Genetic Dissection of Quantitative Trait Loci for Substances of Abuse

It has been reported that an individual’s initial level of response to a drug might be predictive of his or her future risk of becoming dependent, thus basal gene expression profiles underlying those drug responses may be informative for both predicting addiction susceptibility and determining targets for intervention. This dissertation research aims to elucidate genetic risk factors underlying acute alcohol and nicotine dependence phenotypes using mouse genetic models of addiction. Phenotyping, brain region-specific mRNA expression profiling, and genetic mapping of a recombinant inbred panel of over 25 mouse strains were performed in order to identify quantitative trait loci (QTL) harboring candidate genes that may modulate these phenotypes. Previous BXD (B6 x D2) behavioral studies performed in our laboratory identified an ethanol-induced anxiolysis-like QTL (Etanq1) in the light dark box (LDB). We hypothesized that genetic variation within Nin (a gene within the Etanq1 support interval involved in microtubule-anchoring) may modulate anxiolytic-like responses to acute ethanol in the LDB as well as other preclinical models of anxiety, the elevated plus maze (EPM), and marble burying (MB) task. Molecular studies have allowed us to confirm cis regulation of Nin transcript levels in the NAc. To elucidate potential mechanisms mediating Etanq1, the pharmacological tools, diazepam and HZ166 (a benzodiazepine derivative) were utilized to interrogate whether GABAA receptor activation modulates ethanol’s anxiety-like behaviors in the LDB. We show that the LDB phenotype, percent time spent (PTS) in the light following a brief restraint stress, is not being modulated through direct activation of GABAA α2/α3 receptor subunits. To genetically dissect Etanq1 as well as parse the ethanol anxiolytic-like phenotype, we have assayed 8 inbred strains, selected based on genotypes at Nin, in various preclinical models of anxiety. Principal components analysis of these behavioral data suggests that the gene(s) modulating the ethanol anxiolytic-like component in the LDB do not overlap with similar phenotypes in the elevated plus maze (EPM), nor the MB phenotype. Furthermore, site-specific delivery of an sh-Nin lentivirus into the NAc of D2 mice revealed that Nin may modulate one LDB endophenotype, latency to enter the light side of the LDB, which loaded as a part of the “anxiolysis” principal component. These data strongly imply that basal neuronal Nin expression in the NAc is important for acute ethanol anxiolytic-like behavior, perhaps through a novel mechanism involving synaptic remodeling. In separate behavioral QTL mapping studies, we hypothesized that genetic variation regulating expression of Chrna7 modulates the reward-like phenotype, conditioned place preference (CPP), for nicotine. We provide evidence for genetic regulation of Chrna7 across the BXD panel of mice and through pharmacological and genetic behavioral studies, confirm Chrna7 as a quantitative trait gene modulating CPP for nicotine in mice. Microarrays, followed by network analyses, allowed us to identify a genetically co-regulated network within the nucleus accumbens (NAc), differentially expressed in mice null for Chrna7, which was similarly correlated in the BXD panel of mice. Our network and molecular analyses suggest a putative role for Chrna7 in regulating insulin signaling in the NAc, which together, may contribute to the enhanced sensitivity to nicotine observed in strains of mice that lack or have low mRNA levels of Chrna7 in the NAc. Overall, this research has elucidated and confirmed new genetic risk factors underlying alcohol and nicotine dependence phenotypes and has enabled a better understanding of the neurogenomic bases of alcohol and nicotine addiction. Future studies that further investigate the signaling pathways and/or gene interactions involving Nin and Chrna7 may lead the field to new candidates for pharmacotherapies that may be tailored for use in individuals with susceptible genotypes. Supported by NIAAA grants P20AA017828 and R01AA020634 to MFM, NIDA T32DA007027 to WLD, and NIDA R01DA032246 to MFM and MID

Transcriptomic profiling of 39 commonly-used neuroblastoma cell lines

Neuroblastoma cell lines are an important and cost-effective model used to study oncogenic drivers of the disease. While many of these cell lines have been previously characterized with SNP, methylation, and/or mRNA expression microarrays, there has not been an effort to comprehensively sequence these cell lines. Here, we present raw whole transcriptome data generated by RNA sequencing of 39 commonly-used neuroblastoma cell lines. These data can be used to perform differential expression analysis based on a genetic aberration or phenotype in neuroblastoma (e.g., MYCN amplification status, ALK mutation status, chromosome arm 1p, 11q and/or 17q status, sensitivity to pharmacologic perturbation). Additionally, we designed this experiment to enable structural variant and/or long-noncoding RNA analysis across these cell lines. Finally, as more DNase/ATAC and histone/transcription factor ChIP sequencing is performed in these cell lines, our RNA-Seq data will be an important complement to inform transcriptional targets as well as regulatory (enhancer or repressor) elements in neuroblastoma

Genetic Analysis of the Neurosteroid Deoxycorticosterone and Its Relation to Alcohol Phenotypes: Identification of QTLs and Downstream Gene Regulation

