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Prediction of eye and hair colour from DNA can be an important investigative tool in forensic cases if conventional DNA profiling fails to match DNA from any known suspects or cannot obtain a hit in a DNA database. The HIrisPlex model for simultaneous eye and hair colour predictions was developed for forensic usage. To genotype a DNA sample, massively parallel sequencing (MPS) has brought new possibilities to the analysis of forensic DNA samples. As part of an in-house validation, this study presents the genotyping and predictive performance of the HIrisPlex SNPs in a Norwegian study population, using Verogen's ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx system and the HIrisPlex webtool. DNA-profiles were successfully typed with DNA input down to 125 pg. In samples with DNA input < 125 pg, false homozygotes were observed with as many as 92 reads. Prediction accuracies in terms of AUC were high for red (0.97) and black (0.93) hair colours, as well as blue (0.85) and brown (0.94) eye colours. The AUCs for blond (0.72) and brown (0.70) hair colour were considerably lower. None of the individuals was predicted to have intermediate eye colour. Therefore, the error rates of the overall eye colour predictions were 37% with no predictive probability threshold (pmax) and 26% with a probability threshold of 0.7. We also observed that more than half of the incorrect predictions were for individuals carrying the rs12913832 GG genotype. For hair colour, 65% of the individuals were correctly predicted when using the highest probability category approach. The main error was observed for individuals with brown hair colour that were predicted to have blond hair. Utilising the prediction guide approach increased the correct predictions to 75%. Assessment of phenotype-genotype associations of eye colours using a quantitative eye colour score (PIE-score), revealed that rs12913832 AA individuals of Norwegian descent had statistically significantly higher PIE-score (less brown eye colour) than individuals of non-northern European descent. To our knowledge, this has not been reported in other studies. Our study suggests that careful assessment of the target population prior to the implementation of forensic DNA phenotyping to case work is beneficial.

Single nucleotide polymorphisms (SNPs) are the most abundant genetic markers in the human genome. SNPs are used in forensics for predicting the externally visible characteristics of a given individual based on a sample of DNA alone. In this study, we tested degraded DNA samples to determine the eye color prediction accuracy of the IrisPlex system. We used five years old bloodstains and UVC exposed (30 and 60 min) DNA samples. PCR and electrophoresis were performed. The multinomial logistic regression model statistics is applied for eye color prediction. We observed allele and locus drop-outs on both bloodstains stored for five years and UVC exposed samples. The brown and blue eye colors were correctly predicted for reference and degraded samples. The intermediate eye colors were predicted to be brown or inconclusive. The degradation by aging bloodstains or UVC exposure, is differently affected the prediction accuracy depending on the informativeness of the SNPs. This study showed that the prediction of the eye color is highly accurate for the blue and brown eye colored individuals. However, the IrisPlex prediction accuracy could be influenced by the old and degraded samples. Istanbul University, Scientific Research FundIstanbul University [41343, 26105] This study was supported by Istanbul University, Scientific Research Fund (Project No: 41343 and 26105).

