DOT/FAA/AM-08/5Office of Aerospace MedicineWashington, DC 20591

Functional Genomics Group—Program Description

Dennis Burian

Civil Aerospace Medical InstituteOklahoma City, OK 73125

February 2008

Final Report

NOTICE

This document is disseminated under the sponsorship of the U.S. Department of Transportation in the interest

of information exchange. The United States Government assumes no liability for the contents thereof.

___________

This publication and all Office of Aerospace Medicine technical reports are available in full-text from the Civil Aerospace Medical Institute’s publications Web site:

www.faa.gov/library/reports/medical/oamtechreports/index.cfm

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Technical Report Documentation Page 1. Report No. 2. Government Accession No. 3. Recipient's Catalog No.

DOT/FAA/AM-08/5 4. Title and Subtitle 5. Report Date

February 2008 Functional Genomics Group-Program Description 6. Performing Organization Code

7. Author(s) 8. Performing Organization Report No. Burian, D

9. Performing Organization Name and Address 10. Work Unit No. (TRAIS) FAA Civil Aerospace Medical Institute P.O. Box 25082 11. Contract or Grant No. Oklahoma City, OK 73125

12. Sponsoring Agency name and Address 13. Type of Report and Period Covered Office of Aerospace Medicine Federal Aviation Administration 800 Independence Ave., S.W. Washington, DC 20591 14. Sponsoring Agency Code

15. Supplemental Notes Work was accomplished under approved task AM- 16. Abstract Regulation of gene expression is a complex process that exquisitely responds to the environment to maintain cellular and, ultimately, organismal homeostasis. Gene expression research is undertaken at the Federal Aviation Administration as a means of discovering sets of biomarkers that change in response to environmental factors that affect aviation safety. This article reviews mechanisms of gene regulation and discusses how genomics is changing the way medicine is practiced today as a means of demonstrating that molecular medicine is here to stay. Next, the protocols that have been developed into a cohesive workflow to perform gene expression analysis are presented. Environmental factors currently under investigation are delineated followed by a discussion of other factors of interest for future research. We believe this research will benefit the aviation industry by improving the accuracy of the data used to write regulation, thus improving the already remarkable safety record of the aviation industry and decreasing medical risks to flight crew.

17. Key Words 18. Distribution Statement

Gene Expression; Gene Regulation; Molecular Medicine; Aerospace Medicine; Microarray; Messenger RNA

Document is available to the public through the Defense Technical Information Center, Ft. Belvior, VA 22060; and the National Technical Information Service, Springfield, VA 22161

19. Security Classif. (of this report) 20. Security Classif. (of this page) 21. No. of Pages 22. Price Unclassified Unclassified 15

Form DOT F 1700.7 (8-72) Reproduction of completed page authorized

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Contents

IntroductIon. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

class�cal.Modes.of.Inher�tance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1non-class�cal.Modes.of.Inher�tance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Stay�ng.Ahead.of.the.curve. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2Marker.d�scovery.Projects.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Gene.exPreSSIon.At.the.FAA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

Workflow. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Array.des�gn. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4Analys�s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

current.Gene.exPreSSIon.StudIeS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Moderate.Alcohol.use. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Fat�gue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8rad�at�on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8hypox�a/Alt�tude.exposure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

GenotyPInG.StudIeS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

Future.StudIeS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

concluSIonS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

reFerenceS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1

Functional Genomics Group-proGram Description

INTrOduCTION

Structurally,.dnA.�s.a.double.stranded.polymer.of.molecules. called. deoxy-nucleot�des .. deoxynucleot�des.conta�n.two.chem�cal.mo�et�es,.the.base.and.a.deoxy-r�-bose.sugar.that.�s.mod�fied.w�th.a.phosphate.group ..Four.nucleot�des.are.used.�n.the.synthes�s.of.dnA.del�neated.by.the.�dent�ty.of.the.base,.aden�ne.(A),.thym�d�ne.(t),.cyt�d�ne.(c).or.guan�d�ne.(G) ..the.polymer.forms.by.the.enzymat�c.add�t�on.of.the.phosphate.group.from.a.free.nucleot�de.to.one.of.the.hydroxyl.groups.of.the.term�nal.deoxy-r�bose.of.the.nascent.molecule ..dur�ng.nucle�c.ac�d.synthes�s,.the.synthes�s.mach�nery.“reads”.the.sequence.of.bases.on.the.oppos�te.strand ..In.th�s.way,.the.synthes�s.mach�nery. �s.d�rected. to.add.the.complementary.base.to.the.newly.formed.nucle�c.ac�d.strand,.preserv�ng.the.sequence.�nformat�on.present.�n.the.or�g�nal.molecule ..A.and.t.are.complementary,.as.are.c.and.G ..

Classical Modes of Inheritancethe.“class�cal”.central.dogma.of.B�ology.holds.that.

dnA.�s.a.storage.molecule.for.genes ..In.the.nucleus,.genes.are.transcr�bed.�nto.rnA.that.�s.processed.and.exported.to.the.cytoplasm.where.r�bosomes.translate.the.messenger.rnA.(mrnA).�nto.a.s�ngle.prote�n ..Also.known.as.the.“one.Gene,.one.Prote�n”.theory,.the.central.dogma,.as.or�g�nally.hypothes�zed,.held.that.each.gene.led.to.one.and.only.one.prote�n.(F�g ..1A) ..however,.the.central.dogma. has. undergone. major. rev�s�on. �n. recent. years.(F�g .. 1B). as. understand�ng. of. the. many. mechan�sms.that.regulate.gene.express�on.has.expanded ..there.�s.an.

�ncreas�ng.awareness.that.the.human.genome.encodes.far.fewer.genes. than.or�g�nally. thought;.current.est�mates.range.between.20,000.and.25,000.genes.�n.contrast.to.est�mates.that.were.as.h�gh.as.100,000.genes.ten.years.ago ..the.d�screpancy.�s.due,.�n.part,.to.alternat�ve.spl�c�ng.of.transcr�bed.rnA.result�ng.�n.mult�ple.gene.products.from.a.s�ngle.gene ..Alternat�ve.spl�c�ng.was.the.first.departure.from.the.central.dogma.to.be.d�scovered .

After. a. gene. �s. transcr�bed. from. genom�c. dnA. to.rnA,.�t.�s.spl�ced.to.form.the.messenger.rnA.(mrnA).sequence ..dur�ng. the.course.of. spl�c�ng,.p�eces.of. the.or�g�nal.transcr�pt.are.enzymat�cally.removed.by.a.spec�al-�zed.set.of.prote�ns,.and.the.p�eces.that.eventually.w�ll.be.translated.�nto.prote�n.are.rejo�ned.to.make.the.mature.mrnA ..the.p�eces. that.are.kept.are.known.as.exons;.the.p�eces.that.are.d�scarded,.or.�nterven�ng.sequence,.are.called.�ntrons ..Spl�c�ng.�s.sequence.dependent.and.t�ghtly.controlled ..Alternat�ve.spl�c�ng.events.ar�se.when.certa�n.exons.�n.the.gene.of.�nterest.are.e�ther.kept.or.lost. depend�ng. on. env�ronmental. factors. or. cell. type ..the.prote�ns.that.result.from.alternat�ve.spl�c�ng.do.not.have.the.same.sequence.and.w�ll.have.altered.act�v�t�es.compared.to.the.same.prote�n.that.�s.not.alternat�vely.spl�ced ..It.should.be.noted.that.because.alternat�ve.spl�c-�ng.can.be.cell.type.spec�fic,.the.defin�t�on.of.“normal”.becomes.blurred;.the.mrnA.�n.one.cell.type.would.be.an.aberrat�on.�n.another.cell.type ..Further.compl�cat�ng.the.p�cture.�s.the.recent.d�scovery.that.gene.dupl�cat�ons.have.occurred.�n.human.evolut�on.such.that.certa�n.eth-n�c.groups.have.more.cop�es.of.some.genes.than.other.ethn�c�t�es.(1) ..

Figure 1. The Central Dogma of Biology. A. (above). As originally put forth, held that one gene at the DNA level is transcribed into one RNA molecule that is translated into one protein. B. (right). A more current view of the Central Dogma accounts for chemical modification of DNA modifying the transcriptional activity of a gene through epigenetic mechanisms (1).

DNA

1. EpigeneticRegulation

2. Alternative Splicing

DNA RNA Protein

3. Micro RNAs

RNA Protein

Figure 1A.

