Non-Radical

Diagnostic

Biopsies

Do

Not

Negatively

Influence

Melanoma

Patient

Survival

Background

In

fair-skinned

Caucasian

populations

both

the

incidence

and

mortality

rates

of

cutaneous

melanoma

have

been

increasing

over

the

past

decades

With

adjuvant

therapies

still

being

under

investigation

early

detection

is

the

only

way

to

improve

melanoma

patient

survival

The

influence

of

incisional

biopsies

on

melanoma

patient

survival

has

been

discussed

for

many

years

This

study

investigates

both

the

influence

of

diagnostic

biopsy

type

and

the

presence

of

residual

tumor

cells

in

the

re-excision

specimen

on

disease

free

and

overall

survival

In

fair-skinned

Caucasian

populations

cutaneous

melanoma

is

an

important

growing

public

health

problem

causing

a

heavy

burden

on

healthcare

services

Both

its

incidence

and

mortality

rates

have

been

increasing

in

Europe

over

the

past

decades

The

absolute

total

number

of

new

cases

of

melanoma

in

the

Netherlands

is

expected

to

be

more

than

in

compared

with

around

in

The

Netherlands

as

many

other

countries

has

a

two-tiered

medical

care

system

in

which

patients

need

to

seek

a

medical

opinion

initially

from

a

general

practitioner

-LRB-

GP

-RRB-

before

referral

if

necessary

to

a

specialist

The

mean

number

needed

to

treat

-LRB-

NNT

-RRB-

defined

as

the

mean

number

of

pigmented

lesions

needed

to

be

excised

to

identify

one

melanoma

among

GPs

in

Perth

Australia

was

and

ranged

from

in

the

youngest

patients

-LRB-

years

-RRB-

to

in

the

oldest

patients

-LRB-

years

-RRB-

Assuming

that

for

each

new

case

of

melanoma

another

patients

with

pigmented

skin

lesions

will

visit

the

GP

the

demand

for

detection

will

increase

quite

markedly

With

adjuvant

therapies

still

being

under

investigation

early

detection

is

the

only

way

to

improve

melanoma

patient

survival

Dutch

guidelines

recommend

pigmented

skin

lesions

suspect

for

melanoma

to

be

removed

through

a

diagnostic

excision

biopsy

with

a

minimal

lateral

clearance

of

mm

This

recommendation

is

inline

with

the

advice

of

the

National

Institutes

of

Health

-LRB-

NIH

-RRB-

to

remove

any

suspicious

lesion

through

excisional

biopsy

with

a

narrow

margin

of

normal-appearing

skin

The

influence

of

an

incisional

biopsy

on

melanoma

patient

survival

has

been

discussed

for

many

years

and

different

investigators

have

found

contradicting

results

Two

recent

publications

concluding

incisional

biopsies

not

to

interfere

with

melanoma

patient

survival

were

not

able

to

end

this

discussion

since

patient

groups

were

not

fully

or

follow

up

-LRB-

FU

-RRB-

was

short

This

study

investigated

both

the

influence

of

diagnostic

biopsy

type

and

the

presence

of

residual

tumor

cells

in

the

re-excision

specimen

on

melanoma

patient

disease

free

survival

-LRB-

DFS

-RRB-

and

overall

survival

-LRB-

OS

-RRB-

in

patients

with

a

mean

FU

of

more

than

years

Survival

analysis

was

done

using

Cox

's

proportional

hazard

model

adjusted

for

gender

age

site

of

primary

melanoma

Breslow

thickness

type

of

melanoma

ulceration

lymphatic

invasion

and

sentinel

node

-LRB-

SN

-RRB-

status

Both

the

diagnostic

biopsy

type

and

the

presence

of

tumor

cells

in

the

re-excision

specimen

were

found

not

to

influence

melanoma

patient

survival

METHODS

Patients

Between

August

and

September

patients

were

diagnosed

with

clinical

stage

I/II

cutaneous

melanoma

according

to

criteria

of

the

American

Joint

Committee

on

Cancer

-LRB-

AJCC

-RRB-

and

underwent

re-excision

of

the

primary

melanoma

site

and

a

SN

biopsy

If

patients

were

referred

to

us

from

another

institution

the

pathologic

characteristics

of

the

primary

melanoma

were

reviewed

in

our

hospital

before

the

SN

procedure

All

patients

were

treated

according

to

the

same

protocol

re-excision

margins

were

cm

for

melanomas

with

a

Breslow

thickness

of

mm

and

cm

for

melanomas

with

a

Breslow

thickness

of

mm

To

identify

and

retrieve

the

SN

the

triple

technique

was

used

as

described

previously

In

short

the

day

before

surgery

patients

underwent

a

dynamic

and

static

lymphoscintigraphy

to

determine

the

lymphatic

drainage

pattern

Just

prior

to

surgery

Patent

Blue

V

-LRB-

Laboratoire

Guerbet

Aulnay-sous-Bois

France

-RRB-

was

injected

intradermally

next

to

the

initial

site

of

the

melanoma

During

surgery

guided

by

a

hand

held

gamma

probe

and

the

blue

staining

of

the

draining

tissues

the

SN

was

removed

To

investigate

the

influence

on

survival

patients

were

divided

both

according

to

their

diagnostic

biopsy

type

wide

excisional

biopsy

-LRB-

lateral

clearance

mm

-RRB-

narrow

excisional

biopsy

-LRB-

lateral

clearance

mm

-RRB-

excisional

biopsy

with

positive

margins

and

incisional

biopsy

-LRB-

includes

punch

-RRB-

and

the

presence

of

residual

tumor

cells

in

their

re-excision

specimen

Statistical

Analysis

Data

were

processed

with

the

Statistical

Package

for

the

Social

Sciences

software

for

Windows

-LRB-

SPSS

Chicago

IL

-RRB-

Cox

's

proportional

hazard

model

was

used

for

survival

analysis

P

values

were

considered

significant

RESULTS

Patient

Population

Between

August

and

September

patients

were

diagnosed

with

clinical

stage

I/II

cutaneous

melanoma

male

-LRB-

-RRB-

and

female

-LRB-

-RRB-

with

a

mean

age

of

years

-LRB-

Table

-RRB-

Most

primary

melanomas

were

located

on

the

trunk

-LRB-

-RRB-

or

on

the

lower

extremities

-LRB-

-RRB-

Breslow

thickness

was

categorized

into

four

groups

-LRB-

mm

mm

mm

mm

-RRB-

but

due

to

spontaneous

regression

of

the

primary

lesion

remained

unknown

in

patients

The

majority

of

patients

had

a

superficial

spreading

melanoma

-LRB-

-RRB-

or

a

nodular

melanoma

-LRB-

-RRB-

In

patients

the

type

of

melanoma

was

different

or

remained

unknown

-LRB-

-RRB-

Ulceration

defined

as

the

absence

of

intact

epidermis

overlying

the

major

portion

of

primary

melanoma

was

diagnosed

in

patients

-LRB-

-RRB-

unknown

in

patient

-LRB-

-RRB-

and

absent

in

patients

-LRB-

-RRB-

Lymphatic

invasion

was

present

in

patients

-LRB-

-RRB-

absent

in

patients

-LRB-

-RRB-

and

