flippant-An R package for the automated analysis of fluorescence-based scramblase assays.

BACKGROUND: The lipid scrambling activity of protein extracts and purified scramblases is typically measured using a fluorescence-based assay. While the assay has yielded insight into the scramblase activity in crude membrane preparations, functional validation of candidate scramblases, stoichiometry of scramblase complexes as well as ATP-dependence of flippases, data analysis in its context has remained a task involving many manual steps. RESULTS: With the extension package "flippant" to R, a free software environment for statistical computing and graphics, we introduce an integrated solution for the analysis and publication-grade graphical presentation of dithionite scramblase assays and demonstrate its utility in revisiting an originally manual analysis from the publication record, closely reproducing the reported results. CONCLUSIONS: "flippant" allows for quick, reproducible data analysis of scramblase activity assays and provides a platform for review, dissemination and extension of the strategies it employs.

Connecting genetic risk to disease end points through the human blood plasma proteome.

Genome-wide association studies (GWAS) with intermediate phenotypes, like changes in metabolite and protein levels, provide functional evidence to map disease associations and translate them into clinical applications. However, although hundreds of genetic variants have been associated with complex disorders, the underlying molecular pathways often remain elusive. Associations with intermediate traits are key in establishing functional links between GWAS-identified risk-variants and disease end points. Here we describe a GWAS using a highly multiplexed aptamer-based affinity proteomics platform. We quantify 539 associations between protein levels and gene variants (pQTLs) in a German cohort and replicate over half of them in an Arab and Asian cohort. Fifty-five of the replicated pQTLs are located in trans. Our associations overlap with 57 genetic risk loci for 42 unique disease end points. We integrate this information into a genome-proteome network and provide an interactive web-tool for interrogations. Our results provide a basis for novel approaches to pharmaceutical and diagnostic applications.

Complementarity of SOMAscan to LC-MS/MS and RNA-seq for quantitative profiling of human embryonic and mesenchymal stem cells.

Dynamic range limitations are challenging to proteomics, particularly in clinical samples. Affinity proteomics partially overcomes this, yet suffers from dependence on reagent quality. SOMAscan, an aptamer-based platform for over 1000 proteins, avoids that issue using nucleic acid binders. Targets include low expressed proteins not easily accessible by other approaches. Here we report on the potential of SOMAscan for the study of differently sourced mesenchymal stem cells (MSC) in comparison to LC-MS/MS and RNA sequencing. While targeting fewer analytes, SOMAscan displays high precision and dynamic range coverage, allowing quantification of proteins not measured by the other platforms. Expression between cell types (ESC and MSC) was compared across techniques and uncovered the expected large differences. Sourcing was investigated by comparing subtypes: bone marrow-derived, standard in clinical studies, and ESC-derived MSC, thought to hold similar potential but devoid of inter-donor variability and proliferating faster in vitro. We confirmed subtype-equivalency, as well as vesicle and extracellular matrix related processes in MSC. In contrast, the proliferative nature of ESC was captured less by SOMAscan, where nuclear proteins are underrepresented. The complementary of SOMAscan allowed the comprehensive exploration of CD markers and signaling molecules, not readily accessible otherwise and offering unprecedented potential in subtype characterization. SIGNIFICANCE: Mesenchymal stem cells (MSC) represent promising stem cell-derived therapeutics as indicated by their application in >500 clinical trials currently registered with the NIH. Tissue-derived MSC require invasive harvesting and imply donor-to-donor differences, to which embryonic stem cell (ESC)-derived MSC may provide an alternative and thus warrant thorough characterization. In continuation of our previous study where we compared in depth embryonic stem cells (ESC) and MSC from two sources (bone marrow and ESC-derived), we included the aptamer-based SOMAscan assay, complementing LC-MS/MS and RNA-seq data. Furthermore, SOMAscan, a targeted proteomics platform developed for analyzing clinical samples, has been benchmarked against established analytical platforms (LC-MS/MS and RNA-seq) using stem cell comparisons as a model.

readat: An R package for reading and working with SomaLogic ADAT files.

BACKGROUND: SomaLogic's SOMAscan™ assay platform allows the analysis of the relative abundance of over 1300 proteins directly from biological matrices such as blood plasma and serum. The data resulting from the assay is provided in a proprietary text-based format not easily imported into R. RESULTS: readat is an R package for working with the SomaLogic ADAT file format. It provides functionality for importing, transforming and annotating data from these files. The package is free, open source, and available on Bioconductor and Bitbucket. CONCLUSIONS: readat integrates into both Bioconductor and traditional R workflows, rendering it easy to make use of ADAT files.

