Development of an epigenetic age predictor for costal cartilage with a simultaneous somatic tissue differentiation system.

Age prediction from DNA has been a topic of interest in recent years due to the promising results obtained when using epigenetic markers. Since DNA methylation gradually changes across the individual's lifetime, prediction models have been developed accordingly for age estimation. The tissue-dependence for this biomarker usually necessitates the development of tissue-specific age prediction models, in this way, multiple models for age inference have been constructed for the most commonly encountered forensic tissues (blood, oral mucosa, semen). The analysis of skeletal remains has also been attempted and prediction models for bone have now been reported. Recently, the VISAGE Enhanced Tool was developed for the simultaneous DNA methylation analysis of 8 age-correlated loci using targeted high-throughput sequencing. It has been shown that this method is compatible with epigenetic age estimation models for blood, buccal cells, and bone. Since when dealing with decomposed cadavers or postmortem samples, cartilage samples are also an important biological source, an age prediction model for cartilage has been generated in the present study based on methylation data collected using the VISAGE Enhanced Tool. In this way, we have developed a forensic cartilage age prediction model using a training set composed of 109 samples (19-74 age range) based on DNA methylation levels from three CpGs in FHL2, TRIM59 and KLF14, using multivariate quantile regression which provides a mean absolute error (MAE) of ± 4.41 years. An independent testing set composed of 72 samples (19-75 age range) was also analyzed and provided an MAE of ± 4.26 years. In addition, we demonstrate that the 8 VISAGE markers, comprising EDARADD, TRIM59, ELOVL2, MIR29B2CHG, PDE4C, ASPA, FHL2 and KLF14, can be used as tissue prediction markers which provide reliable blood, buccal cells, bone, and cartilage differentiation using a developed multinomial logistic regression model. A training set composed of 392 samples (n = 87 blood, n = 86 buccal cells, n = 110 bone and n = 109 cartilage) was used for building the model (correct classifications: 98.72%, sensitivity: 0.988, specificity: 0.996) and validation was performed using a testing set composed of 192 samples (n = 38 blood, n = 36 buccal cells, n = 46 bone and n = 72 cartilage) showing similar predictive success to the training set (correct classifications: 97.4%, sensitivity: 0.968, specificity: 0.991). By developing both a new cartilage age model and a tissue differentiation model, our study significantly expands the use of the VISAGE Enhanced Tool while increasing the amount of DNA methylation-based information obtained from a single sample and a single forensic laboratory analysis. Both models have been placed in the open-access Snipper forensic classification website.

Development and evaluations of the ancestry informative markers of the VISAGE Enhanced Tool for Appearance and Ancestry.

The VISAGE Enhanced Tool for Appearance and Ancestry (ET) has been designed to combine markers for the prediction of bio-geographical ancestry plus a range of externally visible characteristics into a single massively parallel sequencing (MPS) assay. We describe the development of the ancestry panel markers used in ET, and the enhanced analyses they provide compared to previous MPS-based forensic ancestry assays. As well as established autosomal single nucleotide polymorphisms (SNPs) that differentiate sub-Saharan African, European, East Asian, South Asian, Native American, and Oceanian populations, ET includes autosomal SNPs able to efficiently differentiate populations from Middle East regions. The ability of the ET autosomal ancestry SNPs to distinguish Middle East populations from other continentally defined population groups is such that characteristic patterns for this region can be discerned in genetic cluster analysis using STRUCTURE. Joint cluster membership estimates showing individual co-ancestry that signals North African or East African origins were detected, or cluster patterns were seen that indicate origins from central and Eastern regions of the Middle East. In addition to an augmented panel of autosomal SNPs, ET includes panels of 85 Y-SNPs, 16 X-SNPs and 21 autosomal Microhaplotypes. The Y- and X-SNPs provide a distinct method for obtaining extra detail about co-ancestry patterns identified in males with admixed backgrounds. This study used the 1000 Genomes admixed African and admixed American sample sets to fully explore these enhancements to the analysis of individual co-ancestry. Samples from urban and rural Brazil with contrasting distributions of African, European, and Native American co-ancestry were also studied to gauge the efficiency of combining Y- and X-SNP data for this purpose. The small panel of Microhaplotypes incorporated in ET were selected because they showed the highest levels of haplotype diversity amongst the seven population groups we sought to differentiate. Microhaplotype data was not formally combined with single-site SNP genotypes to analyse ancestry. However, the haplotype sequence reads obtained with ET from these loci creates an effective system for de-convoluting two-contributor mixed DNA. We made simple mixture experiments to demonstrate that when the contributors have different ancestries and the mixture ratios are imbalanced (i.e., not 1:1 mixtures) the ET Microhaplotype panel is an informative system to infer ancestry when this differs between the contributors.