Introducción: El color de los ojos, rasgo hereditario poligénico, es una de las características físicas más visibles y diferenciables entre humanos. Por ello, la predicción del color del iris es ampliamente utilizada en medicina forense. Variaciones en genes involucrados en la pigmentación han sido asociadas a la variación en la pigmentación del iris entre individuos, especialmente para discernir entre ojos azules y marrones. Sin embargo, poco se conoce acerca de los determinantes genéticos que definen el color de ojos verde, así como el anillo peripupilar. Objetivos: Analizar las variaciones genéticas que definen los patrones de pigmentación en el iris. Metodología: Se obtuvo una muestra de ADN de 829 voluntarios para determinar el genotipo de 6 genes involucrados en pigmentación humana. La asociación entre color de ojos y genotipo se analizó mediante regresión logística. Resultados: Se confirma que el SNP rs12913832 localizado en HERC2/OCA2 es el mayor determinante de la variación en el color de ojos (p < 0,001). Aunque el alelo derivado C de rs12913832 se asocia al color de ojos azul, la mayoría de individuos con ojos verdes son portadores de dicho alelo. Polimorfismos en IRF4 y OCA2 también parecen influir en tener el iris verde. También observamos una asociación entre la presencia de anillo peripupilar y variaciones en el gen SLC24A4, aunque únicamente en individuos heterocigotos para rs12896399. Conclusión: Las herramientas de predicción de características fenotípicas como el color de ojos deberían analizar los modelos de herencia, ya que el número de copias del alelo derivado parece influir en el patrón de pigmentación del iris. Introduction: The colour of eyes, a polygenic hereditary trait, is one of the most visible and distinguishable physical characteristics among humans. For that reason, the prediction of iris colour is widely used in forensic medicine. Genetic variations in pigmentation-related genes have been associated with the variation of iris pigmentation between individuals, particularly to distinguish between blue and brown eyes. However, little is known about the genetic determinants responsible for the green colour, as well as the presence of pigmented ring on the iris. Objectives: To analyse the genetic variations that determine the patterns of iris pigmentation. Methodology: DNA samples from 829 volunteers were obtained in order to analyse the genotype of 6 genes involved on human pigmentation. The association between eye colour and genotype was analysed using logistic regression. Results: Our results confirmed that the SNP rs12913832 located on HERC2/OCA2 is the major determinant of the natural eye colour variation (p < 0.001). Although the derived C allele of rs12913832 is associated with blue eye colour, most individuals with green eyes are carriers of that allele. Polymorphisms in IRF4 and OCA2 also seem to influence on having green iris. We also observed an association between the presence of pigmented ring on the iris and genetic variations in SLC24A4, although only in individuals heterozygous for rs12896399. Conclusion: Prediction tools for phenotypic characteristics such as eye colour should take into account inheritance models, since the copy number of the derived allele carried seems to influence the pigmentation pattern of the iris.

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2017/2018 Introduction: The expression of the PDZK1 gene has s een to be increased in melasma via t he action of estrogens, causing an increase in the activity of tyrosinase and the synthesis of melanin. The aim of this study is to determine the expression and genetic variability of the PDZK1 gene in normal skin and its role in human pigmentation. Metho dology: We analyzed the expression of the PDZK1 gene and the genotype of 8 genetic variants of PDZK1 in 90 skin biopsies. Information on pigmentation traits and solar sensitivity was coll ected through a questionnaire in 90 volunteers. Results: The results show that there are differences in the level of expression of PDZK1 according to the zone of the skin analyzed, a chronically or intermittently photoexposed zone (p - value = 0.0023), and according to the sex of the individuals (p - value = 0.021). The varian t rs11576685 * C of PDZK1 seems to increase the basal expression of the gene (p - value = 0.03), a nd shows an association with dark skin colo u r (p - value = 0.02). Conclusion: The results of this study show that there are differences in expression of the PDZK 1 gene that are influenced by sun exposure and by genetic factors, and not only by hormonal factors as seen previously. Introducción: La expresión del gen PDZK1 se ha visto aumentada en melasma vía la acción de los estrógenos, provocando un incremento de la actividad de la tirosinasa y la síntesis de m elanina. En este estudio se pretende conocer la expresión y variabilidad genética del gen PDZK1 en piel normal y su papel en la pigmentación humana. Metodología: Se analizó la expresión del gen PDZK1 y el genotipo de 8 variantes genéticas de PDZK1 en 90 biopsias cutáneas. Se recogió información sobre los rasgos de pigmentación y sensibilidad solar mediante un cuest ionario en los 90 voluntarios . Resultados: Los resultados muestran que existen diferencias en el nivel de expresión de PDZK1 dependiendo de si la muestra procedía de una zona crónicamente o intermitentemente fotoexpu esta (p - valor = 0.0023), y dependiendo d el sexo de los individuos (p - valor = 0.021). La variante rs11576685*C de PDZK1 parece incrementar la expresión basal del gen (p - valor = 0.03 ), y muestra una asociación con el color de piel oscuro (p - valor = 0.02). Conclusión: Los resultados de este estudio evidencian que existen diferencias de expresión del gen PDZK1 que están influenciadas por la exposición solar y por factores genéticos, y no solo por factores hormonales como se había visto anteriormente.

