Browsing by Author "Hargreaves, Katherine A."

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    Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders
    (Springer Nature, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
    Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.
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    Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.
    (Springer, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, Yunlong
    Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.
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    Improving the Patency of Jugular Vein Catheters in Sprague-Dawley Rats by Using an Antiseptic Nitrocellulose Coating
    (Ingenta, 2018-09-01) De Luca, Thomas; Szilágyi, Keely L; Hargreaves, Katherine A.; Collins, Kimberly S.; Benson, Eric A.; Department of Medicine, Indiana University School of Medicine
    Preclinical studies in animals often require frequent blood sampling over prolonged periods. A preferred method in rats is the implantation of a polyurethane catheter into the jugular vein, with heparinized glycerol as a lock solution. However, analysis of various biologic compounds (for example, microRNA) precludes the use of heparin. We used sodium citrate as an alternative to heparin but observed more frequent loss of catheter patency. We hypothesized that this effect was due to evaporation of lock solution at the exteriorized portion of the catheter, subsequent blood infiltration into the catheter, and ultimately clot formation within the catheter. We therefore tested evaporation and its variables in vitro by using 5 common catheter materials. We used the migration of dye into vertically anchored catheters as a measure of lock displacement due to evaporation. Exposure to dry room-temperature air was sufficient to cause dye migration against gravity, whereas a humid environment and adding glycerol to the lock solution mitigated this effect, thus confirming loss of the lock solution from the catheter by evaporation. We tested 4 catheter treatments for the ability to reduce lock evaporation. Results were validated in vivo by using male Sprague-Dawley rats (n = 12) implanted with polyurethane jugular vein catheters and randomized to receive a nitrocellulose-based coating on the exteriorized portion of the catheter. Coating the catheters significantly improved patency, as indicated by a Kaplan-Meier log-rank hazard ratio greater than 5 in untreated catheters. We here demonstrate that a simple nitrocellulose coating reduces evaporation from and thus prolongs the patency of polyurethane catheters in rats.
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    RegSNPs-intron: a computational framework for predicting pathogenic impact of intronic single nucleotide variants
    (BMC, 2019-11-28) Lin, Hai; Hargreaves, Katherine A.; Li, Rudong; Reiter, Jill L.; Wang, Yue; Mort, Matthew; Cooper, David N.; Zhou, Yaoqi; Zhang, Chi; Eadon, Michael T.; Dolan, M. Eileen; Ipe, Joseph; Skaar, Todd C.; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
    Single nucleotide variants (SNVs) in intronic regions have yet to be systematically investigated for their disease-causing potential. Using known pathogenic and neutral intronic SNVs (iSNVs) as training data, we develop the RegSNPs-intron algorithm based on a random forest classifier that integrates RNA splicing, protein structure, and evolutionary conservation features. RegSNPs-intron showed excellent performance in evaluating the pathogenic impacts of iSNVs. Using a high-throughput functional reporter assay called ASSET-seq (ASsay for Splicing using ExonTrap and sequencing), we evaluate the impact of RegSNPs-intron predictions on splicing outcome. Together, RegSNPs-intron and ASSET-seq enable effective prioritization of iSNVs for disease pathogenesis.
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    Tracheal Aspirate as an Alternative Biologic Sample for Pharmacogenomics Testing in Mechanically Ventilated Pediatric Patients
    (Wiley, 2021-03) Hargreaves, Katherine A.; Pratt, Victoria M.; Medeiros, Elizabeth B.; Lynnes, Ty C.; Granfield, Caitlin A.; Skaar, Todd C.; Iwata-Otsubo, Aiko; Tillman, Emma M.; Medical and Molecular Genetics, School of Medicine
    Patients in the pediatric intensive care unit are exposed to multiple medications and are at high risk for adverse drug reactions. Pharmacogenomic (PGx) testing could help decrease their risk of adverse reactions. Although whole blood is preferred for PGx testing, blood volume in this population is often limited. However, for patients on mechanical ventilation, tracheal secretions are abundant, frequently suctioned, and discarded. Thus, the aim of this pilot study was to determine if tracheal aspirates could be used as a source of human genomic DNA for PGx testing. We successfully extracted DNA from tracheal secretions of all 23 patients in the study. The samples were successfully genotyped for 10 clinically actionable single nucleotide variants across 3 cytochrome P450 genes (CYP2D6, CYP2C19, and CYP3A5). Using DNA from whole blood samples in 11 of the patients, we confirmed the accuracy of the genotyping with 100% concordance. Therefore, our results support the use of tracheal aspirates from mechanically ventilated children as an adequate biospecimen for clinical genetic testing.