DNA

1. EpigeneticRegulation

2. Alternative Splicing

DNA RNA Protein

3. Micro RNAs

RNA Protein

Figure 1B. Further regulation at the RNA level occurs: (2) by alternative splicing of a transcript diversifying the possible mRNAs that can arise from a single gene or (3) the activity of micro-RNAs upon target transcripts such that a RNA regulates a RNA.

2

Non-classical Modes of Inheritanceep�genet�cs.�s.the.non-Mendel�an.�nher�tance.of.a.tra�t,.

or.�nher�tance.that.�s.not.dependent.on.the.passage.of.genet�c.sequence.from.parents.to.offspr�ng.(F�g ..1B) ..there.are.numerous.mechan�sms.of.ep�genet�c.gene.regulat�on;.one.example.�nvolves.her�table.changes.to.the.chem�stry.of.genom�c.dnA.by.methylat�on.of.cytos�ne.res�dues ..c-methylat�on.has.great.effect.when.�t.occurs.w�th�n.the.genom�c.dnA.�mmed�ately.upstream.of.a.gene.known.as.the.promoter ..Methylat�on.w�th�n.promoters.decreases.the.b�nd�ng.affin�ty.of.prote�ns.necessary.for.the.synthes�s.of.mrnA.result�ng.�n.decreased.levels.of.mrnA.and.the.result�ng.prote�n.(2) ..two.other.examples.of.ep�genet�c.regulat�on.are.post-translat�onal.h�stone.mod�ficat�on.(3).and.the.depos�t�on.of.h�stone.var�ants.�n.genom�c.dnA.(4) ..e�ther.of.these.can.change.the.ava�lab�l�ty.of.genom�c.dnA.for.b�nd�ng.of.the.regulatory.and/or.transcr�pt�on.mach�nery.and.result.�n.altered.mrnA.levels .

A.second.example.of.non-Mendel�an. �nher�tance. �s.the.presence.of.a.regulatory.pattern.based.on.the.parental.or�g�n.of.the.gene.�n.quest�on ..th�s.mode.of.�nher�tance.has.been.demonstrated.�n.numerous.�nstances ..the.most.general.of.these.�s.known.as.�mpr�nt�ng,.where.gene.ex-press�on.�s.monoallel�c.due.to.transcr�pt�onal.repress�on.of.one.copy.of.the.gene.�n.quest�on ..th�s.mechan�sm.has.been.shown.to.regulate.genes.�nvolved.�n.development,.neural.d�sorders.(rev�ewed.�n.refs ..5-7).and.cancer.(re-v�ewed.�n.refs ..8-10) ..A.more.spec�fic.case.of.�mpr�nt�ng.�s.gene.s�lenc�ng.by.x-chromosome.�nact�vat�on.�n.mam-mal�an.females.(11) ..th�s.regulat�on.�s.necessary.because.females.have.two.x-chromosomes.whereas.males.have.one .. x-chromosome. �nact�vat�on. turns. off. express�on.from.one.x-chromosome,.thereby.decreas�ng.x-chromo-some.der�ved.transcr�pt.levels.to.normal ..transcr�pt�on.from.both.chromosomes.�n.mammal�an.females.�s.very.detr�mental.to.the.organ�sm ..

A.more.recently.d�scovered.form.of.gene.regulat�on.�s.the.b�nd�ng.of.a.small.rnA,.known.as.a.m�cro-rnA,.to.an.mrnA.�n.a.sequence.spec�fic.manner.(rev�ewed.�n.(12)and.F�g .1B) ..M�cro-rnA.b�nd�ng.occurs.�n.a.complex.cons�st�ng.of.the.mrnA,.the.m�cro-rnA,.and.a.prote�n.complex.known.as.the.rnA-�nduced.s�lenc�ng.complex.(rISc) ..B�nd�ng.of.m�cro-rnAs.to.mrnAs.leads.to.gene.down-regulat�on.by.one.of.two.mechan�sms ..In.the.first.mechan�sm,.b�nd�ng.of.the.m�cro-rnA/rISc.complex.to.mrnA.ster�cally.prevents.r�bosomes.from.translat�on.of.the.mrnA.(F�g ..2A) ..the.m�cro-rnA.sequence.�n.th�s. regulatory. mechan�sm. �s. not. completely. comple-mentary.to.the.mrnA.sequence ..Alternat�vely,.b�nd�ng.of.the.m�cro-rnA/rISc.complex.targets.the.transcr�pt.for.destruct�on.by.a.nuclease.�n.the.rISc.complex ..th�s.mechan�sm.requ�res. that. the.m�cro-rnA.sequence.be.perfectly. complementary. to. the. mrnA .. M�cro-rnA.

based. gene. regulat�on. has. been. demonstrated. �n. taxa.from.yeast.and.fung�.to.mammals.and.�s.w�despread.�n.plants.(rev�ewed.�n.ref ..13) ..In.A. thaliana, a.mustard.plant,. more. than. 1 .5. m�ll�on. m�cro-rnAs. have. been.character�zed ..the.human.genome.�s.est�mated.to.en-code.400.m�cro-rnAs.that.regulate.~5000.genes ..th�s.mode.of.regulat�on.has.been.shown.to.be.�mportant.�n.the.development.of.almost.all.human.t�ssues,.and.recent.reports. have. demonstrated. a. role. for. m�cro-rnAs. �n.cancer. (14). and. neurolog�cal. d�sorders. (15) .. clearly,.m�cro-rnA.gene.regulat�on.mechan�sms.greatly.expand.the.central.dogma.�n.that.an.rnA.molecule.regulates.an.rnA.transcr�pt .

Staying Ahead of the CurveMed�cal.sc�ence.�s.rap�dly.progress�ng.toward.the.day.

when.“personal�zed.med�c�ne”.w�ll.be.the.norm ..Genet�c.analys�s.of.�nd�v�dual.pat�ents.�s.at.the.forefront.of.th�s.evolut�on ..the.cancer.Genome.Anatomy.Project.(http://cgap .nc� .n�h .gov/;.accessed.January.2006).�s.a.publ�cly.ava�lable.data.warehouse.conta�n�ng.gene.express�on.pro-files,.chromosomal.gene.mapp�ng.�nformat�on.and.tools.used.�n.cancer.research ..Informat�on.be�ng.gathered.there.compares.express�on.profiles.and.sequence.�nformat�on.from.cancerous,.precancerous,.and.normal.t�ssues.�n.an.effort. to. further. character�ze. target. molecules. aga�nst.wh�ch.to.des�gn.cancer.treatments .

B�omarkers.have.been.defined.by.the.nat�onal.Inst�tutes.of.health.as.“…an.�nd�cator.of.a.d�sease.process,.and.could.replace.hard.cl�n�cal.end.po�nts.as.a.measure.of.the.effect.of.new.therap�es”.(16) ..th�s.document.�s.a.request.for.grant.appl�cat�ons.to.develop.b�omarkers.for.“well-defined.human.d�seases.of.l�ver,.k�dney,.urolog�cal.tract,.d�gest�ve.and.hematolog�c.systems,.and.endocr�ne.and.metabol�c.d�sorders,.d�abetes.and.�ts.compl�cat�ons,.and.obes�ty”.�nd�cat�ve.of.the.w�de.array.of.d�seases.that.b�omarker.d�scovery.�s.expected.to.�mpact ..Gene.express�on.research.by.m�croarray.�s.a.pr�mary.component.of.b�omarker.d�s-covery.because.�t.�s.h�gh.throughput,.scann�ng.thousands.of.cand�date.genes.�n.a.s�ngle.exper�ment ..

Another.use.of.m�croarray.technology.�s.to.�dent�fy.s�ngle-base. d�fferences. (S�ngle. nucleot�de. Polymor-ph�sms;.SnPs).as.well.as.other.genet�c.polymorph�sms.(�nsert�ons,.delet�ons,.or.dupl�cat�ons).�n.a.gene.sequence ..roche. �s.ut�l�z�ng. th�s. capab�l�ty. �n.a.m�croarray. that.screens.for.a.panel.of.polymorph�sms.�n.the.cytochrome.P450.fam�ly.of.drug-process�ng.prote�ns.(17) ..Polymor-ph�sms.�n.th�s.fam�ly.of.prote�ns.result.�n.d�ffer�ng.drug.metabol�sm.rates ..

