Changes in mouse epidermal DNA methylation during development of squamous cell carcinoma in response to UVR.

Squamous cell carcinoma (SCC) is a common skin cancer, often caused by exposure to ultraviolet radiation (UVR). Recent studies have shown that changes in DNA methylation play a crucial role in the development of cancers. However, methylation patterns of SCC are not well characterised. Identifying biomarkers for the risk of developing SCC could be helpful for early detection and diagnosis and can potentially improve treatment and prevention strategies. This study aimed to investigate methylation changes in the epidermis of mice exposed to UVR for 24 weeks. We examined the DNA methylation levels of 260 199 CpGs using the Illumina Infinium Mouse Methylation BeadChip and studied the epidermis of UVR-exposed and unexposed mice every 4 weeks for 24 weeks (n = 39). We identified CpGs with large differences in methylation levels (ß-values) between UVR-exposed and unexposed mice. We also observed differences in the epigenetic age of these mice. We identified CpGs in Rev, Ipmk, Rad51b, Fgfr2, Fgfr3 and Ctnnb1 that may serve as potential biomarkers for SCC risk and could be helpful for the early detection and prevention of SCC. Further investigations are necessary to determine the biological functions and clinical significance of these CpGs.

Analysis of Skin Pigmentation and Genetic Ancestry in Three Subpopulations from Pakistan: Punjabi, Pashtun, and Baloch.

Skin pigmentation is one of the most prominent and variable phenotypes in humans. We compared the alleles of 163 SNPs and indels from the Human Pigmentation (HuPi) AmpliSeq™ Custom panel, and biogeographic ancestry with the quantitative skin pigmentation levels on the upper arm, lower arm, and forehead of 299 Pakistani individuals from three subpopulations: Baloch, Pashtun, and Punjabi. The biogeographic ancestry of each individual was estimated using the Precision ID Ancestry Panel. All individuals were mainly of mixed South-Central Asian and European ancestry. However, the Baloch individuals also had an average proportion of Sub-Saharan African ancestry of approximately 10%, whereas it was <1% in the Punjabi and Pashtun individuals. The pairwise genetic distances between the Pashtun, Punjabi, and Baloch subpopulations based on the ancestry markers were statistically significantly different. Individuals from the Pashtun subpopulation had statistically significantly lower skin pigmentation than individuals from the Punjabi and Baloch subpopulations (p < 0.05). The proportions of European and Sub-Saharan African ancestry and five SNPs (rs1042602, rs10831496, rs1426654, rs16891982, and rs12913832) were statistically significantly associated with skin pigmentation at either the upper arm, lower arm or forehead in the Pakistani population after correction for multiple testing (p < 10-3). A model based on four of these SNPs (rs1426654, rs1042602, rs16891982, and rs12913832) explained 33% of the upper arm skin pigmentation. The four SNPs and the proportions of European and Sub-Saharan African ancestry explained 37% of the upper arm skin pigmentation. Our results indicate that the four likely causative SNPs, rs1426654, rs1042602, rs16891982, and rs12913832 located in SLC24A5, TYR, SLC45A2, and HERC2, respectively, are essential for skin color variation in the admixed Pakistani subpopulations.

Development of an automated AmpliSeq™ library building workflow for biological stain samples on the Biomek® 3000.

Here, we present the development of an automated AmpliSeq™ (ThermoFischer, MA, USA) workflow for library building using the Biomek® 3000 Laboratory Automation Workstation (Beckman Coulter Inc., CA, USA), in which the total volume of PCR reagents and reagents for library preparation are reduced by one-half. The automated AmpliSeq workflow was tested using 43 stain samples (blood, bone, muscle tissue, semen, swab, nail scrape and cigarette butts) collected from crime scenes. The sequencing data were evaluated for locus balance, heterozygous allele balance and noise. The performance of libraries built with the automated AmpliSeq workflow using one-half of the recommended reagent volumes were similar to the performance of libraries built with the recommended (full) volumes of the reagents.