remained

unknown

in

patients

-LRB-

-RRB-

The

SN

was

negative

in

patients

-LRB-

-RRB-

and

positive

in

patients

-LRB-

-RRB-

In

patients

the

SN

was

not

removed

and

the

SN

status

remained

unknown

-LRB-

-RRB-

In

total

there

were

missing

variables

in

patients

all

were

excluded

from

the

study

SN

Identification

In

of

the

patients

the

SN

status

remained

unknown

-LRB-

-RRB-

in

of

these

patients

the

SN

was

located

in

the

deep

lobe

of

the

parotid

gland

and

in

one

patient

the

SN

was

located

high

in

the

left

axilla

in

all

cases

the

decision

was

made

not

to

remove

the

SN

to

avoid

potential

morbidity

associated

with

the

intervention

The

SN

was

not

identified

in

cases

due

to

non-visualization

by

preoperative

lymphoscintigraphy

In

one

patient

the

SN

was

located

in

the

right

axilla

and

could

not

be

removed

because

the

patient

was

suffering

from

frozen

shoulder

syndrome

the

physical

condition

of

another

patient

did

not

allow

further

treatment

Therefore

the

success

rate

of

SN

identification

was

-LRB-

of

patients

-RRB-

Two

of

the

patients

with

the

SN

located

in

the

deep

lobe

of

the

parotid

gland

experienced

metastasis

of

the

parotid

gland

one

patient

is

still

alive

with

disease

and

one

patient

is

dead

of

disease

The

patient

whose

physical

condition

did

not

allow

further

treatment

passed

away

soon

after

re-excision

of

the

primary

melanoma

site

from

massive

hematogenic

and

lymphogenic

metastasis

The

remaining

patients

have

shown

no

evidence

of

disease

Diagnostic

Biopsy

Type

and

Survival

The

influence

of

diagnostic

biopsy

type

on

DFS

and

OS

was

tested

in

patients

with

a

mean

FU

of

more

than

years

patients

-LRB-

-RRB-

underwent

a

wide

excision

biopsy

patients

-LRB-

-RRB-

a

narrow

excision

biopsy

patients

-LRB-

-RRB-

an

excision

biopsy

with

positive

margins

and

patients

-LRB-

-RRB-

an

incision

biopsy

-LRB-

Table

-RRB-

TABLE

Patient

characteristicsCharacteristicsPatients

-LRB-

n

-RRB-

Follow

up

-LRB-

years

-RRB-

Mean

-LRB-

SD

-RRB-

-LRB-

-RRB-

Gender

-LRB-

-RRB-

-LRB-

-RRB-

Age

-LRB-

years

-RRB-

Mean

-LRB-

SD

-RRB-

-LRB-

-RRB-

Site

of

primary

melanoma

Lower

-LRB-

-RRB-

Upper

extremity

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Breslow

thickness

-LRB-

mm

-RRB-

x

-LRB-

-RRB-

x

-LRB-

-RRB-

x

-LRB-

-RRB-

-LRB-

-RRB-

Unknown

-LRB-

regression

-RRB-

-LRB-

-RRB-

Type

of

melanoma

Superficial

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Ulceration

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Lymphatic

invasion

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

Sentinel

node

status

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

TABLE

Patient

distribution

according

to

-LRB-

A

-RRB-

diagnostic

biopsy

type

and

-LRB-

B

-RRB-

the

presence

of

residual

tumor

cells

in

the

re-excision

specimenAPatients

-LRB-

n

-RRB-

Follow

up

-LRB-

years

mean

SD

-RRB-

Diagnostic

biopsy

type

Wide

excision

biopsy

-LRB-

mm

-RRB-

-LRB-

-RRB-

Narrow

excision

biopsy

-LRB-

x

mm

-RRB-

-LRB-

-RRB-

Excision

biopsy

with

positive

-LRB-

-RRB-

Incision

-LRB-

-RRB-

BPatients

-LRB-

n

-RRB-

Follow

up

-LRB-

years

mean

SD

-RRB-

Re

excision

specimen

No

residual

tumor

-LRB-

-RRB-

Residual

tumor

-LRB-

-RRB-

In

patients

-LRB-

-RRB-

the

SN

was

positive

patients

-LRB-

-RRB-

after

a

wide

excision

biopsy

patients

-LRB-

-RRB-

after

a

narrow

excision

biopsy

patients

-LRB-

-RRB-

after

an

excision

biopsy

with

positive

margins

and

patients

-LRB-

-RRB-

after

an

incisional

biopsy

In

patients

-LRB-

-RRB-

a

recurrence

was

found

during

FU

patients

-LRB-

-RRB-

after

a

wide

excision

biopsy

-LRB-

locoregional

skin

SN

basin

and

systemic

-RRB-

patients

-LRB-

-RRB-

after

a

narrow

excision

biopsy

-LRB-

locoregional

skin

SN

basin

and

systemic

-RRB-

patients

-LRB-

-RRB-

after

an

excision

biopsy

with

positive

margins

-LRB-

locoregional

skin

SN

basin

and

systemic

-RRB-

and

patients

-LRB-

-RRB-

after

an

incision

biopsy

-LRB-

locoregional

skin

and

systemic

-RRB-

But

confounding

factors

were

not

equally

distributed

between

the

diagnostic

biopsy

groups

Therefore

survival

analysis

was

done

using

Cox

's

proportional

hazard

model

adjusted

for

gender

age

site

of

primary

melanoma

Breslow

thickness

type

of

melanoma

ulceration

lymphatic

invasion

and

sentinel

node

-LRB-

SN

-RRB-

status

In

univariate

analysis

gender

age

Breslow

thickness

type

of

melanoma

ulceration

lymphatic

invasion

and

SN

status

were

all

significantly

related

to

both

DFS

and

OS

-LRB-

Table

-RRB-

Site

of

primary

melanoma

on

the

head/neck

was

in

univariate

analysis

only

significantly

related

to

DFS

-LRB-

Table

-RRB-

Diagnostic

biopsy

type

did

not

have

a

significant

relation

with

either

DFS

or

OS

-LRB-

Table

-RRB-

TABLE

Univariate

and

multivariate

Cox

regression

analysis

of

disease-free

survival

and

overall

survival

according

to

diagnostic

biopsy

typeDisease

Free

SurvivalOverall

SurvivalUnivariateMultivariateUnivariateMultivariateTested

CIPDiagnostic

biopsy

type

Wide

excision

Narrow

excision

Excision

biopsy

with

positive

Incision

Age

-LRB-

years

-RRB-

Site

of

primary

melanoma

Lower

Upper

Breslow

thickness

-LRB-

mm

-RRB-

x

x

x

Type

of

melanoma

Ulceration

Lymphatic

invasion

Sentinel

node

status

Even

though

diagnostic

biopsy

type

did

not

have

a

significant

relation

with

either

DFS

or

OS

in

univariate

Cox

regression

analysis

it

was

also

tested

in

multivariate

Cox

regression

analysis

together

with

all

significant

variables

from

univariate

analysis

to

investigate

its

influence

on

DFS

and

OS

after

correction

for

the

confounding

factors

In

multivariate

analysis

the

only

significant

and

independent

predictors

of

both

DFS

and

OS

were

age

Breslow

thickness

lymphatic

invasion

and

SN

status

-LRB-

Table

-RRB-

Site

of

primary

melanoma

on

the

lower

extremities

or

head/neck

and

ulceration

were

in

multivariate

analysis

only

significant

and

independent

predictors

of

DFS

-LRB-

Table

-RRB-

Also

in

multivariate

analysis

diagnostic

biopsy

type

did

not

have

a

significant

relation

with

either

DFS

or

OS

-LRB-

Fig.

B

-RRB-

FIG.