It.�s.a.common.hypothes�s.that.�n.the.future,.genet�c.profiles.w�ll.be.used.as.a.preventat�ve.or.pred�ct�ve.mea-sure.of.a.d�sease.state ..the.Food.and.drug.Adm�n�stra-t�on. has. released. a. gu�dance. document. deta�l�ng. how.

3

Figure 2. Translational regulation by the RISC complex. A. Binding of the RISC complex physically blocks the ribo-

some from translating the mRNA into protein (see text). B. RISC complex binding results in mRNA degradation

(see text).

GC CG3‘ – GAAGACU AGUCAUUUA – 5’ GC CG3‘ – GAAGACU AGUCAUUUA – 5’Ribosome

GCGGCAUGGGCCAAUAUCUUCUGA GGUU UCAGUAAAUCGGUAAUGACUCUUCUGA UCAA UCAGUAAAUCCGCUGGGC

RISC RISC

Figure 2A.

3‘ – GAAGACUCCAAAGUCAUUUA – 5’

RISC

3‘ – GAAGACUCCAAAGUCAUUUA – 5’

GCGGCAUGGGCCAAUAUCUUCUGAGGUUUCAGUAAAUCGGUAAUGACUCUUCUGAGGUUUCAGUAAAUCCGCUGGGC

mRNA cleavage by the RISC complex

RISC

Figure 2B.

and.when.genet�c.test�ng.or.gene.express�on.data.may.be.used.�n.appl�cat�ons.for.l�cens�ng.of.new.drugs.(18) ..th�s.would.breach.a.major.regulatory.obstacle.to.drug.development.for.pat�ents.exh�b�t�ng.spec�fic.genet�c.pro-files ..the.marketplace.�s.dr�v�ng.th�s.change.�n.the.way.drugs.are.developed.by.collect�ng.genet�c.data. to.find.target.molecules.and.then.screen�ng.huge.repos�tor�es.of.small.molecules.for.b�olog�cal.act�v�ty.aga�nst.these.new.targets ..recogn�t�on.of.these.market.forces.�s.found.�n.the.gu�dance.document.(18),.“ . . .some.pharmacogenet�c.tests.-.pr�mar�ly.those.related.to.drug.metabol�sm.-.have.well-accepted.mechan�st�c.and.cl�n�cal.s�gn�ficance.and.are. currently. be�ng. �ntegrated. �nto. drug. development.dec�s�on.mak�ng.and.cl�n�cal.pract�ce .”.health.care.prog-nost�cators.foresee.a.day.when.an.�nd�v�dual.w�ll.carry.h�s.or.her.genet�c.profile.on.a.computer-readable.card.or.ch�p ..Phys�c�ans.w�ll.prescr�be. therap�es. spec�fically.ta�lored.to.treat.d�sease.based.on.polymorph�sms.�n.that.�nd�v�dual’s.genet�c.code .

Marker discovery Projects two.projects.are.underway.to.survey.sequence.var�ab�l-

�ty.w�th�n.the.human.populat�on ..the.first,.tak�ng.place.outs�de.the.un�ted.States,.�s.a.determ�nat�on.of.sequence.var�ab�l�ty.�n.large.nat�onal.cohort.groups ..the.head.of.

the.human.Genome.Project,.Franc�s.coll�ns,.recently.called.for.a.comparable.states�de.effort.to.survey.human.heterogene�ty.at.the.sequence.level.w�th�n.the.un�ted.States.(19).as.a.means.of.determ�n�ng.how.env�ronmental.factors.�nterplay.w�th.genet�c.sequence.�n.d�sease ..he.proposed.that.the.dnA.sequenc�ng.capac�ty,.or�g�nally.focused.on.decod�ng.the.human.genome,.be.appl�ed.to.sequenc�ng.a.human.mult�-thousand.cohort.from.all.ethn�c�t�es.to.determ�ne.the.underly�ng.genet�cs.of.Amer�can.pheno-typ�c.var�ab�l�ty ..Add�t�onal.background.�nformat�on.of.all.types.would.be.collected.and.used.to.find.corollar�es.between.d�sease.and.genet�cs,.based.on.factors.such.as.d�et,.l�festyle.and.exposure.to.env�ronmental.factors ..

the.second.survey,.the.hapMap.project,.�s.an.�nter-nat�onal.effort.to.�nvest�gate.human.heterogene�ty ..the.first.phase.has.recently.been.publ�shed.(20) ..to.ach�eve.the.goals.of.the.project,.common.SnPs.were.sequenced.from.269.people.who.or�g�nated.from.four.geograph�cally.constra�ned.areas.around.the.world ..the.goal.of.the.project.was.“to.create.a.resource.that.would.accelerate.the.�dent�fi-cat�on.of.genet�c.factors.that.�nfluence.med�cal.tra�ts .”(20).Phase.II.of.the.project.w�ll.delve.�nto.rare.SnP.sequence.var�ab�l�ty.�n.the.same.populat�on.(20) ..the.results.from.these.efforts.w�ll.allow.med�cal.profess�onals.to.treat.d�sease.based.on.the.genet�c.code.of.the.pat�ent .

4

Ind�cat�ve. of. the. appl�cab�l�ty. of. results. from. bas�c.sc�ence. to. the. cl�n�c. �s. the. recent. announcement. that.the.cancer.drug.camptosar®.from.Pfizer.w�ll.soon.be.re-labeled. to. reflect. add�t�onal. r�sks. assoc�ated. w�th. a.spec�fic.mutat�on. �n. the.uGt1A1.gene. (21),.and.the.lung.cancer.drug.IreSSA.w�ll.be.marketed.heav�ly. �n.As�an. populat�ons. reflect�ng. �ts. �ncreased. efficacy. �n.that. populat�on. (22) .. these. two. �nstances. h�ghl�ght.the.grow�ng.trend.toward.ta�lor�ng.spec�fic.treatments.to.genet�c.backgrounds ..Wh�le.�t.�s.less.l�kely.that.gene.sequence.�nformat�on.w�ll.ever.have.broad.appl�cab�l�ty.�n.an.av�at�on.sett�ng,.b�omarker.d�scovery.has.clear.and.present.appl�cat�on.to.aerospace.med�c�ne.for.a.var�ety.of.env�ronmental.st�mul�.�nclud�ng.fat�gue.and.hypox�a ..

GENE ExPrESSION AT ThE FAA

the.Federal.Av�at�on.Adm�n�strat�on.�s.a.regulatory.agency.respons�ble.for.av�at�on.safety ..In.add�t�on.to.�ts.tra�n�ng.and.regulatory.respons�b�l�t�es,.�t.�s.respons�ble.postmortem.analys�s.of.acc�dent.v�ct�ms ..As.med�cal.sc�-ence.enters.the.post-genome-sequenc�ng.era,.the.agency.has. the. respons�b�l�ty. to. �nvest�gate. new. technolog�es.that.can.lead.to.better.forens�c.test�ng,.regulat�on,.and.cert�ficat�on ..W�th�n.the.Funct�onal.Genom�cs.group.at.the.FAA’s.c�v�l.Aerospace.Med�cal.Inst�tute.(cAMI).�n.oklahoma.c�ty,.th�s.effort.has.begun.at.the.level.of.d�scov-er�ng.gene.express�on.changes.�n.response.to.factors.that.affect.av�at�on.safety ..the.most.effic�ent.method.currently.ava�lable.for.screen�ng.the.ent�re.transcr�pt�onal.output.(transcr�ptome).of.a.t�ssue.�s.m�croarray.analys�s ..

nucle�c.ac�d.m�croarrays.quant�tat�vely.detect.b�nd�ng.of.nucle�c.ac�ds.w�th.complementary.sequence ..Publ�shed.stud�es.show.that.m�croarray.analyses.are.sens�t�ve.enough.to. d�fferent�ate. not. just. between. normal. and. d�sease.states,.but.also.between.d�fferent.subtypes.of.a.complex.d�sease.w�th.mult�ple.presentat�ons.such.as.leukem�a ..In.add�t�on.to.S�ngle.nucleot�de.Polymorph�sm.detect�on,.sequence.changes.of.one.base,.a.common.use.of.m�croar-ray.technology.has.been.to.determ�ne.d�fferences.�n.gene.express�on.patterns.between.d�sease.and.healthy.states ..therefore,.wh�le.genet�c.background.cannot.be.�gnored.�n.the.search.for.therapeut�c.targets,.express�on.�nforma-t�on.y�elds. tremendous. �ns�ghts. �nto. the.phys�olog�cal.response.to.an.env�ronmental.st�mulus ..As.an.express�on.screen�ng.tool,.m�croarrays.determ�ne.changes.�n.gene.express�on.or.the.funct�on.of.the.regulatory.mach�nery,.not. sequence.var�ab�l�ty ..the.ab�l�ty.of.m�croarrays. to.detect.subtle.express�on.changes.bodes.well.for.our.goal.to.character�ze.phys�olog�cally.heterogeneous.human.fac-tors.such.as.fat�gue.and.hypox�a.or.exposure.to.moderate.levels.of.rad�at�on,.tox�ns,.or.m�crobes .

