mRNA-based skin identification for forensic applications

Although the identification of human skin cells is of important relevance in many forensic cases, there is currently no reliable method available. Here, we present a highly specific and sensitive messenger RNA (mRNA) approach for skin identification, meeting the key requirements in forensic analyses. We examined 11 candidate genes with skin-specific expression, as ascertained from expression databases and the literature, as well as five candidate reference genes ascertained from previous studies, in skin samples and in other forensically relevant tissues. We identified mRNA transcripts from three genes CDSN, LOR and KRT9, showing strong over-expression in skin samples relative to samples from forensic body fluids, making them suitable markers for skin identification. Out of the candidate reference genes tested, only ACTB showed similarly high expression in skin and body-fluid samples, providing a suitable reference marker for quantitative real-time PCR (qPCR) analysis of skin. Analyses of palmar and thumbprint skin samples indicate that our qPCR approach for the three skin-targeted mRNA markers, as well as the reference mRNA marker ACTB, is highly sensitive, allowing successful detection of minute amounts of skin material including full, half and quarter thumbprints, albeit with decreased success in decreasing print material. Furthermore, thumbprints stored for 6.5 months provided similar results relative to freshly analysed samples implying reasonable time-wise stability of the three skin-targeted mRNAs as well as the ACTB reference mRNA. Our study represents the first attempt towards reliable mRNA-based skin identification in forensic applications with particular relevance for future trace/touched object analyses in forensic case work. Although the approach for skin identification introduced here can be informative when applied on its own, we recommend for increased reliability the integration of (one or more of) the skin-targeted mRNA markers presented here into multiplex assays additionally including mRNA markers targeting alternative cell types expected in forensic samples

Genetics of skin color variation in Europeans: genome-wide association studies with functional follow-up

In the International Visible Trait Genetics (VisiGen) Consortium, we investigated the genetics of human skin color by combining a series of genome-wide association studies (GWAS) in a total of 17,262 Europeans with functional follow-up of discovered loci. Our GWAS provide the first genome-wide significant evidence for chromosome 20q11.22 harboring the ASIP gene being explicitly associated with skin color in Europeans. In addition, genomic loci at 5p13.2 (SLC45A2), 6p25.3 (IRF4), 15q13.1 (HERC2/OCA2), and 16q24.3 (MC1R) were confirmed to be involved in skin coloration in Europeans. In follow-up gene expression and regulation studies of 22 genes in 20q11.22, we highlighted two novel genes EIF2S2 and GSS, serving as competing functional candidates in this region and providing future research lines. A genetically inferred skin color score obtained from the 9 top-associated SNPs from 9 genes in 940 worldwide samples (HGDP-CEPH) showed a clear gradual pattern in Western Eurasians similar to the distribution of physical skin color, suggesting the used 9 SNPs as suitable markers for DNA prediction of skin color in Europeans and neighboring populations, relevant in future forensic and anthropological investigations

From GWAS to Function: Transcriptional regulation of pigmentation genes in humans Transcriptional regulation of pigmentation genes in humans

Human pigmentation is one of the most explicit visual traits, which therefore has been subject of many research studies. With the emergence of large-scale genetic association studies like GWASs, numerous SNPs have been associated with a phenotype of interest, such as human eye, hair and skin color. Many of the identified pigmentation-associated SNPs have been implemented in forensic and/or anthropological applications that are developed to predict human pigmentation traits. The work described in this thesis aims to understand the functional biology underlying several of these highly associated pigmentation SNPs. This thesis starts with a general overview of the current knowledge on human pigmentation in Chapter 1, including its evolutionary history and biochemistry, the mechanisms of melanogenesis, and genetic variation of pigmentation genes. It also summarizes the essentials of transcriptional regulation and the key players involved in this complex process. Chapters 2-5 contain the experimental work performed during the course of this PhD study. Herein I focus on the biological function of SNPs that are strongly associated with human pigmentation phenotypes. In Chapter 2, I describe a detailed analysis of the regulatory function of an enhancer element that contains the intronic SNP rs12913832 which is strongly associated with human skin, eye and hair color, and controls expression of the pigmentation gene OCA2. Due to the original design of GWASs and the SNP arrays used, the genetic association signals prioritized in these studies are not necessarily the actual causal or functional SNPs. These causal SNPs need to be identified in order to study the functional biology underlying the detected genetic association signals. This is exemplified in Chapter 3, in which I describe the identification of the actual functional SNP (rs12350739) responsible for the detected skin pigmentation-associated signal in the BNC2 gene, followed by a detailed analysis of the transcriptional regulation of that gene and the involvement of rs12305739 therein. Regulatory elements, such as enhancers are typically located at large distances from their target genes, however this generally does not restrict the activity of these elements, as they are able to regulate transcription over large distances through long-range interactions. Chapter 4 focuses on chromatin structure to characterize the allele-specific regulatory mode-of-action of an intronic enhancer in which the pigmentation-associated SNP rs12203592 is located, and controls expression of the IRF4 gene. In Chapter 5 I investigate the genetic basis of human skin color by combining a series of GWASs. This is followed by functional analyses of one of the five genomic regions harboring skin-color associated SNPs detected in these GWASs. At the time the work on this thesis was done, this genomic region represented the least understood genetic association signal at the functional molecular level. Finally, Chapter 6 summarizes the results of the experimental research described in Chapters 2-5, and I discuss the in silico and experimental workflow as employed in Chapters 2-5 to unravel the functional biology underlying genetic association signals in a more general context

Human skin color is influenced by an intergenic DNA polymorphism regulating transcription of the nearby BNC2 pigmentation gene