A

Disease

free

survival

rates

and

B

overall

survival

rates

according

to

the

initial

biopsy

type

The

same

analysis

was

done

after

combining

the

groups

the

wide

excision

biopsy

group

was

joined

with

the

narrow

excision

biopsy

group

and

compared

to

the

excision

biopsy

group

with

positive

margins

joined

with

the

incision

biopsy

group

Still

diagnostic

biopsy

type

did

not

have

a

significant

influence

on

either

DFS

or

OS

-LRB-

data

not

shown

-RRB-

Residual

Tumor

Cells

in

the

Re-Excision

Specimen

and

Survival

Patients

-LRB-

-RRB-

underwent

re-excision

of

the

primary

melanoma

site

and

in

patients

-LRB-

-RRB-

the

diagnostic

biopsy

was

found

to

be

sufficient

In

patients

-LRB-

-RRB-

residual

tumor

cells

were

found

in

the

re-excision

specimen

-LRB-

Table

-RRB-

All

patients

with

residual

tumor

cells

in

the

re-excision

specimen

underwent

either

an

excision

biopsy

with

positive

margins

or

an

incision

biopsy

In

of

the

patients

with

a

wide

or

narrow

excision

biopsy

residual

tumor

cells

were

found

In

univariate

analysis

gender

age

Breslow

thickness

type

of

melanoma

ulceration

lymphatic

invasion

and

SN

status

were

all

significantly

related

to

both

DFS

and

OS

-LRB-

Table

-RRB-

Site

of

primary

melanoma

on

the

lower

extremity

or

head/neck

was

in

univariate

analysis

significantly

related

to

DFS

and

site

of

primary

melanoma

on

the

head/neck

or

trunk

was

significantly

related

to

OS

-LRB-

Table

-RRB-

Residual

tumor

cells

in

the

re-excision

specimen

did

not

have

a

significant

relation

with

either

DFS

or

OS

-LRB-

Table

-RRB-

TABLE

Univariate

and

multivariate

Cox

regression

analysis

of

disease-free

survival

and

overall

survival

according

to

the

presence

of

residual

tumor

cells

in

the

re-excision

specimenDisease

Free

SurvivalOverall

CIPResidual

tumor

Age

-LRB-

years

-RRB-

Site

of

primary

melanoma

Lower

Upper

Breslow

thickness

-LRB-

mm

-RRB-

x

x

x

Type

of

melanoma

Ulceration

Lymphatic

invasion

Sentinel

node

status

Even

though

residual

tumor

cells

in

the

re-excision

specimen

did

not

have

a

significant

relation

with

either

DFS

or

OS

in

univariate

Cox

regression

analysis

it

was

also

tested

in

multivariate

Cox

regression

analysis

together

with

all

significant

variables

from

univariate

analysis

to

investigate

its

influence

on

DFS

and

OS

after

correction

for

the

confounding

factors

In

multivariate

analysis

the

only

significant

and

independent

predictors

of

both

DFS

and

OS

were

age

site

of

primary

melanoma

on

the

head/neck

Breslow

thickness

lymphatic

invasion

and

SN

status

-LRB-

Table

-RRB-

Also

in

multivariate

analysis

residual

tumor

cells

in

the

re-excision

specimen

did

not

have

a

significant

relation

with

either

DFS

or

OS

-LRB-

Fig.

B

-RRB-

FIG.

A

Disease

free

survival

rates

and

B

overall

survival

rates

according

to

the

presence

of

residual

tumor

cells

in

the

re-excision

specimen

Consistent

Confounders

of

Melanoma

Patient

Survival

Age

Breslow

thickness

lymphatic

invasion

and

SN

status

were

the

most

consistent

independent

and

significant

confounders

of

melanoma

patient

DFS

and

OS

-LRB-

Table

-RRB-

Site

of

the

primary

melanoma

was

not

always

an

independent

and

significant

confounder

of

melanoma

patient

survival

but

location

on

the

head/neck

region

consistently

carried

the

highest

hazard

ratio

-LRB-

HR

-RRB-

-LRB-

Table

-RRB-

Surprisingly

ulceration

was

not

an

independent

and

significant

confounder

of

OS

-LRB-

Table

-RRB-

DISCUSSION

Numerous

investigators

have

studied

the

influence

of

incisional

biopsy

on

melanoma

patient

survival

and

found

contradicting

results

Fitzpatrick

et

al.

found

a

five-year

OS

rate

of

in

the

incisional

biopsy

group

as

compared

to

in

the

excisional

biopsy

group

but

the

different

biopsy

groups

were

not

matched

for

important

prognostic

factors

Epstein

et

al.

found

a

more

favorable

ten-year

OS

in

the

biopsy

group

-LRB-

-RRB-

as

compared

to

the

primary

wide

excision

group

-LRB-

-RRB-

but

biopsy

was

loosely

defined

as

less

than

optimal

or

complete

surgical

excision

Ironside

et

al.

found

a

five-year

OS

rate

of

in

the

excision

and

in

the

incision

biopsy

group

but

failed

to

describe

the

distribution

of

prognostic

factors

between

both

groups

Rampen

et

al.

also

found

a

worse

prognosis

for

patients

after

an

incision

biopsy

-LRB-

patients

-RRB-

but

the

study

was

small

and

retrospective

Griffiths

et

al.

found

no

difference

in

survival

but

incisional

biopsy

patients

were

excluded

because

of

missing

histopathological

data

Survival

rates

were

not

significantly

different

between

the

incision

and

excision

biopsy

groups

of

Lederman

et

al.

but

patient

groups

were

matched

for

Breslow

thickness

only

Lees

et

al.

also

indicated

no

significant

averse

effect

of

incisional

biopsy

in

patients

but

of

the

histopathological

data

was

not

assessable

Austin

et

al

did

find

a

significantly

reduced

survival

in

the

incisional

biopsy

group

but

patients

in

the

incisional

biopsy

group

were

also

significantly

older

The

two

most

recent

publications

found

no

negative

influence

of

incisional

biopsies

on

melanoma

patient

survival

but

in

Bong

et

al.

patients

groups

were

not

fully

matched

and

in

Martin

et

al.

median

FU

was

only

months

This

study

not

only

investigated

the

influence

of

diagnostic

biopsy

type

but

also

the

presence

of

residual

tumor

cells

in

the

re-excision

specimen

on

melanoma

patient

survival

in

patient

groups

adjusted

for

important

confounders

of

survival

with

a

mean

FU

years

Excision

biopsies

with

positive

margins

and

incisional

biopsies

were

found

not

to

influence

melanoma

patient

survival

Interestingly

DFS

and

OS

even

seemed

slightly

better

in

the

non-radical

biopsy

groups

-LRB-

Fig.

B

-RRB-

In

line

with

this

patients

with

residual

tumor

cells

in

their

re-excision

specimen

also

had

a

slightly

better

survival

as

compared

to

patients

without

residual

tumor

cells

in

their

re-excision

specimen

-LRB-

Fig.

B

-RRB-

Melanoma

is

the

most

immunogenic

tumor

identified

to

date

melanoma

specific

T

cells

are

detectable

both

in

the

blood

and

in

tumor

draining

lymph

nodes

from

melanoma

patients

and

their

frequency

can

be

increased

by

specific

vaccination

This

intrinsic

immunogenicity

makes

melanoma

lesions

particularly

amenable

to

therapeutic

approaches

aimed

at

strengthening

tumor

immune

surveillance

Did

residual

tumor

cells

combined

with

biopsy

induced

wound

healing

trigger

the

melanoma

patient

's

immunesystem

In

non-melanoma

skin

cancer

-LRB-

NMSC

-RRB-

biopsy

induced

tumor

regression

has

previously

been

described

Swetter

et

al

reported

that

of

NMSCs

transected

on

the

initial

biopsy

showed

no

residual

tumor

in

the

excision

specimens

and

suggested

biopsy

induced

wound

healing

to

play

an

important

role

Even

though

non-radical

diagnostic

biopsies

and

residual

tumor

cells

in

the

re-excision

specimen

do

not

have

a

negative

influence

on

melanoma

patient

survival

the

routine

use

of

incision

biopsies

is

not

recommended

Incisional

biopsies

often

consist

only

of

a

small

percentage

of

the

surface

area

of

the

pigmented

skin

lesion

making

it

difficult

to

sample

a

representative

area

within

the

tumor

Somach

et

al

found

that

of

the

histopathological

features

were

more

pronounced

in

the

re-excision

specimen

as

compared

to

the

incision

biopsy

and

in

areas

of

invasive

melanoma

not

detected

in

the

incisional

biopsy

were

observed

in

the

re-excision

specimen

Further

more

when

melanoma

is

diagnosed

attempting

to

evaluate

the

depth

of

invasion

in

an

incisional

biopsy

is

treacherous

and

may

lead

to

over

or

underestimation

of

the

invasion

Of

course

these

problems

are

less

prominent

in

excision

biopsies

with

positive

margins

here

the

majority

of

the

lesion

has

been

removed

and

only

the

outer

borders

are

compromised

making

a

sampling

error

highly

unlikely

In

conclusion

age

Breslow

thickness

lymphatic

invasion

and

SN

status

were

the

most

consistent

independent

and

significant

confounders

of

melanoma

patient

DFS

and

OS

The

site

of

primary

melanoma

and

ulceration

were

also

important

confounders

of

survival

Both

the

diagnostic

biopsy

type

and

the

presence

of

residual

tumor

cells

in

the

re-excision

specimen

did

not

have

a

negative

influence

on

melanoma

patient

DFS

and

OS

With

melanoma

incidence

rates

and

early

detection

of

melanoma

still

being

the

only

way

to

improve

melanoma

patient

survival

it

is

important

for

all

physicians

to

feel

confident

about

removing

a

pigmented

skin

lesion

suspect

for

melanoma

Incisional

biopsies

are

not

recommended

but

there

is

no

cause

for

concern

when

an

excision

biopsy

turns

out

to

have

positive

margins

O B O O O O O B O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O B O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O B O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O B O O O O O O O B O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O