Association between prenatal alcohol exposure and children's facial shape: a prospective population-based cohort study.

STUDY QUESTION: Is there an association between low-to-moderate levels of prenatal alcohol exposure (PAE) and children's facial shape? SUMMARY ANSWER: PAE before and during pregnancy, even at low level (<12 g of alcohol per week), was found associated with the facial shape of children, and these associations were found attenuated as children grow older. WHAT IS KNOWN ALREADY: High levels of PAE during pregnancy can have significant adverse associations with a child's health development resulting in recognizably abnormal facial development. STUDY DESIGN, SIZE, DURATION: This study was based on the Generation R Study, a prospective cohort from fetal life onwards with maternal and offspring data. We analyzed children 3-dimensional (3D) facial images taken at ages 9 (n = 3149) and 13 years (n = 2477) together with the data of maternal alcohol consumption. PARTICIPANTS/MATERIALS, SETTING, METHODS: We defined six levels of PAE based on the frequency and dose of alcohol consumption and defined three tiers based on the timing of alcohol exposure of the unborn child. For the image analysis, we used 3D graph convolutional networks for non-linear dimensionality reduction, which compressed the high-dimensional images into 200 traits representing facial morphology. These 200 traits were used for statistical analysis to search for associations with PAE. Finally, we generated heatmaps to display the facial phenotypes associated with PAE. MAIN RESULTS AND THE ROLE OF CHANCE: The results of the linear regression in the 9-year-old children survived correction for multiple testing with false discovery rate (FDR). In Tier 1 where we examined PAE only before pregnancy (exposed N = 278, unexposed N = 760), we found three traits survived FDR correction. The lowest FDR-P is 1.7e-05 (beta = 0.021, SE = 0.0040) in Trait #29; In Tier 2b where we examine any PAE during first trimester (exposed N = 756; unexposed N = 760), we found eight traits survived FDR correction. The lowest FDR-P is 9.0e-03 (beta = -0.013, SE = 0.0033) in Trait #139. Moreover, more statistically significant facial traits were found in higher levels of PAE. No FDR-significant results were found in the 13-year-old children. We map these significant traits back to the face, and found the most common detected facial phenotypes included turned-up nose tip, shortened nose, turned-out chin, and turned-in lower-eyelid-related regions. LIMITATIONS, REASONS FOR CAUTION: We had no data for alcohol consumption more than three months prior to pregnancy and thus do not know if maternal drinking had chronic effects. The self-reported questionnaire might not reflect accurate alcohol measurements because mothers may have denied their alcohol consumption. WIDER IMPLICATIONS OF THE FINDINGS: Our results imply that facial morphology, such as quantified by the approach we proposed here, can be used as a biomarker in further investigations. Furthermore, our study suggests that for women who are pregnant or want to become pregnant soon, should quit alcohol consumption several months before conception and completely during pregnancy to avoid adverse health outcomes in the offspring. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Erasmus Medical Centre, Rotterdam, the Erasmus University Rotterdam, and the Netherlands Organization for Health Research. V.W.V.J. reports receipt of funding from the Netherlands Organization for Health Research (ZonMw 90700303). W.J.N. is a founder, a scientific lead, and a shareholder of Quantib BV. TRIAL REGISTRATION NUMBER: N/A.

