Candidate genes in child psychiatry: What do we know and where to from here?

Examples from ADHDMark A. Bellgrove

School of Behavioural Science, University of Melbourne;Institute of Neuroscience, Trinity College Dublin

Overview

• Symptoms and Etiology of ADHD• Candidate gene approach to ADHD• Rationale behind the endophenotype

approach• Genotype/Phenotype studies of ADHD

– Spatial Attention– Sustained Attention/Response Inhibition

Symptoms of ADHD

• ADHD is a behavioural disorder of childhood– Age inappropriate levels of inattention, hyperactivity and

impulsivity

• Onset is early (before 7 years of age), 3-6% of school-aged children affected.

• 30-60% carry psychopathology into adulthood

• 70% respond well to stimulant medications– Methylphenidate

Etiology of ADHD

• Exact etiology is uncertain

• Family, twin and adoption studies suggest strong genetic component– E.g., concordance rates in MZ twins of 68-81%

• Imaging studies consistently implicate dysfunction to frontal and sub-cortical regions, particularly within the right-hemisphere

• Neuropsychology shows deficit in sustained attention, spatial working memory and inhibition-all frontally mediated functions

Candidate Gene Approach

• Dysfunction to catecholamine (e.g., DA and NA) systems seems likely, since stimulants act on these systems

• Candidate gene approach seeks to determine whether genetic variants are associated with ADHD at a greater than chance frequency

• Candidate genes for ADHD include those coding for receptors, enzymes or transporters, amongst others, involved in catecholamine function

TransporterBlocked byMPH,particularly instriatum

DA NA

Tyrosine

DOPADopamineBeta Hydroxylase

Feedback Inhibition

DACOMTDegrades DAin PFC

Potential sites of expressionfor genetic effects

Rationale behind the endophenotype approach

Castellanos and Tannock (2002)

Neuropsychological endophenotypes should be related to symptoms but be closer to the site of gene action

ADHD SymptomatologySymptoms

Neuropsych

Brain pathology

Genetic Factors DAT1 DBH DRD4

Left-spatial inattentionSustained Attention

Spatial Working Memory

Right Striatal DysfunctionPrefrontal dysfunction

DA and NA dysfunction

Molecular Biology

Exon-coding region for amino acid sequence

Intron-non-coding region

Promoter-affects gene expression

Gene Structure

5’ 3’

DNA sequencesSNP- single nucleotide polymorphism (point mutation)

VNTR- variable number of tandem repeats

Genotype/Phenotype studies of ADHDDAT1 and left-spatial inattention

• Dopamine Transporter (DAT1)– 10-repeat allele of a VNTR of the DAT1 gene

associated with ADHD in a number of studies (Cook et al, 1995; Gill et al, 1997; Daly et al, 1999)

– 10-repeat allele associated with an enhanced therapeutic response to MPH (Kirley et al, 2003)

– The 10-repeat allele affects expression levels of the transporter

• DAT1 Hypothesis: greater density of transporter or activity associated with the 10-repeat DAT1 allele leads to a reduction of available dopamine in the striatum. MPH normalises this.

Genotype/Phenotype studies of ADHDDAT1 and left-spatial inattention

• Voeller and Heilman (1988) first proposed that ADHD could be a “neglect syndrome”– ADHD children made more left-sided errors resembling

patients with right-hemisphere lesions• Supporting evidence has been inconsistent.• Evidence that attentional asymmetries may be

normalised with MPH (Sheppard et al, 1999; Nigg et al, 1997)

• Neglect is also consequent upon lesions to striatum and DA pathways– Hypothesis: Left-spatial inattention in ADHD would

relate to DAT1 genotype

Left-spatial Inattention in ADHD

The Landmark Task

Subject is presented with a pre-bisected lineWhich end is the shortest?

Left-spatial Inattention in ADHD

The Landmark Task

Healthy subjects show a leftward bias and relative inattention to the rightward extent

“The right end of the line is the shortest!”

Left-spatial Inattention in ADHD

The Landmark Task

In left-neglect subjects show a rightward bias and relative inattention to the leftward extent

“The left end of the line is the shortest!”

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Group

Mag

nitu

de a

nd D

irect

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of M

ean

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mm

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Indi

ces Controls

Low-Risk-DAT1 ADHDHigh-Risk-DAT1 ADHD

High-Risk DAT1 ADHD group display left spatial inattentionBellgrove et al (2005), Neuropsychopharmacology

Exogenous Orienting Task

Exogenous Orienting Task

• If the observed relationship between DAT1 and left-sided inattention reflects dysfunction to spatial attentional systems then:

– Would predict a reorienting deficit for LVF targets (invalidly cued to the RVF) in DAT1 10-repeat homozygotes

Left-spatial inattention is related to Inattentive symptoms butcloser to the site of gene action (DAT1)

ADHD Inattentive SymptomsSymptoms

Neuropsych

Brain pathology

Genetic Factors DAT1

Left-spatial inattention

Right Striatal DysfunctionOveractive DAT

Pharmacogenetics:Left-spatial inattention as predictor

of therapeutic response to MPH

10-repeat DAT1 allele

Left-spatialinattention

Enhanced responseto MPH

Kirley et al, 2003Study 1

?

