DNA Phenotyping in Forensic Science PDF

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Research and Reports in Forensic Medical Science Dovepress open access to scientific and medical research Open Access Full Text Article REVIEW DNA phenotyping: current application in forensic science Research and Reports in Forensic Medical Science downloaded from https://www.dovepress.com/ Leonardo Arduino Marano 1 Abstract: DNA analysis for forensic investigations is based on the idea that each individual is Cintia Fridman 2 genetically unique, except in cases of monozygotic twins. DNA obtained from biological samples 1 is able to individualize this material by direct comparison of short tandem repeats genetic profile, Laboratório de Genética Molecular Forense, Polícia Científica do Estado obtained from biological samples of unknown origin to a reference sample profile. One of the For personal use only. do Paraná, Curitiba, PR, Brazil; major limitations of this approach is the need for a reference sample for comparison. Numer- 2 Departamento de Medicina Legal, Ética Médica e Medicina Social e do ous studies seeking to understand the relationship between certain polymorphisms and certain Trabalho, Faculdade de Medicina phenotypic characteristics are increasing and have generated promising results in aiding forensic FMUSP, Universidade de São Paulo, sciences. The process of inferring externally visible characteristics (EVCs) with forensic purpose São Paulo, SP, Brazil – eg, the color of skin, iris and hair, height, facial features, and male baldness pattern – from biological samples is known as forensic DNA phenotyping (FDP). Therefore, FDP provides more details about the subject to which a given biological sample belongs, without the need for a reference sample for comparative analysis. Some ethical and legal aspects should be taken into account so that this new technology does not promote segregation or ethnic persecution of certain population groups. Despite this, several real cases have benefited from these methods to orientate investigations to identify both suspects and victims. Keywords: forensic DNA phenotyping, DNA, externally visible characteristics, forensic genetics Introduction )RUHQVLFKXPDQLGHQWLÀFDWLRQ Human identification based on genetic profiles obtained from DNA polymorphisms (short tandem repeats [STRs]) is considered to be the gold standard among forensic science techniques. STRs are polymorphisms generated by a sequence (in tandem) of copies of small DNA segments (ranging from 2 to 6 base pairs). The most informative STRs may present more than a dozen alleles, and thousands of these polymorphisms have already been identified in humans; some estimations point to the existence of about one million STRs distributed among the human genome.1,2 The biological samples collected at crime scenes are processed to obtain a DNA profile that will be compared to the suspect’s profile, assisting investigation to establish Correspondence: Cintia Fridman a connection between the offender and the crime scene, or even eliminating suspects.3 Departamento de Medicina Legal, The same approach can be used to identify missing persons or unidentified bodies, Ética Médica e Medicina Social e do Trabalho, Faculdade de Medicina FMUSP, comparing their profiles in familial searches. Universidade de São Paulo, Rua Teodoro The main advantage of STR markers is due to their high allele diversity, making Sampaio, 115 CEP 05405000 - Cerqueira César, São Paulo, SP, Brazil such markers highly informative. The most polymorphic STRs have a high discriminat- Email [email protected] ing power (probability that two randomly selected individuals have distinct genotypes) submit your manuscript | www.dovepress.com Research and Reports in Forensic Medical Science 2019:9 1–8 1 Dovepress © 2019 Marano and Fridman. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. http://dx.doi.org/10.2147/RRFMS.S164090 php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Marano and Fridman Dovepress and low probability of match (probability that two randomly DNA phenotyping selected individuals have identical genotypes).1,2 In practical Eye color terms, these values ensure that each individual in the world Eye color can be considered as one of the human traits with population (except for identical twins) may have a unique the most color variability, ranging from light shades of blue genetic profile. to dark shades of brown or black, through intermediate colors The main disadvantage lies in the fact that this type of such as gray, hazel, yellow, and green. This color difference examination is uniquely comparative, requiring a pair of follows a pattern similar to the pigmentation of the skin and unknown/reference samples to be compared. In the absence hair, being defined by the amount of melanin and number of such a pair, the only possibility would be searching a DNA of melanosomes in the outer layer of the iris: blue eyes have database containing suspects’ profiles. Another disadvantage less melanin/melanosomes than brown eyes, for example.7 is the structure of the STR markers, which are composed of One of the first phenotyping tools developed and validated repetitions from ∼100 to 300 base pairs in length. There are was the Irisplex System8 consisting of six SNPs distributed situations in which biological material from the crime scene