Development and inter-laboratory evaluation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA.

Responding to the growing scientific and practical interest in forensic DNA phenotyping, the VISible Attributes through GEnomics (VISAGE) Consortium was founded in 2017 with the main goal of developing and validating new and reliable molecular and statistical tools to predict appearance, ancestry and age from DNA. Here, we describe the development and inter-laboratory evaluation and validation of the VISAGE Enhanced Tool for Appearance and Ancestry inference from DNA. The VISAGE Enhanced Tool for Appearance and Ancestry is the first forensic-driven genetic laboratory tool that comprises well-established markers for eye, hair and skin color with more recently discovered DNA markers for eyebrow color, freckling, hair shape and male pattern baldness and bio-geographic ancestry informative DNA markers. The bio-geographic ancestry markers include autosomal SNPs (bi- and tri-allelic SNPs), X-SNPs, Y-SNPs and autosomal Microhaplotypes. In total, primers targeting 524 SNPs (representing a 97.6% assay conversion rate) were successfully designed using AmpliSeq into a single primer pool (i.e., one multiplex assay) and sequenced with the Ion S5. In a collaborative framework, five VISAGE laboratories tested the VISAGE Enhanced Tool for Appearance and Ancestry on reproducibility, sensitivity, genotyping concordance, mixtures, species specificity and performance in relevant forensic conditions, including inhibitor-spiked, mock casework and artificially degraded samples. Based on our results, the VISAGE Enhanced Tool for Appearance and Ancestry is a robust, reproducible, and - for the large SNP number - fairly sensitive MPS assay with high concordance rates. With the VISAGE Enhanced Tool for Appearance and Ancestry introduced here, the VISAGE Consortium delivers the first single DNA-test for combined appearance prediction based on seven traits together with bio-geographic ancestry inference based on major continental regions for separated bi-parental and paternal ancestry, which represents the most comprehensive validated laboratory tool currently available for Forensic DNA Phenotyping.

Genetics of facial telangiectasia in the Rotterdam Study: a genome-wide association study and candidate gene approach.

BACKGROUND: The severity of facial telangiectasia or red veins is associated with many lifestyle factors. However, the genetic predisposition remains unclear. OBJECTIVES: We performed a genome-wide association study (GWAS) on facial telangiectasia in the Rotterdam Study (RS) and tested for replication in two independent cohorts. Additionally, a candidate gene approach with known pigmentation genes was performed. METHODS: Facial telangiectasia were extracted from standardized facial photographs (collected from 2010-2013) of 2842 northwestern European participants (median age 66.9, 56.8% female) from the RS. Our GWAS top hits (P-value <10-6 ) were tested for replication in 460 elderly women of the SALIA cohort and in 576 additional men and women of the RS. Associations of top single nucleotide polymorphisms (SNPs) with expression quantitative trait loci (eQTL) in various tissues were reviewed (GTEx database) alongside phenotype associations in the UK biobank database. SNP-based associations between known pigmentation genes and facial telangiectasia were tested. Conditional analysis on skin colour was additionally performed. RESULTS: Our most significant GWAS signal was rs4417318 (P-value 5.38*10-7 ), an intergenic SNP on chromosome 12 mapping to the SLC16A7 gene. Other suggestive SNPs tagged genes ZNF211, ZSCAN4, ICOS and KCNN3; SNP eQTLs and phenotype associations tagged links to the vascular system. However, the top signals did not pass significance in the two replication cohorts. The pigmentation genes KIAA0930, SLCA45A2 and MC1R, were significantly associated with telangiectasia in a candidate gene approach but not independently of skin colour. CONCLUSION: In this GWAS on telangiectasia in a northwestern European population, no genome-wide significant SNPs were found, although suggestive signals indicate genes involved in the vascular system might be involved in telangiectasia. Significantly associated pigmentation genes underline the link between skin colour and telangiectasia.

