ExonMicroarray Analysis of Human DorsolateralPrefrontal Cortex in Alcoholism

Ann M. Manzardo, Sumedha Gunewardena, KunWang, and Merlin G. Butler

Background: Alcohol abuse is associated with cellular and biochemical disturbances that impactupon protein and nucleic acid synthesis, brain development, function, and behavioral responses. To fur-ther characterize the genetic influences in alcoholism and the effects of alcohol consumption on geneexpression, we used a highly sensitive exon microarray to examine mRNA expression in human frontalcortex of alcoholics and control males.

Methods: Messenger RNA was isolated from the dorsolateral prefrontal cortex (dlPFC; Brodmannarea 9) of 7 adult alcoholic (6 males, 1 female, mean age 49 years) and 7 matched controls. AffymetrixHuman Exon 1.0 ST array was performed according to standard procedures and the results analyzed atthe gene level. Microarray findings were validated using quantitative reverse transcription polymerasechain reaction, and the ontology of disturbed genes characterized using Ingenuity Pathway Analysis(IPA).

Results: Decreased mRNA expression was observed for genes involved in cellular adhesion (e.g.,CTNNA3, ITGA2), transport (e.g., TF, ABCA8), nervous system development (e.g., LRP2, UGT8,GLDN), and signaling (e.g., RASGRP3, LGR5) with influence over lipid and myelin synthesis (e.g.,ASPA, ENPP2, KLK6). IPA identified disturbances in network functions associated with neurologicaldisease and development including cellular assembly and organization impacting on psychological dis-orders.

Conclusions: Our data in alcoholism support a reduction in expression of dlPFC mRNA for genesinvolved with neuronal growth, differentiation, and signaling that targets white matter of the brain.

Key Words: Affymetrix, ExonMicroarray, Alcoholism, Frontal Cortex, Myelin.

ALCOHOLISM IS A chronic, severe relapsing disorderwith global impact on individuals and society contrib-

uting to significant illness, injury, and death worldwide eachyear (Schuckit, 2009a; Whiteford et al., 2010). The compul-sive use of alcohol and other substances arises from genetic,neurobiological and developmental, environmental, and psy-chosocial influences that direct both the likelihood (and level)of alcohol exposure and biologic response (Knop et al.,2003; Manzardo and Penick, 2006; Manzardo et al., 2005,2011; Schuckit, 2009b; Spanagel, 2009; Yan et al., 2013).Genetic factors are particularly important and contribute anestimated 40 to 60% of the risk of developing alcoholism(Goodwin et al., 1974; Prescott and Kendler, 1999; Schuckit,

2009b; Yan et al., 2013). Additionally, prolonged alcoholexposure can cause significant damage and measurable dis-tortions in brain structure with associated cognitive dysfunc-tion observed in the clinical setting (Harper, 2009; Harperand Matsumoto, 2005; Mukherjee, 2013; M€uller-Oehringet al., 2013; Pfefferbaum et al., 2009). Alcohol-relatedgenetic and molecular changes precipitate the developmentof tolerance, physiological dependence, craving, psychiatric,and other behavioral changes that may propagate abusebehavior (Contet, 2012; Hashimoto et al., 2011; Mukherjee,2013; M€uller-Oehring et al., 2013). The identification ofgenes and proteins associated with predisposition, develop-ment, and maintenance of alcoholism is an important area ofintense research to facilitate effective treatments includingpharmacotherapy and small molecule discovery.

Brain regions of primary interest in the etiology of addic-tions encompass components and projections of the meso-limbic dopamine circuitry including the ventral tegmentalarea, nucleus accumbens, amygdala, and prefrontal cortex(PFC) which mediate brain reward responses to endoge-nously and exogenously derived substrates (Mayfield et al.,2002, 2008; Mukherjee, 2013; M€uller-Oehring et al., 2013;Ross and Peselow, 2009). Disturbances in the mesolimbicdopamine circuitry in response to exposure to addictive sub-stances are widely reported in human and animal literature(Koob, 2003; Koob and Le Moal, 2001; M€uller-Oehringet al., 2013; Ross and Peselow, 2009). The PFC is believed to

From the Department of Psychiatry and Behavioral Sciences(AMM, KW, MGB), University of Kansas School of Medicine, KansasCity, Kansas; Department of Molecular and Integrative Physiology(SG), University of Kansas School of Medicine, Kansas City, Kansas;Department of Biostatistics (SG), University of Kansas School of Medi-cine, Kansas City, Kansas; and Department of Pediatrics (MGB), Uni-versity of Kansas School ofMedicine, Kansas City, Kansas.

