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.     

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.     

Association between copy number variations in the OCA2-HERC2 locus and human eye colour

Human eye colour variation is strongly associated with single nucleotide polymorphisms (SNPs) in the OCA2-HERC2 locus, especially rs12913832 that is found in an enhancer element of OCA2. In a previous study we found that 43 out of 166 individuals in a Norwegian population with the brown eye colour genotype HERC2 rs12913832:AA or AG, did not have the expected brown eye colour. To investigate if duplications or deletions in the OCA2-HERC2 locus could explain the blue eye colour in these individuals, we analysed massively parallel sequencing (MPS) data for copy number variations (CNVs) in the OCA2-HERC2 region. The ∼500 kb long OCA2-HERC2 locus was sequenced in 94 individuals with the rs12913832:AG and AA genotypes. Of these, 43 were observed to have blue eye colour and 51 were observed to have brown eye colour. CNVs were analysed using R and the R-package panelcn.MOPS - CNV detection tool for targeted NGS panel data. In rs12913832:AG individuals, CNVs in 32 regions were significantly associated with blue eye colour (Benjamini-Hochberg adjusted p-value ≤ 0.05). In rs12913832:AA individuals, CNVs in 14 regions were associated with blue eye colour using raw p-values (p ≤ 0.05). The functional effects of these CNVs on OCA2 expression are yet to be investigated. However, this study suggests that CNVs in the OCA2-HERC2 locus might explain why some of the rs12913832:AG and AA individuals have unexpectedly blue eyes.     

Investigating the morphology and genetics of scalp and facial hair characteristics for phenotype prediction

Microscopic traits and ultrastructure of hair such as cross-sectional shape, pigmentation, curvature, and internal structure help determine the level of variations between and across human populations. Apart from cosmetics and anthropological applications, such as determining species, somatic origin (body area), and biogeographic ancestry, the evidential value of hair has increased with rapid progression in the area of forensic DNA phenotyping (FDP). Individuals differ in the features of their scalp hair (greying, shape, colour, balding, thickness, and density) and facial hair (eyebrow thickness, monobrow, and beard thickness) features. Scalp and facial hair characteristics are genetically controlled and lead to visible inter-individual variations within and among populations of various ethnic origins. Hence, these characteristics can be exploited and made more inclusive in FDP, thereby leading to more comprehensive, accurate, and robust prediction models for forensic purposes. The present article focuses on understanding the genetics of scalp and facial hair characteristics with the goal to develop a more inclusive approach to better understand hair biology by integrating hair microscopy with genetics for genotype-phenotype correlation research.     

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.     

Predicting Phenotype from Genotype: Normal Pigmentation*

Abstract: Genetic information in forensic studies is largely limited to CODIS data and the ability to match samples and assign them to an individual. However, there are circumstances, in which a given DNA sample does not match anyone in the CODIS database, and no other information about the donor is available. In this study, we determined 75 SNPs in 24 genes (previously implicated in human or animal pigmentation studies) for the analysis of single‐ and multi‐locus associations with hair, skin, and eye color in 789 individuals of various ethnic backgrounds. Using multiple linear regression modeling, five SNPs in five genes were found to account for large proportions of pigmentation variation in hair, skin, and eyes in our across‐population analyses. Thus, these models may be of predictive value to determine an individual’s pigmentation type from a forensic sample, independent of ethnic origin.     

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.     

Genetic polymorphisms of 20 STR loci in Chinese Han population in Tianjin,North China

The PowerPlex^® 21 System PCR Amplification Kit was a new PCR Amplification Kit developed for forensic laboratories, but there was a lack of data about this kit in Chinese people in Tianjin, North China. This kit contained 20 STR loci, D3S1358, D1S1656, D6S1043, D13S317, Penta E, D16S539, D18S51, D2S1338, CSF1PO, Penta D, TH01, vWA, D21S11, D7S820, D5S818, TPOX, D8S1179, D12S391, D19S433 and FGA. In order to evaluate this kit and to get basic population data for its use in forensic practice in Chinese Han population, 360 unrelated Chinese Han individuals from Tianjin were typed using the Kit. Allele frequencies of the 20 STR loci and further population forensic genetic parameters were obtained. The observed genotype frequencies and expected genotype frequencies were evaluated by χ² test. No significant deviation from the Hardy–Weinberg equilibrium was observed in the population sample for the 20 STR loci. The population data in the present study can be used for routine forensic practice in Tianjin, North China.     