Evaluation

of

the

Serotonergic

Genes

and

in

Aggressive

Behavior

of

Golden

Retriever

Dogs

Aggressive

behavior

displays

a

high

heritability

in

our

study

group

of

Golden

Retriever

dogs

Alterations

in

brain

serotonin

metabolism

have

been

described

in

aggressive

dogs

before

Here

we

evaluate

whether

four

genes

of

the

canine

serotonergic

system

coding

for

the

serotonin

receptors

and

and

the

serotonin

transporter

could

play

a

major

role

in

aggression

in

Golden

Retrievers

We

performed

mutation

screens

linkage

analysis

an

association

study

and

a

quantitative

genetic

analysis

There

was

no

systematic

difference

between

the

coding

DNA

sequence

of

the

candidate

genes

in

aggressive

and

non-aggressive

Golden

Retrievers

An

affecteds-only

parametric

linkage

analysis

revealed

no

strong

major

locus

effect

on

human-directed

aggression

related

to

the

candidate

genes

An

analysis

of

single

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

in

the

Mb

regions

flanking

the

genes

in

unrelated

human-directed

aggressive

and

unrelated

non-aggressive

dogs

did

not

show

association

of

SNP

alleles

genotypes

or

haplotypes

with

aggression

at

the

candidate

loci

We

completed

our

analyses

with

a

study

of

the

effect

of

variation

in

the

candidate

genes

on

a

collection

of

aggression-related

phenotypic

measures

The

effects

of

the

candidate

gene

haplotypes

were

estimated

using

the

Restricted

Maximum

Likelihood

method

with

the

haplotypes

included

as

fixed

effects

in

a

linear

animal

model

We

observed

no

effect

of

the

candidate

gene

haplotypes

on

a

range

of

aggression-related

phenotypes

thus

extending

our

conclusions

to

several

types

of

aggressive

behavior

We

conclude

that

it

is

unlikely

that

these

genes

play

a

major

role

in

the

variation

in

aggression

in

the

Golden

Retrievers

that

we

studied

Smaller

phenotypic

effects

of

these

loci

could

not

be

ruled

out

with

our

sample

size

Introduction

Dogs

have

been

living

in

close

proximity

to

humans

for

at

least

years

-LRB-

Clutton-Brock

-RRB-

Behavior

has

been

a

strong

selective

factor

in

the

domestication

and

breeding

of

dogs

According

to

the

breed

standard

Golden

Retriever

dogs

should

have

a

friendly

character

-LRB-

link

accessed

March

-RRB-

However

there

are

reports

of

very

aggressive

Golden

Retrievers

-LRB-

Galac

and

Knol

Heath

-RRB-

We

recently

described

the

behavioral

phenotype

of

Golden

Retrievers

referred

to

our

clinic

for

aggression

problems

and

Golden

Retrievers

that

were

recruited

because

they

were

related

to

one

or

more

of

the

aggressive

dogs

-LRB-

van

den

Berg

et

al.

-RRB-

The

phenotypes

were

based

on

mail

questionnaires

and

on

personal

interviews

with

dog

owners

In

a

quantitative

genetic

study

including

Golden

Retrievers

we

found

a

heritability

of

for

the

traits

of

human-directed

aggression

and

dog-directed

aggression

-LRB-

Liinamo

et

al.

-RRB-

The

influence

of

serotonin

-LRB-

-RRB-

on

aggressive

behavior

has

been

studied

extensively

-LRB-

reviewed

by

Berman

and

Coccaro

Gingrich

and

Hen

Lesch

and

Merschdorf

-RRB-

There

is

evidence

for

a

role

of

the

system

in

canine

aggression

as

well

For

instance

Reisner

and

colleagues

-LRB-

-RRB-

reported

decreased

levels

of

acid

-LRB-

the

major

metabolite

of

-RRB-

in

cerebrospinal

fluid

of

dominant

aggressive

dogs

Badino

et

al.

-LRB-

-RRB-

found

modifications

of

receptor

concentrations

in

brains

of

aggressive

dogs

Domestication

of

silver

foxes

which

are

taxonomically

close

relatives

of

dogs

seems

to

cause

modifications

in

the

system

-LRB-

see

Trut

for

a

review

-RRB-

The

role

of

in

canine

aggression

is

further

supported

by

two

small

clinical

studies

where

pharmacological

or

dietary

intervention

in

the

system

was

shown

to

modulate

aggressive

behavior

-LRB-

DeNapoli

et

al.

Dodman

et

al.

-RRB-

Four

genes

that

code

for

factors

involved

in

serotonergic

neurotransmission

are

particularly

good

candidates

for

the

regulation

of

aggressive

behavior

the

serotonin

receptor

genes

-LRB-

-RRB-

-LRB-

-RRB-

and

-LRB-

-RRB-

and

the

serotonin

transporter

gene

-LRB-

-RRB-

Serotonin

receptor

plays

a

role

in

anxiety

stress

response

and

aggression

-LRB-

Olivier

et

al.

-RRB-

knockout

mice

show

increased

anxiety

and

stress

response

and

an

antidepressant-like

phenotype

-LRB-

Heisler

et

al.

Ramboz

et

al.

-RRB-

In

the

above-mentioned

studies

of

silver

foxes

the

researchers

observed

a

lower

density

of

receptors

in

the

hypothalamus

of

tame

foxes

compared

to

their

wild

counterparts

-LRB-

Popova

et

al.

-RRB-

Many

studies

have

suggested

involvement

of

in

the

etiology

of

mental

disorders

For

instance

Huang

et

al.

-LRB-

-RRB-

and

Sanders

et

al.

-LRB-

-RRB-

reported

an

association

between

one

of

the

polymorphisms

in

the

human

gene

and

alcoholism

suicidality

and

obsessive-compulsive

disorder

Knockout

mice

lacking

display

increased

aggression

-LRB-

Saudou

et

al.

-RRB-

A

mutation

in

the

human

gene

is

associated

with

altered

binding

which

has

been

implicated

in

schizophrenia

suicidal

behavior

impaired

impulse

control

and

aggression

history

-LRB-

Abdolmaleky

et

al.

Bjork

et

al.

Khait

et

al.

-RRB-

Peremans

and

colleagues

-LRB-

-RRB-

found

an

increased

binding

index

of

serotonin

receptors

in

cortical

brain

regions

of

impulsive

aggressive

dogs

A

polymorphism

in

the

promoter

region

of

influences

serotonin

transporter

density

in

the

brain

and

is

associated

with

mental

disorders

in

humans

-LRB-

Anguelova

et

al.

Hariri

et

al.

Lesch

et

al.

-RRB-

knockout

mice

show

reduced

aggression

-LRB-

Holmes

et

al.

-RRB-

In

this

paper

we

test

the

hypothesis

that

there

is

a

strong

effect

of

variation

in

these

genes

on

the

variation

in

aggression

in

Golden

Retrievers

We

performed

mutation

screens

of

the

coding

DNA

sequence

in

unrelated

aggressive

Golden

Retrievers

In

addition

we

used

linkage

analysis

to

determine

the

likelihood

of

the

presence

of

a

strong

aggression

locus

in

or

close

to

the

genes

in

several

dog

families

Third

we

used

unrelated

aggressive

Golden

Retrievers

and

unrelated

non-aggressive

Golden

Retrievers

to

search

for

association

of

alleles

of

SNPs

flanking

the

candidate

genes

with

the

trait

of

human-directed

aggression

To

complete

our

analyses

we

evaluated

the

effects

of

variation

in

the

genes

on

a

range

of

aggression-related

phenotypes

using

the

same

models

as

in

Liinamo

et

al.

