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

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

Obsessive compulsive disorder with and without hoarding symptoms: Characterizing differences

Objective: In recent years there has been some ambiguity about the way hoarding and OCD are related to each other. The present study examines the differences between persons with OCD/hoarding and OCD/non-hoarding and examines which characteristics are associated with the OCD/hoarding group. Information is established about prevalences, socio-demographical characteristics, OCD and related characteristics, OCD subtypes, comorbidity (depression, anxiety disorders and PTSD) and personality traits. Methods: Data from baseline assessment of The Netherlands Obsessive Compulsive Disorder Association (NOCDA) study are used. The NOCDA sample consists of 419 participants between 18 and 79 years of age, including participants with current or remitted full DSM-IV-TR criteria for OCD. Results: Results show that 58 persons (14.3%) are classified as persons with OCD/hoarding and 349 persons (85,7%) are classified as persons with OCD/non-hoarding. OCD/hoarding is independently associated with severity of autism symptoms (p<.001), living without a partner (p<.05) and being less conscientious (p<.05). Persons with OCD/hoarding are not associated with childhood trauma (p=.31), PTSD (p=.91) and AD(H)D, inattentive type (p=.22) and hyperactive type (p=.57). Limitations: Causal interferences about associations between the risk indicators and hoarding symptoms were precluded since results were based on cross-sectional data. Conclusion: This study confirmed differences between persons with OCD/hoarding and persons with OCD/non-hoarding. The most relevant outcome of this study was the association between persons with OCD/hoarding and the increased severity of autism symptoms. These results provide a better understanding of persons with OCD/hoarding and have the potential to improve treatment