Massively parallel sequencing and the emergence of forensic genomics: Defining the policy and legal issues for law enforcement

Use of DNA in forensic science will be significantly influenced by new technology in coming years. Massively parallel sequencing and forensic genomics will hasten the broadening of forensic DNA analysis beyond short tandem repeats for identity towards a wider array of genetic markers, in applications as diverse as predictive phenotyping, ancestry assignment, and full mitochondrial genome analysis. With these new applications come a range of legal and policy implications, as forensic science touches on areas as diverse as ‘big data’, privacy and protected health information. Although these applications have the potential to make a more immediate and decisive forensic intelligence contribution to criminal investigations, they raise policy issues that will require detailed consideration if this potential is to be realised. The purpose of this paper is to identify the scope of the issues that will confront forensic and user communities.     

中国朝鲜族H19基因上游差异甲基化区SNP分布

目的调查中国朝鲜族群体中H19基因上游差异甲基化区(differentially methylated region,DMR)SNP及单倍型分布,为法医学应用及群体遗传学研究提供基础数据。方法收集中国朝鲜族101份无关个体血样和14份来自5个亲缘关系已知的两代家系血样,用PCR-循环测序、McrBC消化DNA后PCR方法检测H19基因上游DMR的SNP,并用亲源印记等位基因(parentally imprinted allele,PIA)分型法进行单倍型检测,再计算相关遗传学参数。结果在H19基因上游DMR 1174bp的目的基因扩增产物中,共检出13个SNP(rs10840167、rs2525883、rs12417375、rs4930101、rs2525882、rs2735970、rs2735971、rs11042170、rs2735972、rs10732516、rs2071094、rs2107425、rs4930098)和5种单倍型,有9个SNP属于高鉴别能力的遗传标记,其单倍型具有较高的个人识别率,单倍型平均基因多样性(GD)为0.714。应用McrBC酶消化基因组DNA的PIA分型法确定了家系子代样本的母源单倍型。结论 H19基因上游DMR在中国朝鲜族群体中具有很高的遗传多态性,母源单倍型的确定进一步提高了印记基因的法医学鉴定效能。     

MtSNP typing before mtDNA sequencing: Why do it?

Analysis of control mitochondrial DNA (mtDNA) hypervariable regions is sometimes the only available method to study hair evidence in forensic casework although being a laborious technique. Nowadays there is a huge interest in new genetic markers such as single nucleotide polymorphisms (SNPs) to type degraded forensic samples. For that purpose, a 10-Plex mitochondrial SNP for haplogroup typing, chosen from several SNP studies and useful to study the most common populations in our laboratory was applied in forensic casework. Hair shafts from three forensic cases with different ethnic backgrounds were studied with mtDNA sequencing and compared with mitochondrial SNPs (mtSNPs) study. Coding mtSNP typing prior to sequencing can allow for a rapid screening in forensic casework, which is emphasized in the first two cases. Moreover, in cases in which mtDNA sequencing fails, mtSNPs can still be detected. This 10 SNP loci multiplex provides a less expensive and simpler method for mitochondrial typing compared to control region mtDNA sequencing, especially when used as a fast screening method.     

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.     

Accumulation of population polymorphisms in the mitonuclear genome with probable adaptive effect to extreme environments as altitude for forensic purposes

We analyzed the accumulation of population polymorphism in 2504 individuals - nuclear genomes (nDNA) of 26 populations (81 genes associated to extreme environments) and 3295 mitochondrial genomes (mtDNA) of 47 populations with the aim to found mitonuclear relationship associated an extremes environment as altitude. For that, we use an algorithm developed by us to determine the accumulation of polymorphisms by segments in the genome and thus be able to perform the multivariate analysis to found SNPs differences and similarities among populations. The results showed in Peruvian population a statistically significant mitonuclear relationship for 113/293970 nDNA SNPs in 16/81 genes. In the case of the mtDNA, we found a statistically significant mitonuclear relationship for 6/22 mtDNA positions – Gene. Additionally for the Peruvian population, the MRPP3 had the greatest polymorphism contribution with respect to other populations. Then, these nDNA and mtDNA SNPs in genetically close populations to Peru can be applied to forensic genomic phenotyping to identify groups likely adapted to extreme conditions (such as altitude) or make individualization between low and high altitude populations.     

