both sexes and different skin colours. DNA was extracted from peripheral blood and genotyped using the TaqMan® OpenArray® Real-Time PCR System (ThermoFischer Scientific) technique. Statistical analyses were carried out with the R software (version 4.0.2). As for the results obtained, three SNPs were shown to be statistically associated to the freckling, rs12203592, rs1800404 and rs222847, with CT, AG and AA genotypes being the main contributors, respectively. Variables such as sex of the individuals and skin colour were found to also contribute to the manifestation of this pigmentation trait. Further statistical analyses will be carried out to evaluate the possibility of using the SNPs in this study for phenotyping prediction of the Brazilian population, improving existing DNA phenotyping models in forensic sciences.     

What makes your “eyes” look different?

Facial morphology is the most distinguishable appearance and represents a promising subfield of Forensic DNA phenotyping. Located in the center of the face, eyes are important for facial recognition. In this study, epicanthal fold and palpebral fissure were selected for a preliminary genotype-phenotype analysis. Two SNPs were genotyped with SNaPshot method in 156 Chinese Han adults. A significant increased incidence of high ratio of palpebral fissure width to height was observed in the populations with AG genotype for SNP rs2074612 on gene HBEGF: AG versus GG, OR = 2.36, 95%CI = 1.08–5.18, p <0.05; AG versus GG/AA, OR = 2.07, 95%CI = 1.03–4.16, p < 0.05. No significant genotype difference was detected between epicanthal fold positive/negative populations for both SNPs (p > 0.05). Further study including more samples should be conducted to discover SNPs associated with epicanthal fold and palpebral fissure traits.     

A comparison of hair colour measurement by digital image analysis with reflective spectrophotometry

While reflective spectrophotometry is an established method for measuring macroscopic hair colour, it can be cumbersome to use on a large number of individuals and not all reflective spectrophotometry instruments are easily portable. This study investigates the use of digital photographs to measure hair colour and compares its use to reflective spectrophotometry. An understanding of the accuracy of colour determination by these methods is of relevance when undertaking specific investigations, such as those on the genetics of hair colour. Measurements of hair colour may also be of assistance in cases where a photograph is the only evidence of hair colour available (e.g. surveillance). Using the CIE L^*a^*b^* colour space, the hair colour of 134 individuals of European ancestry was measured by both reflective spectrophotometry and by digital image analysis (in V++). A moderate correlation was found along all three colour axes, with Pearson correlation coefficients of 0.625, 0.593 and 0.513 for L^*, a^* and b^* respectively (p-values = 0.000), with means being significantly overestimated by digital image analysis for all three colour components (by an average of 33.42, 3.38 and 8.00 for L^*, a^* and b^* respectively). When using digital image data to group individuals into clusters previously determined by reflective spectrophotometric analysis using a discriminant analysis, individuals were classified into the correct clusters 85.8% of the time when there were two clusters. The percentage of cases correctly classified decreases as the number of clusters increases. It is concluded that, although more convenient, hair colour measurement from digital images has limited use in situations requiring accurate and consistent measurements.     

Population genetic data of 38 autosomal InDels in San Basilio de Palenque,the first free town in America

In order to increase the information about Indels, we report allele frequencies and statistical parameters of forensic efficiency obtained typing a sample of 114 unrelated healthy individuals living in San Basilio de Palenque – Colombia using a panel of 38 autosomal InDels. No significant deviations from Hardy–Weinberg expectations were found except in the marker rs10629077 (p = 0.0002). The present database will be useful for forensic and paternity purposes for the region studied. Moreover, these additional markers can help forensic laboratories to solve parentage testing as well as to improve the analysis of degraded DNA samples.     

The importance of forensic evidence for decisions on criminal guilt

Recent studies have found that the general public perceives forensic evidence to be relatively inaccurate and to involve high levels of human judgement. This study examines how important the general public finds forensic evidence by comparing decisions on guilt and punishment in criminal cases that involve forensic versus eyewitness testimony evidence and examining whether a CSI effect exists. Specifically, this experimental survey study utilized a 2 (crime type: murder or rape) × 4 (evidence type: DNA, fingerprint, victim eyewitness testimony, or bystander eyewitness testimony) ? 1 (no victim testimony for murder scenario) design, yielding seven vignettes scenarios to which participants were randomly assigned. Results indicate that forensic evidence was associated with more guilty verdicts and higher confidence in a guilty verdict. Forensic evidence did not change the expected sentence length and did not generally affect the ideal sentence length. However, for rape, respondents believed that the defendant should receive a longer sentence when forensic evidence was presented but forensic evidence did not alter likely sentence that respondents expected the defendant to receive. The results of this study did not support a CSI effect. Overall, this study suggests that forensic evidence – particularly DNA – has a stronger influence during the verdict stage than the sentencing stage.     

