Andrew Brooks

A genome wide association study of alcohol dependence symptom counts in extended pedigrees identifies C15orf53

Several studies have identified genes associated with alcohol-use disorders (AUDs), but the variation in each of these genes explains only a small portion of the genetic vulnerability. The goal of the present study was to perform a genome-wide association study (GWAS) in extended families from the Collaborative Study on the Genetics of Alcoholism to identify novel genes affecting risk for alcohol dependence (AD). To maximize the power of the extended family design, we used a quantitative endophenotype, measured in all individuals: number of alcohol-dependence symptoms endorsed (symptom count (SC)). Secondary analyses were performed to determine if the single nucleotide polymorphisms (SNPs) associated with SC were also associated with the dichotomous phenotype, DSM-IV AD. This family-based GWAS identified SNPs in C15orf53 that are strongly associated with DSM-IV alcohol-dependence symptom counts (P=4.5 × 10−8, inflation-corrected P=9.4 × 10−7). Results with DSM-IV AD in the regions of interest support our findings with SC, although the associations were less significant. Attempted replications of the most promising association results were conducted in two independent samples: nonoverlapping subjects from the Study of Addiction: Genes and Environment (SAGE) and the Australian Twin Family Study of AUDs (OZALC). Nominal association of C15orf53 with SC was observed in SAGE. The variant that showed strongest association with SC, rs12912251 and its highly correlated variants (D′=1, r2⩾ 0.95), have previously been associated with risk for bipolar disorder.

Inhibition of Autoregulated TGFβ Signaling Simultaneously Enhances Proliferation and Differentiation of Kidney Epithelium and Promotes Repair Following Renal Ischemia

We studied autocrine transforming growth factor (TGF)beta signaling in kidney epithelium. Cultured proximal tubule cells showed regulated signaling that was high during log-phase growth, low during contact-inhibited differentiation, and rapidly increased during regeneration of wounded epithelium. Autoregulation of signaling correlated with TGFbeta receptor and Smad7 levels, but not with active TGFbeta, which was barely measurable in the growth medium. Confluent differentiated cells with low receptor and high Smad7 levels exhibited blunted responses to saturating concentrations of exogenously provided active TGFbeta, suggesting that TGFbeta signaling homeostasis was achieved by cell density-dependent modulation of signaling intermediates. Antagonism of Alk5 kinase, the TGFbeta type I receptor, dramatically accelerated the induction of differentiation in sparse, proliferating cultures and permitted better retention of differentiated features in regenerating cells of wounded, confluent cultures. Alk5 antagonism accelerated the differentiation of cells in proximal tubule primary cultures while simultaneously increasing their proliferation. Consequently, Alk5-inhibited primary cultures formed confluent, differentiated monolayers faster than untreated cultures. Furthermore, treatment with an Alk5 antagonist promoted kidney repair reflected by increased tubule differentiation and decreased tubulo-interstitial pathology during the recovery phase following ischemic injury in vivo. Our results show that autocrine TGFbeta signaling in proliferating proximal tubule cells exceeds the levels that are necessary for physiological regeneration. To that end, TGFbeta signaling is redundant and maladaptive during tubule repair by epithelial regeneration.

Transcriptional profiling of C57 and DBA strains of mice in the absence and presence of morphine

BackgroundThe mouse C57BL/6 (C57) and DBA/2J (DBA) inbred strains differ substantially in many aspects of their response to drugs of abuse. The development of microarray analyses represents a genome-wide method for measuring differences across strains, focusing on expression differences. In the current study, we carried out microarray analysis in C57 and DBA mice in the nucleus accumbens of drug-naïve and morphine-treated animals.ResultsWe identified mRNAs with altered expression between the two strains. We validated the mRNA expression changes of several such mRNAs, including Gnb1, which has been observed to be regulated by several drugs of abuse. In addition, we validated alterations in the enzyme activity of one mRNA product, catechol-O-methyltransferase (Comt). Data mining of expression and behavioral data indicates that both Gnb1 and Comt expression correlate with aspects of drug response in C57/DBA recombinant inbred strains. Pathway analysis was carried out to identify pathways showing significant alterations as a result of treatment and/or due to strain differences. These analyses identified axon guidance genes, particularly the semaphorins, as showing altered expression in the presence of morphine, and plasticity genes as showing altered expression across strains. Pathway analysis of genes showing strain by treatment interaction suggest that the phosphatidylinositol signaling pathway may represent an important difference between the strains as related to morphine exposure.ConclusionmRNAs with differing expression between the two strains could potentially contribute to strain-specific responses to drugs of abuse. One such mRNA is Comt and we hypothesize that altered expression of Comt may represent a potential mechanism for regulating the effect of, and response to, multiple substances of abuse. Similarly, a role for Gnb1 in responses to multiple drugs of abuse is supported by expression data from our study and from other studies. Finally, the data support a role for semaphorin signaling in morphine effects, and indicate that altered expression of genes involved in phosphatidylinositol signaling and plasticity might also affect the altered drug responses in the two strains.