Development and optimization of the VISAGE basic prototype tool for forensic age estimation.

The VISAGE (VISible Attributes through GEnomics) consortium aims to develop, optimize and validate prototype tools to broaden the use of DNA intelligence methods in forensic routine laboratories. This includes age estimation based on the quantification of DNA methylation at specific CpG sites. Here, we present the VISAGE basic prototype tool for age estimation targeting 32 CpGs from five genes ELOVL2, MIR29B2CHG (herein, MIR29B2C), FHL2, TRIM59 and KLF14. The assay interrogates these well described age markers by multiplex PCR for bisulfite converted DNA and massively parallel sequencing on a MiSeq FGx instrument. We describe protocol optimizations including tests on five bisulfite conversion kits and an evaluation of the assay's reproducibility and sensitivity with artificially methylated DNA standards. We observed robust quantification of methylation levels with a mean standard deviation of 1.4 % across ratios. Sensitivity tests showed no increase of variability down to 20 ng DNA input into bisulfite conversion with a median difference below 1.6 % between technical replicates.

Principal component analysis of seven skin-ageing features identifies three main types of skin ageing.

BACKGROUND: The underlying phenotypic correlations between wrinkles, pigmented spots (PS), telangiectasia and other related facial-ageing subphenotypes are not well understood. OBJECTIVES: To analyse the underlying phenotypic correlation structure between seven features for facial ageing: global wrinkling, perceived age (PA), Griffiths photodamage grading, PS, telangiectasia, actinic keratosis (AK) and keratinocyte cancer (KC). METHODS: This was a cross-sectional study. Facial photographs and a full-body skin examination were used. We used principal component analysis (PCA) to derive principal components (PCs) of common variation between the features. We performed multivariable linear regressions between age, sex, body mass index, smoking and ultraviolet radiation exposure and the PC scores derived from PCA. We also tested the association between the main PC scores and 140 single-nucleotide polymorphisms (SNPs) previously associated with skin-ageing phenotypes. RESULTS: We analysed data from 1790 individuals with complete data on seven features of skin ageing. Three main PCs explained 73% of the total variance of the ageing phenotypes: a hypertrophic/wrinkling component (PC1: global wrinkling, PA and Griffiths grading), an atrophic/skin colour component (PC2: PS and telangiectasia) and a cancerous component (PC3: AK and KC). The associations between lifestyle and host factors differed per PC. The strength of SNP associations also differed per component with the most SNP associations found with the atrophic component [e.g. the IRF4 SNP (rs12203592); P-value = 1·84 × 10-22 ]. CONCLUSIONS: Using a hypothesis-free approach, we identified three major underlying phenotypes associated with extrinsic ageing. Associations between determinants for skin ageing differed in magnitude and direction per component. What's already known about this topic? Facial ageing is a complex phenotype consisting of different features including wrinkles, pigmented changes, telangiectasia and cancerous-related growths; it is not clear how these phenotypes are related to each other and to other phenotypes. A few studies have described two main clinical phenotypes for photoageing, namely hypertrophic ageing and atrophic ageing, which have been based solely on the clinical assessment of photoageing characteristics. What does this study add? We are the first to use epidemiology data to identify three main components associated with photoageing, namely a hypertrophic component (global wrinkling; perceived age; Griffiths grading) and atrophic component (pigmented spots; telangiectasia) and a cancer component (actinic keratosis; keratinocyte cancer). Association analysis showed different effects and direction of environmental determinants and genetic associations with the three components, with the most significant gene variants associations found for the atrophic component.

Epidemiology and determinants of facial telangiectasia: a cross-sectional study.

