Biomarkers in the Diagnosis of ADHD – Promising Directions

ATTENTION-DEFICIT DISORDER (A ROSTAIN, SECTION EDITOR)

Biomarkers in the Diagnosis of ADHD – Promising Directions

Stephen V. Faraone & Cristian Bonvicini &Catia Scassellati

# Springer Science+Business Media New York 2014

Abstract The etiology and pathogenesis of attention-deficit/hyperactivity disorder (ADHD) are unclear and a more validdiagnosis would certainly be welcomed. Starting from theliterature, we built an hypothetical pyramid representing aputative set of biomarkers where, at the top, variants inDAT1 and DRD4 genes are the best candidates for theirassociations to neuropsychological tasks, activation in specificbrain areas, methylphenidate response and gene expressionlevels. Interesting data come from the noradrenergic system(norepinephrine transporter, norepinephrine, 3-methoxy-4-hydroxyphenylglycol, monoamine oxidase, neuropeptide Y)for their altered peripheral levels, their association with neu-ropsychological tasks, symptomatology, drugs effect andbrain function. Other minor putative genetic biomarkers couldbe dopamine beta hydroxylase and catechol-O-methyltrans-ferase. In the bottom, we placed endophenotype biomarkers.A more deep integration of “omics” sciences along with moreaccurate clinical profiles and new high-throughput computa-tional methods will allow us to identify a better list of bio-markers useful for diagnosis and therapies.

Keywords Biomarkers . Attention-deficit/hyperactivitydisorder . Dopaminergic pathway . Noradrenergic pathway .

Metabolism enzymes . CNS developmental network .

Environmental risk factors . Endophenotypes . SLC6A3 .

DRD4 . DBH . COMT . Reaction time variability .

Vigilance/sustained attention . Executive functions .

Workingmemory . EEG Theta/Beta ratio

Introduction

The aetiology and pathogenesis of attention-deficit/hyperac-tivity disorder (ADHD) is not yet fully understood [1, 2].ADHD is also a highly heritable disease, with estimatedheritability rates of up to 80% [3]. A recent review on geneticsof ADHD summarized that all variants associated with thepathology explain only a small fraction of heritability: pheno-type complexity and variants of small effect contribute to themissing heritability issue [4••]. To gain more insight into themechanisms leading from a genetic/biological basis of thedisease to the full clinical phenotype, endophenotypes are apromising strategy [5].

The DSM and ICD diagnoses of ADHD are based on aconsensus about clusters of clinical symptoms. However, thisdiagnostic procedure has been criticized for not allowingsufficiently reliable and valid diagnoses [6]. NIMH recentlyhas approved the Research Domain Criteria (RDoC) projectwhere a set of assumptions permit to found a new classifica-tion system, by integrating genetics, imaging and cognitiveinformation [6]. The RDoC approach suggests that a biomarkerapproach to diagnosis may be a more valid way to classifycomplexmental disorders such as ADHD.Biomarkers offer theopportunity to standardize and improve diagnostic assessmentwhile providing insights into etiological mechanisms.

What is a Biomarker?

The United States food and drug administration (FDA) de-fines a biomarker as an objective measure of normal process-es, pathological processes or pharmacological response [7].For psychiatry, biomarkers could be used to screen for, diag-noses, or predict the development of, not only psychiatricdisorders but also of personality or behavioral traits and

This article is part of the Topical Collection on Attention-Deficit Disorder

S. V. FaraoneDepartment of Psychiatry, SUNY Upstate Medical University,Syracuse, NY, USA

S. V. FaraoneDepartment of Neuroscience and Physiology, SUNY UpstateMedical University, Syracuse, NY, USA

C. Bonvicini : C. Scassellati (*)Genetic Unit - IRCCS “Centro S. Giovanni di Dio” Fatebenefratelli,Via Pilastroni 4, 25123 Brescia, Italye-mail: [email protected]

Curr Psychiatry Rep (2014) 16:497DOI 10.1007/s11920-014-0497-1

RDoC domains. Biomarkers could potentially be used toinform treatment decisions.

Because no single biomarker candidate may be sufficientfor accurate and reliable diagnosis, the current trend in psy-chiatry has shifted towards identifying sets of biomarkers [8,9••, 10••, 11]. For example, Pies et al. defined a set of bio-markers for schizophrenia comprising:-neuregulin-1 muta-tions; -abnormal smooth-pursuit eye movements; -reducedanterior cingulate volumes, enlarged lateral and third ventric-ular volumes and white matters abnormalities [12].

Despite their promise, the search for biomarkers of psychiatricdisorders has largely proven elusive. In a comprehensive review[10••], the authors concluded that according to their stringentcriteria, no biomarker is available for diagnosing ADHD.

The main goal of this review is to present evidence for aputative signature set of biomarkers for ADHD using geno-mic, neurophysiological/neuropsychological, neuroimaging,pharmacogenomic and proteomic knowledge to diagnoseADHD. In Table 1 we report data from genomics and meta-bolomics classified according to neurophysiological/neuro-psychological, neuroimaging, pharmacogenomic, biochemi-cal and gene expression findings. Table 2 presents data fromendophenotypes studies to define a further potential signatureset of biomarkers useful for ADHD diagnosis.

Potential Dopaminergic Biomarkers for ADHD

Genomics

Deficiencies of dopaminergic and noradrenergic neurotrans-mitter systems have been associated with ADHD and the mainsymptoms of ADHD, but the dopamine system has been themain focus of genomic research [69].

Dopamine Transporter Gene (DAT1, SLC6A3)

There are at least four main reasons for a central role of DAT1 inADHD genetics: 1) dopaminergic neurotransmission is con-trolled by the DAT1 protein; 2) the DAT1 protein is the maintarget for two ADHDmedications: methylphenidate (MPH) andamphetamine (AMP); 3) knockout mice for DAT1 show hyper-activity and deficits in inhibitory behavior [69]; 4) DAT1 hasbeenmapped near to a susceptibility locus for ADHD, 5p13 [70].The most studied DAT1 variant is a variable number of tandemrepeats (VNTR) of 40 base pairs located at the 3′-untranslatedregion (3′-UTR) of the gene. The ten repeat (10R) and ninerepeat (9R) alleles are the most common. It has been demon-strated that the DAT density measured in vivo using neuroimag-ing was significantly higher in ADHD children with homozy-gosity for the 10R in basal ganglia, a brain area participating ininhibitory behaviors. This suggests a functional effect of the 10Rallele [71]. A recent meta-analysis demonstrated an association

between the 10R allele and ADHD [13], although a reverseassociation has been seen for ADHD adults [72].

Kebir and Joober and Barnes et al. summarized DAT1 studiesof neuropsychological tasks [21, 22]. As regards the associationbetween attention tasks and the 10R allele, some studies showedhigher commission errors, more impulsive responses and greaterreaction time variability (RTV) using the continuous performancetest (CPT) and sustained attention to response test (SART). Otherstudies reported no association or opposite results [21]. Moreoverboys scoring high on a teacher-rated report of ADHD symptomsand who were homozygous for the 10R allele displayed poorerresponse inhibition on the opposite word task. Concerning execu-tive functions, the 10/10 genotype predicted better performance onthe Tower of London (TOL), the self-order pointing task, and theWechsler Intelligence Scale for Children-III (WISC-III) arithmeticand digit span subtests. Associations between a neurocognitivephenotype-spatial attentional bias and 10R allele was reported inchildren with ADHD [73, 74]. The attentional phenotype (left-sided inattention) was related to higher symptom severity [74].