WorkflowGene.express�on.analys�s.exper�ments.beg�n.by.�solat-

�ng.the.rnA.from.test.subjects.(F�g ..3) ..Most.stud�es.performed.at.cAMI.use.human. subjects. so. the.rnA.source. �s.whole.blood.due.the.s�mpl�c�ty.of.collect�on.and.the.demonstrated.efficacy.of.whole.blood.as.a.t�ssue.that.�s.respons�ve.to.cond�t�ons.affect�ng.other.sol�d.t�s-sues.(23-28) ..A.commerc�al.whole.blood.collect�on.and.mrnA.stab�l�zat�on.system.from.PaxGene.(PreAnalyt�x,.hombrecht�kon,. ch). �s. used. for. sample. collect�on ..After.mrnA.pur�ficat�on,.a.ser�es.of.molecular.b�ology.steps.y�elds.a.set.of.target.molecules.that.ma�nta�ns.the.relat�ve.gene.express�on.levels.of.the.subject.at.the.t�me.of. collect�on .. dur�ng. synthes�s,. the. target. molecules.are.b�ot�n-labeled.for.post-hybr�d�zat�on.detect�on.w�th.streptav�d�n/phycoerythr�n .. Streptav�d�n. t�ghtly. b�nds.b�ot�n. w�th. very. h�gh. affin�ty. and. spec�fic�ty .. Phyco-erythr�n.fluoresces.�n.response.to.exc�tat�on.w�th.l�ght ..the.system.allows.the.hybr�d�zat�on.step.to.be.separated.from.the.detect�on.step.such.that.the.bulky.fluorescent.molecule.does.not.�nterfere.w�th.hybr�d�zat�on.between.the.target.and.the.probe .

target.molecules.are.hybr�d�zed. to. the.arrays. �n.an.overn�ght. �ncubat�on. step ..After. a.wash�ng.and.SAPe.label�ng.protocol.�s.performed,.the.arrays.are.v�sual�zed.on. a. fluorescent. scanner. and. the. data. collected. on. a.computer ..the.data.from.these.exper�ments.are.the.raw.fluorescence.�ntens�t�es.from.the.phycoerythr�n ..Genes.w�th.h�gher.express�on.levels.�n.the.sample.exh�b�t.h�gher.s�gnal.�ntens�t�es.on.the.array ..the.Affymetr�x.system.d�f-fers.from.other.m�croarray.systems.�n.that.each.sample.�s.�nd�v�dually.hybr�d�zed.to.a.s�ngle.array ..Most.other.systems.ut�l�ze.a.compet�t�ve.hybr�d�zat�on.where�n.two.samples,.� .e .,.one.each.from.a.normal.and.a.d�sease.subject,.are.hybr�d�zed.to.an.array ..each.of.the.two.samples.has.a.d�fferent.color.fluorescent.dye.conjugated.to.�t ..As.a.result,.the.data.from.these.arrays.�s.a.comparat�ve.analys�s.of.the.relat�ve.fluorescence.�ntens�t�es.of.each.sample.aga�nst.the.other ..th�s.method.has.several.techn�cal.d�sadvantages.related.to.the.d�fferent.spectral.character�st�cs,.stab�l�t�es,.and.�ncorporat�on.rates.of.the.two.dyes .

Array designthe.arrays.are.made.up.of.probe.sequence.covalently.

attached.to.a.sol�d.substrate,.usually.glass.or.s�l�ca.(F�g ..4) ..each.probe.sequence.�n.a.part�cular.locat�on.on.the.substrate.�s.from.a.s�ngle.transcr�pt,.as.annotated.�n.an.external. sequence. database .. Arrays. w�th. probes. repre-sent�ng. all. human. genes. encod�ng. known.or. putat�ve.prote�ns.and.funct�onal.rnAs.are.ava�lable.from.many.commerc�al.sources ..our.lab.uses.arrays.from.Affymetr�x.(Santa.clara,.cA).due.to.the�r.ease.of.use,.demonstrated.

5

Sample Collection,Whole Blood From Volunteers

RNA Isolation

Molecular Biology Steps to MakeLabeled Target RNA

Hybridization

Scanning

Figure 3. Microarray analysis workflow. Samples, usually blood, are collected from volunteer study participants. Total RNA is purified, amplified to make target material, hybridized to chips and the data gathered by scanning the chips.

mRNA Sequence 3’ACGGUGCGUGCUGACUUGUGUCUGU

Probe Sequence 5’TGCCACGCACGACTGAACACAGACA

Sample probe from mammaglobin gene

Subs

trate

AttachmentLinker

Figure 4. Affymetrix GeneChip® design. The Affymetrix GeneChip® design strategy involves multiple short probes to a single transcript instead of a single longer probe to each transcript as with most other manufactur-ers. The redundancy of the Affymetrix strategy allows the system to detect expression even if hybridization to a single probe is not efficient.

6

reproduc�b�l�ty.of.results,.and.h�gh.data.qual�ty ..current.array.arch�tectures.are.based.on.e�ther.detect�on.of.an.ent�re.gene.or.detect�on.of.each.exon.w�th�n.a.gene ..Both.arch�tectures.ut�l�ze.mult�ple.probes.to.the.sequence.of.�nterest,.whether.�t.�s.a.gene.or.just.an.exon ..Both.ch�ps.can.be.used.to.detect.an.ent�re.gene,.as.well ..the.d�ffer-ence.�s.that.the.exon.arrays.gather.data.from.four.probes.to.each.exon.and.detect.express�on.at.the.exon.level ..the.gene.arrays.�nterrogate.a.gene.across.the.ent�re.length.of.the.transcr�pt.but.lack.the.redundancy.of.the.exon.arrays.at.the.exon.level.mak�ng.them.unsu�table.for.exon-level.express�on. analys�s .. As. an. example,.tMeM8,. a. gene.that.was.d�fferent�ally.expressed.�n.our.study.of.moder-ate.alcohol.use,.has.15.exons ..the.Affymetr�x.human.exon.1 .0.St.array.�nterrogates.these.15.exons.w�th.28.probe. sets. and.compr�s�ng.108.probes ..the.probe. set.for.the.same.gene.on.the.human.Gene.1 .0.St.arrays.�ncludes.33.probes,.an.average.coverage.of.just.over.two.probes.per.exon ..

In.add�t�on.to.d�fferences.�n.probe.dens�ty,.the.two.ar-ray.types.were.des�gned.w�th.d�fferent.ph�losoph�es.based.on.the.transcr�pts.that.were.�ncluded.�n.the�r.des�gn ..the.gene.arrays.�nterrogate.only.well.annotated.genes.to.the.spec�es.of.�nterest ..the.human.Gene1 .0.St.arrays.have.about.28,000.probe.sets.from.known.human.genes.w�th.several.sources.of.val�dat�on.as.to.the�r.�dent�ty ..conversely,.the.exon1 .0.St.arrays.are.much.more.exper�mental.�n.nature ..Probe.sets.�ncluded.�n.these.arrays.can.be.based.on.as.l�ttle.as.sequence.homology.w�th.a.known.gene.from.another.organ�sm,.even.�f.that.gene.has.never.been.shown.

to.be.act�ve.�n.the.spec�es.of.�nterest ..As.a.result,.there.are.numerous.hypothet�cal.genes.�ncluded.on.the.exon.arrays.that.are.not.�ncluded.on.the.gene.arrays ..clearly,.the.two.ch�ps.are.des�gned.to.answer.very.d�fferent.ques-t�ons.necess�tat�ng.careful.exper�mental.des�gn .