-LRB-

-RRB-

extended

to

include

the

effects

of

the

most

common

candidate

gene

haplotypes

Materials

and

methods

Animals

DNA

isolation

and

phenotyping

We

have

collected

behavioral

information

of

privately

owned

Golden

Retrievers

This

group

includes

dogs

that

were

referred

to

our

clinic

because

of

their

aggressive

behavior

-LRB-

probands

-RRB-

and

relatives

of

probands

DNA

samples

were

available

for

of

these

dogs

In

addition

we

collected

DNA

of

a

cohort

of

random

privately

owned

Goldens

that

were

born

between

July

and

February

No

phenotypes

were

available

for

these

random

dogs

Genomic

DNA

was

isolated

from

whole

blood

leucocytes

using

a

standard

protocol

-LRB-

Miller

et

al.

-RRB-

For

each

type

of

analysis

-LRB-

mutation

analysis

linkage

analysis

association

analysis

and

quantitative

genetic

analysis

-RRB-

we

selected

a

study

group

that

was

suited

for

the

study

design

The

study

groups

are

described

in

the

Supplementary

Information

I

We

have

collected

various

quantitative

measures

of

aggressiveness

for

the

dogs

-LRB-

van

den

Berg

et

al.

-RRB-

In

the

linkage

and

association

analysis

we

focused

on

one

of

these

measures

the

dog

owners

impression

on

human-directed

aggression

Owner

impressions

were

collected

in

a

personal

interview

We

asked

the

owners

if

their

dog

was

aggressive

towards

humans

and

the

status

of

the

dog

was

coded

in

three

classes

non-aggressive

-LRB-

score

-RRB-

threatens

-LRB-

score

-RRB-

or

bites

-LRB-

score

-RRB-

We

focused

on

human-directed

aggression

because

the

majority

of

the

probands

were

referred

to

our

clinic

for

human-directed

aggressive

behavior

Owner

impressions

were

available

for

all

dogs

and

the

quantitative

genetic

analyses

showed

that

the

heritability

of

this

trait

was

high

in

our

population

of

dogs

-LRB-

Liinamo

et

al.

-RRB-

Mutation

screening

We

analyzed

the

coding

DNA

sequence

-LRB-

CDS

-RRB-

of

the

four

candidate

genes

in

seven

-LRB-

and

-RRB-

or

eight

-LRB-

and

-RRB-

probands

The

CDS

were

amplified

and

sequenced

using

overlapping

primer

pairs

as

described

previously

-LRB-

van

den

Berg

et

al.

-RRB-

Possible

functional

effects

of

polymorphisms

were

predicted

with

POLYPHEN

-LRB-

-RRB-

Effects

of

polymorphisms

close

to

splice

sites

were

predicted

with

three

splice

prediction

programs

-LRB-

Brunak

et

al.

-RRB-

Splice

Prediction

by

Neural

Network

-LRB-

Reese

et

al.

-RRB-

and

SpliceSiteFinder

-LRB-

Shapiro

and

Senapathy

-RRB-

Linkage

analysis

We

used

nine

families

for

linkage

analysis

-LRB-

Figs

of

Supplementary

Information

I

-RRB-

DNA

samples

were

available

for

affected

and

unaffected

dogs

from

these

families

We

converted

the

owner

impression

about

human-directed

aggression

into

a

dichotomous

variable

for

the

linkage

analysis

-LRB-

see

Supplementary

Information

I

-RRB-

We

have

described

polymorphic

markers

for

the

candidate

genes

before

-LRB-

van

den

Berg

et

al.

-RRB-

We

selected

three

microsatellite

markers

and

seven

single

nucleotide

polymorphisms

for

linkage

analysis

-LRB-

see

Table

-RRB-

Microsatellite

markers

were

genotyped

after

PCR

on

an

ABI

Genetic

Analyzer

-LRB-

Applied

Biosystems

Foster

City

CA

-RRB-

PCR

conditions

were

described

by

van

den

Berg

et

al.

-LRB-

-RRB-

GENESCAN

software

was

used

for

genotype

assessment

Single

nucleotide

polymorphism

genotyping

was

performed

by

DNA

sequencing

of

PCR

products

on

the

ABI

Genetic

Analyzer

-LRB-

van

den

Berg

et

al.

-RRB-

The

DNA

sequence

chromatograms

were

inspected

using

LASERGENE

software

-LRB-

DNASTAR

Inc.

