Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Massively Parallel Reporter Assay (MPRA) of 175 bp human genomic fragments containing variants in high LD with lead variants from asthma GWAS.

ABSTRACT: We performed a massively parallel reporter assay (MPRA) of 2,034 genomic regions containing single nucleotide polymorphisms (4,587 regions tested with 96,328 barcodes) that are in high linkage disequilibrium with lead variants from an asthma GWAS (PMID: 31036433). Test sequences were transfected into 16HBE14o- Human Bronchial Epithelial Cells, and assessed for enhancer activity by comparing RNA counts to DNA input counts. The processed files contain the MPRA barcodes, read counts and activities.

INSTRUMENT(S): Illumina NovaSeq 6000

ORGANISM(S): Homo sapiens

SUBMITTER:

PROVIDER: E-MTAB-14273 | biostudies-arrayexpress |

SECONDARY ACCESSION(S): ERP162197

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The identification of causal variants responsible for disease associations from genome-wide association studies (GWAS) facilitates functional understanding of the disease mechanisms implicated by GWAS. One of the earliest GWAS associations to COPD spans an intragenic region within FAM13A, but the causal variants at this loci have not yet been identified. Massively parallel reporter assays (MPRA) can be used to prioritize functional regulatory variants in a high-throughput manner. Methods: We used an integrated approach using fine-mapping in over 10,000 subjects from COPD GWAS studies, two MPRA experiments, traditional reporter assays, chromatin conformation capture, and CRISPR-based gene editing to characterize COPD-associated regulatory variants in FAM13A in human bronchial epithelial cell lines. Results: Conditional genetic association and fine mapping analyses identified two independent COPD association signals in FAM13A. MPRA identified 45 common functional regulatory variants, and six COPD-associated putative functional variants were prioritized for further functional investigation. Three variants demonstrated significant activity in traditional reporter assays, and one variant, rs2013701, was selected for further testing in the endogenous genomic context based on a direction of effect consistent with postulated mechanisms of FAM13A-mediated COPD susceptibility. CRISPR-based genome editing for this variant confirmed allele-specific effects on FAM13A expression and altered rates of cellular proliferation, providing multiple levels of functional characterization for this COPD-associated variant. Conclusions: Comprehensive screening for regulatory variants near FAM13A identified the presence of extensive functional regulatory variation within a 250kb window of FAM13A in HBECs. Focused functional evaluation of the COPD-associated functional variants in LD with the two independent association signals in this region prioritized the common variant rs2013701, for which multiple parallel lines of functional evidence confirm allelic effects on FAM13A regulation.

Project description:Although genetic studies have identified many hundreds of loci associated with human traits and diseases, pinpointing the causal alleles remains difficult, particularly for non-coding variants. To address this challenge, we have enhanced the sensitivity and reproducibility of the massively parallel reporter assay (MPRA), adapting it to identify variants that directly modulate gene expression. We then applied it to over 29,000 single nucleotide and insertion/deletion polymorphisms from 3,965 cis-expression quantitative trait loci (eQTL). We demonstrate strong correlation between our MPRA approach and existing measures of regulatory function, and determine an approximate sensitivity of ~20% with a positive predictive value of 60-65% to detect an eQTL causal allele. We identify 842 variants showing differential expression between alleles, including 53 well-annotated variants associated with diseases and traits. Thus, we have created a resource of concrete leads for understanding the genetic basis of specific phenotypes and illustrate the promise of this kind of approach for comprehensively interrogating how non-coding polymorphism shapes human biology. The study consists of two separate MPRA experiments, a 78,958 oligo (79k study) and a 7,500 oligo library (7.5k). For each library we processed independent transfections into two lymphoblastoid cell lines; in total we completed 5 replicates of NA12878 and 3 replicates of NA19239. For the 79k library we also performed 5 replicate transfections into the hepatocarcinoma cell line HepG2. Raw data is provided as Illumina reads of the 20 bp barcode from the RNA extracted 24 hours post transfection as well as from the plasmid library used for transfection. We also provide oligo/barcode combinations from the âgfp vector in the form of a tab delimited file containing the raw sequence reads of the barcode (column 1) and genomic sequence (column 2). Processed count files are unnormalized counts for each oligo acquired by summing all barcode matches together for each replicate.

Dataset Information

Massively Parallel Reporter Assay (MPRA) of 175 bp human genomic fragments containing variants in high LD with lead variants from asthma GWAS.

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