Analysisthere.are.numerous.sources.of.s�gnal.var�at�on.on.a.

m�croarray ..there.�s.s�gnal.from.the.array.substrate.and.from.non-spec�fic.hybr�d�zat�on.of.target.molecules.to.probe ..Sample.to.sample.var�at�on.�n.s�gnal.�ntens�ty.ar�ses.from.d�fferences.�n.rnA.pur�ty,.effic�ency.of.the.enzy-mat�c.steps.to.make.target,.and.hybr�d�zat�on.effic�ency.of.target.to.probe ..F�nally,.d�fferences.�n.s�gnal.�ntens�ty.are.due.to.the.parameter.the.exper�ment.�s.des�gned.to.measure,.a.b�olog�cal.d�fference.�n.the.gene.express�on.level.that.�s.attr�butable.to.the.adm�n�stered.treatment.or.cond�t�on.of.�nterest ..Further.confus�ng.the.unamb�guous.�dent�ficat�on.of.genes. that.are.d�fferent�ally.expressed.between.treatment.groups.�s.the.cons�derable.b�olog�cal.var�at�on.between.�nd�v�duals .

the.raw.data.cons�sts.of.total.s�gnal.�ntens�ty.values.from. each. �nd�v�dual. probe .. A. pre-process�ng. step. to.“summar�ze”.the.data.�s.performed ..the.funct�on.of.th�s.step.�s.to.subtract.the.background.�ntens�t�es,.est�mated.from.s�gnal.on.a.blank.ch�p.reg�on,.and.then.condense.or.“summar�ze”.the.data.from.each.set.of.probe.�nto.a.s�gnal.�ntens�ty.value.across.the.probe.set.(F�g ..5) ..the.exon.array.software.can.return.summar�zat�on.values.at.both.the.exon.and.the.gene.level .

Data files containing raw intensities from arrays

Normalization of summarized data across all chips

Summarization of raw data toderive an expression value for

each probe set

Differential expression testing by t-test, ANOVA, and/or time course

analysis

Other analysis, ie, clustering pathway analysis

Figure 5. The analysis workflow used with Affymetrix GeneChips® (see text).

7

Background/summar�zed.probe.set.�ntens�t�es.are.then.“normal�zed .”.normal�zat�on.methods.computat�onally.adjust.s�gnal.�ntens�t�es.across.the.exper�ment.such.that.between. arrays,. most. s�gnal. �ntens�t�es. are. stat�st�cally.equal ..the.effect.of.normal�zat�on.�s.to.adjust.the.data.for.b�olog�cal.d�vers�ty.between.subjects.or.samples ..there-fore,.after.normal�zat�on,.d�fferences.�n.s�gnal.�ntens�ty.are.due.to.treatment.related.changes.�n.express�on.(29) ..normal�zat�on.has.been.shown.to.�ncrease.the.number.of.genes.called.d�fferent�ally.expressed.over.non-normal-�zed.datasets .

due.to.the.normal.b�olog�cal.var�at�on.between.�nd�-v�duals,.stat�st�cal.tools.such.as.t-tests.and.AnoVA.are.used.to.determ�ne.wh�ch.genes.are.d�fferent�ally.expressed.between.treatment.groups ..Because.m�croarrays.test.the.express�on.of.tens.of.thousands.of.genes.at.a.t�me,.the.stat�st�cal.analys�s.�s.computat�onally.�ntens�ve ..Spec�al�zed.software.has.been.developed.to.perform.these.analyses.on.m�croarray.data.and.del�ver.an.output.that.reflects.b�ol-ogy.and.�s.stat�st�cally.val�d.desp�te.the.necessar�ly.small.sample.s�zes ..Furthermore,.downstream.“data.m�n�ng”.tools.such.as.cluster�ng.and.pathway.analys�s.have.been.developed.that.allow.�nvest�gators.to.find.genes.that.change.express�on.levels.as.a.group.and.can.be.expected.to.be.s�m�larly.regulated.and/or.be.part.of.the.same.b�olog�cal.pathway ..We.use.ArrayAnalyzer.(Ins�ghtful.corporat�on,.Seattle.WA).and.B�oconductor.(www .b�oconductor .org).for.analys�s.of.our.data.from.the.Affymetr�x.system ..th�s.software.has.shown.�tself.to.be.capable.of.handl�ng.large.datasets,.l�m�ted.only.by.ava�lable.computat�onal.power ..Pathway. Analys�s. (Ingenu�ty. Systems,. Inc .;. redwood.c�ty,.cA),.a.Internet-based.knowledge.env�ronment,.�s.used.to.group.d�fferent�ally.expressed.genes.by.b�olog�-cal.pathway .

early.�n.the.development.of.m�croarray.technology,.m�croarray.results.were.found.to.be.somewhat.�rreproduc-�ble.from.lab.to.lab.(29-31) ..however,.recent.carefully.controlled.exper�ments.have.shown.some.�mprovement.(30-32),.and.�ncons�stency.�s.thought.to.be.due.to.the.gene.reg�ons.be�ng.�nterrogated.�n.d�fferent.platforms ..In.any.event,.other.molecular.b�ology.techn�ques.are.used.to.ver�fy.the.results ..real-t�me.quant�tat�ve.polymerase.cha�n. react�on. (Q-Pcr). �s. the. method. of. cho�ce. for.�n�t�al.val�dat�on.of.m�croarray.data.and.�s.w�dely.used.for.�n�t�al.val�dat�on.of.m�croarray.results ..It.�s.s�mple.to.use,.very.reproduc�ble.and,.as.the.name.�mpl�es,.can.be.quant�tat�ve.w�th.respect.to.the.transcr�pt.copy.number.�n.the.start�ng.sample .

once. a. set. of. genes. that. change. �n. response. to. a.part�cular.factor.has.been.�dent�fied.by.m�croarray.and.val�dated.by.Q-Pcr,.the.gene.products.w�ll.be.�nvest�-gated.further ..In�t�ally,.a.change.�n.prote�n.level.would.be.confirmed.us�ng.methods.that.could.�nclude.prote�n.

arrays,.or.two-d�mens�onal.gels ..Further.work.to.�nvest�-gate.pathways.affected.by.a.part�cular.st�mulus.would.be.performed.by.under-.or.over-express�on.�n.t�ssue.culture.or.an�mal.models .

As. d�scussed. above,. mult�ple. mod�ficat�ons. to. the.central. dogma. have. recently. been. d�scovered .. the.analys�s.techn�que.outl�ned.here.�s.l�m�ted.to.detect�ng.poly-A.conta�n�ng.mrnA.express�on ..however,.meth-ods.are.becom�ng.ava�lable.to.assess.the.effects.of.other.genet�c.regulatory.mechan�sms.and.could.be.ut�l�zed.at.the.FAA.w�th.only.m�nor.mod�ficat�ons.to.the.workflow.deta�led.above ..currently,.two.methods.for.the.detect�on.of.m�cro-rnAs.are.ava�lable ..A.quant�tat�ve.Pcr.assay.spec�fic.for.m�cro-rnAs.from.Appl�ed.B�osystems.�s.newly.ava�lable.and.Affymetr�x.markets.whole.genome.t�l�ng.arrays.that.enable.�nterrogat�on.of.the.ent�re.genome.for.novel.reg�ons.of.express�on.w�thout.rel�ance.on.an.outs�de.database.of.express�on.�nformat�on ..rather,.a.probe.from.both.dnA.strands.represent�ng.the.genom�c.sequence.at.predeterm�ned.�ntervals.�s.bound.to.the.array.substrate ..these.arrays.requ�re.a.mod�ficat�on.�n.the.ampl�ficat�on.of.target.mater�al;.random.pr�mers.are.used.for.ampl�ficat�on.of.total.rnA.�n.order.to.ampl�fy.all.rnAs,.�rrespect�ve.of.the.presence.of.a.poly-A.ta�l ..other.methods.that.take.advantage.of. the. t�l�ng.arrays. for.detect�on.but.ut�l�ze.alternat�ve.methods.for.target.�solat�on.are.ava�lable.for.d�scovery.of.reg�ons.of.ep�genet�c.control ..