Received for publication January 2, 2014; acceptedMarch 20, 2014.Reprint requests: Ann M. Manzardo, PhD, Department of Psychiatry

and Behavioral Sciences, MS 4015, University of Kansas Medical Center,3901 Rainbow Blvd, Kansas City, KS 66160; Tel.: 913-588-6473; Fax:913-588-1305; E-mail: amanzardo@kumc.edu

Copyright© 2014 by the Research Society on Alcoholism.

DOI: 10.1111/acer.12429

1594 Alcohol Clin Exp Res, Vol 38, No 6, 2014: pp 1594–1601

ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH Vol. 38, No. 6June 2014

play a part in the development and expression of alcoholismthrough its role in guiding executive functions impactingjudgment and decision making. Neuronal loss of PFC grayand white matter in addition to region selective disturbancesin gene, noncoding RNA, and protein expression has beenreported in postmortem brain samples taken from alcoholics(Alexander-Kaufman et al., 2007; Contet, 2012; Mayfieldet al., 2002). The dorsolateral PFC (dlPFC) which includesBrodmann area 9 has reciprocal connections to many corti-cal regions and is known to play a role in the regulation ofmotivated behaviors. It has been identified as a brain regionparticularly susceptible to the effects of long-term alcoholabuse associated with significant volume loss in alcoholics—particularly of white matter tissue (Alexander-Kaufmanet al., 2007). Structural abnormalities and dlPFC dysfunc-tion is associated with impulse deregulation and cognitivedysfunction, substance abuse including alcoholism and maycontribute to pathology.High throughput microarray technology has been used to

examine gene expression in human brain of alcoholics rela-tive to controls (Flatscher-Bader et al., 2005, 2006, 2010;Lewohl et al., 2000; Liu et al., 2004, 2006; Mayfield et al.,2002; Sokolov et al., 2003). These studies show remarkableconcordance in alcohol responsive gene disturbances in thePFC which impact upon cellular functioning including mye-lination, cellular signaling, and energy production with anoverrepresentation of down-regulated versus up-regulatedgenes exhibited at the mRNA level (Lewohl et al., 2000; Liuet al., 2004, 2006; Mayfield et al., 2002; Sokolov et al.,2003). Comparative studies of gene expression in the nucleusaccumbens and ventral tegmental area have reported distur-bances in genes associated with vesicle formation and regula-tion of cellular architecture, including pathways regulatingthe actin cytoskeleton, possibly impacting upon neuroplas-ticity and influenced by tobacco use (Flatscher-Bader et al.,2005, 2006, 2010). Liu and colleagues (2006) found signifi-cant down-regulation of astrocyte specific genes in the supe-rior frontal cortex as well as genes associated with cellularadhesion and protein trafficking (Liu et al., 2006). These dif-

ferences were exaggerated among alcoholics complicatedwith cirrhotic liver disease (Liu et al., 2007).Microarray technology and computational capabilities

have continued to increase in the years following these earlystudies. High resolution mapping using >2 million probe setsis now possible at the whole genome mRNA and exon level.Additionally, advancements in computational programs withgenes linked by accession numbers (e.g., Ingenuity PathwayAnalysis [IPA]) have elevated the capabilities and sophistica-tion of mapping of disturbed gene networks. We present anexamination of dlPFC gene expression patterns and func-tional analysis of human alcoholics and controls using ahighly sensitive exon microarray platform.