Human pigmentation is a highly diverse and complex trait among populations and has drawn particular attention from both academic and non-academic investigators for thousands of years. Previous studies detected selection signals in several human pigmentation genes, but few studies have integrated contribution from multiple genes to the evolution of human pigmentation. Moreover, none has quantified selective pressures on human pigmentation over epochs and between populations. Here, we dissect dynamics and differences of selective pressures during different periods and between distinct populations with new approaches. We use genotype data of 19 genes associated with human pigmentation from 17 publicly available datasets and obtain data for 2346 individuals of six representative population groups from across the world. Our results quantify the strength of natural selection on light pigmentation not only in modern Europeans (0.0259/generation) but also in proto-Eurasians (0.00650/generation). Our results also suggest that several derived alleles associated with human dark pigmentation may be under positive directional selection in some African populations. Our study provides the first attempt to quantitatively investigate the dynamics of selective pressures during different time periods in the evolution of human pigmentation. This article has an associated First Person interview with the first author of the article., Summary: This study developed a novel approach to estimate selection strength over time and applied it on the evolution of human pigmentation.

Introducción: La sensibilidad a la radiación ultravioleta está determinada por característicaspigmentarias influenciadas por varios genes. Ciertos rasgos de la pigmentación,como tener un color de piel claro, tener muchas efélides y/o nevus y una dificultadde bronceado, son indicadores de una mayor susceptibilidad a esta radiación solary, por tanto, al cáncer de piel. Sin embargo, poco se conoce sobre los determinantesgenéticos asociados a la sensibilidad solar, a excepción del gen MC1R. Este estudioanaliza las bases genéticas de la sensibilidad solar y la predisposición al cáncer cutáneoen una población española. Metodología: En el estudio participaron 300 voluntarios.Mediante un cuestionario estandarizado, se recogieron características fenotípicasde pigmentación, historia de quemaduras solares y hábitos de exposición solar. Paracada individuo, se determinó el genotipo de genes involucrados en la ruta de la pigmentaciónhumana – MC1R, ASIP, TYR, OCA2, HERC2, SLC24A4, BNC2, IRF4,KITLG, y SLC45A2. El análisis de asociación entre las características fenotípicas y elgenotipo se realizó con una regresión logística, utilizando el programa SPSS v24. Resultados:El mayor determinante genético de los rasgos de pigmentación y sensibilidadsolar es el gen MC1R. Polimorfismos en IRF4, ASP y SLC45A2 también pareceninfluir en la pigmentación cutánea basal, así como en la aparición de lesiones hiperpigmentadasbenignas. Conclusión: Los individuos con fenotipo de riesgo tienensignificativamente mayor número de polimorfismos asociados con una peor toleranciaal sol. Individuos portadores de estos polimorfismos tendrían una mayor sensibilidad ala radiación solar y, por tanto, a padecer cáncer cutáneo. Introduction: Sensitivity to ultraviolet radiation is determined by pigmentation traits, which are influenced by several genes. Particular pigmentation characteristics, such as having fair skin, having ephelides and/or naevi, and the inability to tan, are associated with a high sensitivity to sunlight and, therefore, a predisposition to skin cancer. However, little is known about the genetic determinants associated with sunlight sensitivity, apart from MC1R gene. This study analyses the genetic basisof sunlight sensitivity and skin cancer susceptibility in a Spanish population. Methodology: Three-hundred volunteers participated in the study. Phenotypic characteristics of pigmentation, history of sunburn and sun exposure behaviour were collectedby using a standardized questionnaire. For each participant, we examined the genotype of the following genes involved in the human pigmentation pathway: MC1R, ASIP, TYR, OCA2, HERC2, SLC24A4, BNC2, IRF4, KITLG,and SLC45A2. To determine the association with phenotypic characteristics, a logistic regression was performed for each polymorfism. Statistical analysis were perfomed by using SPSS v24 software. Results: The major genetic determinant of pigmentation and sunlight sensitivity traits is the MC1R gene. Polymorphisms in IRF4, ASIP and SLC45A4 also seems to impacton basal skin pigmentation, as well as on the appearance of benign hyperpigmented lesions. Conclusion: Individuals having a risk phenotype carry significantly a higher number of genetic variants associated with reduced tolerance to sun exposure. Individuals carrying these polymorphisms would have a higher sensitivity to UV radiation and, therefore, an increased susceptibility todevelop skin cancer.