CurrENT GENE ExPrESSION STudIES

the.av�at�on.acc�dent.rate.has.been.stead�ly.decl�n-�ng.over.the.last.several.decades ..Sen�or.FAA.leadersh�p.recogn�zes. that. further. decreases. �n. the. acc�dent. rate.w�ll.be.�ncrementally.more.d�fficult.than.what.has.been.ach�eved.to.date ..FAA.employees.have.been.challenged.to.become.ever.more.creat�ve.�n.the�r.approach.to.�m-prove.av�at�on.safety ..A.determ�nat�on.of.gene.express�on.profiles.�n.response.to.human.factors.that.affect.av�at�on.safety. could. lead. to.better. fact-based. regulat�ons ..the.area.of.forens�cs.could.benefit.greatly.from.determ�n�ng.the.gene.express�on.or.metabol�c.profile.of.related.fac-tors.w�th.the.goal.of.be�ng.able.to.ass�gn.causat�on.to.acc�dent.�nvest�gat�ons ..In.add�t�on,.there.are.�ssues.that.affect.the.work�ng.env�ronment.of.a�rcraft.personnel.that.part�cularly.lend.themselves.to.�nvest�gat�on.by.m�croar-ray.or.other.genet�c.methodolog�es ..exper�mental.des�gn.typ�cally.�ncludes.human.performance.data.�n.order.to.l�nk.gene.express�on.to.performance.levels .

Moderate Alcohol useWe.are.study�ng.the.effects.of.moderate.blood.alcohol.

levels.on.gene.express�on ..A.current.study.�n.collaborat�on.

8

w�th.the.un�vers�ty.of.utah.and.the.center.for.human.tox�cology.�s.look�ng.at.express�on.changes.�n.response.to.blood.alcohol.levels.up.to.0 .08%,.the.legal.l�m�t.for.automob�le.operat�on.�n.most.states ..data.from.s�x.men.�n.the�r.early.20’s.have.been.collected,.and.prel�m�nary.data. analys�s. has. found. about. 400. genes. that. change.express�on.level.at.some.po�nt.between.basel�ne,.0 .08%,.and.return.to.0 .00%.blood.alcohol ..Further.data.analys�s.and. ver�ficat�on. are. underway,. and. add�t�onal. exper�-ments.are.be�ng.planned. to.expand.th�s.work.beyond.the.current.data.set .

Fatiguethe. nat�onal. transportat�on. Safety. Board. �s. very.

�nterested.�n.fat�gue.as.a.potent�al.causat�ve.factor.�n.ac-c�dents.from.all.forms.of.transportat�on.acc�dents,.not.just.av�at�on ..In.add�t�on,.there.�s.support.at.both.the.state.and.federal.government.levels.to.�nvest�gate.fat�gue.as.a.factor.�n.automob�le.safety ..In.collaborat�on.w�th.the.u .S ..A�r.Force,.Brooks.c�ty-Base.�n.San.Anton�o,.texas,.we.are.�nvest�gat�ng.the.effects.of.fat�gue.on.gene.expres-s�on ..th�s.study.compares.gene.express�on.levels.at.up.to.35.hours.of.sleeplessness.w�th.a.well-rested.basel�ne ..

radiationA.cond�t�on.that.�s.somewhat.un�que.to.the.av�at�on.

�ndustry.�s.cosm�c.rad�at�on ..Passengers.have.not.been.shown.to.be.at.r�sk.due.the.short.durat�on.of.�ncreased.exposure.to.rad�at�on.dur�ng.a.s�ngle.fl�ght ..however,.there.�s.ev�dence.that.fl�ght.crews.have.an.�ncreased.�nc�-dence.of.chromosomal.aberrat�ons,.melanomas.and.breast.cancers.(33-36),.presumably.from.�ncreased.exposure.to.solar. rad�at�on .. other. researchers. have. demonstrated.changes.�n.gene.express�on.levels.due.to.�on�z�ng.rad�a-t�on.(37-41) ..A.recent.study.d�scovered.d�fferent.sets.of.markers. to. three. levels.of. �on�z�ng. rad�at�on.exposure.�n.m�ce.(42) ..

hypoxia/Altitude Exposuredecreased. blood. oxygen,. or. hypox�a,. �s. a. human.

factor. that. also. �s. un�que. to. the. av�at�on. �ndustry ..As.such,.we.are.pos�t�oned.to.�nvest�gate.changes.�n.gene.express�on.from.hypox�c.cond�t�ons.that.may.ar�se.due.to.�ncreas�ng.alt�tude ..the.necessary.equ�pment.to.m�m�c.�ncreas�ng.alt�tude.cond�t�ons.�s.ava�lable.at.the.cAMI ..one.current.study.�s.�nvest�gat�ng.the.effects.of.exposure.to.12,400.feet.�n.a.male.populat�on ..A.second.study.at.a�rl�ne.cab�n.alt�tudes.of.6,000.and.7,000.feet.�s.�n.the.plann�ng.stages .

GENOTyPING STudIES

W�th�n.the.Funct�onal.Genom�cs.group,.a.protocol.has.been.developed.(43)to.genotype.acc�dent.samples.us�ng.the.Ag�lent.B�oAnalyzer.to.separate.Pcr.product.from.the.Federal.Bureau.of.Invest�gat�on’s.approved.codIS.marker. set. (44) .. others. have. used. th�s. �nstrument. to.genotype.pap�llomav�rus.(45),.markers.on.the.human.y.chromosome.(46),.and.markers.of.m�tochondr�al.d�sease.(47) ..our.capab�l�t�es.are.l�m�ted.to.comparat�ve.analyses;.nonetheless,.th�s.ab�l�ty.�s.useful.to.the.Forens�c.tox�col-ogy.research.team.on.several.fronts ..F�rst,.�t.�s.useful.for.confirm�ng.sample.�dent�ty.�n.cases.where.samples.are.co-m�ngled.at.the.acc�dent.s�te ..Second,.genotyp�ng.has.allowed.the.confirmat�on.of.chem�cal.tox�cology.results.that. found. d�st�nct. d�fferences. between. two. samples.that. were. documented. as. hav�ng. been. from. the. same.acc�dent.v�ct�m ..th�rd,.the.ab�l�ty.to.perform.th�s.level.of.genotyp�ng.“�n.house”.�s.a.great.cost.saver.to.the.federal.government;.th�s.assay.performed.on.the.B�oAnalyzer.�s.done.at.one-tenth.the.cost.of.send�ng.�t.out.to.a.forens�c.genotyp�ng.laboratory ..

FuTurE STudIES

All.of.the.work.�n.progress.�n.the.Funct�onal.Genom�cs.lab.at.cAMI.�s.among.the.first.to.�nvest�gate.the.affects.of.these.part�cular.aerospace.env�ronmental. factors.on.gene.express�on ..Before.any.global.conclus�ons.can.be.reached,.each.of.these.stud�es.w�ll.need.to.be.extended.and.val�dated ..As.an.example,.the.fat�gue.study.�s.be�ng.performed.w�th.samples.from.A�r.Force.personnel.who.have.all.passed.fl�ght.phys�cals ..W�ll.the.data.gathered.from.th�s.study.translate.broadly.across.genders.or.to.an.older.and/or.less.fit.populat�on?.cand�date.genes.d�scovered.by.m�croarray. analys�s. on. l�m�ted.pat�ent.populat�ons.w�ll.be.further.explored.by.Q-Pcr.on.groups.of.subjects.from.a.populat�on.base.that.more.generally.reflects.the.overall.populat�on ..

the.power.of.the.ava�lable.software.to.find.genes.that.are.regulated.coord�nately.w�ll.allow.us.to.explore.ent�re.b�olog�cal.pathways ..In.th�s.way,.genes.that.may.not.appear.by.m�croarray.analys�s.but.play.a.role.�n.the.factor.be�ng.�nvest�gated.w�ll.become.v�s�ble ..t�ssue.culture.exper�-ments.are.expected.to.play.a.role.�n.th�s.work.because.they.w�ll.allow.for.cells.from.many.d�fferent.t�ssues.to.be.�nvest�gated,.thereby.expand�ng.the.work.�nto.t�ssues.other.than.blood ..Mult�ple.avenues.of.�nvest�gat�on.be-come.ava�lable.�n.t�ssue.culture ..overexpress�on.of.genes.of. �nterest. �n.t�ssue.culture.can.then.be.extended.�nto.an�mal.models.to.confirm.the.find�ngs.in vivo .