Several studies have been conducted on neuroimaging and 3′UTR VNTR variant in ADHD. Genotype 10/10 individualswere found to generate more activations corresponding to inhi-bition tasks, showing also its interaction with diagnostic status(ADHD vs. controls) [15]. Moreover on non-ADHD siblings ofADHD probands and controls, an influence was observed be-tween the variant and the activation in striatum and cerebellumvermis, with its interaction with familial risk for ADHD [18].Similarly in another study, 10/10 individuals were more activat-ed in left striatum, right dorsal premotor cortex, and bilateraltemporoparietal junction [20]. In contrast, Congdon et al. ob-served greater neural activation associated with carriers of the 9R[23]. Finally caudate volumes were overall smaller in 10/10 than9/10 children, particularly in the left than right hemisphere [14].

Positron emission tomography (PET) and single photonemission tomography (SPECT) have been used to determineif ADHD patients show abnormal activity of the DAT instriatum. In a meta-analysis of these studies, Fusar-Poli et al.concluded that DAT activity was 14 % higher in ADHDpatients compared with controls [24]. A meta-analysis of neu-roimaging studies of humans found that the 9R allele wasassociated with increased DAT activity in striatum [75]. Thisstudy was conducted considering different neuropsychiatricillnesses and this strengths the issue that VNTR polymorphismregulates dopamine activity in the striatal brain regions not onlyin presence of ADHD but also of other disorders

A recent review on neuroimaging and pharmacogeneticsreported some studies on DAT1 [25••]. In particular an asso-ciation with 10R was found with higher regional cerebralblood flow in medial frontal and left basal ganglia regions,and with poorer treatment response, in children with ADHDreceiving MPH for 4 days before SPECT imaging session.Moreover this allele was associated with greater DAT avail-ability in basal ganglia following treatment and poorer

497, Page 2 of 20 Curr Psychiatry Rep (2014) 16:497

Tab

le1

Datafrom

genomicandmetabolom

icstudiesclassified

accordingto

neurophysiological/neuropsychological,neuroimaging,pharmacogenom

ic,biochem

icalandgene

expression

findings

System

sPo

tentialb

iomarker

Genefunctio

n/rationale

Location,polymorphism,

risk

allele,functionality

Meta-analysis(O

R,χ

2,

pvalue)

GWAS/

CNVs

studies

Neuropsychological

endophenotypes

Genom

ics

Dopam

inergicsystem

Dopam

inetransporter

(DAT1,SL

C6A

3)3′-U

TR,V

NTR,10R

,higher

expression

levels

ofthetransporter

1.12,3.66,0.028[13]

-Sustained

Attention:

some

studiesshow

edassociation

10Randhigher

commission

errors,m

oreim

pulsive

responses,greaterRTVon

CPT

,SART.

Other

studies

reported

noassociationor

oppositeresults.

Encodes

atransm

embrane

transportp

rotein

mediating

theactiv

ereuptake

ofDA

from

thesynapseandserves

asacriticalregulator

ofdopaminergic

neurotransmission

inthe

brain.

-Executive

function

s:association10

Rwith

good

performance

onTOL,SOPT,WISC-III,

digitspan

subtests.

1)dopaminergicneurotransmission

iscontrolled

bytheDAT1;

2)itisthemaintargetforMPH

;

-Responseinhibitio

n:association10Ralleleand

poorer

response

inhibitio

non

theOpposite

WordTask;

3)DAT1knockout

micelead

tosuggestiv

ebehavior

ofADHD;

-Association10Randspatial

attentionalbias[reviews21,

22].

4)mapsnearasusceptib

ility

locus.

Intron

VIII,VNTR,3R

1.25,3.35,0.034[13]

Intron,rs403636,Intron,

rs463379,Intron,

rs393795,Intron,

rs37020

-Associationhaplotype

rs403636

(G)/rs463379

(C)/

rs393795

(C)/rs37020(G

)andspatialw

orking

mem

ory

[28].

Exon,rs6350

-Associationwith

alertin

gand

executivecontrol

performance

[29].

Dopam

ineD4receptor

(DRD4)

Amem

berof

thedopamineD2-

likereceptor

family

inhibitin

gadenylyl

cyclase.Expressed

infrontalloberegions

(orbitofrontalcortexand

anterior

cingulated

regions).

ExonIII,VNTR,7R,less

responsive

toDA.

1.35,5.10,<0.0001

[13,30••]

-Association7R

with

speedof

processing,setshiftin

g,and

cognitive

impulsiveness;

Itwas

thefirstassociatedwith

novelty-seeking

personality

trait

-Associationabsence7R

with

high

RTV;

-no

effecton

response

inhibition[reviews21,22].

Dopam

ineD2receptor

(DRD2)

Amem

berof

thedopamineD2-

likereceptor

family.

3′Flank,rs18000497

(TaqI),

“T”(A

1allele),reduced

DRD2expression.

1.65,2.56,0.11

[13];

[34]

Expressed

inneostriatum,

olfactorytubercle,substantia

1.65,2.18,0.03

[30••]

Curr Psychiatry Rep (2014) 16:497 Page 3 of 20, 497

Tab

le1

(contin

ued) Sy

stem

sPo

tentialb

iomarker

Genefunction/rationale


risk


Meta-analysis(O

R,χ

2 ,pvalue)

GWAS/

CNVs

studies

Neuropsychological

endophenotypes

nigra,ventraltegmentalarea,

andnucleusaccumbens.

Itplaysarolein

regulatin

gthemesolim

bicreward

pathways.


rs1079596

-Associatio

nwith

greater

commission

errorrates

[review21].

Dopam

ineD5receptor

(DRD5)

Amem

berof

thedopamine

D1-lik

ereceptorsfamily.

5′Flank,Dinucleotide

repeat,136

bp0.58,−

2.13,0.0329[30••]

Expressed

inam

ygdala,frontal

cortex,hippocampus,

striatum

,basalforebrain,

hypothalam

us,cerebellum,

andthalam

us.Itisim

plicated

inhippocam

palm

emory

form

ation.

5′Flank,Dinucleotide

repeat,148

bp1.26,2.91,0.0036

[30••]

-Associationwith

commission

errors,omission

errors,R

Ts,

RTV[36];

-Noassociationwith

cognitive

tests(Stop-signaltask,

Stroop

task,T

MT,

FDI,IQ

)[38].

Metabolom

ics

Phenylethylamine(PEA)

ADAreceptor

agonist,

synthesizedby

the

decarboxylationof

phenylalanine

Dopam

ine(D

A)

InADHDtheturnover

isreducedwith

excessivere-

uptake

andintra-synaptic

monoamineconcentrations

aredecreased.

Hom

ovanillicacid(H

VA)

MainDAmetabolite.

Genom

ics

Noradrenergicsystem

Norepinephrine

transporterprotein1

NET(SLC6A

2)

Codes

foraproteinresponsible

forthereuptake

ofNEfrom

thesynapticcleftb

ackinto

thepresynaptic

neuron.

Exon9,rs5569,“A”

1.06,1.17,0.279[13]

[34]

-Noassociationwith

CPT

performance

[review21].

Expressed

inthefrontallobes.

5′Flank,rs28386840

(A-3081T

),Tallele,

reducedlevelsof

NET

with

inthebrain.

-Associatio

nTallelewith

greaterdecrease

inmean

commission

errorsscores

[40,41].

Itisactiv

elyinvolved

inboth

noradrenergicand

dopaminergicreuptake

and

regulatio

nin

thisregion.


rs3785157

-Associatio

nwith

high

performance

onthe

similaritiessubtesto

fthe

WISCIII[42].