Evaluation of skin-related variants in African ancestry populations and their role in personal identification

Pigment-related genetic variants point out their role in personal identification as they can be considered predictors suitable for Forensic DNA Phenotyping (FDP) and mounting evidence suggest also their bio-geographic inferential power for gaining information about the individual geographical origin. As they could be regarded as AIMs (Ancestry Informative Markers) they are powerful tools for inferring genetic composition of admixed population. Despite the huge range of skin tones across our species, little is known about genetic basis in global population and particularly our knowledge is less precise for those showing a complex historical and genomic background. The current research aims to explore the allelic status in several SNPs mapped in selected genes known to be involved in skin pigmentation: OCA2, HERC2, SLC45A2, SLC24A5 and two intergenic regions between BEND7/PRPF18 and EIF2S2/ASIP. The genetic evaluation has been performed on selected African and African derived populations: Fon, Dendi, Bariba and Berba communities from Benin, and Afroecuadorians. Data integration has been made up merging genotypic results with available information from major biological data warehouse as Phase 3–1000 Genomes Project or International HapMap Project in order to obtain a selected populations panel useful for their use as inferential model training set to test the likelihood of correct assignment to geographically differentiated human groups. The proposed variants panel seems to properly interpret the geographic variation and some new interesting evidence could be pointed out in African mixed populations, that seem to be differentially distributed if the total panel is considered. Understanding human pigmentation architecture can provide fundamental insight into genetic interaction of complex traits and the relationship between environmental adaptation and population history. In addition, the results support the use of phenotypic inference along with bio-geographical ancestry information as valid auxiliary tools in personal identification.     

A preliminary study investigating the overlay method in forensic podiatry for comparison of insole footprints

The overlay method is used in forensic podiatry to compare the shape, position, and overall fit of features between questioned and reference footprint evidence. However, the scientific foundation underpinning its validity in the comparison of insole footprints is not fully understood because of a lack of published data defining its statistical operating parameters. A review of literature revealed its subjective nature with little scientific validation, limited only to face validity.The aim of this study investigated strength of agreement between three expert footprint examiners’ overlay comparisons of ten reference insole footprints using Cohen’s weighted kappa (K_w). Validity of method was explored using measures of sensitivity, specificity, false positive rate (FPR), false negative rate (FNR), receiver operating characteristic (ROC) curve and area under the ROC curve (AUC). Results showed two examiners demonstrated high inter-rater consistency between their overlay comparisons (K_w: 0.981; 95 %CI: 0.943 to 1.020 for Rater1 v Rater2) whilst one examiner did not (K_w: 0.340; 95 %CI: 0.148 to 0.532 for Rater1 v Rater3; K_w: 0.310; 95 %CI: 0.100 to 0.519 for Rater2 v Rater3). Validity was investigated using a conclusion scale in a verbal expressions table to indicate support or rejection of compatibility of footprints between one questioned insole and ten reference insoles. Data analysis indicated validity as follows: Sensitivity: 77.8 %, Specificity: 61.9 %, FPR: 38.1 % and FNR: 22.2 %. ROC analysis corroborated this finding. AUC was calculated at 0.762 or 76.2 % indicating an ‘acceptable’ measure of overall accuracy of the overlay method for insole footprint comparison. Findings provide novel data supporting previous suggestions that the overlay method should not be used in isolation to compare insole footprints. Data also offers insight into the scientific foundation of this method, whilst highlighting its limitations and providing some implications and recommendations for forensic podiatry practice.     

Comparative performance of the Phadebas® Forensic Press Test at room temperature and 37 °C for the detection of saliva stains on fabric exhibits

The Phadebas® Forensic Press Test (PFPT) is an enzyme-based colorimetric test used to visualise and locate latent saliva stains on forensic exhibits. The test relies upon the presence of the enzyme α-amylase which is present in high levels in saliva. Even though the optimal in vitro temperature for α-amylase activity is 37 °C, the PFPT manufacturer’s protocol specifies that the PFPT should be carried out at room temperature (RT). In this study, we compared the performance of the PFPT at RT and 37 °C using combinations of four fabric types (cotton, polyester, acrylic and a cotton/polyester blend), three saliva dilutions (neat, 1:10 and 1:100) and stains aged for four time periods (1 day, 1 week, 1 month and 3 months). The intensity of the PFPT colour reactions at RT and 37 °C were not statistically different across all fabric types, saliva concentrations and stain ages, indicating that maximum sensitivity and performance of the PFPT can be achieved at RT.     

Stature estimation using foot dimensions via 3D scanning in Taiwanese male adults

In forensic identification, the use of foot dimensions to predict stature is a requisite. For Taiwanese male stature prediction, available information about stature estimation using foot dimensions is still lacking. Therefore, the primary purpose of this study was to establish Taiwanese adult male stature prediction models for various foot dimensions. A total of 2,072 male 3D foot models were collected and the subjects' ages ranged from 18 to 59 years old. Thirteen foot dimensions for each foot were utilized in this study. Bilateral differences were tested using paired t-tests. Stature predictions based on different foot dimensions were conducted using simple linear and stepwise regression methods. The results showed significant bilateral differences in the majority of the selected foot dimensions (p < 0.001). Foot length had the highest positive correlation with stature in the right and left foot. Using length-related dimensions generated better prediction accuracy (0.356 < R² < 0.533, 4.193 < SEE < 4.928), whereas the worst accuracy was observed with angle-related dimensions (0.001 < R² < 0.023; 6.072 < SEE < 6.137) when employing a single foot dimension to estimate Taiwanese male stature. Stepwise regression analysis results indicated that the coefficient of determination was improved to 0.590 and 0.593 by applying five and six foot parameters to predict stature, respectively. The findings of the present study suggest that these stature estimation equations can be applied to predict Taiwanese male stature in the forensic science field.     