Hypothesis: Performance on the Landmark Task will predict an enhanced therapeutic response to MPH

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Mea

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sym

met

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sLow-Risk DAT1/ Mediocre ResponseLow-Risk DAT1/ Verygood responseHigh-Risk DAT1/ Mediocre responseHigh-Risk DAT1/ Verygood response

10-repeat DAT1 homozygotes who achieved a Very GoodResponse to MPH, displayed left-spatial inattention

Bellgrove et al (2005), Neuropsychopharmacology

Summary• Results support the existence of a subgroup of

ADHD that is associated with the 10-repeat DAT1 allele and is defined– 1) in neuropsychological terms, by left-spatial inattention.– 2) in symptomatological terms, by inattentive

symptomatology– 3) in pharmacogenomic terms, by an enhanced therapeutic

response to MPH.

• Left spatial inattention might predict therapeutic response to MPH because it acts as a proxy for DAT1 genotype and so transporters that are overactive, perhaps within the right striatum.

• MPH might be most efficacious for those children presenting with left-spatial inattention, because it indexes a hypodopaminergic state

DBH and DRD4 and Sustained Attention and Response Inhibition

• Children with ADHD experience problems with sustained attention and response inhibition

• Response inhibition deficits show familial risk profiles

• Response inhibition deficits are ameliorated by MPH– DBH- A2 allele of Taq I polymorphism associated with

ADHD– DRD4- 7-repeat allele of a VNTR associated with ADHD

– -521 SNP A allele shows trend towards association

– A-, relative to G-allele, reduces transcription levels of the DRD4 gene by up to 40%

The Sustained Attention to Response Test (SART)

Fixed Sequence Version

1,2,3,4,5,6,7,8,9,1,2,3

Sustained Attention

SART

Random Sequence Version

2,8,9,4,3,5,7,9,3,2,8,5

Sustained Attention +Response Inhibition

0

2

4

6

8

10

12

14

16

18

20

no high riskDBH (n=15)

one high riskDBH (n=24)

two high riskDBH (n=20)

Mea

n Fi

xed

SAR

T Er

rors

Mean Fixed SARTCommission ErrorsMean Fixed SARTOmission ErrorsMean Fixed SART TotalErrors

Fixed SART and Taq 1 DBH

Two high-risk DBH Group had sustained attention deficits on the Fixed SART

Bellgrove et al (in press), Cortex

Fixed SART and Taq 1 DBH: Healthy Undergraduates

DBH Taq I

2 A2 alleles0 or 1 A2 allele

Mea

n To

tal E

rror

s

22

20

18

16

14

12

10

8

6

4

-DBH Taq I

•As in ADHD, the A2 allele impairs sustained attention

Random SART and DRD4 GenotypeDRD 4 VNTR and Random SART

51015202530

7-absent (n=30) 7-present (n=20)

VNTR Group

Mea

n To

tal E

rror

sVNTR

•7-present group outperformed7-absent group in terms of Total errors and Variability only on the Random SART

•No effects on the Fixed SARTDRD4 -521 SNP and Random SART

51015202530

<2 A alleles (n=36) 2 A alleles (n=16)

-521 SNP Group

Mea

n To

tal E

rror

s -521 SNP

•A allele homozygotes performed worse than A allele heterozygotes in terms of Total errors and Variability

Bellgrove et al, (2005), AJMG, Part B

Random SART and DRD4 -521 SNP

DRD4 521 SNP

2 A alleles1 A allele0 A alleles

Mea

n To

tal E

rror

s

18

16

14

12

10

8

Random SART and DRD4 Genotype: Healthy Undergraduates

-521 SNP

•Parametric effect of the A allele on response inhibition

AG MFG

IFG

Inhibition Performance: Block 1

Disrupted Site

Control (Sham) IFG MFG AG

% C

orre

ct In

hibi

tions

40

45

50

55

60

65

70

Left Hand Right Hand

Response inhibition depends upon inferior frontal areas

* *

How do DRD4 variants influence activity within prefrontal regions, such as IFG?

Chambers, Bellgrove et al (in press), Journal of Cog Neuro

Summary

• Sustained attention shows genetic variation in ADHD– When inhibitory demands are minimal, DBH affects the ability

to endogenously maintain an alert state in ADHD– Response inhibition appears related to variation in the DRD4

gene.• DRD4 VNTR 7-repeat does not impair cognition• -521 SNP A allele does impair cognition

• DBH and DRD4 may confer risk to ADHD because of their varying effects on the development of fronto-parietal networks

Spatial Working Memory

• Spatial Working Memory deficits are reliably observed in ADHD– Meta-analysis shows large effect sizes

(Storage d’=0.85; Central Executive d’=1.06– Human and animal lesion studies show

regions in the prefrontal and parietal cortices are critical for SWM

– Dopamine and noradrenaline modulate SWM– Candidate endophenotype for ADHD?

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3 sec delay

Same or different?

+

SWM and DBH -1021 C-T

DBH -1021-C-T

2 T alleles1 T allele0 T alleles

Mea

n Ac

cura

cy

.85

.84

.83

.82

.81

.80

-DBH -1021 C-T•Parametric effect of the T allele on SWM•T allele is associated with lower plasma levels of DβH•Suggests that T allele lowers expression of DβH•Role in ADHD?

Conclusions

• Our studies provide evidence that molecular genetics can assist in dissecting complex phenotypes such as ADHD.– Inconsistencies in the neuropsychology of

ADHD may be clarified by this approach.

• Molecular genetics may provide a powerful new tool for studying individual differences and testing models of cognitive function.

Acknowledgements

• Work funded by the Irish Health Research Board (HRB) & Science Foundation Ireland (SFI)

• Currently supported under a NHMRC Howard Florey Centenary Fellowship

• Collaborators in Melbourne:– Alasdair Vance, Jason Mattingley, Ross Cunnington,

Chris Chambers• Collaborators in Dublin:

– Ian Robertson, Michael Gill, Ziarah Hawi, Katherine Johnson & Ciara Greene.