Intron,rs3785155

-Associatio

nwith

RTV[43].

Metabolom

ics

Norepinephrine(N

E)

Itisreleased

bynoradrenergic

neuronslocatedin

the

specificcentraln

ervous

system

apartfrom

dopaminergicneurons.

Normetanephrine(N

M)

Mainmetabolite

ofNE.

3-methoxy-4-

hydroxyphenylg

lycol

(MHPG

)

Mainmetabolite

ofNE.


Tab

le1

(contin

ued) Sy

stem

sPo

tentialb

iomarker



risk


Meta-analysis(O

R,χ

2 ,pvalue)

GWAS/

CNVs

studies

Neuropsychological

endophenotypes

NeuropeptideY(N

PY)

Colocalizes

with

catecholam

ine

system

s.Itparticipatesinthe

regulationoffeeding,circadian

rhythm

s,reproductionand

thermoregulation.

[44]

Genom

ics

Adrenergicsystem

Alpha-2A-adrenergic

receptor

(ADRA2A

)5′Flank,rs1800544(M

spI),

“G”,functio

nal

0.99,0.01,0.542[13];

1.077,0.20,0.65[45]

-AssociationCallelewith

TOL,

TMT,RTVon

differenttasks

[review21].

Encodes

alpha-2A

-adrenergic

receptorsregulatin

gneurotransmitter

releasefrom

sympatheticnerves

andfrom

adrenergicneurons.

Metabolom

ics

Influences

executivefunctions

intheprefrontalcortex.

3′-U

TR,rs553668(D

raI),

“T”

0.94,0.12,0.638[13]

-Associationwith

performance

inTMT[review21].

Epinephrine

(EPI)

Neuroim

agingandanim

alstudiessupportalterationin

EPI

inADHD.

Metanephrine(M

)Mainmetabolite

ofEPI.

Metabolism

enzymes

Catechol-O-

methyltransferase

(COMT)

Enzym

ecatalyzing

the

inactiv

ationof

DAwithin

the

PFC.E

xpressed

infrontal

lobe

regionsof

brain.

ExonIV,rs4680(158Val/

Met),valine,higher

activ

ity

-,1.02,0.7267[48••]

-interaction:

COMT(val/val)-

MAOA(3R)andhigh

intelligence[49].

ItregulatessynapticDAlevels.

Genom

ics/

metabolom

ics

MonoamineoxidaseA

(MAOA)

Encodes

aproteininvolved

inthemetabolism

ofDA,5-H

T,andNE.

Prom

oter,30-bp

VNTR,2-

and3-Rareconsidered

‘low

-activity

’alleles

1.02,0.01,0.464[13]

-Associatio

n4-

and5R

alleles

with

morecommission

errors[54];

-Interactio

n:COMT(val/val)-

MAOA(3R)andhigh

intelligence[49].

Itisassociated

toim

pulsive,

aggressive

behavior.

Intron,rs12843268,“A

”,-Gender-specificassociationATT

haplotypeandpoor

motor

control(boys),visuo-spatial

working

mem

ory(girls)[55];

MAOAknockout

mouse

show

edincreasedlevelsof

aggressive

behavior

and

monoaminergic

neurotransmitterlevels.

Intron,rs3027400,“T”,

Exon,rs1137070,“T

”-A

ssociationof

rs12843268

andsymptom

sandreward

deficiency

orinsufficient

response

inhibitio

n[56].

Dopam

ineBeta

Hydroxylase

(DBH)

Catalyzes

theconversion

ofDA

into

NE.

Intron

V,rs2519152

(TaqI),

“T”/A2allele,associated

with

reducedDBH

activ

ity

1.12,0.43,0.206[13]

-AssociationA2/Tallelewith

poorer

performanceson

temporalo

rder

judgment

task,m

orecommission

and

omission

errorsandgreater

RTV(ontheSA

RT),more

errorson

theWCST

(problem

-solving)andthe

MFF

T(cognitiv

eim

pulsiveness).


Tab

le1

(contin

ued) Sy

stem

sPo

tentialb

iomarker



risk


Meta-analysis(O

R,χ

2 ,pvalue)

GWAS/

CNVs

studies

Neuropsychological

endophenotypes

Expressed

inthePF

C.

[reviews21,22].

CNSdevelopm

ent

pathways

5′Flank,rs1611115(-1021

C/T),Talleleassociated

with

reducedDBH

activ

ity,reductio

nof

DA-to-NEconversion

1.05,0.16,0.692[13]

-Executivefunction:Association

Calleleandglobalexecutive

functionandmoreerrorsof

commission

andom

ission

ontheCPT

[57].

Genom

ics/

metabolom

ics

Brain-D

erived

NeurotrophicFactor

(BDNF)

Encodes

aneurotrophin

prom

otingneurogenesis,

developm

ent,functio

nal

maintenance,and

plasticity

ofneurons.

Prom

oter,rs6265,“G

”,alleleValassociated

with

increasedactiv

ity-

dependentsecretionof

BDNF

1.01,0.02,0.406[13]

[34]

-Noassociationwith

Digitspan

[review21].

Expressed

inpons,

hippocam

pus,frontalcortex,

colliculi,

andolfactorytract.


rs11030104

-Noassociationwith

Digitspan

[review21].

Genom

ics

Synaptosom

al-associated

protein25

(SNAP-25)

Encodes

aproteininvolved

inaxonalgrow

th,synaptic

plasticity,docking,and

fusion

ofsynapticvesicles

inpresynaptic

neurons.

3′-U

TR,rs3746544,

unknow

n1.15,4.71,0.03

[13]

Expressed

inhippocam

pus.

3′-U

TR,rs1051312,“T”

1.06,0.30,0.298[13]

Metabolom

ics

Environmentalrisk

factors

Ferritin(ironstore)

Coenzym

eof

tyrosine

hydroxylaseandMAO.Iron

deficiency

isassociated

with

ADHDsymptom

s.Zinc(Zn)

Cofactorformetabolism

relevant

toneurotransmitters,

anditaffectsDAmetabolism.

Zincdeficiency

isassociated

with

behavior

problemsin

ADHD.

Oxidativ

estress

Somestudiesshow

edsome

treatm

entefficacyforanti-

oxidantcom

pounds.

Potentialb

iomarker

Neurophysiological

endophenotypes

Variations

inbrainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

Biochem

ical/proteom

ics

endophenotypes

Geneexpression

Pharmacogenetics/drugseffect

Dopam

inetransporter

(DAT1,SL

C6A

3)-A

ssociatio

n10Rwith

smallercaudatevolumes

particularly

intheleft

than

righth

emisphere

[14].

-Associatio

n10Randmore

activations

correspondingto

inhibitio

ntasks[15];

-Association10Randincreased

MPH

response,but

also

contrastingresults

[reviews

16•,17••];

-Association10Randactiv

ation

instriatum

andcerebellu

mverm

is[18];

-Noassociation10RandMPH

response

[meta-analysis

[19••].

-Association10Randactiv

ation

inleftstriatum

,right

dorsal


Tab

le1

(contin

ued)

Potentialb

iomarker

Neurophysiological

endophenotypes

Variations

inbrainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

Biochem

ical/proteom

ics

endophenotypes

Geneexpression


prem

otor

cortex,and

bilateral

temporoparietaljunction

[20];

-Associatio

n9R

andgreater

neuralactiv

ation[23];

-Associatio

n9R

andincreased

DATactiv

ityinstriatum

[24];

-Neuroim

aging/

Pharmacogenetics[review25••]:

a)Association10Ralleleand

higher

regionalcerebral

bloodflow

andwith

poorer

MPH

treatm

entresponse;

b)Associatio

n10Randgreater

DATavailabilityin

basal

ganglia

aftertreatm

entand

MPH

poorer

treatm

ent

response.