Comprehensive transcriptomic and proteomic characterization of human mesenchymal stem cells reveals source specific cellular markers.

Mesenchymal stem cells (MSC) are multipotent cells with great potential in therapy, reflected by more than 500 MSC-based clinical trials registered with the NIH. MSC are derived from multiple tissues but require invasive harvesting and imply donor-to-donor variability. Embryonic stem cell-derived MSC (ESC-MSC) may provide an alternative, but how similar they are to ex vivo MSC is unknown. Here we performed an in depth characterization of human ESC-MSC, comparing them to human bone marrow-derived MSC (BM-MSC) as well as human embryonic stem cells (hESC) by transcriptomics (RNA-seq) and quantitative proteomics (nanoLC-MS/MS using SILAC). Data integration highlighted and validated a central role of vesicle-mediated transport and exosomes in MSC biology and also demonstrated, through enrichment analysis, their versatility and broad application potential. Particular emphasis was placed on comparing profiles between ESC-MSC and BM-MSC and assessing their equivalency. Data presented here shows that differences between ESC-MSC and BM-MSC are similar in magnitude to those reported for MSC of different origin and the former may thus represent an alternative source for therapeutic applications. Finally, we report an unprecedented coverage of MSC CD markers, as well as membrane associated proteins which may benefit immunofluorescence-based applications and contribute to a refined molecular description of MSC.

Reprint of PopGen: A virtual human population generator.

The risk assessment of environmental chemicals and drugs is moving towards a paradigm shift in approach which seeks the full replacement animal testing with high throughput, mechanistic, in vitro systems. This new vision will be reliant on the measurement in vitro, of concentration-dependent responses where prolonged excessive perturbations of specific biochemical pathways are likely to lead to adverse health effects in an intact organism. Such an approach requires a framework, into which disparate data generated using in vitro, in silico and in chemico systems, can be integrated and utilised for quantitative in vitro-to-in vivo extrapolation (QIVIVE), ultimately to the human population level. Physiologically based pharmacokinetic (PBPK) models are ideally suited for this and are obligatory in order to translate in vitro concentration-response relationships to an exposure or dose, route and duration regime in people. In this report we describe PopGen a virtual human population generator which is a user friendly, open access web-based application for the prediction of realistic anatomical, physiological and phase 1 metabolic variation in a wide range of healthy human populations. We demonstrate how PopGen can be used for QIVIVE by providing input to a PBPK model, at an appropriate level of detail, to reconstruct exposure from human biomonitoring data. We discuss how the process of exposure reconstruction from blood biomarkers, in general, is analogous to exposure or dose reconstruction from concentration-response measurements made in proposed in vitro cell based systems which are assumed to be surrogates for target organs.

The Human Variome Project: ensuring the quality of DNA variant databases in inherited renal disease.

A recent review identified 60 common inherited renal diseases caused by DNA variants in 132 different genes. These diseases can be diagnosed with DNA sequencing, but each gene probably also has a thousand normal variants. Many more normal variants have been characterised by individual laboratories than are reported in the literature or found in publicly accessible collections. At present, testing laboratories must assess each novel change they identify for pathogenicity, even when this has been done elsewhere previously, and the distinction between normal and disease-associated variants is particularly an issue with the recent surge in exomic sequencing and gene discovery projects. The Human Variome Project recommends the establishment of gene-specific DNA variant databases to facilitate the sharing of DNA variants and decisions about likely disease causation. Databases improve diagnostic accuracy and testing efficiency, and reduce costs. They also help with genotype-phenotype correlations and predictive algorithms. The Human Variome Project advocates databases that use standardised descriptions, are up-to-date, include clinical information and are freely available. Currently, the genes affected in the most common inherited renal diseases correspond to 350 different variant databases, many of which are incomplete or have insufficient clinical details for genotype-phenotype correlations. Assistance is needed from nephrologists to maximise the usefulness of these databases for the diagnosis and management of inherited renal disease.

Coping with esophageal cancer approaches worldwide.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on approaches to the epidemiology, diagnosis, and treatment of esophageal cancer in Europe, South Africa, Kenya, Australia, and China; the molecular classification of esophageal cancers (including cancers at the gastroesophageal junction); the Japanese classification; the scope of the Human Variome Project; and the topographic-anatomic subclassification of adenocarcinomas of the gastroesophageal junction.

Capture of somatic mtDNA point mutations with severe effects on oxidative phosphorylation in synaptosome cybrid clones from human brain.

Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT-RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT-ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function.