among pigmentation genes (HERC2, OCA2, SLC24A4, is so degraded that DNA samples obtained are not feasible of SLC45A2, TYR, and IRF4). This tool allows the differentiation obtaining enough data for an accurate identification.4 between blue and brown eyes with high accuracy (>90%), In face of such difficulties using traditional STR mark- confirmed both in homogeneous and admixed populations.9–13 ers protocols in some situations, various researches have Despite these results, the tool did not demonstrate the same allowed the use of genetic predictions of externally visible accuracy in samples from Asian populations, suggesting that characteristics (EVCs) to be of assistance in police investi- more studies should be performed on more distinct popula- gations, in both tracking suspects and identifying victims. tion samples.14 Several advances in genetics appear to have identified poten- However, intermediate eye colors are still a problem, tially useful markers for the prediction of various physical requiring further research to identify new genetic variants, characteristics. since the accuracy in their predictions is still much lower when compared with blue and brown eyes.15–17 Despite the Phenotype prediction from DNA difficulty in predicting these intermediate colors, a study by markers PoĞpiech et al18 demonstrated gene–gene interaction between Several variations (Insertion/Deletion [InDels] and single three of the main pigmentation genes (HERC2, OCA2, and nucleotide polymorphisms [SNPs]) located in DNA coding TYRP1) related to green eye color, thus aiding in the elabora- or regulatory regions can lead to amino acid substitutions, tion of future prediction models. altering the functional properties of the translated protein and Another debated aspect contemplates gender as a possible consequently being expressed in distinct phenotypes, some influencing factor in the determination of eye pigmentation. of them being the visible characteristics of the individual. In It has been observed that women tend to have darker eyes an attempt to obtain information about the physical features (predominantly brown and green) than men (predominantly of individuals from DNA extracted from biological material, blue and gray) in some European countries.11,19,20 However, no such as blood drops, hair strands, or small body fragments, genetic factor has yet been found to explain this difference, the scientific community has intensively researched the and more studies will be needed to evaluate this correlation. association between genetic markers and physical traits. Some studies have already evaluated the existence of poly- Hair color morphisms associated with skin, hair and eye color, facial Hair color, as well as eye and skin colors, is among the most forms, height, and baldness. noticeable EVCs with a wide range of phenotypes. The main In this context, it is expected that the genotyping of differences observed in hair color are the result of two types of such genetic markers in crime scene evidence, or in an melanin: brown/black eumelanin and red/yellow pheomela- unidentified body, can contribute significantly to increase nin.21 Individuals with red hair display a relative increase in the accurate information on the physical characteristics of the amount of pheomelanin compared to eumelanin, whereas those involved.5,6 Although some following inferences may in dark hair the amount of eumelanin prevails and in blond not present definitive value as forensic evidence, they may hair there is little of each kind of melanin.22 This variability in be an important factor leading police investigation and hair color has probably arisen in Europe, evolving from a dark reducing the number of suspects to a small set.5 ancestral color, resulting from human mating preferences.23 2 submit your manuscript | www.dovepress.com Research and Reports in Forensic Medical Science 2019:9 Dovepress Dovepress Marano and Fridman Among several genes involved in the melanogenesis have the same discriminatory power in more homogeneous process, MC1R was one of the first to demonstrate a strong populations, such as Europeans,28 other studies developed discriminating power for red hair, fair skin, and freckles. using homogeneous populations could not differentiate skin Subsequently, associations were made with other genes, such colors between discrete groups of Asian, African, and Native as SLC45A2, SLC24A5, and HERC2 and a predictive model Americans.29 based on 22 SNPs was created, reaching 81%–93% accuracy Taking into account this evolutionary obstacle, a global for each hair color category.24 prediction model was developed based on 36 markers distrib- A new system was then developed in 2013, adding 18 uted among 16 pigmentation genes.17 This model was created hair color markers to the 6 preexisting Irisplex SNPs, called taking into account three (light, dark, dark–black) or five the HIrisplex System.16 HIrisplex encompasses markers (very pale, pale, intermediate, dark, dark–black) skin tones, from MC1R, HERC2, OCA2, SLC45A2, KITLG, EXOC2, obtaining prediction accuracies ranging from 83%–97% for TYR, SLC24A4, IRF4, ASIP, and TYRP1 genes and, even the three-category scale to 72%–97% for the five-category though it has fewer markers than the model previously cre- scale. Some of these associations have previously been ated by Branicki et al,24 it can