Minimum information and guidelines for reporting a Multiplexed Assay of Variant Effect

Multiplexed Assays of Variant Effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines has led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs

Spatial and Temporal Localization of the Melanocortin 1 Receptor and Its Ligand α–Melanocyte-Stimulating Hormone during Cutaneous Wound Repair

Growing evidence indicates that the melanocortin 1 receptor (MC1R) and its ligand α–melanocyte-stimulating hormone (α-MSH) have other functions in the skin in addition to pigment production. Activation of the MC1R/α-MSH signaling pathway has been implicated in the regulation of both inflammation and extracellular matrix homeostasis. However, little is known about the role of MC1R/α-MSH signaling in the regulation of inflammatory and fibroproliferative responses to cutaneous injury. Although MC1R and α-MSH localization has been described in uninjured skin, their spatial and temporal expression during cutaneous wound repair has not been investigated. In this study, the authors report the localization of MC1R and α-MSH in murine cutaneous wounds, human acute burns, and hypertrophic scars. During murine wound repair, MC1R and α-MSH were detected in inflammatory cells and suprabasal keratinocytes at the leading edge of the migrating epithelial tongue. MC1R and α-MSH protein levels were upregulated in human burn wounds and hypertrophic scars compared to uninjured human skin, where receptor and ligand were absent. In burn wounds and hypertrophic scars, MC1R and α-MSH localized to epidermal keratinocytes and dermal fibroblasts. This spatiotemporal localization of MC1R and α-MSH in cutaneous wounds warrants future investigation into the role of MC1R/α-MSH signaling in the inflammatory and fibroproliferative responses to cutaneous injury. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials

MC1R gene polymorphisms are associated with dysfunctional immune responses and wound infection after burn injury.

BACKGROUND: Systemic inflammatory response syndrome (SIRS) is associated with organ failure and infectious complications after major burn injury. Recent evidence has linked melanocortin signaling to anti-inflammatory and wound-repair functions, with mutations in the melanocortin 1 receptor (MC1R) gene leading to increased inflammatory responses. Our group has previously demonstrated that MC1R gene polymorphisms are associated with postburn hypertrophic scarring. Thus, we hypothesized that MC1R single nucleotide polymorphisms (SNPs) would be associated with increased burn-induced SIRS and increased infectious complications. METHODS: We performed a retrospective cohort study of adults (\u3e18 y of age) admitted to our burn center with \u3e20% total body surface area (TBSA) partial/full thickness burns between 2006 and 2013. We screened for five MC1R SNPs (V60L, V92M, R151C, R163Q, T314T) by polymerase chain reaction from genomic DNA isolated from blood samples. We performed a detailed review of each patient chart to identify age, sex, race, ethnicity, %TBSA burned, burn wound infections (BWIs), and 72-hr intravenous fluid volume, the latter a surrogate for a dysfunctional inflammatory response to injury. Association testing was based on multivariable regression. RESULTS: Of 106 subjects enrolled, 82 had complete data for analysis. Of these, 64 (78%) were male, with a median age of 39 and median burn size of 30% TBSA. A total of 36 (44%) subjects developed BWIs. The median total administered IV crystalloid in first 72h was 24.6 L. In multivariate analysis, the R151C variant allele was a significant independent risk factor for BWI (adjusted prevalence ratio 2.03; 95% CI: 1.21-3.39; P = 0.007), and the V60L variant allele was independently associated with increased resuscitation fluid volume (P = 0.021). CONCLUSIONS: This is the first study to demonstrate a significant association between genetic polymorphisms and a nonfatal burn-induced SIRS complication. Our findings suggest that MC1R polymorphisms contribute to dysfunctional responses to burn injury that may predict infectious and inflammatory complications

Race Does Not Predict Melanocyte Heterogeneous Responses to Dermal Fibroblast-Derived Mediators

<div><p>Introduction</p><p>Abnormal pigmentation following cutaneous injury causes significant patient distress and represents a barrier to recovery. Wound depth and patient characteristics influence scar pigmentation. However, we know little about the pathophysiology leading to hyperpigmentation in healed shallow wounds and hypopigmentation in deep dermal wound scars. We sought to determine whether dermal fibroblast signaling influences melanocyte responses.</p><p>Methods and Materials</p><p>Epidermal melanocytes from three Caucasians and three African-Americans were genotyped for single nucleotide polymorphisms (SNPs) across the entire genome. Melanocyte genetic profiles were determined using principal component analysis. We assessed melanocyte phenotype and gene expression in response to dermal fibroblast-conditioned medium and determined potential mesenchymal mediators by proteome profiling the fibroblast-conditioned medium.</p><p>Results</p><p>Six melanocyte samples demonstrated significant variability in phenotype and gene expression at baseline and in response to fibroblast-conditioned medium. Genetic profiling for SNPs in receptors for 13 identified soluble fibroblast-secreted mediators demonstrated considerable heterogeneity, potentially explaining the variable melanocyte responses to fibroblast-conditioned medium.</p><p>Discussion</p><p>Our data suggest that melanocytes respond to dermal fibroblast-derived mediators independent of keratinocytes and raise the possibility that mesenchymal-epidermal interactions influence skin pigmentation during cutaneous scarring.</p></div