MATERIALS ANDMETHODS

Samples

Messenger RNA (exon) expression profiles were obtained fromtotal RNA isolated using the Qiagen (Qiagen Inc, Gaithersburg,MD) kit from postmortem human dlPFC (Brodmann area 9) of 7alcoholics (6 males, 1 female; mean [�SD] age = 49.0 [�6.7] years,range 41 to 57 years) and 7 age-matched control subjects (5 males, 2female; mean [�SD] age = 49.4 [�7.1] years, range 37 to 56 years)characterized in Table 1. The average RNA integrity number (RIN)was 6 for the alcoholics and 5 for the controls and considered ade-quate for microarray analysis. The gender composition of the sam-ple reflects the sex ratio distribution found in the general populationof alcoholics. Whole genome DNA methylation and miRNAexpression for these samples have been previously reported (Man-zardo et al., 2012, 2013). Samples were procured from the NewSouth Wales Brain Bank (NSWBB, Sydney, NSW, Australia) andcollected according to a standardized protocol (Sheedy et al., 2008)in compliance with ethical guidelines established by the SydneySouth West Area Health Service Human Ethics Committee (X03-0074). Informed written consent was obtained from the nearest liv-ing relative. The mean (�SD) postmortem interval (PMI) for oursubjects shown in Table 1 was 27.5 (�10.3) hours with a range of13 to 43 hours. The mean (�SD) sample pH was 6.6 (�0.22). Allsamples tested were negative for viral hepatitis and for the humanimmunodeficiency virus.

All subjects were of European descent and alcoholic subjects metthe criteria described in the Diagnostic and Statistical Manual forMental Disorders, Fourth Edition and National Health and

Table 1. Characteristics of Alcoholic and Control Subjects

IDAge

(years) Sex GroupAbuse(years)

Smokingstatus

Liverpathology Cause of death PMI pH RIN

201 46 Male Dependent >10 Unknown Cirrhosis Alcohol toxicity 24 6.5 7.5259 56 Male Abuse 20 Unknown Steatosis Ischemic heart disease; emphysema 15 6.7 5.0430 43 Male Abuse 20 Ex smoker Steatosis Sepsis 29 6.3 6.6466 57 Male Dependent 10 Current Cirrhosis Ischemic heart disease 43 6.5 6.5586 55 Male Dependent 30 Nonsmoker Steatosis Asphyxia 17 6.8 4.4628 41 Male Abuse 20 Current Steatosis Alcohol/methadone toxicity 38 6.5 6.7596 45 Female Dependent 10 Current Cirrhosis Coronary atherosclerosis 41 6.8 6.4225 56 Male Control 0 Current Steatosis Coronary artery atheroma 24 6.5 6.4236 43 Male Control 0 Current Normal Thrombotic coronary artery occlusion 13 6.4 6.5239 37 Male Control 0 Unknown Steatosis Electrocution 24 6.4 6.3339 56 Male Control 0 Current Normal Cardiomegaly 37 6.8 4.0636 54 Male Control 0 Ex smoker Normal Coronary artery disease 28 6.4 4.0460 49 Female Control 0 Nonsmoker Normal Arrhythmogenic right ventricular dysplasia 15 6.9 4.0551 51 Female Control 0 Nonsmoker Steatosis Myocardial infarction 37 6.9 5.8

ID, subject identifier from the New South Wales Tissue Resource Centre; PMI, postmortem interval; RIN, RNA integrity number.

EXON EXPRESSION IN ALCOHOLISM 1595

Medical Research Council/World Health Organization criteria.Control subjects were social drinkers (nonabstainers for alcoholuse) and did not meet criteria for alcohol abuse or dependence. Theaverage estimated duration of alcohol dependence for case subjectswas 19.5 (�8.1) years (range 10 to 30 years). All alcoholic subjectswere diagnosed with hepatic complications of steatosis or cirrhosiswhile controls showed normal liver function to moderate steatosis.The individual causes of death varied across participants with themost common causes due to cardiovascular and respiratory prob-lems or infection. Direct alcohol toxicity or overdose was indicatedin 2 deaths of alcoholic subjects. Family history of alcohol problemswas either negative or unknown for all subjects. Previous reportsexamining gene expression profiles in the frontal cortex reported byLiu and colleagues (2004, 2006) and Flatscher-Bader and colleagues(2005) with brain tissue procured from the NSWBB utilized a differ-ent collection of alcoholic and nonalcoholic individuals which is evi-dent by comparison of age, sex, and PMI data.

Microarray

The Human Exon 1.0 ST (sense target) array (Affymetrix, Inc.,Santa Clara, CA) was used to examine dlPFC mRNA expressiondifferences between alcoholics and control subjects. Array specifica-tions include the following: Array Type – Human Exon 1.0 ST;Source – cDNA-based content including the more establishedhuman RefSeq mRNAs, GenBank� mRNAs, and ESTs fromdbEST. Additional annotations were created by mapping synteniccDNAs to the human, mouse, and rat genomes using genome synte-ny maps from the UCSC Genome Bioinformatics group. Predictedgene structure sequences from GENSCAN; Ensembl; Vega; geneidand sgp; TWINSCAN; Exoniphy; microRNA Registry; MITO-MAP; and structural RNA predictions; Build – all probe locationsused the human genome reference GRCh36/hg19 assembly (http://genome.ucsc.edu/cgi-bin/hgGateway?db=hg19). Probe Length –25mer or greater. The Human Exon 1.0 ST Array uses 1.4 millionprobe sets to interrogate exons at 28,869 well-annotated genes.