reach similar accuracy values described in admixed populations for some of these genes, (75%–92%).25 such as SLC24A5,30,31 HERC2,31 and SLC45A2,32 making However, current hair prediction models face a challenge: these seemingly promising for future applications. the accurate prediction of hair colors from individuals who The results from the IrisPlex, HIrisPlex, and HIrisPlex-S have had hair color changes throughout life (eg, darker hair systems were compiled into a publically available interac- after childhood). Most studies do not contemplate the sam- tive tool used to predict eye, hair, and skin color from DNA pling of younger individuals, or question adult subjects about data. From https://hirisplex.erasmusmc.nl/, one can use the distinct phenotypes in early childhood. Therefore, prediction tool to insert genotype data from the 41 markers available models are only elaborated with phenotypic information and obtain probabilities for three eye, four hair, and five skin observed in adults, without taking into account informative color categories to get individual probabilities through their markers for age-dependent phenotypes, partially explaining prediction model. the lower accuracy value for blond hair (only 69.5% against 78.5%, 80%, and 87.5% for brown, red, and black colors, Height respectively) prediction by HIrisplex. A study done with Until 2008, only a few genes have been described as associ- young individuals found that hair darkening usually occurs ated with human height. Subsequent association studies were between 6 and 13 years of age and that HIrisplex model performed in 2008 (in which 54 loci with direct correlation incorrectly predicts hair phenotypes for those individuals who with height variation were observed) and in 2010, increas- were blond only during early childhood, advising for the need ing the number of genetic markers to 180 and subsequently to identify new markers that could reduce this error rate.26 reaching almost 700 markers in 2014.33–37 Most of these genes are involved with growth-signaling pathways, such as Skin color the fibroblast growth factor, as well as genes expressed in Skin color has been one of the most complex pigmentation important tissues such as the growth plate38 – oddly though, phenotypes studied. It is believed that the skin pigmentation many of these markers are not directly involved in human variability emerged as an evolutionary response to the inten- growth pathways. sity of ultraviolet radiation among different planet regions. Even with significant increase in the number of height- Regions closer to the Equator line, with higher luminous related variants, there are still no significant values for intensity (high UV), would present a higher selective pres- prediction tests. While the initial studies obtained accuracy sure, keeping dark skin with high frequency, while more values of ∼65%, the most current studies failed to raise this distant regions, with less luminous intensity, would exert value >75%, demonstrating the large number of SNPs still less selective pressure, allowing the appearance of lighter to be discovered and how complex this trait may be.39 More- skin tones.27 over, human height may have a different etiology other than This evolutionary factor makes the genotype/phenotype genetic aspects, such as gestational (placental features and associations in mapping studies difficult, as well as resulting maternal health aspects such as nutrition, pathologies, and in correlations that only apply to a specific population group. drugs), hormonal, and environmental factors (nutrition and While associations found in admixed populations did not lifestyle) mainly during childhood.40 Research and Reports in Forensic Medical Science 2019:9 submit your manuscript | www.dovepress.com 3 Dovepress Marano and Fridman Dovepress Facial features Age estimation Among all EVCs, the facial shape prediction is one of the Using a different approach from those previously shown major objectives when studying phenotyping, glimpsing the here (SNP typing mostly), epigenetics studies using DNA final “DNA facial composite”. The face morphology is stud- methylation detection technologies have been shown to be ied from the distances between facial landmarks, as nostrils useful to the age estimation of an individual. The age estima- width, lips width, distance between eyes and face height. tion has great importance in the forensic context, since it is Some of the genetic markers associated with facial fea- complementary to the data obtained by the EVCs mentioned tures are initially found in syndromes and facial deformities here: besides reducing the number of suspects by predicting diseases studies (such as cleft palate, cleft lip, and other the age group of the sample donor, it can also complement craniofacial dysplasias). Some of these markers are then the obtained facial composite.48 correlated to craniofacial development and consequently DNA methylation changes throughout an individual’s linked to the normal variation of facial shape.41 For example, life – levels of methylation increase in childhood and then PAX3 gene encodes a transcription factor present in neural decrease after reaching adulthood.49 These changes can be crest cells, which was also related to Waardenburg syndrome measured and used to calculate an age estimate of an indi- and was later associated