9

We.expect.that.�n.response.to.the.factor.of.�nterest,.there.w�ll.be. some.genes. that.are.up-regulated.and.others.w�ll.be.down-regulated ..A.molecular.s�gnature.for.a.part�cular.factor.w�ll.therefore.cons�st.of.mult�ple.genes. and. the�r. �nterrelat�onsh�ps .. In. add�t�on,. the.factors.we.are.�nterested.�n.explor�ng.are.b�olog�cally.not.on.or.off.but. are. expected. funct�on.on.a. con-t�nuum ..therefore,. mult�ple. s�gnatures. are. needed.to.determ�ne.where.on.the.cont�nuum.an.unknown.sample.l�es.(F�g ..6) ..

rnA. �s. an. espec�ally. lab�le. molecule .. As. such,.assays.for.the.more.stab�le.gene.products.(prote�ns).are.l�kely.to.be.the.end.goal.of.th�s.body.of.work ..A.potent�al.assay.for.forens�c.test�ng.�s.a.custom.prote�n.array. that.would.assay.prote�n. levels.of.a. subset.of.prote�ns.selected.for.the�r.spec�fic�ty.across.a.range.of.cond�t�ons.of. �nterest ..A.sample.would.be.brought.�nto.the.lab.and.hybr�d�zed.to.the.array ..the.s�gnature.seen.�n.the.unknown.sample.would.be.compared.w�th.normals.and.the.known.s�gnature.cont�nuums.for.all.known.human.factors ..the.unknown.sample.would.be.categor�zed.as.be�ng.e�ther.normal.or.�mpa�red.and.the.cause.of.the.�mpa�rment.determ�ned.�n.th�s.way .

In.the.post.9-11.world,.terror�sm.has.moved.to.the.forefront.of.our.nat�onal.consc�ousness ..W�th�n.funct�onal.genom�cs,. numerous. avenues. of. �nvest�gat�on. present.themselves.�n.the.war.on.terror ..Wh�le.opportun�t�es.to.develop.pathogen.�dent�ficat�on.methods.ex�st,.the.real.power. of. gene. express�on. technology. can. be. brought.to.bear.on. two. fronts ..F�rst,.by.determ�n�ng. the.gene.express�on.changes.w�th�n.the.pathogen,.new.angles.to.attack.the.organ�sm.and.treat.the.�nfect�on.can.be.deter-m�ned ..Second,.host.gene.express�on.changes.that.occur.

post-�nfect�on.can.be.used.to.d�scover.new.host.target.pathways.for.treatment.of.the.symptoms.of.the.�nfect�on ..Knowledge.of.gene.express�on.changes.along.the.�nfec-t�on.t�me-l�ne.would.allow.cl�n�c�ans.to.tr�age.pat�ents.and.pr�or�t�ze.treatment.to.those.who.are.at.greatest.r�sk ..F�nally,.most.of.these.same.quest�ons.could.be.answered.for.exposure.to.chem�cal.agents .

CONCluSIONS

the. FAA. �s. respons�ble. for. publ�c. safety. through.regulat�on.and.overs�ght.of.the.av�at�on.�ndustry ..there.has. been. a. dramat�c. decrease. �n. acc�dent. rates. to. the.po�nt. that.Amer�can. sk�es. are. the. safest. �n. the.world ..however,.acc�dents.st�ll.occur.and.�t.�s.the.goal.of.the.agency.to.further.decrease.the.acc�dent.rate ..to.that.end,.cAMI.has.been.tasked.to.�nvest�gate.the.human.equat�on.from.all.angles.as.�t.perta�ns.to.av�at�on ..the.Funct�onal.Genom�cs.group.at.cAMI.was.formed.�n.part.to.br�ng.av�at�on.med�c�ne.�nto.the.molecular.age.and.d�scover.the.un�que.molecular.profiles.that.result.from.an.aerospace.safety-related.st�mulus ..Factors.we.are.�nvest�gat�ng.or.w�ll.�nvest�gate.have.been.shown.to.�mpact.av�at�on.safety ..Factors.such.as.fat�gue.and.hypox�a.are.of.great.�nterest.�n.all.branches.of.av�at�on;.alcohol.can.be.a.factor.�n.general.av�at�on ..In.add�t�on,.cosm�c.rad�at�on.poses.a.long-term.r�sk.to.the.health.and.well.be�ng.of.p�lots.and.a�rcrew,.espec�ally.as.space.fl�ght.becomes.real�ty ..An.�ncreased.understand�ng.of.these.factors.at.the.molecular.level.w�ll.�ncrease.safety.for.the.fly�ng.publ�c.and.a�rcrews.through.better.regulat�on,.development.of.targeted.therapeut�c.�ntervent�ons.and.�ncreased.understand�ng.of.the.r�sks.posed.by.fl�ght.�n.general .

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Figure 6. Resolution of a set of differentially expressed genes into two expression patterns, or clusters. Clustering of differentially expressed genes demonstrates two expression patterns which, when taken together resolve into a cohesive whole that can be used to differentiate between the timepoints in this study of sleeplessness. Addition of time points and correlation of expression data to performance data will fine-tune the ability of the assay to differentiate among levels of impairment.

10

rEFErENCES

(1). Perry.Gh,.dom�ny.nJ,.claw.KG,.et.al ..d�et.and.the.evolut�on.of.human.amylase.gene.copy.number.var�at�on ..nat.Genet ..2007;39(10):1256-60 .

(2). Wolffe.A ..dnA.methylat�on.and.chromat�n ..Chro-matin, structure and function,.2.ed ..San.d�ego,.cA:.Academ�c.Press.1995:94-6 .

(3). Wolffe.A ..Post-translat�onal.mod�f�cat�on.of.core.h�stones ..Chromatin, structure and function,.2.ed ..San.d�ego.cA:.Academ�c.Press.1995:72-7 .

(4). Wolffe.A ..h�stone.var�ants ..Chromatin, structure and function ..2.ed ..San.d�ego,.cA:.Academ�c.Press.1995:68-72 .

(5). Soej�ma.h,.Wagstaff.J ..Impr�nt�ng.centers,.chro-mat�n.structure,.and.d�sease ..J.cell.B�ochem ..2005.May.15;95(2):226-33 .

(6). Keverne. eB .. Genom�c. �mpr�nt�ng. �n. the. bra�n ..curr.op�n.neurob�ol ..1997.Aug.7;(4):463-8 .

(7). cass�dy.SB,.Schwartz.S ..Prader-W�ll�.and.Angel-man.syndromes ..d�sorders.of.genom�c.�mpr�nt�ng ..Med�c�ne.(Balt�more) ..1998.Mar;77.(2):140-51 .

(8). Gerald. Wl .. the. molecular. genet�cs. of. W�lms.tumor:. A. parad�gm. of. heterogene�ty. �n. tumor.development ..cancer.Invest ..1994;12(3):350-9 .

(9). chung.SW,.dan�el.r,.Wong.By,.Wong.P,.M ..the.ABl.genes.�n.normal.and.abnormal.cell.develop-ment ..cr�t.rev.oncog ..1996;7(1-2):33-48 .

(10). he. h,. olesnan�k. K,. nagy. r,. l�yanarachch�. S,.Prasad.Ml,.et.al ..Allel�c.var�at�on.�n.gene.express�on.�n.thyro�d.t�ssue ..thyro�d ..2005.Jul;15(7):660-7 .

(11). Wolffe.A ..heterochromat�n,.pos�t�on.effect,.locus.control.reg�ons.and.�nsulators ..Chromatin, structure and function,.2.ed ..San.d�ego.cA:.Academ�c.Press.1995:84-94 .

(12). Zamore. Pd,. haley. B .. r�bo-gnome:. the. b�g.world. of. small. rnAs .. Sc�ence .. 2005. Sep.2;309(5740):1519-24 .

(13). Vaughn. MW,. Mart�enssen. r .. It’s. a. small.rnA. world,. after. all .. Sc�ence .. 2005. Sep.2;309(5740):1525-6 .

(14). he.h,.Jazdzewsk�.K,.l�.W,.et.al ..the.role.of.m�-crornA. genes. �n. pap�llary. thyro�d. carc�noma ..PnAS ..2005.dec.19;19075-80 .

(15). Kos�k. KS,. Kr�chevsky. AM ..the. elegance. of. the.m�crornAs:.A.neuronal.perspect�ve ..neuron ..2005.Sep.15;47(6):779-82 .

(16). development.of.d�sease.B�omarkers ..2005.March.14,.2006.(c�ted.2007.october.15);.Program.an-nouncement ..Ava�lable.from:.grants .n�h .gov/grants/gu�de/pa-f�les/PA-05-098 .html .

(17). roche.d�agnost�cs ..Background.�nformat�on:.the.technology.beh�nd.Ampl�ch�p.m�croarrays ..Techni-cal Bulletin ..Basel,.Sw�tzerland .