Treball Final de Grau en Medicina. Codi: MD1158. Curs acadèmic: 2016/2017 Introducción: Las características pigmentarias que determinan la sensibilidad solar están influenciadas por diversos genes. Tener un elevado número de efélides o nevus y una tendencia a quemarse indican mayor susceptibilidad a padecer daños causados por la radiación ultravioleta y a desarrollar un cáncer de piel. A excepción del gen MC1R, poco se conoce sobre los determinantes genéticos asociados a la sensibilidad solar. Este estudio analiza dichas bases genéticas en una población española. Metodología: En el estudio participaron 304 voluntarios españoles. Se recogieron características de pigmentación, historia de quemaduras solares y exposición solar. Se determinó el genotipo de genes involucrados en la pigmentación humana – MC1R, ASP, TYR, OCA2, HERC2, SLC24A4, BNC2, IRF4, KITLG y SLC45A2 – de cada individuo. El análisis de asociación entre las características fenotípicas y el genotipo se realizó con una regresión logística. Resultados: El principal determinante genético de la pigmentación y sensibilidad solar es el gen MC1R. Sin embargo, polimorfismos en IRF4, ASP y SLC45A2 también parecen influir en la capacidad de bronceado y en la aparición de lesiones hiperpigmentadas benignas. Conclusión: Los individuos portadores de polimorfismos en estos genes tendrán un fenotipo de riesgo asociado a una mayor sensibilidad solar y, por tanto, a padecer cáncer cutáneo. Introduction: Pigmentation traits related to cutaneous sensitivity to sunlight are influenced by several genes. Having ephelides or a high naevus count and a tendency to burn indicate a greater susceptibility to the damaging effects of ultraviolet radiation and to develop skin cancer. With the exception of the MC1R gene, little is known about the genetic determinants associated with sun sensitivity. The aim of this study is to analyse the genetic basis of sun sensitivity in a Spanish population. Methodology: A total of 304 Spanish volunteers were included in the study. Pigmentation traits, history of sunburns and sun exposure were collected. The genotype of several genes involved in human pigmentation - MC1R, ASIP, TYR, OCA2, HERC2, SLC24A4, BNC2, IRF4, KITLG and SLC45A2 - was examined in each participant. To determine the association with phenotypic traits, a logistic regression was performed for each polymorphism. Results: The main genetic determinant of pigmentary traits and sun sensitivity is the MC1R gene. However, polymorphisms in IRF4, ASIP and SLC45A2 also seem to have an impact on the ability to tan and on the appearance of benign hyperpigmented lesions. Conclusion: Individuals carrying polymorphisms in these genes would have a risk phenotype associated to an increased sensitivy to sunlight and, therefore, greater susceptibility to develop skin cancer.