Deoxycorticosterone (DOC) is an endogenous neurosteroid found in brain and serum, precursor of the GABAergic neuroactive steroid (3α,5α)-3,21-dihydroxypregnan-20-one (tetrahydrodeoxycorticosterone, THDOC) and the glucocorticoid corticosterone. These steroids are elevated following stress or ethanol administration, contribute to ethanol sensitivity, and their elevation is blunted in ethanol dependence.To systematically define the genetic basis, regulation, and behavioral significance of DOC levels in plasma and cerebral cortex we examined such levels across 47 young adult males from C57BL/6J (B6)×DBA/2J (D2) (BXD) mouse strains for quantitative trait loci (QTL) and bioinformatics analyses of behavior and gene regulation. Mice were injected with saline or 0.075 mg/kg dexamethasone sodium salt at 8:00 am and were sacrificed 6 hours later. DOC levels were measured by radioimmunoassay. Basal cerebral cortical DOC levels ranged between 1.4 and 12.2 ng/g (8.7-fold variation, p<0.0001) with a heritability of ∼0.37. Basal plasma DOC levels ranged between 2.8 and 12.1 ng/ml (4.3-fold variation, p<0.0001) with heritability of ∼0.32. QTLs for basal DOC levels were identified on chromosomes 4 (cerebral cortex) and 14 (plasma). Dexamethasone-induced changes in DOC levels showed a 4.4-fold variation in cerebral cortex and a 4.1-fold variation in plasma, but no QTLs were identified. DOC levels across BXD strains were further shown to be co-regulated with networks of genes linked to neuronal, immune, and endocrine function. DOC levels and its responses to dexamethasone were associated with several behavioral measures of ethanol sensitivity previously determined across the BXD strains by multiple laboratories.Both basal and dexamethasone-suppressed DOC levels are positively correlated with ethanol sensitivity suggesting that the neurosteroid DOC may be a putative biomarker of alcohol phenotypes. DOC levels were also strongly correlated with networks of genes associated with neuronal function, innate immune pathways, and steroid metabolism, likely linked to behavioral phenotypes

Cross-cohort analysis identifies a TEAD4-MYCN positive-feedback loop as the core regulatory element of high-risk neuroblastoma

High-risk neuroblastomas show a paucity of recurrent somatic mutations at diagnosis. As a result, the molecular basis for this aggressive phenotype remains elusive. Recent progress in regulatory network analysis helped us elucidate disease-driving mechanisms downstream of genomic alterations, including recurrent chromosomal alterations. Our analysis identified three molecular subtypes of high-risk neuroblastomas, consistent with chromosomal alterations, and identified subtype-specific master regulator proteins that were conserved across independent cohorts. A 10-protein transcriptional module—centered around a TEAD4–MYCN positive feedback loop—emerged as the regulatory driver of the high-risk subtype associated with MYCN amplification. Silencing of either gene collapsed MYCN -amplified (MYCNAmp) neuroblastoma transcriptional hallmarks and abrogated viability in vitro and in vivo. Consistently, TEAD4 emerged as a robust prognostic marker of poor survival, with activity independent of the canonical Hippo pathway transcriptional coactivators YAP and TAZ. These results suggest novel therapeutic strategies for the large subset of MYCN-deregulated neuroblastomas. SIGNIFICANCE: Despite progress in understanding of neuroblastoma genetics, little progress has been made toward personalized treatment. Here, we present a framework to determine the downstream effectors of the genetic alterations sustaining neuroblastoma subtypes, which can be easily extended to other tumor types. We show the critical effect of disrupting a 10-protein module centered around a YAP/TAZ-independent TEAD4–MYCN positive feedback loop in MYCNAmpneuroblastomas, nominating TEAD4 as a novel candidate for therapeutic intervention

International Comparison of Enumeration-Based Quantification of DNA Copy-Concentration Using Flow Cytometric Counting and Digital Polymerase Chain Reaction

Enumeration-based determination of DNA copy-concentration was assessed through an international comparison among national metrology institutes (NMIs) and designated institutes (DIs). Enumeration-based quantification does not require a calibration standard thereby providing a route to “absolute quantification”, which offers the potential for reliable value assignments of DNA reference materials, and International System of Units (SI) traceability to copy number 1 through accurate counting. In this study, 2 enumeration-based methods, flow cytometric (FCM) counting and the digital polymerase chain reaction (dPCR), were compared to quantify a solution of the pBR322 plasmid at a concentration of several thousand copies per microliter. In addition, 2 orthogonal chemical-analysis methods based on nucleotide quantification, isotope-dilution mass spectrometry (IDMS) and capillary electrophoresis (CE) were applied to quantify a more concentrated solution of the plasmid. Although 9 dPCR results from 8 laboratories showed some dispersion (relative standard deviation [RSD] = 11.8%), their means were closely aligned with those of the FCM-based counting method and the orthogonal chemical-analysis methods, corrected for gravimetric dilution factors. Using the means of dPCR results, the RSD of all 4 methods was 1.8%, which strongly supported the validity of the recent enumeration approaches. Despite a good overall agreement, the individual dPCR results were not sufficiently covered by the reported measurement uncertainties. These findings suggest that some laboratories may not have considered all factors contributing to the measurement uncertainty of dPCR, and further investigation of this possibility is warranted