DNA variant databases improve test accuracy and phenotype prediction in Alport syndrome.

X-linked Alport syndrome is a form of progressive renal failure caused by pathogenic variants in the COL4A5 gene. More than 700 variants have been described and a further 400 are estimated to be known to individual laboratories but are unpublished. The major genetic testing laboratories for X-linked Alport syndrome worldwide have established a Web-based database for published and unpublished COL4A5 variants ( https://grenada.lumc.nl/LOVD2/COL4A/home.php?select_db=COL4A5 ). This conforms with the recommendations of the Human Variome Project: it uses the Leiden Open Variation Database (LOVD) format, describes variants according to the human reference sequence with standardized nomenclature, indicates likely pathogenicity and associated clinical features, and credits the submitting laboratory. The database includes non-pathogenic and recurrent variants, and is linked to another COL4A5 mutation database and relevant bioinformatics sites. Access is free. Increasing the number of COL4A5 variants in the public domain helps patients, diagnostic laboratories, clinicians, and researchers. The database improves the accuracy and efficiency of genetic testing because its variants are already categorized for pathogenicity. The description of further COL4A5 variants and clinical associations will improve our ability to predict phenotype and our understanding of collagen IV biochemistry. The database for X-linked Alport syndrome represents a model for databases in other inherited renal diseases.

Comparison of urinary thallium levels in non-occupationally exposed people and workers.

PURPOSE: To determine a reference background urinary thallium level; to compare urinary thallium data from workers to this background level; to investigate factors affecting these levels and whether creatinine correction is appropriate. METHODS: Urine samples from non-occupationally exposed people (n = 273, from 113 individuals) and workers (n = 896, from 447 individuals) were analysed for thallium by ICP-MS. A reference background level was calculated, defined as the 95th percentile value of a non-occupationally exposed population. Worker data were divided into two subsets: thallium workers (those who work directly with thallium or its compounds) and general workers; and compared to the background level. Bayesian linear mixed effects modelling was used to investigate factors affecting urinary thallium concentration and the efficacy of creatinine correction for the determination of urinary thallium. RESULTS: The reference background urinary thallium level is 0.27 µmol/mol creatinine (creatinine-corrected) or 0.40 µg/l (uncorrected). Median values were 0.11 µmol/mol creatinine or 0.17 µg/l for non-occupationally exposed people, 0.12 µmol/mol creatinine or 0.20 µg/l for general workers and 0.19 µmol/mol creatinine or 0.41 µg/l for thallium workers. Variation was lower in creatinine-corrected models. Nine per cent of samples from general workers and 39 % of samples from thallium workers exceeded the creatinine-corrected background level. By 2010, 90 % of all workers had urinary thallium levels below the 95th percentile reference background level. CONCLUSIONS: Urinary thallium concentrations were higher in thallium workers than non-occupationally exposed people and general workers. Creatinine correction is appropriate.

PopGen: A virtual human population generator.

The risk assessment of environmental chemicals and drugs is moving towards a paradigm shift in approach which seeks the full replacement animal testing with high throughput, mechanistic, in vitro systems. This new vision will be reliant on the measurement in vitro, of concentration-dependent responses where prolonged excessive perturbations of specific biochemical pathways are likely to lead to adverse health effects in an intact organism. Such an approach requires a framework, into which disparate data generated using in vitro, in silico and in chemico systems, can be integrated and utilised for quantitative in vitro-to-in vivo extrapolation (QIVIVE), ultimately to the human population level. Physiologically based pharmacokinetic (PBPK) models are ideally suited for this and are obligatory in order to translate in vitro concentration-response relationships to an exposure or dose, route and duration regime in people. In this report we describe PopGen, a virtual human population generator which is a user friendly, open access web-based application for the prediction of realistic anatomical, physiological and phase 1 metabolic variation in a wide range of healthy human populations. We demonstrate how PopGen can be used for QIVIVE by providing input to a PBPK model, at an appropriate level of detail, to reconstruct exposure from human biomonitoring data. We discuss how the process of exposure reconstruction from blood biomarkers, in general, is analogous to exposure or dose reconstruction from concentration-response measurements made in proposed in vitro cell based systems which are assumed to be surrogates for target organs.

Tetrahydrobiopterin, its mode of action on phenylalanine hydroxylase, and importance of genotypes for pharmacological therapy of phenylketonuria.