Allelic frequencies and statistical data obtained from 48 AIM INDEL loci in an admixed population from the Brazilian Amazon

Allelic frequencies of 48 informative insert-delete (INDEL) loci were obtained from a sample set of 130 unrelated individuals living in Macapá, a city located in the northern Amazon region, in Brazil. The values of heterozygosity (H), polymorphic information content (PIC), power of discrimination (PD), power of exclusion (PE), matching probability (MP) and typical paternity index (TPI) were calculated and showed the forensic efficiency of these genetic markers. Based on the allele frequency obtained for the population of Macapá, we estimated an interethnic admixture for the three parental groups (European, Native American and African) of, respectively, 50%, 21% and 29%. Comparing these allele frequencies with those of other Brazilian populations and the parental populations, statistically significant distances were found. The interpopulation genetic distance (F_ST coefficients) to the present database ranged from F_ST = 0.0431 (p < 0.00001) between Macapá and Belém to F_ST = 0.266 (p < 0.00001) between Macapá and the Native American group.     

Hair characteristics of four Indian bear species

Dorsal guard hairs of four species of bear (family: Ursidae) found in India were characterized using light microscopy by studying features including colour, hair thickness, cuticle pattern, medulla pattern, medullary index, cross-section and scale count index. The gross cuticular pattern was quite similar for the four species but a distinct difference was observed in the scale count index. Medulla type was narrow and amorphous with a very low medullary index (< 0.15) except for brown bear which showed a comparatively higher medullary index (0.38) and a vacuolated medulla. By combining together the parameters mentioned above it was possible to characterize bear species on the basis of their hair characteristics.     

Comparison of the three age estimation methods: Which is more reliable for Turkish children?

BackgroundThree atlases—the GÖK, the Greulich–Pyle (GP), and the Tanner–Whitehouse (TW3)—are used frequently for age determination in Turkey. The purpose of this study was to evaluate the applicability of these three methods related to the skeletal age assessment for Turkish adolescents.Materials and methodsThe conventional roentgenograms of the left hands and wrists, elbows, shoulders, and pelvises of 333 healthy Caucasian children (164 females, 169 males) who fit the study and the criteria of each atlas were obtained. The mean differences (± standard deviation [S.D.] in years) between the chronologic age (CA) and the skeletal age (BA), which were obtained by using each age estimation method, were calculated and tested using t-test.ResultsFor girls, the most accurate method was the TW3 (mean differences (d): ?0.21 (p < 0.05)), following by the GP (d: 0.66 (p < 0.001), and the GÖK (d: 2.99 (p < 0.001)). For boys, the most accurate method was the GP (d: ?0.02 (p > 0.05)), followed by the TW3 (d: ?0.18 (p < 0.05)) and GÖK (d: 1.05 (p < 0.001)).Discussion and conclusionsResults show that the TW3 (for girls) and the GP (for boys) methods are more appropriate than the GÖK atlas for estimating the BA. GÖK could be used for boys aged 11–14 years but it should not be used for girls.     

The identification of individuals by observational gait analysis using closed circuit television footage

This study investigated the ability of individuals with experience in gait analysis to identify people by observing features of gait recorded by closed circuit television cameras (CCTV). Seven experienced analysts each viewed five samples of footage. Each sample showed a “target walker” and five “suspect walkers.” The task of the experienced analysts was to determine which, if any, of the “suspect walkers” was the “target walker.” All of the participant “walkers” wore identical loose fitting clothing to mask anatomical and body contour features, and balaclavas to obscure facial features. The overall results showed that the experienced analysts made a correct decision in 124 of 175 cases (71%), significantly better than would have been expected to have occurred by chance (p < 0.05). A significant variation in correct decisions (p < 0.05) was shown to occur between the various angles from which the footage was recorded, footage recorded in the saggital plane showing the highest number of correct decisions. Significantly more correct decisions (p < 0.05) were also shown to occur when the footage of the “target walker” and that of the “suspect walker” were taken from the same angle. The results suggest that individuals with experience in gait analysis perform well in the comparative identification of suspects from CCTV footage, and therefore do have a role to play as expert witnesses in this field.     

A data collection of vehicle topcoat colours. 2. The measurement of colour samples used in the vehicle refinishing industry

Four collections of painted cards, of the type used as reference samples in the vehicle refinishing industry, have been examined for suitability for incorporation in a data base of vehicle topcoat colours. One of these was considered more suitable for forensic science applications than the others and tristimulus values (X,Y,Z) were obtained for over 6000 samples. The data were then mathematically transformed to a uniform colour space and the sample distribution was plotted according to three colour axes. The evidential value of the plots is briefly discussed.