-Haplotype

studies:

a)Association10/6

and

dopaminergicreward-

processing

circuitry[26];

b)Associatio

n10/3

andspatial

inattention[27];

-Haplotype

studies:

a)Association10/6

and

dopaminergicreward-

processing

circuitry[26];

b)Associatio

n10/3

andspatial

inattention[27];

Dopam

ineD4receptor

(DRD4)

-Associatio

n7R

with

thinnerprefrontaland

parietalcortex;

-low

ermRNAexpression

levels[31].

-Associatio

n7R

andincreased

MPH

response,but

also

contrastingresults;

-Associatio

n7R

with

adelayin

cerebralcortical

maturation[32,33••].

-Associatio

n4R

andpositiv

elin

earim

provem

ento

nmathematicstestoutcom

eswith

increasing

MPH

dose,

absence4R

decreased

responsesto

higher

MPH

doses;

-Associatio

n4R

andpicking

behaviors,7R

with

social


Tab

le1

(contin

ued)

Potentialb

iomarker

Neurophysiological

endophenotypes

Variations

inbrainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

Biochem

ical/proteom

ics

endophenotypes

Geneexpression


with

draw

al,w

ithincreasing

MPH

doses.

[reviews16•,17••].

Dopam

ineD2receptor

(DRD2)

-Noassociationwith

MPH

response

[35].

Dopam

ineD5receptor

(DRD5)

-Noassociationwith

MPH

response

[37].

Phenylethylamine(PEA)

-Reduced

urinelevelsinADHD

[meta-analysis39••].

-NormalizationafterAMPand

MPH

[39••].

Dopam

ine(D

A)

-Noalteratio

nin

urinelevelsin

ADHD[m

eta-analysis39••].

Hom

ovanillicacid(H

VA)

-Noalteratio

nin

urinelevelsin

ADHD[m


Norepinephrine

transporterprotein1

NET(SLC6A

2)

-Smallinfluence

onMPH

response

[review17••];

-Associatio

nGallelewith

greaterdecrease

inmean

omission

errorscores

after

MPH

administration

comparedto

Aallelecarriers

[40,41].

-Noassociationwith

the

cerebralvolumeor

thicknessof

thecortex

[42].

Norepinephrine(N

E)

-HigherurinarylevelsinADHD


-Normalizationafterpolyphenol

complex

(Pyc),fenfluramine,

AMP[39••].

Normetanephrine(N

M)

-Noalteration

sin

urinary

levels

inADHD

after

Bon

ferron

icorrection

[meta-analysis

39••].

3-methoxy-4-

hydroxyphenylg

lycol

(MHPG

)

-Reduced

urinarylevelsin

ADHD[m


-Greater

reductions

afterMPH

andAMPtreatm

ent[meta-

analysis39••].

NeuropeptideY(N

PY)

-Associatio

nwith

gene

dose-

dependentincreases

inNPY

andem

otionprocessing

[44].

-Increased

plasma

concentrations

inADHD

[39••].

Alpha-2A-adrenergic

receptor

(ADRA2A

)-A

ssociatio

nGalleleandMPH

response

[review46];


Tab

le1

(contin

ued)

Potentialb

iomarker

Neurophysiological

endophenotypes

Variations

inbrainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

Biochem

ical/proteom

ics

endophenotypes

Geneexpression


-Haplotype

studies:association

betweenGGhaplotypeand

non-remission

ofADHD

symptom

swith

atom

oxetine

treatm

ent[47].

-Haplotype

studies:association

betweenGGhaplotypeand

non-remission

ofADHD

symptom

swith

atom

oxetine

treatm

ent[47].

Epinephrine

(EPI)

-Noperipheralalteratio

nsin

ADHD[m


Metanephrine(M

)-H

igherurinarylevelsin

ADHD,lostafterbonferroni

correctio

n[m

eta-analysis

39••].

Catechol-O-

methyltransferase

(COMT)

-Associatio

nwith

antisocialand

aggressive

behaviorsof

ADHD[50,51].

-AssociationValalleleandMPH

response

[52,53].

MonoamineoxidaseA

(MAOA)

-Reduced

plateletlevelsin

ADHD[m

eta-analysis[39••].

-NormalizationafterMPH

treatm

ent[39••].

Dopam

ineBeta

Hydroxylase

(DBH)

-Low

eractiv

ities

inserum/urine

inADHD,[39••].

Brain-D

erived

NeurotrophicFactor

(BDNF)

-HigherplasmalevelsinADHD

[58];

-AssociationValalleleandMPH

response

[59].

-noalterationin

serum

levelsin

ADHD[60].

Synaptosomal-associated

protein25

(SNAP-25)

-Associatio

nTallelewith

improved

MPH

responses;

-Associatio

nGallelewith

sleep

difficultiesandirritability

[37].

-Associatio

nTalleleandpoorer

MPH

response;

-Associatio

nCallelewith

tics

andotherabnorm

almovem

ents[37].

Ferritin(ironstore)

-Reportedreducedserum

levels

inADHD,lostafter


treatment response inchildrenwithADHDwhocompleted8weeksof MPH treatment. In particular only a 28.6% of the subjects with10/10 genotype showed good response to MPH treatment [71].

Pharmacogenetic studies have demonstrated increased re-sponse toMPH among homozygous 10R, but there are conflict-ing results [16•, 17••]. However a recent meta-analysis [19••]shows that there is no significant association between thisVNTR and response to MPH treatment. The meta-analysis alsofound no effects on dimensions of hyperactivity/impulsivity andinattention. Thus this finding suggests that VNTR polymor-phism is not a reliable predictor of MPH treatment success inADHD, neither in relation to these specific symptom subscales.

A DAT1 VNTR at intron-8 that contains common 5-repeat(5R) and 6-repeat (6R) alleles has also been associated withADHD susceptibility [13]. Recent evidence suggests that jointconsideration in haplotypes may provide more information thancan be inferred from the analyses of single geneticmarkers. Thereare some neurophysiological studies on the haplotypes of thesetwoDAT1 variants: the 10/6 haplotype (formed by the 10R alleleof the 3’UTRVNTR and the 6R allele of the intron 8 VNTR) isassociated with dopaminergic reward-processing circuitry whosedeficits are linked toADHD [26]. The haplotype 10/3 (formed bythe 10R allele of the 3’UTRVNTR and the 3R allele of the intron8 VNTR) predicts spatial inattention suggesting a functionaleffect of this haplotype on cognitive performance in ADHD [27].

Other variants of DAT1 have been studied. rs6350 wasassociated to alerting and executive control performance usingthe attention network test [29]. Moreover a haplotype(rs403636 (G)/rs463379 (C)/rs393795 (C)/rs37020 (G)) wasassociated with spatial working memory in ADHD [28].

Dopamine D4 Receptor (DRD4)

The DRD4 gene is a good candidate for ADHD because of itshigh expression in brain regions implicated in attention andinhibition such as anterior cingulate cortex [76]. DRD4 wasthe first associated with novelty-seeking, a personality traitwhich is a common in ADHD. It has been demonstrated thatmRNA expression levels ofDRD4were lower in ADHD [31].

