Jeppe Dyrberg Andersen

Genetic analyses of the human eye colours using a novel objective method for eye colour classification

In this study, we present a new objective method for measuring the eye colour on a continuous scale that allows researchers to associate genetic markers with different shades of eye colour. With the use of the custom designed software Digital Iris Analysis Tool (DIAT), the iris was automatically identified and extracted from high resolution digital images. DIAT was made user friendly with a graphical user interface. The software counted the number of blue and brown pixels in the iris image and calculated a Pixel Index of the Eye (PIE-score) that described the eye colour quantitatively. The PIE-score ranged from -1 to 1 (brown to blue). The software eliminated the need for user based interpretation and qualitative eye colour categories. In 94% (570) of 605 analyzed eye images, the iris region was successfully extracted and a PIE-score was calculated. A very high correlation between the PIE-score and the human perception of eye colour was observed. The correlations between the PIE-scores and the six IrisPlex SNPs (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, TYR rs1393350, SLC45A2 rs16891982 and IRF4 rs12203592) were analyzed in 570 individuals. Significant differences (p<10(-6)) in the PIE-scores of the individuals typed as HERC2 rs12913832 G (PIE=0.99) and rs12913832 GA (PIE=-0.71) or A (PIE=-0.87) were observed. We adjusted for the effect of HERC2 rs12913832 and showed that the quantitative PIE-scores were significantly associated with SNPs with minor effects (OCA2 rs1800407, SLC24A4 rs12896399 and TYR rs1393350) on the eye colour. We evaluated the two published prediction models for eye colour (IrisPlex [1] and Snipper[2]) and compared the predictions with the PIE-scores. We found good concordance with the prediction from individuals typed as HERC2 rs12913832 G. However, both methods had difficulties in categorizing individuals typed as HERC2 rs12913832 GA because of the large variation in eye colour in HERC2 rs12913832 GA individuals. With the use of the DIAT software and the PIE-score, it will be possible to automatically compare the iris colour of large numbers of iris images obtained by different studies and to perform large meta-studies that may reveal loci with small effects on the eye colour.

Evaluation of DNA Variants Associated with Androgenetic Alopecia and Their Potential to Predict Male Pattern Baldness

Androgenetic alopecia, known in men as male pattern baldness (MPB), is a very conspicuous condition that is particularly frequent among European men and thus contributes markedly to variation in physical appearance traits amongst Europeans. Recent studies have revealed multiple genes and polymorphisms to be associated with susceptibility to MPB. In this study, 50 candidate SNPs for androgenetic alopecia were analyzed in order to verify their potential to predict MPB. Significant associations were confirmed for 29 SNPs from chromosomes X, 1, 5, 7, 18 and 20. A simple 5-SNP prediction model and an extended 20-SNP model were developed based on a discovery panel of 305 males from various European populations fitting one of two distinct phenotype categories. The first category consisted of men below 50 years of age with significant baldness and the second; men aged 50 years or older lacking baldness. The simple model comprised the five best predictors: rs5919324 near AR, rs1998076 in the 20p11 region, rs929626 in EBF1, rs12565727 in TARDBP and rs756853 in HDAC9. The extended prediction model added 15 SNPs from five genomic regions that improved overall prevalence-adjusted predictive accuracy measured by area under the receiver characteristic operating curve (AUC). Both models were evaluated for predictive accuracy using a test set of 300 males reflecting the general European population. Applying a 65% probability threshold, high prediction sensitivity of 87.1% but low specificity of 42.4% was obtained in men aged <50 years. In men aged ≥50, prediction sensitivity was slightly lower at 67.7% while specificity reached 90%. Overall, the AUC=0.761 calculated for men at or above 50 years of age indicates these SNPs offer considerable potential for the application of genetic tests to predict MPB patterns, adding a highly informative predictive system to the emerging field of forensic analysis of externally visible characteristics.

Evaluation of the predictive capacity of DNA variants associated with straight hair in Europeans.