with the nasion position.42 Other vidual from biological samples of diverse origins (various candidate genes have been identified following patterns tissues and body fluids) and various situations (either from similar to PAX3, such as PRDM16 and TP63. However, human remains or from a crime scene) with high accuracy similar to height determination, each of these genetic mark- (deviation of 3.15 years in relation to the real chronological ers seems to have a small contribution toward the total face age) with as few as seven markers.50 morphology.41 The approach used by Claes et al,43 based primarily on Ancestry data obtained from admixed populations, employs a first step Some specific DNA markers can bring information about in which the sample ancestry and gender are used to create the ancestral composition of an individual, allowing their a base-face, in which data from 24 SNPs will subsequently biogeographic contributions to be detailed (Africa, Europe, be used, to convey nose, lips, face roundness, jaw, chin, and Asia, Amerindian). Thus, the use of ancestry informative supraorbital crest information to this primary face. Other markers (AIMs) allows the inference of an individual’s studies also found significant associations with facial width, ancestry, providing data to reinforce potential witnesses, or eyebrow width, distance between eyes, columella inclination, even bringing new information about crime scene evidence.51 nose bridge width, nostril width, and mouth shape.44,45 However, information about ancestry cannot be used solely as a criterion for determining the appearance of an Baldness individual. One must understand the difference between It is empirically known that male pattern of baldness or ancestry and the mistaken concept of race: the percentage androgenic alopecia has a strong hereditary factor, display- of an individual’s ancestral contribution will not necessar- ing a heritability of ∼80%.46 Among the various loci pos- ily reflect their appearance. This is especially noticeable in sibly involved, the major ones are those on q12 region on X admixed population samples in which AIMs demonstrate that chromosome, containing AR/EDA2R genes directly linked there is no direct correlation between appearance (ethnicity) to the production of androgen receptor and ectodysplasin and ancestral biogeographic origin.52 A2 receptor, respectively, on the 20p11 region, and on genes EBF1, TARDBP, and HDAC9 with predictive potential. Legal and ethical aspects Together, these five SNPs have the best association values In view of the various legal issues surrounding the sample col- to date, with 76.2% accuracy, reaching 86.4% if other 15 lection from suspects for comparison, an advantage of DNA markers are added (rs1041668, rs6625163, rs6625150, phenotyping approach is that it is more focused on obtaining rs962458, rs12007229, rs2180439, rs913063, rs1160312, genetic profiles from crime scene samples, thus not harming rs6113491, rs6461387, rs6945541, rs7349332, rs4679955, dignity or integrity rights. In addition, EVCs are publicly avail- rs9668810, and rs10502861), demonstrating that even low able features and therefore would not involve privacy issues.53 prediction markers can have high accuracy when added to However, all those individuals who share the characteris- stronger ones.47 tics of a facial composite may be interviewed and required to 4 submit your manuscript | www.dovepress.com Research and Reports in Forensic Medical Science 2019:9 Dovepress Dovepress Marano and Fridman donate samples for comparison to the crime sample in ques- EVCs prediction can also be very useful in disaster victim tion. In these cases, it is necessary to raise questions about identification and identification of missing persons, when no harassment that certain groups with a determined physical close relatives or antemortem samples exist for comparison. characteristic could suffer from the moment a phenotype is Forty-nine bone and teeth samples from World War II vic- obtained from evidence. One must question whether such tims found in Slovenia had their DNA extracted and were individuals will receive any protection, since they will now typed for hair and eye pigmentation genetic markers. All 49 belong to a group of suspects solely by their physical appear- samples had a successful prediction of eye and hair color, ance. Therefore, it should be noted if safety of such groups is two of the predictions being confirmed by the living sister being preserved to the detriment of investigation and public of two of the victims.62 safety objectives, and if new legal and ethical regulations In the future, computers may even be able to search should be created to preserve the integrity and intimacy of composites generated from DNA obtained from biological people involved in DNA phenotyping-based investigations. evidences against government databases or even photos from Attention should also be drawn to the use of markers that are social media platforms. Even if this system does not find a neutral in relation to ancestry, since information from some perfect match, at least it will be able to narrow down the AIMs may be erroneously associated to certain phenotypes, subjects of interest.56 resulting in ethnic persecution.54 Evaluation and validation Application and real cases A recent initiative was done by a group of scientists