BACKGROUND: Telangiectasia or red veins are one of the prominent features of facial skin ageing. To date, there are few studies investigating the determinants of telangiectasia. OBJECTIVES: We investigated lifestyle and physiological factors associated with facial telangiectasia in a large prospective Dutch cohort study. METHODS: Telangiectasia was quantified digitally from standardized facial photographs of 2842 North European participants (56.8% female, median age 66.9) from the Rotterdam Study, collected in 2010-2013. Effect estimates from multivariable linear regressions are presented as the percentage difference in the mean value of telangiectasia area per unit increase of a determinant (%Δ) with corresponding 95% CI. RESULTS: Significant determinants were older age [1.7%Δ per year (95% CI 1.4, 2.0)], female sex [18.3%Δ (95% CI 13.2, 23.6)], smoking [current versus never 38.4%Δ (95% CI 30.3, 47.0); former versus never 11.6%Δ (95% CI 6.6, 16.9)], a high susceptibility to sunburn [10.2%Δ (95% CI 5.4, 15.3)] and light skin colour [pale versus white-to-olive 31.4%Δ (95% CI 19.7, 44.1]; white vs. white-to-olive 9.2%Δ (95% CI 2.8, 16.0)]. CONCLUSIONS: In this large cohort study, we confirmed known and described new determinants of facial telangiectasia.

Three-dimensional soft tissue effects of mandibular midline distraction and surgically assisted rapid maxillary expansion: an automatic stereophotogrammetry landmarking analysis.

Studies on mandibular midline distraction (MMD) are mostly performed using conventional research methods. Concerning surgically assisted rapid maxillary expansion (SARME), more research is conducted using three-dimensional (3D) techniques. Research on bimaxillary expansion, the combination of MMD and SARME, is reported sparsely. The main objective of this study was to provide a 3D evaluation of soft tissue effects following SARME and/or MMD. Patients who underwent SARME and/or MMD between 2008 and 2013 were included. Stereophotogrammetry was undertaken at the following time points: preoperative (T1), immediately post-distraction (T2), 1year postoperative (T3). An automatic 3D facial landmarking algorithm using two-dimensional Gabor wavelets was applied for the analysis. Twenty patients who had undergone SARME were included, 12 of whom had undergone bimaxillary expansion. Age at the time of surgery ranged from 16 to 47 years. There was a significant downward displacement of soft tissue pogonion. Furthermore, there was a significant mean increase of 2.20mm in inter-alar width and a non-significant mean increase of 1.77mm in inter-alar curvature point width. In conclusion, automatic stereophotogrammetry landmarking analysis of soft tissue effects showed downward displacement of soft tissue pogonion following bimaxillary expansion and transverse widening of the inter-alar width and a tendency towards an increase in inter-alar curvature point width after SARME.

Body fluid identification using a targeted mRNA massively parallel sequencing approach - results of a EUROFORGEN/EDNAP collaborative exercise.

In a previous study we presented an assay for targeted mRNA sequencing for the identification of human body fluids, optimised for the Illumina MiSeq/FGx MPS platform. This assay, together with an additional in-house designed assay for the Ion Torrent PGM/S5 platform, was the basis for a collaborative exercise within 17 EUROFORGEN and EDNAP laboratories, in order to test the efficacy of targeted mRNA sequencing to identify body fluids. The task was to analyse the supplied dried body fluid stains and, optionally, participants' own bona fide or mock casework samples of human origin, according to specified protocols. The provided primer pools for the Illumina MiSeq/FGx and the Ion Torrent PGM/S5 platforms included 33 and 29 body fluid specific targets, respectively, to identify blood, saliva, semen, vaginal secretion, menstrual blood and skin. The results demonstrated moderate to high count values in the body fluid or tissue of interest with little to no counts in non-target body fluids. There was some inter-laboratory variability in read counts, but overall the results of the laboratories were comparable in that highly expressed markers showed high read counts and less expressed markers showed lower counts. We performed a partial least squares (PLS) analysis on the data, where blood, menstrual blood, saliva and semen markers and samples clustered well. The results of this collaborative mRNA massively parallel sequencing (MPS) exercise support targeted mRNA sequencing as a reliable body fluid identification method that could be added to the repertoire of forensic MPS panels.

