Project description:<p>We hypothesized that genetic variability in the enzymes/proteins involved in drug metabolism, inflammation, and immune function is a major underlying contributor to mucositis incidence and progression and that, based on this variability we can identify variants that influence risk, and develop a mucositis progression prediction model that improves on existing models by incorporating genetic variability along with clinical factors. Using genome wide association study (GWAS) combined with a pathway candidate gene approach and a modified case-control study method, we investigated the hypothesis in a sample of 1092 patients who received a myeloablative dose of melphalan (MEL) followed by autologous hematopoietic stem cell (ASCT) transplantation as treatment for multiple myeloma and for whom banked blood stem cell samples and clinical data were available.</p>
Project description:S. cerevisiae strain S288C was subjected to CRISPR/Cas9 edition in the HMG-CoA reducatase gene using a modified housekeeping promoter ADH1 incorporating the up-stream region of TEF1 promoter (UADH1). Mutant strain was called S288C-H2.
Project description:Amino acid insertions and deletions (indels) are an abundant class of genetic variants. However, compared to substitutions, the effects of indels are not well understood and poorly predicted. Here we address this shortcoming by performing deep indel mutagenesis (DIM) of structurally diverse proteins. Indel tolerance is strikingly different to substitution tolerance and varies extensively both between different proteins and within different regions of the same protein. Although state of the art variant effect predictors perform poorly on indels, we show that both experimentally-measured and computationally-predicted substitution scores can be repurposed as good indel variant effect predictors by incorporating information on protein secondary structures. Quantifying the effects of indels on protein-protein interactions reveals that insertions can be an important class of gain-of-function variants. Our results provide an overview of the impact of indels on proteins and a method to predict their effects genome-wide.
Project description:Fibroblast growth factors (FGF) play important roles during embryonic development as well as in the adult organism in tissue homeostasis, regeneration, repair and metabolism. We recently showed that inhibition of FGF receptor (FGFR) signaling in keratinocytes either pharmacologically or in a mouse FGFR1/2 knockout model, promoted expression of interferon-stimulated genes (ISGs) (Maddaluno et al, 2020). Here we analyzed the transcriptome of human HaCaT keratinocytes in response to treatment with the FGFR kinase inhibitors AZD4547 and BGJ398.
Project description:MicroRNAs (miRNAs) are small regulatory RNAs processed from stem-loop regions of primary transcripts (pri-miRNAs), with the choice of stem-loops for initial processing largely determining what becomes a miRNA. To identify sequence and structural features influencing this choice, we determined cleavage efficiencies of >50,000 variants of three human pri-miRNAs, focusing on the regions intractable to previous high-throughput analyses. Our analyses revealed a mismatched motif in the basal stem region, a preference for maintaining or improving base-pairing throughout the remainder of the stem, and a narrow stem-length preference of 35±1 base pairs. Incorporating these features with previously identified features, including three primary-sequence motifs, yielded a unifying model defining mammalian pri-miRNAs, in which motifs help orient processing and increase efficiency, with the presence of more motifs compensating for structural defects. This model enables generation of artificial pri-miRNAs, designed de novo, without reference to any natural sequence, yet processed more efficiently than natural pri-miRNAs. Three major experiments are included in the submitted data. 1) Pools of synthetic DNA containing barcodes and pri-miRNA variants were sequenced to infer the barcode-pri-miRNA linkages, and the pri-miRNA variants were assayed for their cleavage efficiency ("selection"), which can be quantified by comparing their representations in the input and after cleavage. 2) Artificial miRNAs produced in HEK293T cells were sequenced. 3) Transcriptomes of artificial miRNA-expressed cells were sequenced to quantify mRNA changes.
Project description:The present prospective study included a total number of 9 patients with a final diagnosis of chronic thromboembolic pulmonary hypertension (CTEPH), who were treated by pulmonary endarterectomy (PEA) at the Kerckhoff Heart and Thorax Center between 2016 and 2020. Biopsies of theright ventricularfreewall from9patients were collected at baseline (BL) before PEA surgery (prePEA). Patients were grouped in severe and moderate risk groups according to 1 year mortality risk by applying a modified version of the European Society of Cardiology (ESC) guidelines risk stratification model according to their BL characteristics. The aim of the Study was a comparative characterization of RNA-profiles in CTEPH patients with disease status at baseline prior to PEA therapy
Project description:Genome-wide association studies (GWAS) have identified multiple lung cancer risk loci including those that are distinct in the major histological types. However, most of these loci have not been functionally characterized. Here we employed massively parallel reporter assays (MPRA) to assess allelic transcriptional activity of risk-associated variants en masse. We tested 2,245 variants in 42 loci from 3 recent GWASs of East Asian and European populations in the context of lung adenocarcinoma and squamous cell carcinoma cells while incorporating exposure to a tobacco-smoke carcinogen, benzo[a]pyrene. At FDR < 0.01, we identified 844 MPRA-significant variants with allelic transcriptional activity across 88% of lung cancer loci. Further variant scoring using lung-specific epigenomic annotation demonstrated that 63% of the loci harbored multiple equally functional variants in linkage disequilibrium. Cell-type-specific variants were observed in 72% of the loci, a subset of which aligned with histology-specific association in the GWAS. Distinct subsets of transcription factors were predicted to bind to cell-type-specific variants and those affecting multiple GWAS loci in trans. Linking MPRA-significant variants to target genes based on four different approaches identified candidate susceptibility genes including essential genes for lung cancer cell growth. CRISPR-interference of a high-scoring MPRA-significant variant validated multiple variant-gene connections from different datasets, including RTEL1, SOX18, and ARFRP1. Our data provide a comprehensive catalog of functional characterization of lung cancer GWAS loci and the molecular basis of heterogeneity and polygenicity of lung cancer susceptibility.
Project description:To illustrate the functions of the various cell types in the zebrafish kidney, we sequenced the kidney cells by single-cell messenger RNA sequencing. Six randomly selected zebrafish kidneys were used and obtained about 7,147 cells for RNA sequencing using a modified version of the cell expression by linear amplification and sequencing (CEL-seq) method and incorporating unique molecular identifiers to count the transcripts.
Project description:The KPCA ovarian cancer cell line was generated by S. Iyer in the Weinberg lab at the Whitehead Institute (MIT) (S. Iyer et al., 2020). Multiple clones derived from the KPCA cells were established, presenting different level of sensitivity to immunotherapy. To investigate the tumor microenvironment in this syngeneic mouse model, we implanted the different KPCA clones (3.5 × 10^6 cells) into the peritoneal cavity. Ten days after grafting, we performed single-cell RNA sequencing on harvested tumors and cells from the peritoneal fluid.