DRD4 is located on chromosome 11p15.5. A highly poly-morphic functional VNTR in the third exon has been frequent-ly investigated in association studies. It comprises 11 copies ofa 48-bp repeat sequence, where 4, 7, and 2R repeat alleles arethe most prevalent. A meta-analysis showed that the 7R allelewas associated with ADHD [13, 30••].

Recent reviews on neuropsychological endophenotypes[21, 22] concluded that, although more studies are needed,the 7R allele is associated with some cognitive markerssuch as speed of processing, set shifting, and cognitiveimpulsiveness, but not with response inhibition (the stopand go/no-go tasks). More robust evidence suggests thatthe absence of this allele is linked to high reaction timevariability (RTV).T

able1

(contin

ued)

Potentialb

iomarker

Neurophysiological

endophenotypes

Variations

inbrainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

Biochem

ical/proteom

ics

endophenotypes

Geneexpression


Bonferronicorrection[m

eta-

analysis39••].

Zinc(Zn)

-Reportedreducedserum/

plasma/urinelevelsinADHD


Oxidativ

estress

-Normallevelsof

anti-oxidant

productionin

ADHD,but

theirresponse

tooxidative

stress

isinsufficient

[meta-

analysis61••].

Note:

ADHD:Attention-deficit/h

yperactiv

itydisorder;OR:Odd

Ratio;χ2:chi-square;VNTR:Variablenumbertandem

repeat;UTR:Untranslatedregion;MPH

:methylphenidate;DA:Dopam

ine;

WGAS:Genom

eWideAssociatio

nstudy;CNVs:Copynumbervariatio

ns;M

RI:Magnetic

resonanceim

aging;TMT:T

railmakingtest;R

TV:R

eactiontim

evariability;C

PT:C

ontin

uous

performance

test;

SART:S

ustained

attentiontoresponse

test;T

OL:T

owerof

Londontest;W

ISC-III:W

echslerintelligencescaleforchild

ren-III;SOPT:S

elforderedpointin

gTest;F

DI:Freedomfrom

distractibilityindex;

IQ:Intellig

ence

quotient;A

MP:

Amphetam

ine;MFF

T:M

atchingfamiliar

figurestest


Tab

le2

Dataextractedfrom

endophenotypes

studiesto

define

apotentialsignature

seto

fbiom

arkersuseful

forADHDdiagnosis

Potentialb

iomarker

Rationale

Meta-analysis

Review

Neuropsychological

endophenotypes

Neurophysiological

endophenotypes

Variatio

nsin

brainanatom

y(structuralM

RIfinding)

Variatio

nsin

brainfunctio

n(functionalM

RIfinding)

EEGTheta/Betaratio

(TBR)

ElevatedTheta/Betaratio

(TBR)in

ADHD.

[62••]

[63]

-Prognostic

measure

[62••];

-potential

biom

arker[63].

Reactiontim

evariability

(RTV)

IncreasedRTVinADHD.[100••]

Stablefeatureof

ADHD

butn

ospecific.

Selectiv

eattentionandvigilance/

sustainedattention

Attentionim

pairmentin

ADHD.

[64–66].

[10••]

-Robustfindings[10••];

-Noassociation[64–66].

Verbaland

visuo-spatial

working

mem

ory

Anomaliesin

prefrontal

corticalregiones

and

thebasalg

angliain

ADHD.A

lteratio

nsin

dopaminergic,

noradrenergicand

fronto-striatalsystems.

[64–67]

[10••]

-Robustfindings

[10••,67,68];

-Noassociation[64–66]

Caudate

Neuroimagingstudies

supporteddysfunctionof

severalareasofthebrain.

[102••]

Reduced

gray

mattervolume

Reduced

activ

ityand

functio

nalconnectivity

Thalamus

Reduced

gray

mattervolume

Reduced

activ

ityand

functio

nalconnectivity

Anteriorcingulated

Reduced

gray

mattervolume,

reducedwhitematterintegrity

Reduced

activ

ityand

functio

nalconnectivity

Prefrontalcortex

Reduced

gray

mattervolume,


Reduced

activ

ityand

functio

nalconnectivity

Premotor

andSM

Acortex

Reduced

gray

mattervolume

Reduced

activ

ityand

functio

nalconnectivity

Superiorparietalcortex

Reduced

gray

mattervolume

Reduced

activ

ityand

functio

nalconnectivity

Precuneus,posteriorcingulate,

lateralp

arietalcortex,medial

frontalcortex(default-mode

network)

Reduced

activ

ityand

functio

nalconnectivity

Cerebellum

(posterior

inferior

verm

is)

Reduced

gray

mattervolume,


Reduced

activ

ityand

functio

nalconnectivity

Corpuscallo

sum

(splenium/

isthmus)

Reduced

whitematterintegrity

Fasciculus

longitu

dinalis

superior

Reduced


Anteriorcorona

radiate

Reduced


Note:ADHD:A

ttention-deficit/h

yperactiv

itydisorder;M

RI:Magnetic

resonanceim

aging;

SMA:S

upplem

entary

motor

area


Neuroimaging studies found associations of 7R allele withthinner prefrontal and parietal cortex as well as with delay incerebral cortical maturation [32, 33••].

Pharmacogenetic studies suggest an enhanced response toMPH among 7R carriers even though conflicting results havebeen obtained [16•, 17••]. Moreover some studies have sug-gested that different DRD4 genotypes exhibit different MPHdose–response curves [16•, 17••]. One study demonstrated thatindividuals with one or two copies of the 4R allele had positivelinear improvement on mathematics test outcomes with increas-ing MPH dose, while those lacking a copy showed greaterimprovement at lower doses, and deterioration at higher doses.A second study found that those lacking any 4R allele haddecreasing responses to higherMPHdoses compared with othergenotypes. Both these studies are consistent with the ideathat the 7R codes for a defective dopamine receptor thatresponds less effectively at higher doses, an idea with somepotential for clinical relevance if replicated in larger samples.One interesting finding suggests that DRD4 might play arole in predicting susceptibility to medication side effects. Inthe Preschool ADHD Treatment Study, MPH treated ADHDchildren homozygous for the 4R were three-times morelikely to develop picking behaviors, while those with at leastone copy of the 7R were four-times more likely to developsocial withdrawal with increasing doses. Thus, 7R carriersappear to be a distinct subgroup of ADHD patients whichexperiences differential medication effects.

Other variants in the promoter region have also been stud-ied, focusing on a 120-base pair duplication (120-bp dup),−521C/T (rs1800955), −616C/G (rs747302), −615A/G, and−376C/T, located in the 5′ untranslated region. However twometa-analyses conducted on 120-bp dup and −521C/Tshowed no association with ADHD [13, 30••].

Dopamine D2 receptor (DRD2)

The DRD2 gene is located at chromosome 11q23.1. Therationale for its involvement in ADHD is based on the evi-dence that it is expressed in those brain areas relevant for thepathology and it is implicated in the regulation of mesolimbicreward pathways [77]. A recent genome wide associationstudy reported a nominal association of this gene withADHD susceptibility [34]. The TaqIA (rs1800497) variantof DRD2 was believed to connect with urinary level of theDA metabolite homovanillic acid (HVA) and DRD2 expres-sion levels. According to Wu et al.’s meta-analysis, an asso-ciation of this polymorphism was reported [30••]. However,due to excessive heterogeneity, the authors concluded that thisresult was invalid. Pharmacogenetic studies have found noassociation of this variant with MPH response [35]. Twoother DRD2 variants, rs2075654 and rs1079596, havebeen associated with greater commission error in ADHD[21].