Madison

WI

USA

-RRB-

We

combined

several

markers

into

haplotypes

for

the

genes

and

For

dogs

that

were

heterozygous

for

multiple

markers

we

deduced

the

haplotypes

from

the

data

of

relatives

If

this

was

not

possible

we

assigned

the

most

frequently

observed

possible

haplotypes

to

these

dogs

Table

used

for

linkage

analysisGeneaType

of

markerbPosition

of

markercAlleles

observed

-LRB-

allele

frequency

-RRB-

dHaplotypes

observed

-LRB-

haplotype

frequency

-RRB-

-LRB-

CA

-RRB-

n

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

C

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

G/A

-LRB-

-RRB-

A

-LRB-

-RRB-

T/C

-LRB-

-RRB-

C

-LRB-

-RRB-

G/C

-LRB-

-RRB-

C

-LRB-

-RRB-

-LRB-

GA

-RRB-

n

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

SNPIVS

-LRB-

-RRB-

T

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

CA

-RRB-

n

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

T

-LRB-

-RRB-

C-G

-LRB-

-RRB-

T-A

-LRB-

-RRB-

G/A

-LRB-

-RRB-

A

-LRB-

-RRB-

respectively

serotonin

receptor

and

gene

serotonin

transporter

genebSNP

single

nucleotide

polymorphism

Names

of

microsatellite

markers

have

been

included

in

bracketscPosition

refers

to

the

coding

sequence

of

the

canine

gene

We

used

the

nomenclature

recommended

by

den

Dunnen

and

Antonarakis

-LRB-

-RRB-

the

A

of

the

ATG

start

codon

is

designated

number

the

nucleotide

to

this

A

is

numbered

and

the

nucleotide

of

the

translation

termination

codon

is

Positions

in

introns

refer

to

the

nearest

exon

The

nomenclature

of

the

introns

is

based

on

the

human

gene

structure

IVS

intervening

sequencedAllele

and

haplotype

frequencies

were

determined

in

a

group

of

-LRB-

and

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

parentseThis

polymorphism

is

nonsynonymous

We

performed

a

parametric

affecteds-only

linkage

analysis

to

determine

whether

the

candidate

gene

haplotypes

were

linked

to

aggressive

behavior

in

the

Golden

Retriever

families

Marker

haplotype

frequencies

were

determined

in

a

group

of

-LRB-

and

-RRB-

-LRB-

-RRB-

or

-LRB-

-RRB-

parent

dogs

-LRB-

see

Supplementary

Information

I

-RRB-

The

mode

of

inheritance

of

the

aggressive

phenotype

in

our

families

is

unclear

We

therefore

analyzed

the

data

under

both

autosomal

dominant

and

autosomal

recessive

models

The

penetrance

of

the

genotype

at

risk

was

set

at

In

this

way

affected

dogs

are

assumed

to

have

the

risk

allele

and

the

software

calculated

likelihood

that

aggressive

dogs

share

alleles

by

descent

from

a

common

ancestor

Unaffected

dogs

with

the

genotype

at

risk

have

no

effect

on

the

outcome

of

the

calculation

We

assumed

that

there

were

no

phenocopies

in

the

families

and

we

assumed

genetic

homogeneity

because

all

probands

were

related

to

each

other

within

a

limited

number

of

generations

-LRB-

not

shown

-RRB-

The

frequency

of

the

aggression

allele

was

set

at

to

allow

for

multiple

transmitting

ancestors

in

the

pedigrees

SUPERLINK

software

was

used

to

calculate

two-point

logarithm

of

the

odds

-LRB-

LOD

-RRB-

scores

-LRB-

Fishelson

and

Geiger

-RRB-

In

order

to

estimate

the

power

of

the

pedigrees

we

calculated

the

maximum

obtainable

LOD

scores

Affected

individuals

were

assigned

haplotypes

in

these

calculations

unaffected

parents

were

assigned

haplotypes

and

other

unaffected

individuals

were

assigned

haplotypes

We

assigned

haplotypes

-LRB-

unknown

-RRB-

to

dogs

from

which

we

did

not

have

a

DNA

sample

We

assumed

that

there

were

four

alleles

of

the

hypothetical

marker

with

equal

allele

frequencies

Association

study

To

test

for

a

more

complex

genetic

effect

of

variants

of

the

candidate

genes

we

performed

an

association

study

Fifty

aggressive

Golden

Retrievers

were

selected

from

our

database

The

main

selection

criteria

were

high

estimated

breeding

values

for

human-directed

aggression

and

as

little

interrelationship

among

the

cases

as

possible

Non-aggressive

dogs

were

selected

for

low

estimated

breeding

values

for

both

human

and

dog-directed

aggression

We

avoided

an

excess

of

relationship

within

either

the

case

or

the

control

group

The

non-aggressive

group

was

completed

with

dogs

from

the

random

group

A

more

detailed

description

of

cases

and

controls

is

provided

in

Supplementary

Information

I

We

genotyped

a

total

number

of

SNPs

in

these

Golden

Retrievers

using

customized

Affymetrix

Genotyping

Arrays

The

SNPs

were

selected

for

the

chip

using

a

scoring

system

that

optimized

the

SNPs

accounting

for

low

repeat

content

low

likelihood

of

SNPs

in

the

assay

probe

sequences

and

their

distribution

over

the

genome

as

a

whole

-LRB-

Lindblad-Toh

personal

communication

-RRB-

Twenty

probes

interrogated

each

locus

with

genotyping

calls

made

using

the

algorithm

BRLMM

-LRB-

-RRB-

which

analyses

intensities

for

sets

of

probes

that

interrogate

both

forward

and

reverse

sequences

with

perfect

match

and

mismatched

probes

Dogs

with

call

rates

of

lower

than

were

discarded

from

the

analysis

The

total

set

of

SNPs

was

filtered

for

genotype

call

probability

heterozygosity

rate

and

call

rate

across

a

large

set

of

dogs

reducing

the

dataset

to

SNPs

From

this

set

only

SNPs

within

Mb

of

the

four

candidate

genes

were

used

for

the

analysis

described

in

this

paper

There

were

SNPs

within

Mb

of

within

Mb

of

within

Mb

of

and

within

Mb

of

From

these

SNPs

we

selected

SNPs

with

a

minor

allele

frequency

and

call

rates

of

We

used

Haploview

software

version

-LRB-

Barrett

et

al.

-RRB-

for

the

analysis

of

the

presence

of

Hardy-Weinberg

equilibrium

-LRB-

HWE

-RRB-

the

local

association

analysis

and

the

calculation

of

pairwise

linkage

disequilibrium

-LRB-

LD

-RRB-

between

the

SNPs

A

HWE

P-value

cutoff

of

was

used

We

used

Bonferonni

correction

to

account

for

multiple

testing

in

the

association

analysis

Genotype

frequencies

in

cases

and

controls

were

compared

with

Chi

square

tests

using

SPSS

software

Two

tailed

Fisher

's

exact

tests

were

used

when

the

number

of

expected

cases

was

less

than

in

more

than

of

the

categories

Haplotype

blocks

were

formed

using

three

methods

in

Haploview

-LRB-

confidence

intervals

four

gamete

rule

and

solid

spine

of

LD

-RRB-

For

all

possible

combinations

we

performed

permutations

to

obtain

empirical

P

values

for

haplotype

association

tests

We

used

the

genetic

power

calculator

prepared

by

Purcell

to

estimate

the

power

of

the

association

analysis

-LRB-

-RRB-

The

following

assumptions

were

made

high-risk

allele

frequency

prevalence

The

mean

pairwise

D

between

the

SNPs

flanking

a

candidate

gene

was

used

as

an

estimate

of

the

local

D

in

these

estimations

Calculations

were

performed

for

two

different

genotype

relative

risks

-LRB-

genotype

relative

risk

Aa

genotype

relative

risk

AA

-RRB-

and

-LRB-

genotype

relative

risk

Aa

genotype

relative

risk

AA

-RRB-

Quantitative

genetic

analysis

In

addition

to

the

owner

impression

on

human-directed

aggression

and

dog-directed

aggression

we

collected

a

variety

of

other

aggression-related

behavioral

measures

using

the

canine

behavioral

assessment

and

research

questionnaire

-LRB-

CBARQ

Hsu

and

Serpell

-RRB-

As

described

in

Liinamo

et

al.

-LRB-

-RRB-

these

measures

were

of

three

types

original

CBARQ

items

-LRB-

items

on

the

aggressiveness

of

the

dog

in

various

everyday

situations

-RRB-

shortened

CBARQ

scores

-LRB-

scores

based

on

questions

that

addressed

stranger-directed

owner-directed

and

strange

dog-directed

aggression

-RRB-

and

CBARQ

factors

-LRB-

scores

based

on

questions

about

stranger-directed

owner-directed

and

strange

dog-directed

and

familiar

dog-directed

aggression

-RRB-

For

further

explanation

of

the

measures

and

the

difference

between

shortened

CBARQ

scores

and

CBARQ

factors

see

Liinamo

et

al.

-LRB-

-RRB-

The

effects

of

the

haplotypes

of

the

serotonergic

genes

on

the

different

aggression

measures

were

estimated

with

Restricted

Maximum

Likelihood

-LRB-

REML

-RRB-

method

-LRB-

Patterson

and

Thompson

-RRB-

using

univariate

analyses

and

an

animal

model

with

the

software

-LRB-

Groeneveld

-RRB-

The

analyses

were

an

extension

of

the

analyses

outlined

in

Liinamo

et

al.

-LRB-

-RRB-

using

similar

linear

animal

model

methodology

but

this

time

also

including

the

haplotype

classes

of

the

dogs

for

the

four

studied

loci

as

additional

fixed

effects

in

the

model

For

instance

the

linear

animal

model

that

was

assumed

in

the

analyses

for

owner

impression

traits

was

where

yijklmnois

the

observed

value

for

the

owner

impression

score

for

animal

o

μ

the

general

mean

in

the

population

sexithe

fixed

effect

of

the

reproductive

status

-LRB-

i

with

intact

male

castrated

male

intact

female

and

castrated

female

-RRB-

agej

the

fixed

effect

of

the

age

j

-LRB-

j

with

year

old

years

old

years

old

and

over

years

old

-RRB-

and

the

fixed

effects

of

the

respective

haplotype

classes

aothe

random

additive

genetic

effect

-LRB-

i.e.

polygenic

breeding

value

-RRB-

of

the

animal

o

and

eo

the

random

residual

effect

related

to

the

animal

o

The

age

and

reproductive

status

of

the

dogs

had

been

recorded

at

the

same

time

as

the

owner

impressions

The

haplotype

classes

were

formed

so

that

the

most

common

haplotypes

formed

separate

classes

the

very

rare

haplotypes

were

all

combined

in

one

class

and

the

unknown

haplotypes

were

classified

as

a

separate

class

-LRB-

see

Table

-RRB-

Results

Mutation

screening

of

the

coding

DNA

sequence

The

coding

DNA

sequence

of

each

candidate

gene

was

scanned

for

mutations

in

seven

-LRB-

and

-RRB-

or

eight

-LRB-

and

-RRB-

aggressive

Golden

Retrievers

Analysis

of

the

CDS

in

non-aggressive

Golden

Retrievers

has

been

described

by

van

den

Berg

et

al.