DNA-based prediction of hair morphology, defined as straight, curly or wavy hair, could contribute to an improved description of an unknown offender and allow more accurate forensic reconstructions of physical appearance in the field of forensic DNA phenotyping. Differences in scalp hair morphology are significant at the worldwide scale and within Europe. The only genome-wide association study made to date revealed the Trichohyalin gene (TCHH) to be significantly associated with hair morphology in Europeans and reported weaker associations for WNT10A and FRAS1 genes. We conducted a study that centered on six SNPs located in these three genes with a sample of 528 individuals from Poland. The predictive capacity of the candidate DNA variants was evaluated using logistic regression; classification and regression trees; and neural networks, by applying a 10-fold cross validation procedure. Additionally, an independent test set of 142 males from six European populations was used to verify performance of the developed prediction models. Our study confirmed association of rs11803731 (TCHH), rs7349332 (WNT10A) and rs1268789 (FRAS1) SNPs with hair morphology. The combined genotype risk score for straight hair had an odds ratio of 2.7 and these predictors explained ∼ 8.2% of the total variance. The selected three SNPs were found to predict straight hair with a high sensitivity but low specificity when a 10-fold cross validation procedure was applied and the best results were obtained using the neural networks approach (AUC=0.688, sensitivity=91.2%, specificity=23.0%). Application of the neural networks model with 65% probability threshold on an additional test set gave high sensitivity (81.4%) and improved specificity (50.0%) with a total of 78.7% correct calls, but a high non-classification rate (66.9%). The combined TTGGGG SNP genotype for rs11803731, rs7349332, rs1268789 (European frequency=4.5%) of all six straight hair-associated alleles was identified as the best predictor, giving >80% probability of straight hair. Finally, association testing of 44 SNPs previously identified to be associated with male pattern baldness revealed a suggestive association with hair morphology for rs4679955 on 3q25.1. The study results reported provide the starting point for the development of a predictive test for hair morphology in Europeans. More studies are now needed to discover additional determinants of hair morphology to improve the predictive accuracy of this trait in forensic analysis.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.

The effect of gender on eye colour variation in European populations and an evaluation of the IrisPlex prediction model

In two recent studies of Spanish individuals, gender was suggested as a factor that contributes to human eye colour variation. However, gender did not improve the predictive accuracy on blue, intermediate and brown eye colours when gender was included in the IrisPlex model. In this study, we investigate the role of gender as a factor that contributes to eye colour variation and suggest that the gender effect on eye colour is population specific. A total of 230 Italian individuals were typed for the six IrisPlex SNPs (rs12913832, rs1800407, rs12896399, rs1393350, rs16891982 and rs12203592). A quantitative eye colour score (Pixel Index of the Eye: PIE-score) was calculated based on digital eye images using the custom made DIAT software. The results were compared with those of Danish and Swedish population samples. As expected, we found HERC2 rs12913832 as the main predictor of human eye colour independently of ancestry. Furthermore, we found gender to be significantly associated with quantitative eye colour measurements in the Italian population sample. We found that the association was statistically significant only among Italian individuals typed as heterozygote GA for HERC2 rs12913832. Interestingly, we did not observe the same association in the Danish and Swedish population. This indicated that the gender effect on eye colour is population specific. We estimated the effect of gender on quantitative eye colour in the Italian population sample to be 4.9%. Among gender and the IrisPlex SNPs, gender ranked as the second most important predictor of human eye colour variation in Italians after HERC2 rs12913832. We, furthermore, tested the five lower ranked IrisPlex predictors, and evaluated all possible 3(6) (729) genotype combinations of the IrisPlex assay and their corresponding predictive values using the IrisPlex prediction model [4]. The results suggested that maximum three (rs12913832, rs1800407, rs16891982) of the six IrisPlex SNPs are useful in practical forensic genetic casework.

Importance of nonsynonymous OCA2 variants in human eye color prediction

The color of the eyes is one of the most prominent phenotypes in humans and it is often used to describe the appearance of an individual. The intensity of pigmentation in the iris is strongly associated with one single‐nucleotide polymorphism (SNP), rs12913832:A>G that is located in the promotor region of OCA2 (OMIM #611409). Nevertheless, many eye colors cannot be explained by only considering rs12913832:A>G.