to create In 2010, a woman was sexually assaulted in broad daylight in a project aiming to establish new scientific knowledge, to Florida (USA) and no match was found in DNA databases. develop, validate, and implement analysis tools that allow Seven years later, police contracted a private DNA phenotype the prediction of an individual’s appearance from DNA company, which obtained a facial composite predicting a samples for use in forensic routine. In 2017, the VISible male subject with light brown skin, brown-hazel eyes, and Attributes Through GEnomics (VISAGE) Consortium was black hair. The new DNA facial composite led investigation created, composed by members of academic, police, and to the crime scene neighboring property, a wildlife sanctuary, justice institutions from eight European countries, with the where police found a suspect with matching characteristics. goal to expand the use of DNA as a tool to quickly obtain After a voluntary sample donation, Hugo Giron-Polanco was facial composites from evidence. The limitation of current arrested since a DNA comparison showed him and the semen DNA forensic techniques is expected to be overcome and sample found on the victim shared the same STR profile with new technologies to be developed allowing police investiga- a 1:400,000,000,000 match probability.55 However, Parabon tions to find unknown suspects more quickly through DNA Nanolabs (the private DNA phenotype company contracted information from crime scenes. The VISAGE Consortium in this case) has not scientifically published its methodology intends to use massive parallel sequencing techniques to or any validation tests so far. obtain higher quantitative data about suspect’s appearance, Toronto Police Service has already submitted several cold age, and ancestry predictions, as well as to create software case samples to private DNA phenotyping services, assisting that facilitates the interpretation of the generated data.63 to change the original direction of the investigations, although Furthermore, the European DNA Profiling (EDNAP) no actual arrests occurred.56 Several other cold cases across Group is responsible for promoting meetings and develop- the world are counting on facial composites obtained from ing collaborative comparison exercises to promote critical DNA profiles, with hope some new information will arise evaluations between laboratories around the world, aiming from this new data added to the investigation, hoping to to test the reliability and consistency of new forensic DNA identify both suspects and human remains.57–60 technologies. So far, EDNAP has tested the IrisPlex System On May 15, 2018, the German state of Bavaria approved reproducibility between 21 laboratories, considering it to be a law for police to analyze DNA samples and predict hair, successful64 and has recently been evaluating the age predic- eye, and skin color in addition to ancestry. These same tion through DNA methylation analysis (data not published). DNA predictions have already been used in the Nether- Although several studies cited here have shown significant lands, France, UK, Canada, and several US states, although phenotype–genotype association (Table 1), it should be noted some of these countries have no precise laws regarding the that each target population has its own genetic background, practice.61 and extrapolation of these data should be done with caution. Research and Reports in Forensic Medical Science 2019:9 submit your manuscript | www.dovepress.com 5 Dovepress Marano and Fridman Dovepress Table 1 Summary of main EVCs and some associated genetic markers and references EVCs Nearest genes/chromosome region associated References Eye color HERC2, IRF4, LOC105370627, OCA2, SLC45A2, TYR 8–14, 18, 67, 68 Hair color EXOC2, HERC2, IRF4, KITLG, MC1R, OCA2, SLC24A4, SLC45A2, TYR 16, 24–26 Skin color ANKRD11, ASIP, BNC2, DEF8, HERC2, IRF4, KITLG, MC1R, OCA2, PIGU, RALY, SLC24A4, SLC45A2, TYR, TYRP1, 17, 29–32, SLC24A5 69–71 Height ACAN, DNM3, EFEMP1, FBXW11, GH region, GHSR, GPR126, HHIP, HMGA1, HMGA1, IHH, LCORL, MICA, NOG, 33–39 NPR3, PML, PPIF, SDR16C5, SOCS2 Facial ADAMTS2, ASPH, C5orf50, COL11A1, COL17A1, CTNND2, DNMT3B, EVC2, FBN1, FGFR1, FGFR2, GDF5, LRP6, 41–45 features PAX3, POLR1D, PRDM16, RAI1, RELN, ROR2, SATB2, SEMA3E, SLC35D1, TP63, UFD1L, WNT3 Baldness AR/EDA2R, EBF1, HDAC9, TARDBP, 20 p11 46, 47 Abbreviation: EVCs, externally visible characteristics. 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Hum Mol Genet. intergenic DNA polymorphism regulating transcription of the nearby 2008;17(R2):R156–R165. BNC2 pigmentation gene. Hum Mol Genet. 2014;23(21):5750–5762. Research and Reports in Forensic Medical Science Dovepress Publish your work in this journal Research and Reports in Forensic Medical Science is an international, very quick and fair peer-review system. Visit http://www.dovepress.com/ peer-reviewed, open access journal publishing original research, reports, testimonials.php to read real quotes from published authors. reviews and commentaries on all areas of forensic medical science. The manuscript management system is completely online and includes a Submit your manuscript here: https://www.dovepress.com/research-and-reports-in-forensic-medical-science-journal 8 submit your manuscript | www.dovepress.com Research and Reports in Forensic Medical Science 2019:9 Dovepress