Sutura laparoscópica frente a sutura por laparotomía en úlceras gastroduodenales perforadas / Laparoscopic suture facing laparotomy suture in perforated gastroduodenal ulcers

Resumen Introducción La perforación gastroduodenal continúa siendo una urgencia quirúrgica relativamente frecuente, a pesar de los avances realizados en el tratamiento médico de la enfermedad ulcerosa. Su abordaje laparoscópico ha ido aumentando en los últimos años, aunque no se ha generalizado. Nuestro objetivo es analizar los resultados postoperatorios en pacientes con úlcera perforada tratados mediante sutura laparoscópica, y compararlos con un grupo similar con sutura por laparotomía. Mantenemos la hipótesis de que la sutura laparoscópica es una opción segura y con menor morbilidad que el abordaje por laparotomía. Material y Métodos Análisis retrospectivo comparativo de dos cohortes de pacientes: una tratada mediante sutura laparoscópica durante los años 2014 y 2015, período en el que este abordaje estaba plenamente implantado en la Urgencia en nuestro hospital, y otro grupo comparable tratado mediante sutura por cirugía abierta durante el período 2001-2003. Se analizaron las complicaciones según la clasificación de Clavien-Dindo, tasa de conversión, estancia media y mortalidad. Resultados Los grupos eran comparables en edad, sexo, comorbilidades y riesgo anestésico. Se observó una tendencia a la superioridad a favor del abordaje laparoscópico en ciertas variables analizadas, con una tasa de conversión de un 3%. La presencia de complicaciones postoperatorias precoces fue mayor en el grupo con sutura por laparotomía: shock séptico postquirúrgico (15,2 % vs 6%) e infección de herida (15,2 % vs 3%), así como las complicaciones médicas, aunque de forma no significativa. El grupo tratado con sutura laparoscópica tuvo un mayor tiempo quirúrgico, menor estancia media y menor mortalidad. Conclusión La sutura laparoscópica de la úlcera gastroduodenal en nuestro centro ha tenido una baja tasa de conversión y una morbilidad algo menor a la sutura por laparotomía, con una menor tasa de reintervenciones y menor estancia media, a pesar de un mayor tiempo quirúrgico.

Introduction Gastroduodenal perforation continues to be a relatively frequent surgical emergency, despite advances in the medical treatment of ulcer disease. Its laparoscopic approach has been increasing in the last years, although it has not been generalized. Objective Was to analyze the postoperative results in patients with perforated ulcer treated with laparoscopic suture, and to compare them with a similar group with laparotomy suture. Our hypothesis was that laparoscopic suture is a safe option and with less morbidity than the laparotomy approach. Material and Methods Comparative retrospective analysis of two patient cohorts: one treated with laparoscopic suture during 2014 and 2015, a period in which this approach was fully implanted in the emergency room in our hospital, and another comparable group treated by suture for open surgery during the period 2001-2003. Complications were analyzed according to Clavien-Dindo classification, conversion rate, mean stay and mortality. Results The groups were comparable in age, sex, comorbidities and anesthetic risk. There was a trend towards superiority in favor of the laparoscopic approach in certain variables analyzed, with a conversion rate of 3%. The presence of early postoperative complications was greater in the laparotomy suture group: post-surgical septic shock (15.2% vs 6%) and wound infection (15.2% vs 3%), as well as medical complications, although not significantly. The group with laparoscopic suture had a longer surgical time, lower mean stay and lower mortality. Conclusion The laparoscopic suture of the gastroduodenal ulcer in our center has had a very low conversion rate and a somewhat lower morbidity to the laparotomy suture, with a lower rate of reinterventions and a mean stay, despite a longer surgical time.