An Atlas of Variant Effects to understand the genome at nucleotide resolution

Abstract Sequencing has revealed hundreds of millions of human genetic variants, and continued efforts will only add to this variant avalanche. Insufficient information exists to interpret the effects of most variants, limiting opportunities for precision medicine and comprehension of genome function. A solution lies in experimental assessment of the functional effect of variants, which can reveal their biological and clinical impact. However, variant effect assays have generally been undertaken reactively for individual variants only after and, in most cases long after, their first observation. Now, multiplexed assays of variant effect can characterise massive numbers of variants simultaneously, yielding variant effect maps that reveal the function of every possible single nucleotide change in a gene or regulatory element. Generating maps for every protein encoding gene and regulatory element in the human genome would create an ‘Atlas’ of variant effect maps and transform our understanding of genetics and usher in a new era of nucleotide-resolution functional knowledge of the genome. An Atlas would reveal the fundamental biology of the human genome, inform human evolution, empower the development and use of therapeutics and maximize the utility of genomics for diagnosing and treating disease. The Atlas of Variant Effects Alliance is an international collaborative group comprising hundreds of researchers, technologists and clinicians dedicated to realising an Atlas of Variant Effects to help deliver on the promise of genomics

Dermal fibroblast secretome analysis.

<p>In order to determine which fibroblast-derived mediators might be responsible for the melanocyte responses, we performed a proteome analysis. Densitometry (b) of the protein spots on the antibody array film targeting 102 human cytokines (a) indicated 13 proteins that were unique to the dermal fibroblast-conditioned medium. The growth factors EGF and bFGF are components of the fibroblast medium and are unlikely to be responsible for the observed paracrine effects by the conditioned medium on the melanocytes since the control cells were exposed to fresh fibroblast medium.</p

Dermal fibroblast derived paracrine signaling regulates expression of genes involved in melanin biosynthesis.

<p>Baseline expression of 6 genes involved in melanogenesis varied significantly between the melanocyte samples (<i>p</i><0.01 for all genes); conditioned medium significantly affected gene expression (<i>p</i><0.001 for all genes). The degree of response to conditioned medium was significantly different between melanocyte samples for MITF (<i>p</i><0.0001), TYR (<i>p</i><0.0001), TYRP1 (<i>p</i> = 0.02), and TYRP2 (<i>p<</i>0.0001) and was nearly significant for DKK1 (<i>p</i> = 0.07), but not for MC1R (<i>p</i> = 0.22). Statistical analysis was based on average values across all six melanocyte lineages rather than individual sample responses to fibroblast-conditioned medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139135#sec006" target="_blank">Methods</a>).</p

Genetic heterogeneity in melanogenesis-pathway genes underlies variable melanin production.

<p>Three Caucasian (C1-3) and three African American (AA2-3) melanocyte lineages were genotyped for >500,000 single nucleotide polymorphisms (SNPs). Principal component analysis (PCA) was performed based on a) the entire genetic profile and in sub-analyses limited b) to pigmentation genes alone or c) genes corresponding to receptors / ligands for the mediators identified in the fibroblast conditioned medium. PCA summarizes genetic variability present across all SNPs by transforming the data into a smaller set of uncorrelated variables called principal components. The first principal component accounts for the highest proportion of the total variability, with each subsequent component accounting for the next highest amount of variability. In these biplots (a, b, and c) of the first two principal components, each point represents an individual sample; points near each other have more similar genetic profiles than those that are farther apart. a) PCA based on all SNPs on the genotyping array demonstrates overall genetic similarity between Caucasian samples compared to African American samples. b) PCA based on SNPs in 102 melanogenesis-pathway genes demonstrates considerably more genetic variability across samples, in accordance with variable melanin production even between samples from individuals of the same race. c) PCA based on 433 SNPs in 22 genes for receptors for the 13 identified fibroblast-secreted cytokines demonstrated high genetic variability between samples and did not cluster samples according to race. Melanocyte lineage responsiveness to fibroblast-conditioned medium could be explained by loss of function in one or more of the receptors to the mediators in the fibroblast derived conditioned medium.</p

Melanin production is highly variable across melanocyte lineages.

<p>a) Melanin production varied significantly between cell lineages at baseline (<i>p</i><0.0001) and in response to fibroblast-conditioned medium (<i>p</i> = 0.01); the magnitude of response to fibroblast-conditioned medium varied significantly across samples (<i>p</i> = 0.03). Melanin production was not consistent with either reported race or pigmentation type as assigned by Life Technologies. Statistical analysis was based on average values across all six samples rather than individual sample responses to fibroblast-conditioned medium (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0139135#sec006" target="_blank">Methods</a>). b) Photodocumentation of cell pellets from each of the melanocyte cell lineages demonstrated no dramatic differences in melanin appearance.</p

Minimum information and guidelines for reporting a multiplexed assay of variant effect

Multiplexed assays of variant effect (MAVEs) have emerged as a powerful approach for interrogating thousands of genetic variants in a single experiment. The flexibility and widespread adoption of these techniques across diverse disciplines have led to a heterogeneous mix of data formats and descriptions, which complicates the downstream use of the resulting datasets. To address these issues and promote reproducibility and reuse of MAVE data, we define a set of minimum information standards for MAVE data and metadata and outline a controlled vocabulary aligned with established biomedical ontologies for describing these experimental designs