Quantitative Reverse Transcription Polymerase Chain ReactionMethodology

Three representative genes (UGT8, TF, LRP2) identified by exonmicroarray as differentially expressed in alcoholism were evaluatedby quantitative reverse transcription polymerase chain reaction(RT-qPCR). First strand cDNA was synthesized from 500 ng oftotal mRNA using iScript cDNA Synthesis Kit (BIO-RAD, Hercu-les, CA). Primers for the genes of interest including the referencegene, GAPDH were purchased from Qiagen. Primers spanningexon–exon junctions were selected to avoid potential genomic DNAcontamination. SYBR green PCR assays were performed in 48-wellwhite plates on a MJ Mini Personal Thermal Cycler (BIO-RAD).The reaction cycling parameters for each of the PCR reaction were95°C for 10 minutes, followed by 40 cycles of 95°C for 10 secondsand 60°C for 1 minute. Expression levels of UGT8, TF, and LRP2were normalized to GAPDH. Fold induction values were calculatedusing DDCt method according to manufacturer’s instructions.

Data Analysis

Exon Array Analysis. Gene expression profiling was carried outusing the Affymetrix GeneChip Human Exon 1.0 ST array consist-ing of 1.4 million probe sets, of which around 300,000 are coreexon probe sets supported by putative full-length mRNA (RefSeqand full-length GenBank annotated alignments). These core probesets map to approximately 18,000 genes with high confidence. Geneexpression level was determined by averaging the intensity signalsof multiple probes to the individual exons per gene. The exonarrays are robust multi-array averaging background corrected,

quantile-normalized, and gene-level summarized using the MedianPolish algorithm (Irizarry et al., 2003). The resulting log (base 2)transformed signal intensities (expression values) were used toascertain differentially expressed genes. Fold change statistics forindividual genes were calculated by taking the linear contrastbetween the least square means of the (log) alcoholic and (log) con-trol groups and back transforming the result to a linear scale (thisis the ratio of the geometric mean of the treatment samples to thegeometric mean of the control samples). Corresponding significancescores (p-values) were assigned based on the t-statistic of the linearcontrast. A statistical model including 8 potential confounding fac-tors (age, gender, alcoholism diagnosis, duration of alcohol abuse,hepatic illness, PMI, tissue pH, and sample RIN) was carried outfor genes with ≥3-fold change difference in alcoholics. No signifi-cant factors were identified and thus the factors were excluded fromthe final model.

Our analysis was conducted on brain tissues obtained from bio-logical replicates of 7 alcoholic and 7 control samples, and IPA wasperformed on genes showing a fold change of �1.5, p-value ≤ 0.05,and false discovery rate (FDR) of ≤0.2. Messenger RNAs fromgenes showing a fold change of�2.0, p-value ≤ 0.05, and FDR ≤0.2were hierarchically clustered and visualized in a heat map (Fig. 1).Direct and indirect relationships were examined including endoge-nous chemicals.

RESULTS

Exon (Gene) Expression

Exon and gene expression analysis of mRNAs taken frombrain specimens of alcohol dependent and control subjectsshowed less than or equal to �1.5-fold down-regulation of280 exons corresponding with 248 genes recognized by IPAand ≥1.5-fold up-regulation of 70 exons from 56 IPA recog-nized genes. Consistent with previous reports of gene distur-bances in the frontal cortex in alcoholism, significant down-regulation (>1.5-fold) in gene expression was observed forCLDN11, ENPP2, PMP22, MOG, MAG, MBP, NPY, TF,and UGT8 (Liu et al., 2004, 2006; Mayfield et al., 2002).Table 2 provides a list of selected genes with ≥3-fold down-regulation in alcoholism relative to control subjects (p-value < 0.05, FDR < 0.2) which held the greatest confidenceand will be the focus of this report. Down-regulated geneshad functional roles in cellular adhesion (e.g., CTNNA3,ITGA2), signaling (e.g., RASGRP3, LGR5), and nervoussystem development (e.g., LRP2, UGT8, GLDN). Expres-sion of genes associated with cellular and transmembranetransport of ions and minerals (e.g., TF, SLC5A11, ABCA8)were also reduced. Genes associated with lipid metabolismand myelin synthesis (e.g., LRP2, ASPA, ENPP2, KLK6)appeared to be specifically effected.