Unraveling the Neurobiology of Sleep and Sleep Disorders Using Drosophila.

Sleep disorders in humans are increasingly appreciated to be not only widespread but also detrimental to multiple facets of physical and mental health. Recent work has begun to shed light on the mechanistic basis of sleep disorders like insomnia, restless legs syndrome, narcolepsy, and a host of others, but a more detailed genetic and molecular understanding of how sleep goes awry is lacking. Over the past 15 years, studies in Drosophila have yielded new insights into basic questions regarding sleep function and regulation. More recently, powerful genetic approaches in the fly have been applied toward studying primary human sleep disorders and other disease states associated with dysregulated sleep. In this review, we discuss the contribution of Drosophila to the landscape of sleep biology, examining not only fundamental advances in sleep neurobiology but also how flies have begun to inform pathological sleep states in humans.

A collaborative EDNAP exercise on SNaPshot™-based mtDNA control region typing.

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation.

Chondroitin Sulfate Tetrasaccharides: Synthesis, Three-Dimensional Structure and Interaction with Midkine.

The biological activity of midkine, a cytokine implicated in neuro- and tumourigenesis, is regulated by its binding to glycosaminoglycans (GAGs), such as heparin and chondroitin sulfate (CS). To better understand the molecular recognition of GAG sequences by this growth factor, the interactions between synthetic chondroitin sulfate-like tetrasaccharides and midkine were studied by using different techniques. Firstly, a synthetic approach for the preparation of CS-like oligosaccharides in the sequence GalNAc-GlcA was developed. A fluorescence polarisation competition assay was then employed to analyse the relative binding affinities of the synthetic compounds and revealed that midkine interacted with CS-like tetrasaccharides in the micromolar range. The 3D structure of these tetramers was studied in detail by a combination of NMR spectroscopy experiments and molecular dynamics simulations. Saturation transfer difference (STD) NMR spectroscopy experiments indicate that the CS tetrasaccharides bind to midkine in an extended conformation, with similar saturation effects along the entire sugar chain. These results are compatible with docking studies that suggest an interaction of the tetrasaccharide with midkine in a folded structure. Overall, this study provides valuable information on the interaction between midkine and well-defined, chemically synthesised CS oligosaccharides and these data can be useful for the design of more active compounds that modulate the biological function of this protein.

Forensic ancestry analysis with two capillary electrophoresis ancestry informative marker (AIM) panels: Results of a collaborative EDNAP exercise.

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified.

MC1R gene variants and non-melanoma skin cancer: a pooled-analysis from the M-SKIP project.

BACKGROUND: The melanocortin-1-receptor (MC1R) gene regulates human pigmentation and is highly polymorphic in populations of European origins. The aims of this study were to evaluate the association between MC1R variants and the risk of non-melanoma skin cancer (NMSC), and to investigate whether risk estimates differed by phenotypic characteristics. METHODS: Data on 3527 NMSC cases and 9391 controls were gathered through the M-SKIP Project, an international pooled-analysis on MC1R, skin cancer and phenotypic characteristics. We calculated summary odds ratios (SOR) with random-effect models, and performed stratified analyses. RESULTS: Subjects carrying at least one MC1R variant had an increased risk of NMSC overall, basal cell carcinoma (BCC) and squamous cell carcinoma (SCC): SOR (95%CI) were 1.48 (1.24-1.76), 1.39 (1.15-1.69) and 1.61 (1.35-1.91), respectively. All of the investigated variants showed positive associations with NMSC, with consistent significant results obtained for V60L, D84E, V92M, R151C, R160W, R163Q and D294H: SOR (95%CI) ranged from 1.42 (1.19-1.70) for V60L to 2.66 (1.06-6.65) for D84E variant. In stratified analysis, there was no consistent pattern of association between MC1R and NMSC by skin type, but we consistently observed higher SORs for subjects without red hair. CONCLUSIONS: Our pooled-analysis highlighted a role of MC1R variants in NMSC development and suggested an effect modification by red hair colour phenotype.