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.

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 four automated protocols for extraction of DNA from FTA cards.

Extraction of DNA using magnetic bead-based techniques on automated DNA extraction instruments provides a fast, reliable, and reproducible method for DNA extraction from various matrices. Here, we have compared the yield and quality of DNA extracted from FTA cards using four automated extraction protocols on three different instruments. The extraction processes were repeated up to six times with the same pieces of FTA cards. The sample material on the FTA cards was either blood or buccal cells. With the QIAamp DNA Investigator and QIAsymphony DNA Investigator kits, it was possible to extract DNA from the FTA cards in all six rounds of extractions in sufficient amount and quality to obtain complete short tandem repeat (STR) profiles on a QIAcube and a QIAsymphony SP. With the PrepFiler Express kit, almost all the extractable DNA was extracted in the first two rounds of extractions. Furthermore, we demonstrated that it was possible to successfully extract sufficient DNA for STR profiling from previously processed FTA card pieces that had been stored at 4 °C for up to 1 year. This showed that rare or precious FTA card samples may be saved for future analyses even though some DNA was already extracted from the FTA cards.

Human eye colour and HERC2, OCA2 and MATP.

Prediction of human eye colour by forensic genetic methods is of great value in certain crime investigations. Strong associations between blue/brown eye colour and the SNP loci rs1129038 and rs12913832 in the HERC2 gene were recently described. Weaker associations between eye colour and other genetic markers also exist. In 395 randomly selected Danes, we investigated the predictive values of various combinations of SNP alleles in the HERC2, OCA2 and MATP (SLC45A2) genes and compared the results to the eye colours as they were described by the individuals themselves. The highest predictive value of typing either the HERC2 SNPs rs1129038 and/or rs12913832 that are in strong linkage disequilibrium was observed when eye colour was divided into two groups, (1) blue, grey and green (light) and (2) brown and hazel (dark). Sequence variations in rs11636232 and rs7170852 in HERC2, rs1800407 in OCA2 and rs16891982 in MATP showed additional association with eye colours in addition to the effect of HERC2 rs1129038. Diplotype analysis of three sequence variations in HERC2 and one sequence variation in OCA2 showed the best discrimination between light and dark eye colours with a likelihood ratio of 29.3.

Carrier frequency of a nonsense mutation in the adenosine deaminase (ADA) gene implies a high incidence of ADA-deficient severe combined immunodeficiency (SCID) in Somalia and a single, common haplotype indicates common ancestry.

Inherited adenosine deaminase (ADA) deficiency is a rare metabolic disorder that causes immunodeficiency, varying from severe combined immunodeficiency (SCID) in the majority of cases to a less severe form in a small minority of patients. Five patients of Somali origin from four unrelated families, with severe ADA-SCID, were registered in the Greater London area. Patients and their parents were investigated for the nonsense mutation Q3X (ADA c7C>T), two missense mutations K80R (ADA c239A>G) and R142Q (ADA c425G>A), and a TAAA repeat located at the 3' end of an Alu element (AluVpA) positioned 1.1 kb upstream of the ADA transcription start site. All patients were homozygous for the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7. Among 207 Somali immigrants to Denmark, the frequency of ADA c7C>T and the maximum likelihood estimate of the frequency of the haplotype ADA-7T/ADA-239G/ADA-425G/AluVpA7 were both 0.012 (carrier frequency 2.4%). Based on the analysis of AluVpA alleles, the ADA c7C/T mutation was estimated to be approximately 7,100 years old. Approximately 1 out of 5 - 10000 Somali children will be born with ADA deficiency due to an ADA c7C/T mutation, although within certain clans the frequency may be significantly higher. ADA-SCID may be a frequent immunodeficiency disorder in Somalia, but will be underdiagnosed due to the prevailing socioeconomic and nutritional deprivation.