We observed fewer up-regulated than down-regulatedgenes which were disturbed to a lesser degree (Fig. 1). Inagreement with previous reports of gene expression distur-bances in the frontal cortex in alcoholism, a significant up-regulation (>1.5-fold) in gene expression was observed forGABRG1, MT1L, SLC1A3, and TGFß1 (Flatscher-Baderet al., 2005, 2008; Mayfield et al., 2002). Table 3 providesa list of selected genes with ≥3-fold up-regulation in alco-holism relative to control subjects (p-value < 0.05,

1596 MANZARDO ET AL.

FDR < 0.2) which held the greatest confidence and will bethe focus of this report. A significantly up-regulated genein our study with a previously unreported association withalcoholism includes an important lipid-binding protein,WIF1, which binds to and inhibits WNT signaling, a con-trolling pathway in embryonic development (Hsieh et al.,1999). Other up-regulated and novel genes had functional

Fig. 1. Heat map of mRNAs clustered and based upon significant dis-turbances in the frontal cortex of alcoholics and control subjects (red ordark gray represents increased expression and green or light gray repre-sents decreased expression).

Table 2. Expression of Genes Down-Regulated in Alcoholism

GeneFold

change Biological functionChromosome

band

LRP2 �4.0 Lipid/vitamin/steroid metabolism,cellular proliferation, andforebrain development

2q24-q31

FAM38Ba �3.9 Membrane protein with unknownfunction

18p11.22

ASPA �3.9 Aspartate catabolismcontributing acetyl groups forthe synthesis of lipidsand myelin

17p13.3

TFa,b �3.9 Iron transport/homeostasis,required for cell division

3q22.1

EVI2A �3.7 Transmembrane receptor 17q11.2UGT8b �3.7 Hexosyl transfer, central

and peripheral nervoussystem development

4q26

ST18 �3.6 Regulation of DNA-dependenttranscription, inhibition of RNApolymerase II promoter

8q11.23

ANLN �3.5 Cytokinesis, mitosis 7p15-p14SLC5A11 �3.4 Transmembrane ion transport:

sodium, carbohydrate16pter-p11

ENPP2b �3.4 Lipid catabolism, chemotaxis,regulation of cellular migration

8q24.1

CTNNA3 �3.3 Cell–cell adhesion 10q22.2ABCA8 �3.3 Transmembrane transport 17q24GLDN �3.2 Cell differentiation, nervous system

development15q21.2

C21orf91 �3.1 Unknown 21q21.1RASGRP3 �3.1 Intracellular signaling, MAPKKK

cascade, Ras protein signaltransduction

2p25.1-p24.1

ITGA2 �3.1 Cellular adhesion and migration,integrin-mediated signaling,cellular response mechanism

5q11.2

KLK6 �3.1 Cellular response, regulation ofcellular differentiation in centralnervous system development,myelination, proteinprocessing (e.g., amyloidprecursor protein)

19q13.3

TMEM63A �3.0 Membrane protein with nucleotidebinding and unknown function

1q42.12

SPP1 �3.0 Tissue development, osteoblastdifferentiation, cellular responseto vitamin, hormone or injury,TGF-b signaling

4q22.1

LGR5 �3.0 G-protein receptor coupledsignaling

12q22-q23

aTwo exons for the indicated genes were down-regulated ≥3-fold.bPreviously reported to be down-regulated in the frontal cortex by May-

field and colleagues (2002) or Liu and colleagues (2004, 2006).Exon arrays were robust multi-array averaging background corrected,

quantile-normalized and gene-level summarized using the Median Polishalgorithm followed by linear regression. Results are presented for geneswith exons down-regulated ≥3-fold, p-value < 0.01; false discovery ratecutoff <0.2.

EXON EXPRESSION IN ALCOHOLISM 1597

roles in intracellular transport, metabolism, and detoxifica-tion (RANBP3L, MT1G, GJB6, AGX2L1).