La pigmentación basal es un rasgo de carácter poligénico con alta heredabilidad, influenciada por factores ambientales, genéticos y endocrinos. La incidencia del cáncer de piel revela una clara relación entre ciertos rasgos de pigmentación y los daños causados por la exposición solar. Además, estudios recientes muestran una diferencia tanto en las características de pigmentación así como en la prevalencia e incidencia de melanoma entre sexos. El objetivo principal de esta tesis doctoral es ampliar el conocimiento actual sobre las bases moleculares de la pigmentación humana y la predisposición a melanoma cutáneo. Los análisis genéticos revelan la existencia de efectos genéticos que influyen en la pigmentación humana y el riesgo a melanoma según el sexo. Asimismo, polimorfismos localizados en la región 3’UTR de genes relacionados con la pigmentación estarían implicados en la aparición de lesiones cutáneas benignas relacionadas con el sol. Los estudios in vitro revelan que el riesgo a melanoma observado en individuos portadores de mutaciones en CDKN2A no es consecuencia de un comportamiento anormal del melanocito, siendo necesarias alteraciones genéticas y epigenéticas adicionales para transformar dichos melanocitos., Human pigmentation traits are some of the most visible and differentiable human characteristics. Basal cutaneous pigmentation is a polygenic quantitative trait with high heritability, being influenced by genetic, environmental and sex-endocrine factors. Skin cancer incidence reveals a clear relationship between genetically controlled pigmentation traits and sunlight damage. Besides, sexual disparity in skin pigmentation and melanoma incidence and outcome has been recently shown. The general objective of this thesis is to expand the current knowledge on the molecular bases of human pigmentation and predisposition to cutaneous melanoma. Genetic analyses reveal a strong evidence for sex-differentiated genetic effects in human pigmentation as well as in melanoma risk. Additionally, there is a potential implication of polymorphisms in the 3'UTR regions of pigmentation-associated genes in the appearance of different sun-related benign pigmented skin lesions. In vitro studies show that the increased melanoma risk seen in CDKN2A mutation carriers is not the result of intrinsic abnormalities in melanocytes, being additional somatic genetic or epigenetic changes needed to transform melanocytes.

BACKGROUND: Although genetic studies have reported a number of loci associated with cutaneous melanoma (CM) risk, a comprehensive synopsis of genetic association studies published in the field and systematic meta-analysis for all eligible polymorphisms have not been reported. METHODS: We systematically annotated data from all genetic association studies published in the CM field (n = 145), including data from genome-wide association studies (GWAS), and performed random-effects meta-analyses across all eligible polymorphisms on the basis of four or more independent case-control datasets in the main analyses. Supplementary analyses of three available datasets derived from GWAS and GWAS-replication studies were also done. Nominally statistically significant associations between polymorphisms and CM were graded for the strength of epidemiological evidence on the basis of the Human Genome Epidemiology Network Venice criteria. All statistical tests were two-sided. RESULTS: Forty-two polymorphisms across 18 independent loci evaluated in four or more datasets including candidate gene studies and available GWAS data were subjected to meta-analysis. Eight loci were identified in the main meta-analyses as being associated with a risk of CM (P < .05) of which four loci showed a genome-wide statistically significant association (P < 1 x 10(-7)), including 16q24.3 (MC1R), 20q11.22 (MYH7B/PIGU/ASIP), 11q14.3 (TYR), and 5p13.2 (SLC45A2). Grading of the cumulative evidence by the Venice criteria suggested strong epidemiological credibility for all four loci with genome-wide statistical significance and one additional gene at 9p23 (TYRP1). In the supplementary meta-analyses, a locus at 9p21.3 (CDKN2A/MTAP) reached genome-wide statistical significance with CM and had strong epidemiological credibility. CONCLUSIONS: To the best of our knowledge, this is the first comprehensive field synopsis and systematic meta-analysis to identify genes associated with an increased susceptibility to CM. J Natl Cancer Inst

Aim To analyze two phenotype characteristics – eye and hair color – using single-nucleotide polymorphisms (SNPs) and evaluate their prediction accuracy in Slovenian population. Methods Twelve SNPs (OCA2 – rs1667394, rs7170989, rs1800407, rs7495174; HERC2 – rs1129038, rs12913832; MC1R – rs1805005, rs1805008; TYR – rs1393350; SLC45A2 – rs16891982, rs26722; SLC24A5 – rs1426654) were used for the development of a single multiplex assay. The single multiplex assay was based on SNaPshot chemistry and capillary electrophoresis. In order to evaluate the accuracy of the prediction of eye and hair color, we used the logistic regression model and the Bayesian network model, and compared the parameters of both. Results The new single multiplex assay displayed high levels of genotyping sensitivity with complete profiles generated from as little as 62 pg of DNA. Based on a prior evaluation of all SNPs in a single multiplex, we focused on the five most statistically significant in our population in order to investigate the predictive value. The two prediction models performed reliably without prior ancestry information, and revealed very good accuracy for both eye and hair color. Both models determined the highest predictive value for rs12913832 (P