-LRB-

-RRB-

There

was

no

variation

in

the

CDS

of

and

in

the

Golden

Retrievers

We

observed

five

SNPs

in

the

CDS

of

and

one

SNP

in

the

CDS

of

The

allele

distribution

of

these

SNPs

in

the

two

groups

of

Golden

Retrievers

did

not

indicate

a

role

in

aggressive

behavior

In

conclusion

there

seems

to

be

no

systematic

difference

between

the

CDS

of

the

candidate

genes

in

aggressive

and

non-aggressive

Golden

Retrievers

Linkage

analysis

We

observed

two

alleles

for

marker

-LRB-

Table

-RRB-

The

four

SNPs

in

were

fully

in

LD

-LRB-

D

-RRB-

The

SNPs

displayed

six

haplotypes

in

the

Goldens

three

of

which

were

rare

-LRB-

frequency

-RRB-

We

detected

four

haplotypes

of

in

the

Golden

Retrievers

The

two

SNPs

in

were

fully

in

LD

and

formed

two

haplotypes

in

the

dogs

In

the

nine

families

that

we

used

for

linkage

analysis

haplotypes

were

deduced

with

certainty

in

of

the

dogs

for

for

and

for

We

calculated

the

maximum

achievable

LOD

score

using

hypothetical

genotypes

The

maximum

LOD

score

generated

by

our

pedigrees

was

at

recombination

fraction

θ

assuming

a

dominant

mode

of

inheritance

-LRB-

Table

-RRB-

Under

a

recessive

model

the

maximum

LOD

score

was

at

θ

The

families

are

therefore

theoretically

powerful

enough

to

prove

linkage

under

a

recessive

model

and

powerful

enough

to

provide

a

good

indication

of

the

presence

of

linkage

under

a

dominant

model

There

was

no

significant

linkage

of

any

of

the

candidate

genes

with

the

aggressive

phenotype

-LRB-

Table

-RRB-

LOD

scores

varied

from

to

assuming

dominant

inheritance

and

from

to

assuming

recessive

inheritance

The

highest

LOD

scores

were

obtained

for

-LRB-

under

a

dominant

model

and

under

a

recessive

model

-RRB-

Table

from

the

ODDS

-LRB-

LOD

-RRB-

scoresaGeneAutosomal

dominant

Autosomal

aLOD

scores

were

calculated

with

the

following

assumptions

frequency

of

the

aggression

allele

penetrance

of

the

aggression

allele

θ

Marker

haplotype

frequencies

were

deduced

from

a

group

of

parents

Association

study

Two

dogs

-LRB-

one

case

and

one

control

-RRB-

were

discarded

from

the

association

analysis

because

they

had

call

rates

lower

than

Mean

call

rates

in

the

other

dogs

were

for

SNPs

flanking

for

for

and

for

There

were

SNPs

with

a

minor

allele

frequency

and

call

rates

of

that

occurred

within

Mb

of

the

candidate

genes

The

genotype

frequencies

of

for

and

for

deviated

from

HWE

in

control

dogs

-LRB-

P

and

P

respectively

-RRB-

All

but

one

dog

in

both

case

and

control

group

had

heterozygous

genotypes

for

these

SNPs

We

concluded

that

the

data

for

these

two

SNPs

was

artefactual

and

they

were

excluded

from

further

analyses

The

observed

genotype

frequencies

of

the

other

SNPs

were

in

HWE

in

controls

-LRB-

P

values

are

listed

in

Table

in

Supplementary

Information

II

-RRB-

The

final

SNP

set

used

for

the

association

analysis

consisted

of

SNPs

flanking

flanking

flanking

and

flanking

-LRB-

Table

-RRB-

Table

nucleotide

polymorphisms

-LRB-

SNPs

-RRB-

used

for

the

association

study

their

allele

frequencies

in

aggressive

cases

and

control

dogs

and

results

of

chi-square

tests

for

comparisons

of

case

and

control

allele

frequenciesSNP

nameaChromosomal

locationChromosomal

position

-LRB-

Mb

-RRB-

bMinor

allele

frequency

in

controlsCorresponding

allele

frequency

in

respectively

serotonin

receptor

and

gene

serotonin

transporter

gene

The

genes

are

included

in

the

table

to

show

their

position

relative

to

the

SNPsbSNP

positions

are

based

on

the

second

version

of

the

dog

genome

assembly

released

in

May

-LRB-

-RRB-

as

can

be

viewed

on

of

the

genes

are

based

on

the

second

version

of

the

dog

genome

assembly

-LRB-

-RRB-

as

displayed

in

NCBI

Map

Viewer

for

Canis

familiarishttp

The

power

of

our

association

analysis

depends

on

the

local

extent

of

LD

The

mean

between

the

SNPs

flanking

was

and

the

mean

D

between

these

SNPs

was

Mean

values

were

and

for

and

respectively

Mean

D

values

were

and

for

and

respectively

When

the

marker

allele

frequency

is

the

power

to

detect

a

variant

with

a

relative

risk

of

with

cases

and

controls

would

be

for

for

for

and

for

Additional

power

estimations

are

provided

in

Fig

in

Supplementary

Information

II

The

allele

frequencies

of

the

SNPs

did

not

differ

significantly

between

cases

and

controls

after

correction

for

multiple

testing

-LRB-

Table

-RRB-

Genotype

frequencies

also

did

not

display

significant

differences

between

cases

and

controls

-LRB-

see

Table

in

Supplementary

Information

II

-RRB-

We

also

analyzed

the

association

of

haplotypes

with

the

phenotype

No

significant

associations

were

found

-LRB-

data

not

shown

-RRB-

In

conclusion

there

seemed

to

be

no

association

between

alleles

genotypes

or

haplotypes

of

SNPs

that

flank

the

candidate

genes

and

human-directed

aggression

in

the

Golden

Retrievers

Quantitative

genetic

analysis

We

completed

our

analyses

with

a

study

of

the

effect

of

variation

in

the

candidate

genes

on

a

collection

of

aggression-related

phenotypic

measures

The

haplotype

effects

were

studied

on

owner

impressions

on

human

and

dog-directed

aggression

the

original

CBARQ

items

related

to

stranger

and

owner-directed

aggression

the

shortened

CBARQ

scores

and

the

CBARQ

factors

The

haplotypes

did

not

have

a

significant

effect

on

any

of

the

studied

measures

i.e.

the

heritability

estimates

of

the

measures

remained

similar

to

the

results

presented

in

Liinamo

et

al.