The Affymetrix Human Exon 1.0 ST array possesses ahigh level of resolution and internal validity; for example,expression level analysis of the LRP2 gene was based uponthe average intensity reading of 87 separate probes of LRP2exons. The number obviously varies by gene (e.g.,WIF1 con-tained 12 probes), but remains much more robust than ear-lier microarrays with 1 probe per gene requiring external

validation (i.e., qRT-PCR). However, 3 representative genesdown-regulated in our study: UGT8, TF, and LRP2 werealso selected for further confirmation using qRT-PCR(Fig. 2). The results showed a 3- to 4-fold down-regulationof all 3 genes in alcoholism relative toGAPDH (a housekeep-ing gene) which was not disturbed in our exon microarrayanalysis (Fig. 2).

Ingenuity Pathway Analysis

The functional ontology of impacted genes characterizedusing IPA identified the top biological function disturbancesas those pertaining to neurological disease and nervous sys-tem development and function networks (Table 4a). Anno-tated functions assigned to these networks impact uponmyelination and glial integrity and have been associated withschizophrenia. Correlational analysis considering activationstate of disturbed genes identified a significant suppression ofbiological functions related to cellular morphology, function,maintenance, organization, and assembly impacting uponneuronal outgrowth and cytoskeletal integrity (Table 4b).Top canonical pathways identified by IPA showed a signifi-cant disturbance in several Rho signaling pathways whichalso impact upon cytoskeletal integrity (Table 4c). Consis-tent with previous reports in liver, a significant up-regulationof actin mRNAwas observed in alcoholism (Boujedidi et al.,2012); however, the precise molecular composition of actin

Table 3. Expression of Genes Up-Regulated in Alcoholism

GeneFold

change Biological/molecular functionChromosome

band

WIF1 2.5 Lipid protein that bindsand inhibits WNT signaling

12q14.3

RANBP3L 2.3 Intracellular transport 5p13.2MT1G 2.2 Metal ion binding, storage,

transport, and detoxification16q13

GJB6 2.0 Proliferation, cellular signalingand apoptosis

13q11-q12.1

AGX2L1 2.0 Pyridoxal-phosphate-dependentmetabolism ofphosphoethanolamine toammonia, inorganic phosphate,and acetaldehyde

4q25

Exon arrays were robust multi-array averaging background corrected,quantile-normalized and gene-level summarized using the Median Polishalgorithm followed by linear regression. Results presented for fold changes≥2.0 with a p-value < 0.01 and false discovery rate step-up <0.15.

Gene Fold Change

qRT-PCR Exon Microarray

UGT8 -3.5 -3.7

TF -2.8 -3.9

LRP2 -3.8 -4.0

Amplifica on

0 10 20 30 40

Cycles

101

100

10-1

10-2

RFU

B. TF

Amplifica on

0 10 20 30 40Cycles

101

100

10-1

10-2

RFU

C. LRP2

Amplifica on

0 10 20 30 40Cycles

101

100

10-1

10-2

RFU

100

10-1

A. UGT8

Fig. 2. qRT-PCR amplification of brain mRNA of selected disturbed genes in alcoholism relative to control gene (GAPDH) expression for (A) UDP gly-cosyltransferase 8 (UGT8), (B) transferrin (TF), and (C) low density lipoprotein receptor-related protein 2 (LRP2). GAPDH: Black = control subjects(N = 7), Blue = alcohol dependent subjects (N = 7). Selected disturbed genes: Red = control subjects (N = 7), Gold = alcohol dependent subjects(N = 7).

1598 MANZARDO ET AL.

(e.g., globular, filament, polymer) cannot be determinedbased upon an exon array. Thus, the nature of the distur-bance and its impact upon cytoskeletal integrity and den-dritic spine formation is unclear from these data. In additionto these changes, IPA also identified an underlying structurefor toxology function disturbances pertaining to cardiac,renal, and hepatic systems, and nutritional deficiencies com-monly associated with severe alcoholism but not the focus ofthis article.