Dopamine D5 Receptor (DRD5)

DRD5 maps to chromosome 4p15.3. In situ hybridizationstudies demonstrated that the expression of the gene is higherin hippocampus, a brain area involved in ADHD pathogenesis[78]. Moreover functional studies showed that DRD5 is impli-cated in synaptic strength in hippocampal memory formation[79]. These studies support the rationale on the involvement ofDRD5 gene in ADHD. The association between ADHD and ahighly polymorphic dinucleotide repeat of DRD5 ((CA)n),located in 18.5 kb at the end of the 5′ flanking region, has beenthemost studied. This variant comprises 12 alleles ranging from134 to 156 bps in length, among which the 148-bp and 136-bpalleles are the most common. The 148-bp allele was a riskfactor for ADHD according meta-analyses, while that of 136-bp allele was a protective factor for ADHD [13, 30••].

Interestingly, the 148 bp allele associated to commis-sion errors, omission errors, RTs, and RTV [36], whereasno association was detected with cognitive test scores [38].Pharmacogenetic studies found no association of MPHresponse with the 148-bp allele, whereas the 151-bp allelehas been linked to a favorable MPH response [37].

Metabolomics

Phenylethylamine (PEA)

In dopaminergic neurons of the nigrostriatal system, the dopamine(DA) receptor agonist PEA is synthesized by the decarboxylationof phenylalanine. PEA stimulates the release of DA and, accord-ing to a meta-analysis [39••], urinary levels of PEA are signifi-cantly lower in patients with ADHD compared with controls.Interestingly decreased levels of PEA have also been associatedwith symptoms of inattentiveness and administration ofAMP andMPH markedly increased urinary excretion of PEA [39••].

DA and HVA

DA and its mainmetabolite HVA showed no difference in urineexcretion in ADHD patients compared with controls [39••].

Potential Noradrenergic Biomarkers for ADHD

Genomics

Norepinephrine Transporter Gene (NET1, SLC6A2)

The SLC6A2 gene codes for the norepinephrine transporter,which is responsible for the reuptake of norepinephrine (NE)from the synaptic cleft back into the presynaptic neuron and istargeted by atomoxetine, a medication for ADHD. NET1 ismost highly expressed in the frontal lobes where it plays a role


in noradrenergic and dopaminergic reuptake [80]. A frequent-ly studied SNP (rs5569 or G1287A) located in exon 9 was notsignificant in a meta-analysis of candidate gene studies [13],but a nominal association was observed in a genome-wideassociation study [34]. No association was found between thisvariant and CPT performance [21]. A recent review on phar-macogenetics concluded that NET1 influence on response toMPH is small [17••], although a recent genome-wide associ-ation study found two SNPs in SLC6A2 gene associated withMPH response [81]. In a study rs3785143 was associated withatomoxetine response [47].

Moreover childrenwith theG/G genotype at G1287A showeda greater decrease in mean omission error scores after MPHadministration compared to A allele carriers. Another polymor-phism A-3081T at 5’flank, demonstrated to be functional, wasassociated with T allele with greater decrease in mean commis-sion errors scores, meaning improved impulsive behavior [40,41]. Other variants such as rs998424 and rs3785157 were linkedto high performance on the similarities subtest of the WISCIII,but not with the cerebral volume or thickness of the cortex [42].Another SNP rs3785155 was associated to RTV [43].

Metabolomics

Norepinephrine (NE)

Concerning NE levels, a meta-analysis [39••] indicated higherurinary levels in ADHD compared with controls, and a nor-malization of NE concentrations has been observed aftertreatment with polyphenol complex and with fenfluramineand AMP [39••].

3-Methoxy-4-Hydroxyphenylglycol (MHPG)

MHPG is the main metabolite of NE. Its urinary levelswere lower in ADHD patients and stimulant trialsshowed that decreases in ADHD symptoms with treat-ment were associated with greater reductions in urinaryMHPG excretion [39••].

Neuropeptide Y (NPY)

NPY frequently colocalizes with catecholamine systems. Itparticipates in the regulation of feeding, circadian rhythms,reproduction, and thermoregulation. Oades et al. found in-creased plasma NPY concentrations in ADHD children com-pared with controls [82]. Moreover a recent genome-widecopy number variation analysis [44] found that NPY wasincluded in a rare 3 Mb duplication on chromosome 7p15.2to 15.3 and an association of this duplication was found withincreased NPY plasma concentrations. Moreover an associa-tion was observed between gene dose-dependent increases inNPY and emotion processing [44].

Potential Adrenergic Biomarkers for ADHD

Genomics

Alpha-2A-adrenergic receptor (ADRA2A)

ADRA2A in the prefrontal cortex influences executive func-tions impaired in ADHD [83]. It is also a target for twoADHDmedications: guanfacine and clonidine. A-1291 C>G createsan MspI site in the promoter region and it is a functionalpolymorphism. Two meta-analyses confirmed no associationwith ADHD [13, 45]. However the C allele was associatedwith TOL, trail making test (TMT), and RTV on the stop-signal task as well as the CC genotype was associated withhigh RTV on the CPT test [21]. A recent pharmacogeneticreview [17••] summarized some studies reporting that G alleleis associated with greater reduction of inattentive symptomsover time. Froehlich et al. also found a main effect of thisgenetic variant on MPH response [46]. However, the G allelewas associated with hyperactive-impulsive symptoms on pla-cebo and across doses.

Another variant rs553668, for which the disorder meta-analysis was negative [13], was associated with the perfor-mance on TMT suggesting an involvement of this gene onexecutive functions [21]. Yang et al. described an associationbetween GG haplotype (rs1800544/rs553668) and non-remission of ADHD symptoms with atomoxetine treatment[47].

Metabolomics

Epinephrine (EPI) and Metanephrine (M)

EPI and its main metabolite M levels, according to a recentmeta-analysis [39••], were respectively no different or higherin ADHD patients. The difference for M was lost afterBonferroni correction.

Potential Metabolism Enzymes as Biomarkers for ADHD

Genomics/Metabolomics

Catechol-O-Methyltransferase (COMT)

COMT is an enzyme responsible for the degradation of DAand NE. It is highly expressed in frontal lobe where it regu-lates synaptic DA levels [84]. Studies examining the associa-tion between COMT and ADHD have largely focused on afunctional SNP in exon 4 that creates an amino acid substitu-tion (valine/methionine). This variant affects COMT enzymeactivity, such that homozygotes for the valine allele shows 3–4times greater activity than homozygotes for the methionine


allele. However meta-analysis [48••] indicates no associationbetween ADHD and the 158Val/Met.

A review summarized studies on 158Val/Met and neuropsy-chological endophenotypes [21]. While no association wasobserved between this variant and performance on cognitivetasks, a significant finding was reported between the Met alleleand impairment in sustained attention and fewer commissionerrors. In the neurophysiological studies, this variant was asso-ciated with antisocial and aggressive behaviors of ADHD [50,51]. Pharmacogenetic studies reported a positive associationbetween Val allele and response to MPH [52, 53].

Monoamine Oxidase A (MAOA)

TheMAOA gene encodes a protein involved in themetabolism ofDA, 5-HT, and NE. It has been associated with impulsive,aggressive behavior [85]. Moreover an MAOA knockout mouseshowed increased levels of aggressive behavior and monoamin-ergic neurotransmitter levels [86]. Recent studies have focusedlargely on a functional 30-bp VNTR 1.2 kb upstream of the gene,previously associatedwith impulsivity and aggression. Accordingthe classification system [87], the 2- and 3R are considered ‘low-activity’ alleles, the others are high-activity’ alleles. A recentmeta-analysis reported no association with ADHD [13].According to neuropsychological studies, the 4 and 5R allelesare associated with commission errors on the TOVA test, and aMPH administration attenuated this association [54].