Single nucleotide polymorphisms (SNPs) found to be statistically significant when associated with human diseases, and other phenotypes are most often located in non-coding regions of the genome. One example is rs10765819 located in the first intron of the BNC2 gene previously associated with (saturation of) human skin color. Here, we demonstrate that a nearby intergenic SNP (rs12350739) in high linkage disequilibrium with rs10756819 is likely the causal DNA variant for the observed BNC2 skin color association. The highly conserved region surrounding rs12350739 functions as an enhancer element regulating BNC2 transcription in human-melanocytes, while the activity of this enhancer element depends on the allelic status of rs12350739. When the rs12350739-AA allele is present, the chromatin at the region surrounding rs12350739 is inaccessible and the enhancer element is only slightly active, resulting in low expression of BNC2, corresponding with light skin pigmentation. When the rs12350739-GG allele is present however, the chromatin at the region surrounding rs12350739 is more accessible and the enhancer is active, resulting in a higher expression of BNC2, corresponding with dark skin pigmentation. Overall, we demonstrate the identification of the functional DNA variant that explains the BNC2 skin color association signal, providing another important step towards further understanding human pigmentation genetics beyond statistical association. We thus deliver a clear example of how an intergenic non-coding DNA variant modulates the regulatory potential of the enhancer element it is located within, which in turn results in allele dependent differential gene expression affecting variation in common human traits

Allele-specific transcriptional regulation of IRF4 in melanocytes is mediated by chromatin looping of the intronic rs12203592 enhancer to the IRF4 promoter

The majority of significant single-nucleotide polymorphisms (SNPs) identified with genome-wide association studies are located in non-coding regions of the genome; it is therefore possible that they are involved in transcriptional regulation of a nearby gene rather than affecting an encoded protein's function. Previously, it was demonstrated that the SNP rs12203592, located in intron 4 of the IRF4 gene, is strongly associated with human skin pigmentation and modulates an enhancer element that controls expression of IRF4. In our study, we investigated the allele-specific effect of rs12203592 on IRF4 expression in epidermal skin samples and in melanocytic cells from donors of different skin color. We focused on the characteristics and activity of the enhancer, and on long-range chromatin interactions in melanocytic cells homozygous and heterozygous for rs12203592. We found that, irrespective of the trans-activating environment, IRF4 transcription is strongly correlated with the allelic status of rs12203592, the activity of the rs12203592 enhancer and that the chromatin features depend on the rs12203592 genotype. Furthermore, we demonstrate that the rs12203592 enhancer physically interacts with the IRF4 promoter through an allele-dependent chromatin loop, and suggest that subsequent allele-specific activation of IRF4 transcription is stabilized by another allele-specific loop from the rs12203592 enhancer to an additional regulatory element in IRF4. We conclude that the non-coding SNP rs12203592 is located in a regulatory region and affects a wide range of enhancer characteristics, resulting into modulation of the enhancer's activity, its interaction with the IRF4 promoter and subsequent allele-specific transcription of IRF4. Our findings provide another example of a non-coding SNP affecting skin color by modulating enhancer-mediated transcriptional regulation

HERC2 rs12913832 modulates human pigmentation by attenuating chromatin-loop formation between a long-range enhancer and the OCA2 promoter

Pigmentation of skin, eye, and hair reflects some of the most evident common phenotypes in humans. Several candidate genes for human pigmentation are identified. The SNP rs12913832 has strong statistical association with human pigmentation. It is located within an intron of the nonpigment gene HERC2, 21 kb upstream of the pigment gene OCA2, and the region surrounding rs12913832 is highly conserved among animal species. However, the exact functional role of HERC2 rs12913832 in human pigmentation is unknown. Here we demonstrate that the HERC2 rs12913832 region functions as an enhancer regulating OCA2 transcription. In darkly pigmented human melanocytes carrying the rs12913832 T-allele, we detected binding of the transcription factors HLTF, LEF1, and MITF to the HERC2 rs12913832 enhancer, and a long-range chromatin loop between this enhancer and the OCA2 promoter that leads to elevated OCA2 expression. In contrast, in lightly pigmented melanocytes carrying the rs12913832 C-allele, chromatin-loop formation, transcription factor recruitment, and OCA2 expression are all reduced. Hence, we demonstrate that allelic variation of a common noncoding SNP located in a distal regulatory element not only disrupts the regulatory potential of this element but also affects its interaction with the relevant promoter. We provide the key mechanistic insight that allele-dependent differences in chromatin-loop formation (i.e., structural differences in the folding of gene loci) result in differences in allelic gene expression that affects common phenotypic traits. This concept is highly relevant for future studies aiming to unveil the functional basis of genetically determined phenotypes, including diseases

Genetic variation in regulatory DNA elements: the case of OCA2 transcriptional regulation

Mutations within the OCA2 gene or the complete absence of the OCA2 protein leads to oculocutaneous albinism type 2. The OCA2 protein plays a central role in melanosome biogenesis, and it is a strong determinant of the eumelanin content in melanocytes. Transcript levels of the OCA2 gene are strongly correlated with pigmentation intensities. Recent studies demonstrated that the transcriptional level of OCA2 is to a large extent determined by the noncoding SNP rs12913832 located 21.5kb upstream of the OCA2 gene promoter. In this review, we discuss current hypotheses and the available data on the mechanism of OCA2 transcriptional regulation and how this is influenced by genetic variation. Finally, we will explore how future epigenetic studies can be used to advance our insight into the functional biology that connects genetic variation to human pigmentation

A multiplex (m)RNA-profiling system for the forensic identification of body fluids and contact traces

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using >= 0.05 mu L body fluid starting material whereas full DNA profiles were obtained with >= 0.1 mu L. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA-and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA-and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present. (C) 2012 Elsevier Ireland Ltd. All rights reserved