DISCUSSION

Our results are consistent with previous reports citing dis-turbances in mRNA expression in the PFC emphasizing thedown-regulation of neurodevelopmental mediators in alco-holism. Messenger RNAs from the medial frontal cortex ofalcoholics showing significant down-regulation relative toage- and gender-matched nonalcoholic controls had func-tional roles in cellular adhesion (e.g., CTNNA3, ITGA2),transport (e.g., TF, ABCA8), nervous system development(e.g., LRP2, UGT8, GLDN), and signaling (e.g., RASGRP3,LGR5) with targeted effects on lipid and myelin synthesis(e.g., ASPA, ENPP2, KLK6). Gene expression disturbancesfor several of these genes (e.g., TF, ENPP2, UGT8) havebeen reported in previous studies of frontal cortex in alcohol-

ism (Liu et al., 2004, 2006; Mayfield et al., 2002). IPA analy-sis identified disturbances in biological functions impactingupon neurological disease, nervous system development, andfunction through the down-regulation of genes associatedwith neuronal architecture and outgrowth. Functionally dis-turbed genes did not overlap with any specific susceptibilitygenes linked to the development of alcoholism or relatedphenotypes but several paralogs derived from similar func-tional classes (e.g., cadherin, thrombospondin, semaphorin,and solute carrier proteins) were identified (Rietschel andTreutlein, 2013). Many disturbed genes were drawn fromgene networks (e.g., cation transport, synaptic transmission,and transmission of nerve impulses) believe to impart riskfor alcohol dependence (Han et al., 2013). Some of the genedisturbances that we present are likely to reflect the patho-logic processes of chronic alcohol use and multi-organinvolvement (e.g., inflammation, hematopoiesis, and hepaticdysfunction and metabolism) impacting brain function andarchitecture (e.g., oligodendrocyte production, cell number,adhesion and differentiation, myelination, and actin forma-tion). The specific impact of these disturbances on the propa-gation of abuse behavior itself remains to be elucidated.The number of down-regulated exons (genes) identified in

our sample surpassed the number up-regulated and appear todifferentially impact upon brain white matter development.IPA identified a functional impairment in biological markersassociated with nervous system development and functionwhich is reflected in the down-regulation of several importantdevelopmental mediators (ANLN, ENPP2, ITGA2, TF; Bau-mann and Pham-Dinh, 2001; Dugas et al., 2006).ENPP2 is aphosphodiesterase and phospholipase involved in the pro-duction of the growth enhancer, lysophosphatidic acid, whichstimulates cellular proliferation and chemotaxis (Tokumuraet al., 2002). ITGA2 plays a role in cell attachment and neu-rite outgrowth (Inoue et al., 2003). TF, required for ironhomeostasis and commonly disturbed in alcoholism, is alsoknown to impact cellular division (Baumann and Pham-Dinh, 2001; Dugas et al., 2006). Lipid biochemistry and mye-lin formation are specifically impacted by UGT8 which isinvolved in the biosynthesis of galactocerebrosides, abundantsphingolipids of the myelin membrane of the central andperipheral nervous systems, andGLDN important for forma-tion of the nodes of Ranvier in myelinating cells (Eshed et al.,2005; Mackenzie et al., 2005). These data are consistent withprevious studies of frontal cortex gene expression in alcohol-ismwhich have reported disturbances in developmental medi-ators (e.g., UGT8, ENPP2, CLDN) and myelination (e.g.,MOG, MAG, MBP, and PMP22; Flatscher-Bader et al.,2005; Liu et al., 2004, 2006; Mayfield et al., 2002). Flatscher-Bader and Wilce (2006) also used real-time PCR and micro-array analysis to identify disturbances in selected genesEAAT-1,MDK, TIMP3, andWASF1 in the PFC in alcohol-ismwhich were not found to be disturbed in our study.Biological functions related to neurological disease were

also identified as disturbed by IPA. The most significantdisturbance that was novel to our study and confirmed by

Table 4. Ingenuity Pathway Analysis Summary. (a) Top BiologicalFunction Disturbances (Independent of Gene Expression Directionp-Values ≤ 1.0E-04), (b) Top Biological Function Disturbances with

Correlated Activation States (z-Scores ≤ �3.0), and (c) Top CanonicalPathway Disturbances

(a)

Name Functions annotation p-Value

Nervous systemdevelopment and function

Morphology of neuroglia 6.1E-05Myelination of cells 1.2E-04Morphology of the nervoussystem

2.3E-04

Neurological disease Demyelination of nervous tissue 1.0E-05Schizophrenia 2.3E-04

(b)

NameFunctionsannotation p-Value

Activationz-score

Cellular morphology Formation ofcellular protrusions

0.014 �3.5

Cellular function andmaintenance; cellularorganization and assembly(overlapping pathways)