Moreover a gender-specific association has been reportedwiththe ATT haplotype (rs12843268/rs3027400/rs1137070) [55]. Inboys, the ATT haplotype was associated with motor control; ingirls with visuo-spatial working memory. This suggests thatMAOA genotype influences cognitive and motor functioning inADHD and sex moderates these effects. This may be related tobiological differences between males and females in serotoninneurotransmission. Finally Nymberg et al. reported a sex-specificassociation between ADHD symptomatology and reward defi-ciency or insufficient response inhibition depending onrs12843268 genotype [56]. Concerning blood levels, a recentmeta-analysis indicated reduced levels in ADHD compared withcontrols [39••]. MAO levels were associated with impulsivenessand MPH treatment normalized the reduced levels.

Dopamine Beta Hydroxylase (DBH)

TheDBH gene encodes an enzyme that catalyzes the conversion ofDA into NEwhich is particularly expressed in the prefrontal cortex[88]. A–1021C/T variant (rs1611115) accounts for up to 50 % ofthe variation of plasma DBH activity, but it is the intron 5 TaqIvariant (rs2519152) that has been most often tested. A meta-analyses of these variants reported no associationwithADHD [13].

Two recent reviews of neuropsychological endophenotypessummarized studies of DBH [21, 22]. In particular the A2/Tallele was found associated with poorer performances on a

temporal order judgment task, more commission and omissionerrors and greater RTV on the SART, more errors on theWisconsin Card Sorting Test (WCST) (problem-solving) andthe Matching Familiar Figures Test (MFFT) (cognitiveimpulsiveness).

Finally an association between neuropsychological mea-sures of executive function in children with ADHD and the –1021C/T variant has been reported [57].

A meta-analysis [39••] found that ADHD patients showedlower activities of DBH in serum and urine and it was sug-gested that decreased DBH levels correlate with ADHDsymptoms [39••, 89, 90••].

Potential CNS Development Biomarkers for ADHD

Genomics/Metabolomics

Brain Derived Neurotrophic Factor (BDNF)

BDNF has been mapped to chromosome 11p13. It belongs to afamily of neurotrophins, involved in promoting neurogenesis,neuronal survival, and synaptic plasticity [91]. A genome widestudy supported a nominal association with ADHD susceptibil-ity [34]. A common variant resulting in a valine to methionineamino acid substitution at codon 66 (Val66Met; rs6265) regu-lates the intracellular tracking and activity-dependent secretionof BDNF in brain. A meta-analysis found no association of thisvariant with ADHD [13]. This variant and two others,rs2049046 and rs11030104, were not associated with the DigitSpan test [21]. Moreover better MPH response in ADHD chil-dren who are Val/Val homozygous was reported [59].

Concerning peripheral levels, higher plasma concentrationswere observed in ADHD patients as compared with controls[58], whereas no alterations were observed in serum levels [60].

Synaptosomal-Associated Protein 25 (SNAP-25)

SNAP-25 codes for a protein involved in axonal growth,synaptic plasticity, and in the docking and fusion of synapticvesicles in presynaptic neurons necessary for the regulation ofneurotransmitter release [92]. The major variants studied werers362987 (intron 4), rs363006 (intron 6), rs3746544 (3′UTR),and rs1051312 (3′UTR). A main effect on ADHD suscepti-bility was observed only with rs3746544 [13].

Concerning pharmacogenetic studies, homozygotes forthe T allele of T1065G had moderately improved MPHresponses, whereas homozygotes for the G allele developedsleep difficulties and irritability. Moreover, homozygotes forthe T allele at T1069C exhibited poorer MPH response,whereas homozygotes for the C allele developed tics andother abnormal movements [37].


Potential Biomarkers for Environmental Risk factors

Metabolomics

Iron

It is a coenzyme of tyrosine hydroxylase andMAO, which arecritical in the synthesis and degradation, respectively. Ferritinis a major intracellular iron storage protein and serum ferritinlevels are an indication of iron stores in the body and brain.Children with iron deficiency exhibited ADHD symptoms,such as inattention, hyperactivity, and/or impulsivity [review90••]. Ameta-analysis indicated that serum ferritin levels werelower for ADHD patients compared with controls, althoughthe finding was lost after Bonferroni correction [39••].

Zinc

Zinc is another essential cofactor for neurotransmitter me-tabolism which affects DA metabolism. Zinc deficiency isassociated with behavior problems in children with ADHD[review 90••]. Although a systematic review of randomizedcontrolled clinical trials demonstrated that using zinc, eitheralone or in combination with stimulants, did not improveADHD symptoms [93], a meta-analysis reported reducedserum/plasma/urine levels in ADHD [39••].

Oxidative Stress

The normal oxidation-reduction reactions that create energy in thecell create toxic oxidants or reactive oxygen species. These by-products of normal oxidation-reduction reactions are highly un-stable. Antioxidants counteract the effects of oxidants. Whenantioxidants are not sufficient, oxidative stress occurs and dam-ages cellular proteins, lipids, carbohydrates, and nucleic acids.Brain tissue is especially susceptible to oxidative stress. Indirectevidence for oxidative stress in ADHD comes from studies show-ing some treatment efficacy for anti-oxidant compounds such asomega-3 fatty acids, pycnogenol and N-acetylcysteine [94–97].

A meta-analysis of oxidative stress in ADHD suggestedthat patients with ADHD have normal levels of anti-oxidantproduction but that their response to oxidative stress is insuf-ficient, leading to oxidative damage [61••].

Potential Endophenotypes as Biomarkers for ADHD

Besides indentifying a genetic marker involved in neurophysio-logical/neuropsychological, neuroimaging, pharmacogenetics,biochemical features, a putative signature set of biomarkerscould be integrated with a research on endophenotypes(Table 2).

EEG Theta/Beta Ratio (TBR)

TBR is a measurement of the power (wave amplitude squared)produced by brain waves altering between 3.5 and 28.0 Hz inchildren with ADHD [98]. A genetic overlap between ADHDsymptoms and EEG theta power has been reported [99] andthis supports a recent review suggesting TBR as a potentialbiomarker for ADHD [63]. However a recent meta-analysisconcluded that excessive TBR cannot be considered a reliablediagnostic measure of ADHD but it could have a prognosticvalue [62••]. In June 2013 FDA approved NeuropsychiatricEEG-Based Assessment Aid (NEBA) System, a test recordingthe type and number of brain waves that nerve cells give offeach second.

In summary, as also reported in Thome et al. the neurophys-iological markers must be viewed as promising candidates forbiomarkers of ADHD but further studies are needed [10••].

Reaction Time Variability (RTV)

Individuals with ADHD are described frequently as ubiqui-tously slower and more variable than their unaffected peers,and ADHD-related RTV is considered by many to reflect aunique, stable, and etiologically important characteristic of thedisorder. Results of a recent meta-analysis indicate that, con-trary to contemporary characterizations of individuals withADHD as slower and more variable, ADHD individualsmay be better characterized as more variable but not slowerafter accounting for their increased response variability.However RTV lacks specificity among clinical disorders,and thus is not a viable diagnostic marker of ADHD [100••].