(18). Gu�dance. for. �ndustry:. Pharmacogenom�c. data.subm�ss�ons ..[url].2005.[c�ted.2005.March.22,.2005];. Ava�lable. from:. www .fda .gov/cber/gdlns/pharmdtasub .htm .

(19). coll�ns.FS ..the.case.for.a.uS.prospect�ve.cohort.study.of.genes.and.env�ronment ..nature ..2004.27.May;.429:.475-7 .

(20). the.Internat�onal.hapMap.consort�um ..A.hap-lotype.map.of.the.human.genome ..nature ..2005.27.oct;437:1299-320 .

(21). ndA. 20-571. camptosar®(�r�notecan. hcl);.F�nal. label ..u .S ..Food.and.drug.Adm�n�strat�on.2005:17 .

(22). new. hope. for. cancer. pat�ents:. Iressa. (gef�t�n�b).launched. �n. ch�na. for. treat�ng. advanced. non-small.cell.lung.cancer ..2005.[c�ted.25.Feb.2005];.Ava�lable. from:. http://en .astrazeneca .com .cn/Art�cle/511765 .aspx .

(23). Pahl.A,.Brune.K ..Gene.express�on.changes.�n.blood.after.phlebotomy:.Impl�cat�ons.for.gene.express�on.prof�l�ng ..Blood ..2002;100(3):1094 .

(24). hakonarson. h,. Bjornsdott�r. uS,. halap�. e,. et.al .. Prof�l�ng. of. genes. expressed. �n. per�pheral.blood. mononuclear. cells. pred�cts. glucocort�co�d.sens�t�v�ty. �n. asthma. pat�ents .. PnAS .. 2005. 11.oct;102(41):14789-94 .

(25). connolly.Ph,.ca�ozzo.VJ,.Zald�var.F,.et.al ..effects.of.exerc�se.on.gene.express�on.�n.human.per�pheral.blood.mononuclear.cells ..J.Appl.Phys�ol ..2004.1.oct;97(4):1461-9 .

(26). lee.MS,.hanspers.K,.Barker.cS,.et.al ..Gene.expres-s�on.prof�les.dur�ng.human.cd4+.t.cell.d�fferent�a-t�on ..Int.Immunol ..2004.1Aug;16(8):1109-24 .

(27). n�cholson. Ac,. unger. er,. Mangalathu. r,et. al ..explorat�on.of.neuroendocr�ne.and.�mmune.gene.express�on.�n.per�pheral.blood.mononuclear.cells ..Bra�n.res.Mol.Bra�n.res ..2004;129:193-7 .

11

(28). Q�ng.x,.Putterman.c ..Gene.express�on.prof�l�ng.�n.the.study.of.the.pathogenes�s.of.system�c.lupus.erythematosus ..Auto�mmun.rev ..2004.3.nov;.(7-8):505-9 .

(29). dragh�c�.S ..Array.normal�zat�on ..Data analysis tools for DNA microarrays ..new.york,.ny:.chapman.&.hall/crc.2003:313-8 .

(30). lark�n.Je,.Frank.Bc,.Gavras.h,.et.al ..Independence.and. reproduc�b�l�ty. across. m�croarray. platforms ..nat.Methods ..2005.2.May;.(5):337-44 .

(31). Members.of.the.tox�cogenom�cs.rersearch.consor-t�um:.We�s.BK ..Standard�z�ng.global.gene.express�on.analys�s.between.laborator�es.and.across.platforms ..nat.Methods ..2005.2.May;.(5):351-56 .

(32). Ir�zarry.rA,.Warren.d,.Spencer.F,.et.al ..Mult�ple-laboratory. compar�son. of. m�croarray. platforms ..nat.Methods ..2005.2.May;.(5):345-50 .

(33). hagmar.l,.Stromberg.u,.Bonass�.S,.et.al ..Impacts.of.types.of.lymphocyte.chromosomal.aberrat�ons.on.human.cancer. r�sk:.results. from.nord�c.and.Ital�an.cohorts ..cancer.res ..2004;64:2258-63 .

(34). romano.e,.Ferrucc�.l,.n�cola�.F,. et. al .. Increase.of. chromosomal. aberrat�ons. �nduced. by. �on�s-�ng.rad�at�on.�n.per�pheral.blood.lymphocytes.of.c�v�l.av�at�on.p�lots.and.crew.members ..Mutat.res ..1997;377:89-93 .

(35). hagmar.l,.Brugger.A,.hansteen.I-l,.et.al ..cancer.r�sk.�n.humans.pred�cted.by.�ncreased.levels.of.chro-mosomal.aberrat�ons.�n.lymphocytes:.nord�c.study.group.on.the.health.r�sk.of.chromosome.damage ..cancer.res ..1994.1.Jun;54(11):2919-22 .

(36). hagmar.l,.Bonass�.S,.Stromberg.u,.et.al ..chromo-somal.aberrat�ons.�n.lymphocytes.pred�ct.human.cancer:.A.report.from.the.european.Study.Group.on.cytogenet�c.B�omarkers.and.health.(eSch) ..cancer.res ..1998.15.Sep;58(18):4117-21 .

(37). Grace.MB,.Mcleland.cB,.Blakely.WF ..real-t�me.quant�tat�ve.rt-Pcr.assay.of.GAdd45.gene.expres-s�on.changes.as.a.b�omarker.for.rad�at�on.b�odos�m-etry ..Int.J.rad�at.B�ol ..2002;78(11):1011-21 .

(38). Amundson.SA,.Fornace.Jr ..A .J ..Gene.express�on.prof�les.for.mon�tor�ng.rad�at�on.exposure ..rad�at.Prot.dos�metry ..2001;97(1):11-6 .

(39). Amundson.SA,.do.Kt,.Fornace.Jr ..AJ ..Induct�on.of.stress.genes.by.low.doses.of.gamma.rays ..rad�at.res ..1999;152:225-31 .

(40). Amundson.SA,.do.Kt,.Shahab.S,.et.al ..Ident�f�ca-t�on.of.potent�al.mrnA.b�omarkers.�n.per�pheral.blood.lymphocytes.for.human.exposure.to.�on�z�ng.rad�at�on ..rad�at.res ..2000;154:342-6 .

(41). Amundson.SA,.B�ttner.M,.Meltzer.P,.et.al ..Induc-t�on.of.gene.express�on.as.a.mon�tor.of.exposure.to.�on�z�ng.rad�at�on ..rad�at.res ..2001;156:657-61 .

(42). dressman.hK,.Muramoto.GG,.chao.nJ,. et. al ..Gene.express�on.s�gnatures.that.pred�ct.rad�at�on.exposure. �n. m�ce. and. humans .. PloS. Med�c�ne ..2007.1.Apr;4(4):e106 .

(43). Kupfer.dM,.hugg�ns.M,.cass�dy.B,.et.al ..A.rap�d.and.�nexpens�ve.Pcr-based.Str.genotyp�ng.meth-od.for.�dent�fy�ng.forens�c.spec�mens ..FAA Office of.Aerospace Medicine Technical Reports ..Wash�ngton,.dc ..2006;.report.no ..dot/FAA/AM-06/14 .

(44). Budowle.B,.Morett�.tr,.n�ezgoda.SJ,.Brown.Bl ..codIS.and.Pcr-based.short.tandem.repeat.loc�:.law. enforcement. tools .. Second. european. Sym-pos�um.on.human.Ident�f�cat�on ..Promega.corp;.1998;73-88 .

(45). l�u.ch,.Ma.Wl,.Sh�.r,.et.al ..Appl�cat�on.of.Ag�lent.2100.B�oanalyzer.�n.detect�on.of.human.pap�lloma.v�rus ..d�.y�.Jun.y�.da.xue.xue.Bao ..2003.23.May;.(3):213-5 .

(46). Jabas�n�.M,.Zhang.l,.dang.F,. et. al ..Analys�s. of.dnA.polymorph�sms.on. the.human.y-chromo-some.by.m�croch�p.electrophores�s ..electrophores�s ..2002;23(10):1537-42 .

(47). lu.cy,.tso.dJ,.yang.t,.Jong.yJ,.We�.yh ..detect�on.of.dnA.mutat�ons.assoc�ated.w�th.m�tochondr�al.d�seases.by.Ag�lent.2100.b�oanalyzer ..cl�n.ch�m.Acta ..2002;318(1-2):97-105 .