Formation ofcellular protrusions

0.014 �3.5

Microtubuledynamics

0.019 �3.5

Organizationof cytoskeleton

0.0024 �3.0

Organizationof cytoplasm

0.0075 �3.0

(c)

Name p-Value Ratio (disturbed/total)

Signaling by Rho family GTPases 4.4E-07 18/254Rho A signaling 1.3E-05 11/120Rho GSI signaling 5.3E-04 11/199

EXON EXPRESSION IN ALCOHOLISM 1599

qRT-PCR is the suppression of LRP2 (megalin) a memberof the low density lipoprotein receptor family of importancein brain development that mediates the endocytotic uptakeof vitamin D and steroids (Nykjaer et al., 1999). Geneticdeletion of megalin is associated with Donnai-Barrow syn-drome which is characterized by corpus callosum abnormali-ties, myopia, and sensorineural deafness (Kantarci et al.,2007). The observed suppression of ASPA function is alsonovel and notable for its influence over lipid synthesis inmyelinization as well as the association between ASPA genedeletion and white matter degeneration in Canavan disease(Kumar et al., 2006).

The suppression of white matter formation coincides withdisturbances in signaling pathways influencing cytoskeletalintegrity and neuronal outgrowth including actin formationand is associated with an accumulation of actin mRNA inalcoholism. Disruption of neuronal architecture of multiplebrain regions are widely reported in alcoholism and is consis-tent with the present findings in the dlPFC. Further, Flat-scher-Bader and colleagues (2010) have previously reportedgene disturbances in FN1, RHOA, RHOB, SPARC, and IT-GAV of the Rho-mediated signaling cascade in the nucleusaccumbens impacting upon regulation of actin cytoskeletonassociated with alcohol and tobacco co-abuse. A high fre-quency of tobacco use was also noted in our samples and theRho-mediated signaling cascade was disturbed. However,our findings in the frontal cortex did not consistently overlapat the gene level with findings reported for the nucleusaccumbens. Gene mutations impacting actin polymerizationare associated with several heritable disorders with associ-ated intellectual impairment (e.g., Williams, fragile X, fetalalcohol, and Patau syndromes) and characterized by reduceddendritic arborization and underdeveloped spine structuresimilar to that observed in alcoholism.

The findings in the present investigation are impacted bythe complexity and comorbidities in the study samplesincluding comorbid tobacco use. Hepatic diseases includingcirrhosis identified in alcohol dependent subjects are knownto influence gene expression (Etheridge et al., 2011; Liuet al., 2007; Matsumoto, 2009). Metabolic activities of hepa-tic enzymes have been shown to protect against alcohol med-iated brain damage and compromised hepatic function mayhave significantly enhanced brain injury (Liu et al., 2007;Matsumoto, 2009). The expression of housekeeping genessuch as GAPDH and actin are influenced by hepatic diseasewhich can interfere with qRT-PCR validation (Boujedidiet al., 2012). Examination of both exon microarray andqRT-PCR results showed no evidence of a disturbance inGAPDH expression but as reported actin mRNAwas signifi-cantly elevated. Although tissues samples for several priorstudies were obtained from the same brain bank, the individ-ual alcoholics and control subjects were different and provideadditional support for replicative findings.

The present study results are consistent with previousreports of disturbances in mRNA expression in the PFCemphasizing the down-regulation of neurodevelopmental

mediators in alcoholism (e.g., TF, ENPP2, and UGT8). Bio-logical functions impacting upon neurological disease, ner-vous system development and function were disturbed andgenes associated with neuronal architecture and outgrowthwere down-regulated. Several novel gene disturbances werefound including decreased expression for LRP2, CTNNA3,GLDN, and ITGA2 as well as increased expression for WIF1and MT1G in alcoholism which may influence neurologicaldevelopment, functioning, and behavior. The results of thepresent study further characterize alcoholism-related func-tional abnormalities influencing dlPFC activity and will aidein the development of targeted therapies.

ACKNOWLEDGMENTS

Tissues were received from the New South Wales TissueResource Centre at the University of Sydney which is sup-ported by the National Health and Medical Research Coun-cil of Australia, Schizophrenia Research Institute, and theNational Institute on Alcohol Abuse and Alcoholism (NIH[NIAAA] R24AA012725). This investigation was supportedby a grant from the Hubert & Richard Hanlon Trust andNICHD HD 02528. The authors of this study have no com-peting financial interests pertaining to this work.

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