Higher

Sensitivity

of

Human

Auditory

Nerve

Fibers

to

Positive

Electrical

Currents

Most

contemporary

cochlear

implants

-LRB-

CIs

-RRB-

stimulate

the

auditory

nerve

with

trains

of

amplitude-modulated

symmetric

biphasic

pulses

Although

both

polarities

of

a

pulse

can

depolarize

the

nerve

fibers

and

generate

action

potentials

it

remains

unknown

which

of

the

two

-LRB-

positive

or

negative

-RRB-

phases

has

the

stronger

effect

Understanding

the

effects

of

pulse

polarity

will

help

to

optimize

the

stimulation

protocols

and

to

deliver

the

most

relevant

information

to

the

implant

listeners

Animal

experiments

have

shown

that

cathodic

-LRB-

negative

-RRB-

current

flows

are

more

effective

than

anodic

-LRB-

positive

-RRB-

ones

in

eliciting

neural

responses

and

this

finding

has

motivated

the

development

of

novel

speech-processing

algorithms

In

this

study

we

show

electrophysiologically

and

psychophysically

that

the

human

auditory

system

exhibits

the

opposite

pattern

being

more

sensitive

to

anodic

stimulation

We

measured

electrically

evoked

compound

action

potentials

in

CI

listeners

for

phase-separated

pulses

allowing

us

to

tease

out

the

responses

to

each

of

the

two

opposite-polarity

phases

At

an

equal

stimulus

level

the

anodic

phase

yielded

the

larger

response

Furthermore

a

measure

of

psychophysical

masking

patterns

revealed

that

this

polarity

difference

was

still

present

at

higher

levels

of

the

auditory

system

and

was

therefore

not

solely

due

to

antidromic

propagation

of

the

neural

response

This

finding

may

relate

to

a

particular

orientation

of

the

nerve

fibers

relative

to

the

electrode

or

to

a

substantial

degeneration

and

demyelination

of

the

peripheral

processes

Potential

applications

to

improve

CI

speech-processing

strategies

are

discussed

Introduction

Polarity

sensitivity

of

auditory

nerve

fibers

has

been

extensively

studied

in

animal

and

computational

models

using

both

monophasic

and

biphasic

stimuli

Responses

to

monophasic

cathodic

pulses

show

in

general

lower

thresholds

and

longer

latencies

than

anodic

responses

-LRB-

Miller

et

al.

-RRB-

Similarly

the

most

effective

phase

of

sinusoidal

stimuli

was

shown

to

be

the

cathodic

phase

in

single-fiber

recordings

from

the

cat

-LRB-

Hartmann

et

al.

-RRB-

However

polarity

sensitivity

may

also

depend

on

the

electrode

position

relative

to

the

fibers

and

on

the

nerve

morphology

-LRB-

Ranck

McIntyre

and

Grill

Rubinstein

Rattay

Rattay

et

al.

-RRB-

If

so

then

the

findings

obtained

in

the

vast

majority

of

animal

experiments

may

not

apply

to

human

cochlear

implant

-LRB-

CI

-RRB-

listeners

-LRB-

Miller

et

al.

-RRB-

In

human

CI

stimulation

charge-balanced

stimuli

are

required

to

prevent

damaging

electrochemical

reactions

-LRB-

Brummer

and

Turner

-RRB-

Consequently

the

effect

of

absolute

polarity

which

is

usually

studied

using

monophasic

pulses

remains

largely

unknown

in

humans

Asymmetric

pulses

where

the

second

phase

is

lower

in

amplitude

and

longer

in

duration

than

the

first

have

provided

a

way

to

study

polarity

sensitivity

in

CI

users

In

such

pulses

the

contribution

of

the

short

high-amplitude

phase

is

increased

at

the

expense

of

the

long

low-amplitude

one

while

maintaining

the

charge-balancing

requirement

We

recently

showed

that

inverting

the

polarity

of

an

asymmetric

pulse

stimulus

yielded

differences

in

most

comfortable

levels

-LRB-

MCLs

-RRB-

of

CI

users

giving

lower

MCLs

when

the

short

high-amplitude

phase

was

anodic

than

when

it

was

cathodic

-LRB-

Macherey

et

al.

-RRB-

Because

short

pulses

require

less

charge

to

evoke

the

same

loudness

as

longer

ones

-LRB-

at

least

for

phase

durations

less

than

approximately

μs

Moon

et

al.

Macherey

et

al.

-RRB-

it

is

possible

that

loudness

was

determined

primarily

by

the

higher-amplitude

phase

of

our

asymmetric

pulses

This

result

then

suggests

that

the

human

auditory

nerve

might

be

more

sensitive

to

anodic

stimulation

the

opposite

to

what

one

expects

from

animal

measures

of

auditory-nerve

activity

-LRB-

Hartmann

et

al.

Miller

et

al.

-RRB-

The

following

series

of

experiments

directly

test

this

hypothesis

in

eight

CI

users

using

both

electrophysiological

and

psychophysical

measures

The

experimental

design

relies

on

the

characteristics

of

the

auditory

nerve

fibers

which

after

a

stimulation

pulse

undergo

a

period

of

absolute

refractoriness

lasting

approximately

μs

followed

by

a

progressive

recovery

-LRB-

Stypulkowski

and

van

den

Honert

Miller

et

al.