Selective Attention and Vigilance/Sustained Attention

A prominent multidimensional model of attention differentiatesbetween alertness, divided attention, selective attention,vigilance/sustained attention and shifting. According toThome et al. the most robust findings indicated that childrenwith ADHD displayed difficulties in measures of selectiveattention and vigilance/sustained attention [10••]. Impairmentsof vigilance/sustained attention are the most replicated neuro-psychological finding inADHD.However a considerable num-ber of investigations were unable to find difference betweenADHD patients and controls [64–66]. In addition effect sizesindicated only small to moderate differences between ADHDpatients and controls.

Executive Functions

They are an umbrella term encompassing various functions ofhigher cognitive functioning including planning, problemsolving, concept formation, fluency, cognitive flexibility, andworking memory. The most consistent finding in the domain


of executive functions is that patients with ADHD displayimpairments in working memory. Working memory measuresappear therefore to be the most sensitive indicator of executivedysfunctioning in ADHD [10••]. Additional studies supportthis finding [67, 68]. However, as for vigilance/sustainedattention, no difference was found between patients and con-trols with small to moderate effect sizes [64–66].

In summary, although promising results were obtained forselective attention and vigilance/sustained attention and work-ing memory, no sensitive or specific profile based on neuro-psychological tests assessing cognition in ADHD has as yetbeen found [101••].

Brain Differences in ADHD

Kasparek et al. reported alterations in several areas of thebrain, particularly the anterior cingulum, the dorsolateral aswell as ventrolateral prefrontal cortex, the orbitofrontal cortex,the superior parietal regions, the caudate nucleus, the thala-mus, the amygdala and the cerebellum. Imaging studies pointto the persistence of changes in both brain structure andfunction into adulthood, although there might be a tendencyfor improvement of caudate nucleus pathology. Growing ev-idence suggests that MPH treatment can lead to improvementof brain changes seen in neuroimaging by its positive effect onneuroplasticity [102••].

Conclusion

The study of psychiatric biomarkers is very complex becauseof the heterogeneous nature of psychiatric disorders which areoften referred to as syndromes with several subtypes, notuniform problems. Therefore, a single biomarker is very un-likely to provide enough information to identify cellular andmetabolic pathways involved in a particular individual.Identification of a signature set of biomarkers for disordersubtypes each based on their underlying biological pathwayswill be the most effective for diagnosis and treatmentselection.

Starting from the literature, we have built an hypotheticalpyramid representing a putative set of biomarkers where at thetop there are potentially useful biomarkers for ADHD and atthe bottom those that are probably not useful (Fig. 1).Following Pies et al.’s [12] example, a potential genetic-biomarker could be represented by the variants in DAT1 andDRD4 genes as the best candidates as biomarkers for ADHD.Indeed the 10R allele at the 3′UTR of SLC6A3, is associatedwith specific neuropsychological tasks [21, 22], generatesmore activation in specific brain areas [33••], and is associatedto MPH response [16•, 17••]. Similarly the 7R allele at exon 3ofDRD4 is involved in specific neuropsychological tasks [21,22], brain structure [32, 33••], MPH response [16•, 17••], andexpression levels of DRD4 [31].

Fig. 1 Hypothetical pyramid representation of possible signature sets ofbiomarkers for ADHD diagnosis. Starting from the literature, we built anhypothetical pyramid describing a putative set of biomarkers where, atthe top, variants in DAT1 and DRD4 genes are the best candidates asuseful biomarkers, for their associations to neuropsychological tasks,activation in specific brain areas, methylphenidate response and geneexpression levels. A further level is represented by the noradrenergicsystem (Norepinephrine transporter, Norepinephrine, 3-methoxy-4-hydroxyphenylglycol, Monoamine Oxidase, Neuropeptide Y) for theiraltered peripheral levels, their association with neuropsychologicaltasks, symptomatology, drugs effect and brain function. Other minorputative genetic biomarkers could be Dopamine Beta Hydroxylase and

Catechol-O-methyltransferase. In the bottom, we placed endophenotypebiomarkers. In particular working memory, selective attention vigilance/sustained attention, Theta/beta ratio, Reaction Time Variability andneuropsychological endophenotypes are, in this order, probably notuseful biomarkers for ADHD diagnosis. Notes: Dopamine Transportergene (DAT1, SLC6A3); Dopamine D4 receptor (DRD4);Norepinephrine transporter (NET1, SLC6A2); Norepinephrine (NE);3-methoxy-4-hydroxyphenylglycol (MHPG); Monoamine oxidase(MAO); Neuropeptide Y (NPY); Dopamine beta hydroxylase (DBH);Catechol-O-methyltransferase (COMT); Theta/beta ratio (TBR);Reaction time variability (RTV)


Interesting data also come from the noradrenergic system.In fact, according to metabolomic studies, NET1, NE, itsmetabolite MHPG and MAO along with NPY could representgood candidates as biomarkers for ADHD, based on theirperipheral levels altered in ADHD [39••], their associationwith neuropsychological tasks [21, 40–43, 54–56], symptom-atology, drugs effect [39••], and brain function [44].

Other minor putative genetic biomarkers could be the twoenzymes DBH and COMT. Although the meta-analyses ofTaq1 and -1021 T/C in DBH gene and 158Val/Met inCOMT gene have not identified susceptibility alleles, variantsof these genes are associated with neuropsychological perfor-mance [21, 22, 57], neurophysiological features [50, 51], brainfunction [103], peripheral levels [39••, 89], and MPH re-sponse [52, 53].

Concerning the identification of endophenotypebiomarkers, to date it is not possible to characterize an accu-rate biomarker for ADHD. As described in [10••, 101••, 104],no biomarker has sufficiently high specificity and sensitivityfor the ADHD diagnosis. Currently proposed biomarkers arelimited by age, gender, and drug effects and studies have beenlimited by small sample sizes and poor control for multipletesting.

This work supports the necessity to take into account the deepintegration of “omics” sciences such as “pharmacogenomics”,“phenomics”, “epigenomics,” “proteomics”, “transcriptomics”,and “metabolomics”. In fact, a better understanding of theinteraction network of genes, proteins, and biochemical pro-cesses in relation to more accurate clinical profiles, by usingnew high-throughput computational methods, will allow usto identify a list of biomarkers both for the optimization ofdiagnostic assessment as well as for the personalization oftherapies.

Acknowledgments This research was supported by grants from theFondazione Mariani (RF2006) and from the Italian Ministry of Health(Ricerca Corrente).

Compliance with Ethics Guidelines

Conflict of Interest Cristian Bonvicini Ph.D., Catia Scassellati declarethat they have no conflict of interest.

In the past year, Stephen V. Faraone received consulting income,travel expenses and/or research support from Ironshore, Shire, AkiliInteractive Labs, Alcobra, VAYA Pharma, and SynapDx and researchsupport from the National Institutes of Health (NIH). His institution isseeking a patent for the use of sodium-hydrogen exchange inhibitors inthe treatment of ADHD. In previous years, he received consulting fees orwas on Advisory Boards or participated in continuing medical educationprograms sponsored by: Shire, Alcobra, Otsuka, McNeil, Janssen,Novartis, Pfizer and Eli Lilly. Dr. Faraone receives royalties from bookspublished by Guilford Press: Straight Talk about Your Child’s MentalHealth and Oxford University Press: Schizophrenia: The Facts.

Human and Animal Rights and Informed Consent This article doesnot contain any studies with human or animal subjects performed by anyof the authors.

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http://dx.doi.org/10.1007/s12402-014-0132-3

http://dx.doi.org/10.1177/1087054713505322

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