Towards simultaneous individual and tissue identification: A proof-of-principle study on parallel sequencing of STRs, amelogenin, and mRNAs with the Ion Torrent PGM.

DNA-based individual identification and RNA-based tissue identification represent two commonly-used tools in forensic investigation, aiming to identify crime scene sample donors and helping to provide links between DNA-identified sample donors and criminal acts. Currently however, both analyses are typically performed separately. In this proof-of-principle study, we developed an approach for the simultaneous analysis of forensic STRs, amelogenin, and forensic mRNAs based on parallel targeted DNA/RNA sequencing using the Ion Torrent Personal Genome Machine(®) (PGM™) System coupled with the AmpliSeq™ targeted amplification. We demonstrated that 9 autosomal STRs commonly used for individual identification (CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX, and vWA), the AMELX/AMELY system widely applied for sex identification, and 12 mRNA markers previously established for forensic tissue identification (ALAS2 and SPTB for peripheral blood, MMP10 and MMP11 for menstrual blood, HTN3 and STATH for saliva, PRM1 and TGM4 for semen, CYP2B7P1 and MUC4 for vaginal secretion, CCL27 and LCE1C for skin) together with two candidate reference mRNA markers (HPRT1 and SDHA) can all be successfully combined. Unambiguous mRNA-based tissue identification was achieved in all samples from all forensically relevant tissues tested, and STR sequencing analysis of the tissue sample donors was 100% concordant with conventional STR profiling using a commercial kit. Successful STR analysis was obtained from 1ng of genomic DNA and mRNA analysis from 10ng total RNA; however, sensitivity limits were not investigated in this proof-of-principle study and are expected to be much lower. Since dried materials with noticeable RNA degradation and small DNA/RNA amplicons with high-coverage sequencing were used, the achieved correct individual and tissue identification demonstrates the suitability of this approach for analyzing degraded materials in future forensic applications. Overall, our study demonstrates the feasibility of simultaneously obtaining multilocus STR, amelogenin, and multilocus mRNA information for combined individual and tissue identification from a small sample of degraded biological material. Moreover, our study marks the first step towards combining many DNA/RNA markers for various forensic purposes to increase the effectiveness of molecular forensic analysis and to allow more forensically relevant information to be obtained from limited forensic material.

Validation of image analysis techniques to measure skin aging features from facial photographs.

BACKGROUND: Accurate measurement of the extent skin has aged is crucial for skin aging research. Image analysis offers a quick and consistent approach for quantifying skin aging features from photographs, but is prone to technical bias and requires proper validation. METHODS: Facial photographs of 75 male and 75 female North-European participants, randomly selected from the Rotterdam Study, were graded by two physicians using photonumeric scales for wrinkles (full face, forehead, crow's feet, nasolabial fold and upper lip), pigmented spots and telangiectasia. Image analysis measurements of the same features were optimized using photonumeric grades from 50 participants, then compared to photonumeric grading in the 100 remaining participants stratified by sex. RESULTS: The inter-rater reliability of the photonumeric grades was good to excellent (intraclass correlation coefficients 0.65-0.93). Correlations between the digital measures and the photonumeric grading were moderate to excellent for all the wrinkle comparisons (Spearman's rho ρ = 0.52-0.89) bar the upper lip wrinkles in the men (fair, ρ = 0.30). Correlations were moderate to good for pigmented spots and telangiectasia (ρ = 0.60-0.75). CONCLUSION: These comparisons demonstrate that all the image analysis measures, bar the upper lip measure in the men, are suitable for use in skin aging research and highlight areas of improvement for future refinements of the techniques.