Soluble Urokinase Plasminogen Activator Receptor During Allogeneic Stem Cell Transplantation

Previous studies have found that soluble urokinase plasminogen activation receptor (suPAR) increases during inflammatory and malignant illness and elevated suPAR levels may be associated with poor clinical outcome. The purpose of this study was to investigate plasma levels of suPAR during the course of allogeneic stem cell transplantation (SCT). Twenty SCT patients were included in the study. suPAR was measured by ELISA in daily taken plasma samples during the pretransplant conditioning with chemotherapy and weekly for 1 month after infusion of the graft. suPAR levels before the start of the conditioning were significantly elevated when compared to those of healthy controls. During the conditioning in particular treatment with antithymocyte globulin was associated with significantly increased suPAR levels (P = 0.012). At day +7 after infusion of the graft, suPAR levels had decreased to pretreatment levels. High suPAR levels at day 0 were associated with increased mortality (P = 0.011). The present study found increased suPAR levels during the conditioning in SCT patients. Further, the data indicated that increased suPAR levels may be associated with increased mortality, suggesting suPAR as a candidate for further studies as an outcome predictor in SCT.

Next-generation sequencing of multiple individuals per barcoded library by deconvolution of sequenced amplicons using endonuclease fragment analysis.

The simultaneous sequencing of samples from multiple individuals increases the efficiency of next-generation sequencing (NGS) while also reducing costs. Here we describe a novel and simple approach for sequencing DNA from multiple individuals per barcode. Our strategy relies on the endonuclease digestion of PCR amplicons prior to library preparation, creating a specific fragment pattern for each individual that can be resolved after sequencing. By using both barcodes and restriction fragment patterns, we demonstrate the ability to sequence the human melanocortin 1 receptor (MC1R) genes from 72 individuals using only 24 barcoded libraries.

Genomic and immunohistochemical characterisation of a lacrimal gland oncocytoma and review of literature

The aim of the present study was to report the genetic and immunohistochemical profile of a rare case of lacrimal gland oncocytoma. A 20-year-old male underwent magnetic resonance imaging (MRI) due to viral encephalitis. Notably, the MRI revealed a multicystic tumor in the left lacrimal gland. A lateral orbitotomy was performed and the tumor was completely excised. Four months following surgery, the patient was free of symptoms. Histopathologically, the tumor was composed of large, eosinophilic and polyhedral cells with small round nuclei. The tumor cells stained strongly for antimitochondrial antibody MU213-UC, cytokeratin (CK) 5/6, CK 7, CK 17, CK 8/18 and CK 19. The final diagnosis was an oncocytoma of the lacrimal gland without any signs of malignancy. Array-based comparative genomic hybridisation demonstrated a gain of one copy of chromosome 8 and loss of one copy of chromosome 22 as the sole genomic imbalances. These chromosomal alterations have not previously been identified in oncocytoma and may be specific to lacrimal gland oncocytoma. Sequencing of the mitochondrial genome demonstrated multiple alterations of the NADH-ubiquinone oxidoreductase chain 5 (ND5) gene involved in mitochondrial oxidative phosphorylation. This may support the notion of a common genetic background of oncocytic lesions in the lacrimal gland and other anatomical sites.

Pigmentary Markers in Danes – Associations with Quantitative Skin Colour, Nevi Count, Familial Atypical Multiple-Mole, and Melanoma Syndrome

To investigate whether pigmentation genes involved in the melanogenic pathway (melanogenesis) contributed to melanoma predisposition, we compared pigmentary genetics with quantitative skin pigmentation measurements, the number of atypical nevi, the total nevus count, and the familial atypical multiple mole and melanoma (FAMMM) syndrome. We typed 32 pigmentary SNP markers and sequenced MC1R in 246 healthy individuals and 116 individuals attending periodic control for malignant melanoma development, 50 of which were diagnosed with FAMMM. It was observed that individuals with any two grouped MC1R variants (missense, NM_002386:c. 456C > A (p.TYR152*), or NM_002386:c.83_84insA (p.Asn29Glnfs*14) had significantly (p<0.001) lighter skin pigmentation of the upper-inner arm than those with none or one MC1R variant. We did not observe any significant association of the MC1R variants with constitutive pigmentation measured on the buttock area. We hypothesize that the effect of MC1R variants on arm pigmentation is primarily reflecting the inability to tan when subjected to UVR. A gender specific effect on skin pigmentation was also observed, and it was found that the skin pigmentation of females on average were darker than that of males (p<0.01). We conclude that MC1R variants are associated with quantitative skin colour in a lightly pigmented Danish population. We did not observe any association between any pigmentary marker and the FAMMM syndrome. We suggest that the genetics of FAMMM is not related to the genetics of the pigmentary pathway.