-LRB-

-RRB-

in

spite

of

incorporation

of

the

haplotypes

in

the

mixed

model

The

results

for

owner

impressions

on

human

and

dog-directed

aggression

which

are

the

most

reliable

estimates

due

to

the

largest

number

of

observations

are

presented

in

Table

In

conclusion

the

large

genetic

variability

between

the

dogs

could

not

be

explained

by

the

serotonergic

genes

studied

in

this

paper

Table

effects

of

the

studied

genotypes

-LRB-

-RRB-

or

haplotypes

-LRB-

and

-RRB-

on

owner

impressions

of

human-directed

aggression

and

dog-directed

aggression

in

dogs

Genotype

or

haplotype

classaNumber

of

animalsEffect

relative

to

class

unknown

Human-directed

aggressionDog-directed

Other170.150.36htr2AUnknown1140.000.00132-C

of

the

effects

was

significantaNote

that

there

are

additional

alleles

compared

to

Table

as

a

result

of

the

larger

study

group

The

class

other

contains

genotypes

-LRB-

n

dogs

-RRB-

and

-LRB-

n

dog

-RRB-

For

the

class

other

contains

haplotypes

-LRB-

n

dogs

-RRB-

-LRB-

n

dog

-RRB-

-LRB-

n

dogs

-RRB-

-LRB-

n

dogs

-RRB-

-LRB-

n

dog

-RRB-

-LRB-

n

dogs

-RRB-

and

-LRB-

n

dogs

-RRB-

For

other

contains

haplotypes

-LRB-

n

dogs

-RRB-

-LRB-

n

dog

-RRB-

-LRB-

n

dogs

-RRB-

and

-LRB-

n

dogs

-RRB-

For

other

contains

C-G/T-G

-LRB-

n

dog

-RRB-

and

T-A/T-A

-LRB-

n

dogs

-RRB-

Unknown

genotypes

and

haplotypes

are

the

result

of

failure

of

genotyping

or

of

the

absence

of

a

DNA

sample

-LRB-

n

dogs

-RRB-

Discussion

We

collected

behavioral

information

and

DNA

samples

of

dogs

over

a

period

of

years

Dogs

were

selected

from

this

collection

to

evaluate

four

genes

involved

in

serotonin

metabolism

by

four

methods

DNA

sequence

analysis

of

the

coding

region

of

the

genes

genetic

linkage

analysis

genetic

association

analysis

and

quantitative

genetic

analysis

The

results

indicate

that

it

is

unlikely

that

there

is

a

major

locus

effect

of

one

of

the

genes

on

aggression

in

the

Golden

Retrievers

that

we

studied

The

genetic

study

of

the

variation

of

aggression

in

Golden

Retrievers

is

a

promising

tool

to

identify

the

molecular

systems

involved

in

aggression

The

relative

ease

to

find

disease

loci

in

the

dog

genome

compared

to

the

human

genome

is

the

result

of

the

population

structure

of

dog

breeds

Within

a

breed

Lindblad-Toh

and

colleagues

-LRB-

-RRB-

observed

a

limited

number

of

common

haplotypes

per

genomic

region

In

addition

LD

in

dog

breeds

extends

over

at

least

greater

distances

than

in

human

populations

These

characteristics

make

the

dog

highly

suited

for

molecular

genetic

studies

of

complex

traits

-LRB-

Sutter

and

Ostrander

-RRB-

We

did

not

detect

mutations

in

the

CDS

of

the

genes

specific

for

aggressive

dogs

All

SNPs

except

in

were

synonymous

This

variation

resulting

in

an

isoleucine/leucine

polymorphism

of

amino

acid

was

predicted

to

be

functionally

insignificant

by

POLYPHEN

The

genes

and

contain

SNPs

close

to

splice

sites

-LRB-

at

position

and

respectively

-RRB-

that

could

theoretically

affect

splicing

However

the

polymorphisms

did

not

have

a

large

effect

on

splice

site

prediction

by

three

software

programs

We

performed

mutation

screening

in

a

limited

number

of

dogs

and

it

is

possible

that

we

have

missed

rare

alterations

in

the

genes

Apart

from

this

limitation

we

conclude

that

there

is

no

common

variant

acting

on

protein

structure

that

contributes

to

the

variation

in

aggression

in

our

Golden

Retriever

sample

We

used

linkage

analysis

to

evaluate

the

likelihood

that

there

is

a

major

aggression-influencing

variant

in

the

chromosomal

regions

surrounding

the

coding

exons

The

affecteds

only

parameters

that

we

used

in

the

calculations

are

a

simplification

with

the

assumption

that

all

affected

dogs

of

a

family

have

the

genotype

at

risk

but

unaffected

dogs

can

have

any

genotype

at

the

aggression

locus

A

LOD

score

of

is

usually

considered

as

evidence

for

linkage

whereas

LOD

scores

below

exclude

the

gene

A

power

calculation

with

simulated

genotypes

was

not

feasible

in

this

study

due

to

the

complexity

of

some

pedigrees

with

multiple

loops

Instead

we

calculated

the

maximum

achievable

LOD

scores

to

get

an

impression

of

the

power

embodied

in

the

pedigrees

We

assumed

full

informativeness

of

the

markers

in

these

calculations

but

in

reality

we

expect

the

markers

to

have

limited

informativeness

Realistic

obtainable

LOD

scores

would

then

be

lower

than

the

maximum

values

that

we

presented

in

Table

These

LOD

scores

are

too

low

to

obtain

significant

results

but

they

provide

a

means

to

set

the

obtained

results

into

perspective

The

LOD

scores

for

and

were

low

compared

to

the

maximum

obtainable

scores

A

major

role

of

these

genes

is

unlikely

The

results

for

and

are

less

conclusive

This

is

probably

the

result

of

the

poor

informativeness

of

the

markers

For

both

genes

we

observed

only

two

alleles

or

haplotypes

with

high

frequencies

Typing

of

additional

markers

might

help

to

definitively

exclude

the

genes

However

in

the

light

of

the

observed

low

level

of

variation

it

is

unlikely

that

and

have

a

strong

effect

on

aggression

in

the

Golden

Retriever

families

Our

linkage

analysis

does

not

account

for

genetic

heterogeneity

or

phenocopies

We

have

thus

only

tested

for

a

very

strong

major

locus

effect

In

reality

the

aggressiveness

in

the

Golden

Retrievers

may

be

more

complex

We

therefore

used

a

third

study

design

to

investigate

the

candidate

genes

association

analysis

For

this

analysis

we

used

data

from

a

large-scale

genotyping

project

in

Golden

Retrievers

Our

power

calculations

demonstrate

that

this

sample

size

is

expected

to

be

sufficient

to

detect

variants

that

confer

a

high

relative

risk

for

a

range

of

marker

allele

frequencies

From

the

total

set

of

SNP

genotypes

we

selected

SNPs

that

flank

the

candidate

genes

We

found

no

association

between

alleles

genotypes

or

haplotypes

of

these

SNPs

flanking

the

candidate

genes

and

human-directed

aggression

of

the

Golden

Retrievers

We

focused

on

human-directed

aggression

in

the

affecteds-only

linkage

analysis

and

the

association

study

In

our

quantitative

genetic

analysis

we

studied

additional

types

of

aggression

There

is

no

consensus

in

the

literature

on

how

aggression

should

be

subdivided

-LRB-

Houpt

and

Willis

Jacobs

et

al.

Serpell

and

Jagoe

-RRB-

There

are

indications

that

various

types

of

aggression

have

a

distinct

genetic

basis

For

instance

selection

of

rats

and

silver

foxes

for

reduced

fear-induced

aggression

towards

humans

did

not

change

predatory

or

inter-male

aggression

-LRB-

Naumenko

et

al.

Popova

et

al.

-RRB-

This

suggests

that

molecular

genetic

studies

of

aggressive

behavior

should

focus

on

specific

classes

of

aggression

However

reduced

aggressiveness

towards

man

in

the

rats

and

foxes

was

accompanied

by

reduced

fear

of

novelties

and

irritable

aggression

indicating

that

there

is

overlap

between

classes

As

long

as

the

genetic

roots

of

aggressive

behavior

are

poorly

understood

it

will

remain

impossible

to

design

a

classification

that

reflects

the

genetic

basis

In

conclusion

of

the

four

methods

of

analysis

provided

evidence

for

a

strong

effect

of

variants

of

the

candidate

genes

on

aggression

in

the

Golden

Retrievers

that

we

studied

These

results

seem

to

contradict

reports

of

the

involvement

of

the

candidate

genes

in

the

regulation

of

aggressive

behavior

However

the

study

designs

that

we

used

are

not

powerful

enough

to

detect

variants

of

small

effect

We

can

therefore

not

rule

out

the

possibility

that

variation

in

the

candidate

genes

has

a

smaller

genetic

effect

on

aggression

In

addition

our

results

can

not

be

construed

as

evidence

against

a

major

role

for

these

genes

in

aggression

in

other

dog

breeds

Possibly

other

genes

in

the

serotonin

pathway

play

a

role

With

the

completion

of

the

dog

genome

project

genome-wide

association

studies

have

become

feasible

in

dogs

-LRB-

Lindblad-Toh

et

al.

-RRB-

This

opens

the

opportunity

for

finding

genes

that

have

not

been

associated

with

aggression

up

to

date

Such

studies

are

in

progress

Electronic

supplementary

material

Below

is

the

link

to

the

electronic

supplementary

material

-LRB-

DOC

kb

-RRB-

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O B O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O B O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O

dog
aggression
serotonin
candidate gene
linkage
association