Morsnowski

et

al.

-RRB-

Experiment

provides

an

insight

into

the

morphology

of

anodic

and

cathodic

phase

responses

Experiments

and

measure

the

masking

effectiveness

of

each

individual

polarity

phase

using

electrophysiological

and

psychophysical

masking

respectively

General

methods

Subjects

Eight

users

of

the

CII

or

HiRes

CIs

manufactured

by

Advanced

Bionics

participated

in

this

series

of

electrophysiological

-LRB-

experiment

and

-RRB-

and

psychophysical

-LRB-

experiment

-RRB-

experiments

The

subjects

biographical

data

are

shown

in

Table

Testing

was

approved

by

the

Cambridge

and

Leuven

Local

Research

Ethics

Committees

and

informed

consent

was

obtained

from

all

subjects

TABLE

data

of

the

subjects

including

age

duration

of

deafness

-LRB-

DD

-RRB-

and

duration

of

CI

use

-LRB-

CI

-RRB-

in

years

and

etiologySubjectAge

-LRB-

years

-RRB-

DD

-LRB-

years

-RRB-

CI

-LRB-

years

-RRB-

Electrically

evoked

compound

action

potential

recordings

and

psychophysical

testing

Most

contemporary

CI

systems

allow

the

direct

recording

of

the

auditory

nerve

response

known

as

the

electrically

evoked

compound

action

potential

-LRB-

ECAP

-RRB-

This

is

a

noninvasive

technique

that

consists

of

recording

the

intracochlear

potential

evolution

after

a

stimulation

pulse

To

minimize

electrical

artifacts

we

used

the

masker

probe

paradigm

-LRB-

Brown

et

al.

-RRB-

which

relies

on

the

fact

that

the

neural

response

to

a

probe

pulse

is

reduced

by

an

immediately

preceding

masker

By

presenting

the

probe

and

masker

either

alone

or

in

combination

it

is

possible

to

derive

the

neural

response

to

the

probe

with

the

artifact

subtracted

out

-LRB-

cf.

Fig.

-RRB-

In

experiments

and

ECAPs

were

measured

using

several

combinations

of

masker

and

probe

In

all

cases

masker

and

probe

were

presented

in

monopolar

mode

on

an

electrode

located

in

the

middle

of

the

array

-LRB-

electrode

-RRB-

with

reference

to

the

case

-LRB-

extracochlear

-RRB-

electrode

of

the

implant

The

responses

were

recorded

using

electrode

-LRB-

approximately

mm

apical

to

electrode

-RRB-

with

reference

to

the

ring

ground

electrode

for

and

the

case

electrode

for

all

other

subjects

and

were

averaged

over

sweeps

In

experiment

psychophysical

masking

patterns

were

measured

using

the

APEX

experimental

platform

and

the

BEDCS

software

-LRB-

cf.

Macherey

et

al.

Laneau

et

al.

-RRB-

Masker

and

probe

were

also

presented

in

monopolar

mode

the

masker

on

electrode

and

the

probe

on

an

adjacent

electrode

-LRB-

or

-RRB-

FIG.

Description

of

the

masker

probe

paradigm

for

artifact

cancellation

-LRB-

Brown

et

al.

-RRB-

Four

traces

are

recorded

masker

alone

-LRB-

-RRB-

masker

and

probe

-LRB-

-RRB-

probe

alone

-LRB-

-RRB-

and

zero

stimulation

-LRB-

-RRB-

to

correct

for

any

DC

offset

This

method

is

based

on

the

assumption

that

the

nerve

fibers

are

in

their

absolute

refractory

state

when

the

probe

is

presented

after

the

masker

-LRB-

-RRB-

The

subtraction

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

therefore

yields

the

probe

neural

response

with

the

artifact

removed

Experiments

Experiment

ECAP

responses

to

anodic

and

cathodic

phases

Rationale

and

methods

In

experiment

we

recorded

ECAPs

from

seven

subjects

-LRB-

-RRB-

The

masker

was

a

cathodic-first

symmetric

biphasic

pulse

with

a

phase

duration

presented

at

a

comfortable

level

determined

subjectively

for

each

subject

before

testing

The

probes

were

either

anodic-first

-LRB-

Fig.

-RRB-

or

cathodic-first

-LRB-

Fig.

-RRB-

asymmetric

waveforms

with

a

phase

separation

of

ms.

The

first

-LRB-

short

-RRB-

phase

of

the

pulse

was

long

and

the

second

-LRB-

long

-RRB-

phase

was

eight

times

longer

and

lower

in

amplitude

The

phase

separation

present

in

the

probe

allowed

us

to

measure

the

neural

response

to

the

first

phase

independently

of

the

second

The

two

probes

had

the

same

level

-LRB-

between

most

comfortable

and

comfortably

loud

-RRB-

and

were

each

presented

μs

after

the

end

of

the

masker

Additionally

to

differentiate

neural

responses

from

imperfectly

cancelled

stimulation

artifacts

we

recorded

in

four

of

the

subjects

-LRB-

-RRB-

the

responses

to

several

probe

levels

spanning

the

entire

electrical

dynamic

range

FIG.