In about 20%-30% of phenylketonuria (PKU) patients (all phenotypes of PAH deficiency), Phe levels may be controlled through phenylalanine hydroxylase cofactor tetrahydrobiopterin therapy. These patients can be diagnosed by an oral tetrahydrobiopterin challenge and are characterized by mutations coding for proteins with substantial residual PAH activity. They can be treated with a commercially available synthetic form of tetrahydrobiopterin, either as a monotherapy or as adjunct to the diet. This review article summarizes molecular and metabolic bases of PKU and the importance of the tetrahydrobiopterin loading test used for PKU patients. On the basis of in vitro residual PAH activity, more than 1,200 genotypes from patients challenged with tetrahydrobiopterin were categorized as predictive for tetrahydrobiopterin responsiveness or non-responsiveness and correlated with the loading test, phenotype, and residual in vitro PAH activity. The coexpression of two distinct PAH mutant alleles revealed possible dominance effects (positive or negative) by one of the mutations on residual activity as result of interallelic complementation. The treatment of the transfected cells with tetrahydrobiopterin showed an increase in residual PAH activity with several mutations coexpressed.

Getting ready for the Human Phenome Project: the 2012 forum of the Human Variome Project.

A forum of the Human Variome Project (HVP) was held as a satellite to the 2012 Annual Meeting of the American Society of Human Genetics in San Francisco, California. The theme of this meeting was "Getting Ready for the Human Phenome Project." Understanding the genetic contribution to both rare single-gene "Mendelian" disorders and more complex common diseases will require integration of research efforts among many fields and better defined phenotypes. The HVP is dedicated to bringing together researchers and research populations throughout the world to provide the resources to investigate the impact of genetic variation on disease. To this end, there needs to be a greater sharing of phenotype and genotype data. For this to occur, many databases that currently exist will need to become interoperable to allow for the combining of cohorts with similar phenotypes to increase statistical power for studies attempting to identify novel disease genes or causative genetic variants. Improved systems and tools that enhance the collection of phenotype data from clinicians are urgently needed. This meeting begins the HVP's effort toward this important goal.

Human Variome Project country nodes: documenting genetic information within a country.

The Human Variome Project (http://www.humanvariomeproject.org) is an international effort aiming to systematically collect and share information on all human genetic variation. The two main pillars of this effort are gene/disease-specific databases and a network of Human Variome Project Country Nodes. The latter are nationwide efforts to document the genomic variation reported within a specific population. The development and successful operation of the Human Variome Project Country Nodes are of utmost importance to the success of Human Variome Project's aims and goals because they not only allow the genetic burden of disease to be quantified in different countries, but also provide diagnosticians and researchers access to an up-to-date resource that will assist them in their daily clinical practice and biomedical research, respectively. Here, we report the discussions and recommendations that resulted from the inaugural meeting of the International Confederation of Countries Advisory Council, held on 12th December 2011, during the 2011 Human Variome Project Beijing Meeting. We discuss the steps necessary to maximize the impact of the Country Node effort for developing regional and country-specific clinical genetics resources and summarize a few well-coordinated genetic data collection initiatives that would serve as paradigms for similar projects.

Reconstruction of Exposure to m-Xylene from Human Biomonitoring Data Using PBPK Modelling, Bayesian Inference, and Markov Chain Monte Carlo Simulation.

There are numerous biomonitoring programs, both recent and ongoing, to evaluate environmental exposure of humans to chemicals. Due to the lack of exposure and kinetic data, the correlation of biomarker levels with exposure concentrations leads to difficulty in utilizing biomonitoring data for biological guidance values. Exposure reconstruction or reverse dosimetry is the retrospective interpretation of external exposure consistent with biomonitoring data. We investigated the integration of physiologically based pharmacokinetic modelling, global sensitivity analysis, Bayesian inference, and Markov chain Monte Carlo simulation to obtain a population estimate of inhalation exposure to m-xylene. We used exhaled breath and venous blood m-xylene and urinary 3-methylhippuric acid measurements from a controlled human volunteer study in order to evaluate the ability of our computational framework to predict known inhalation exposures. We also investigated the importance of model structure and dimensionality with respect to its ability to reconstruct exposure.

The Human Variome Project Beijing meeting.

The Human Variome Project Beijing Meeting, a joint meeting of the Human Variome Project Consortium and the Human Variome Project Chinese Node, was held in Beijing, 8th-12th of December, 2011. The aim of the Human Variome Project is to ensure that all information on genetic variation can be collected, curated, interpreted and shared freely and openly. The meeting officially welcomed the Human Variome Project Chinese Node as a partner of the Human Variome Project and focused on those areas where collaborations between China and the global Human Variome Project Consortium are required to develop and extend the coverage of international gene/disease specific databases.