Gaughran vs the UK and public acceptability of forensic biometrics retention

This commentary provides a response to the European Court of Human Rights ruling in the case of Gaughran vs the United Kingdom on 13 February 2020. The Court ruled that the indefinite retention of DNA, fingerprints and facial images from all convicted adults was disproportionate. Using data from a survey on public attitudes, we examine the public acceptability of the police retention of forensic biometrics from the population.     

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.     

法医学二代测序标准化进展与展望

二代测序技术可检测多种法医遗传标记获取海量序列信息,满足法医学实践中精准个体识别、复杂亲缘关系鉴定、特征刻画等应用需求。国内外已开展大量二代测序法医学应用科研工作,但由于缺乏行业标准,二代测序在我国公安实战中并未得到有效应用。目前,法医学二代测序的标准制定面临检测靶标特殊、测序技术流程和结果分析不统一等挑战。本文从核酸标准参考物、测序技术要求、序列多态STR等位基因命名规则等方面,梳理国外法医学二代测序标准化工作进展,并总结国内二代测序检测行业标准现状,希冀为我国法医学二代测序的标准建设提供思路和参考。     

Evaluation of 19 short tandem repeat markers for individualization of Papaver somniferum

Papaver somniferum, commonly known as opium poppy, is the source of natural opiates, which are used as analgesics or as precursors in the creation of semi-synthetic opioids such as heroin. An increase in opioid addiction in the United States has resulted in high rates of illicit opioid use and overdoses. It has recently been shown that P. somniferum DNA suitable for genetic analysis can be recovered from heroin samples. The development of a comprehensive genetic individualization tool for opium poppy could serve to link cases and strengthen programs such as the Drug Enforcement Administration’s (DEA) Heroin Signature Program, which seeks to combat rising opioid use.The purpose of this study was to develop a quantitative real-time PCR (qPCR) method for the quantification of opium poppy DNA, compare three commercial DNA extraction kits for their ability to isolate DNA from poppy seeds, and evaluate nineteen opium poppy short tandem repeat (STR) markers for their use in a forensic identification panel. Such a panel could be used for individualizing samples and determining the geographic origin in heroin or poppy seed tea cases. The qPCR method was proven to be reproducible and reliable, specific for P. somniferum, and sensitive enough for forensic case-type samples. Of the three kits tested, the nexttec™ one-step DNA Isolation Kit for Plants was the optimal method and facilitated rapid extraction of DNA from poppy seeds. The majority of evaluated STR primer sets were unreliable or had low discriminatory power, limiting their use for individualization of poppy samples. A six-locus STR multiplex was developed and evaluated according to Scientific Working Group on DNA Analysis Methods (SWGDAM) and International Society of Forensic Genetics (ISFG) guidelines, including the use of a sequenced allelic ladder. The multiplex was found to have low discriminatory power, with greater than two-thirds of samples analyzed having just two different genotypes. The multiplex was determined to be unsuitable for individualization; however, a genotype map was developed as a proof of concept that these markers may be useful for determining the biogeographical origin of samples. Searching the poppy genome for new STR markers and developing new primer sets may be necessary for the creation of a powerful genetic tool for the individualization of P. somniferum.     

SNP genotyping of forensic casework samples using the 52 SNPforID markers

The analysis of degraded DNA is one of the biggest challenges in forensic casework. SNPs, which can be amplified using small amplicons, have previously been successfully applied to the profiling of forensic evidence that could not be analyzed using conventional STRs. Here we selected the 52 SNPforID SNP markers, with amplicons that ranged in size from 59 bp to 115 bp, and used them to profile a range of casework samples from Malaysia. DNA degradation is a common problem in Malaysia due to the high temperatures and humidity. To carry out the study we modified the 52 SNPforID markers into four 13-plex SNaPshot assays to enable easier interpretation of profiles on the ABI PRISM^® 310 and 3500.Fifty-one crime samples comprising bloodstains on cloth, swabs, and a mat and 2 swabs of trace DNA from 10 crime scenes in Malaysia were profiled after DNA extraction using a phenol–chloroform method. The samples were also subjected to STR analysis using the Powerplex^® 16 system (Promega), which resulted in only 17 full profiles and 9 partial profiles; using SNPs, 36 full profiles and 5 partial profiles could be generated.