Reproductive Aging is Associated With Altered Gene Expression in Human Luteinized Granulosa Cells

Declining reproductive success with aging is attributable to qualitative and quantitative deterioration in oocytes, which are nurtured by granulosa cells (GCs). This prospective study assesses whether reproductive aging is accompanied by differential gene expression in luteinized GCs from in vitro fertilization (IVF) patients. Women with nonovarian infertility etiologies were categorized as younger (≤30, n = 3) or older (≥40, n = 3). During oocyte retrieval, mural GCs were isolated; messenger RNA (mRNA) was extracted and transcribed for complementary DNA (cDNA) microarray analysis. Differential gene expression was confirmed by real-time polymerase chain reaction (PCR). Analysis revealed 120 genes were differentially expressed. Three genes were upregulated and 117 were downregulated (including interleukin [IL]-1β, IL-1R2, and IL-6R) in GCs of older versus younger patients. Our data provide evidence of downregulation in IL-1 and IL-6 gene families in luteinized GCs with advancing age. Given previously recognized roles for the IL gene family in folliculogenesis and ovulation, our findings may partly explain ovulatory and luteal dysfunctions associated with reproductive aging.

A randomized, placebo‐controlled study of the effects of naproxen, aspirin, celecoxib or clopidogrel on gastroduodenal mucosal healing

Backgroundu2002 Many individuals with gastroduodenal ulcers require on‐going, non‐steroidal anti‐inflammatory drug (NSAID) or anti‐platelet therapy.

Borrelia burgdorferi induces a type I interferon response during early stages of disseminated infection in mice.

BackgroundLyme borrelia genotypes differ in their capacity to cause disseminated disease. Gene array analysis was employed to profile the host transcriptome induced by Borrelia burgdorferi strains with different capacities for causing disseminated disease in the blood of C3H/HeJ mice during early infection.ResultsB. burgdorferi B515, a clinical isolate that causes disseminated infection in mice, differentially regulated 236 transcripts (Pu2009<u20090.05 by ANOVA, with fold change of at least 2). The 216 significantly induced transcripts included interferon (IFN)-responsive genes and genes involved in immunity and inflammation. In contrast, B. burgdorferi B331, a clinical isolate that causes transient skin infection but does not disseminate in C3H/HeJ mice, stimulated changes in only a few genes (1 induced, 4 repressed). Transcriptional regulation of type I IFN and IFN-related genes was measured by quantitative RT-PCR in mouse skin biopsies collected from the site of infection 24 h after inoculation with B. burgdorferi. The mean values for transcripts of Ifnb, Cxcl10, Gbp1, Ifit1, Ifit3, Irf7, Mx1, and Stat2 were found to be significantly increased in B. burgdorferi strain B515-infected mice relative to the control group. In contrast, transcription of these genes was not significantly changed in response to B. burgdorferi strain B331 or B31-4, a mutant that is unable to disseminate.ConclusionsThese results establish a positive association between the disseminating capacity of B. burgdorferi and early type I IFN induction in a murine model of Lyme disease.

NSAID‐induced antral ulcers are associated with distinct changes in mucosal gene expression

Backgroundu2002 The basis for individual variation in gastroduodenal vulnerability to NSAIDs is not well understood.

Rodent Genetics, Models, and Genotyping Methods

Laboratory mice (Mus musculus) and rats (Rattus norvegicus) are the most commonly used animals in biomedical and behavioral research. Today, there are many inbred, outbred, hybrid, congenic, and genetically modified mouse and rat models that have made essential contributions to advances in the diagnosis, prevention, and control of human disease. Routine genetic monitoring of these animals is essential to their successful use. Herein, we discuss the basic structure of the mouse and rat genome; common breeding protocols to preserve innate and exogenous genetic backgrounds; and the methods used for creating specific types of mutant model, research applications, and current genotyping methods.

Selective quenching of unhybridized fluorescent probes by gold nanoparticles for rapid SNP genotyping using conventional PCR

We detect single nucleotide polymorphisms (SNP) in genomic DNA that has been amplified using a standard PCR protocol where a short fluorescently tagged probe sequence and a single denaturation and annealing step have been added. We utilize the ability of ionically treated gold nanoparticles to selectively adsorb unhybridized probes in the modified PCR product and quench their fluorescence. The method eliminates the time and expense of gel electrophoresis while retaining the ability to use an ordinary thermal cycler for PCR amplification. We describe an application to genotyping Fatty Zucker rats and validate the method against fluorogenic realtime PCR with double blind studies.