ECAP

responses

to

anodic

and

cathodic

phases

A

Masker

-LRB-

thick

line

-RRB-

and

probe

-LRB-

thin

line

-RRB-

used

to

measure

the

anodic

phase

response

B

Masker

and

probe

used

to

measure

the

cathodic-phase

response

C

and

D

ECAPs

evoked

by

the

first

phases

of

anodic-first

-LRB-

C

-RRB-

and

cathodic-first

-LRB-

D

-RRB-

phase-separated

asymmetric

probes

The

time

reference

corresponds

to

the

onset

of

the

probe

The

second

phase

is

not

displayed

as

it

occurs

ms

after

the

first

one

The

top

panels

show

an

example

of

a

level

series

obtained

with

subject

The

different

curves

correspond

to

probe

levels

ranging

from

a

soft

-LRB-

μA

-RRB-

to

a

comfortably

loud

level

-LRB-

μA

-RRB-

by

steps

of

μA

with

the

size

of

the

fluctuations

increasing

monotonically

with

increasing

current

level

The

other

panels

show

the

responses

to

the

highest

probe

levels

for

all

the

other

subjects

The

peak

of

the

anodic

response

occurs

on

average

μs

after

the

onset

of

the

probe

Results

An

example

of

a

level

series

is

shown

in

the

top

panels

of

Figure

-LRB-

anodic

phase

-RRB-

and

D

-LRB-

cathodic

phase

-RRB-

for

The

first

narrow

short-latency

peak

observed

for

both

anodic-first

and

cathodic-first

probes

almost

certainly

reflects

a

residual

artifact

because

for

most

subjects

-LRB-

with

the

exception

of

and

-RRB-

its

polarity

is

dependent

on

the

probe

polarity

-LRB-

positive

and

negative

for

anodic-first

and

cathodic-first

probes

respectively

-RRB-

and

its

latency

is

similar

for

the

two

probes

For

all

subjects

the

response

to

the

anodic

phase

shows

a

biphasic

shape

with

a

first-negative

peak

followed

by

a

less

robust

positive

peak

Only

one

-LRB-

-RRB-

of

the

seven

subjects

showed

a

measurable

response

to

the

cathodic

phase

and

only

at

the

highest

level

of

the

dynamic

range

For

this

subject

the

latency

of

the

response

was

longer

for

the

cathodic-first

-LRB-

μs

-RRB-

than

for

the

anodic-first

-LRB-

μs

-RRB-

probe

consistent

with

reports

of

animal

auditory

excitation

-LRB-

Miller

et

al.

-RRB-

Most

of

these

animal

data

were

however

obtained

using

a

recording

electrode

located

at

the

nerve

trunk

whereas

our

data

reflect

intracochlear

recordings

The

position

of

the

recording

electrode

may

have

an

important

influence

on

the

response

latencies

and

we

will

further

consider

this

parameter

in

Discussion

For

now

it

is

worth

noting

that

the

major

discrepancy

with

common

animal

data

was

that

the

amplitude

of

the

response

was

in

our

case

always

greater

for

the

anodic

phase

Experiment

masking

effectiveness

of

anodic

and

cathodic

phases

ECAP

measures

Rationale

and

methods

The

reliability

of

the

masker

probe

paradigm

strongly

depends

on

the

effectiveness

of

the

masker

as

the

neural

response

obtained

represents

the

subtraction

of

the

masked

response

from

the

unmasked

one

Instead

of

reflecting

different

neural

responses

the

anodic/cathodic

amplitude

difference

obtained

in

experiment

may

relate

to

the

fact

that

the

biphasic

masker

did

not

effectively

mask

the

cathodic

phase

This

would

be

expected

if

for

example

the

excitation

sites

for

the

biphasic

masker

and

the

cathodic

probe

were

the

central

axon

and

the

peripheral

process

respectively

The

peripheral

process

may

in

such

a

case

not

be

driven

in

its

refractory

state

by

the

masker

and

the

masker

probe

paradigm

would

consequently

subtract

out

the

neural

response

We

performed

in

experiment

another

set

of

ECAP

measures

in

six

subjects

-LRB-

-RRB-

to

verify

that

the

anodic

phase

was

also

a

more

effective

masker

than

the

cathodic

phase

Unlike

the

previous

experiment

we

introduced

a

phase

separation

in

the

symmetric

biphasic

masker

and

assumed

that

the

probe

would

be

mainly

masked

by

the

masker

's

second

phase

-LRB-

denoted

as

the

masking

phase

-RRB-

We

recorded

the

responses

to

anodic-first

and

cathodic-first

symmetric

biphasic

probes

with

no

phase

separation

in

the

two

conditions

anodic

masking

-LRB-

Fig.

-RRB-

and

cathodic

masking

-LRB-

Fig.

-RRB-

phase

and

also

in

a

third

biphasic

masking

-LRB-

Fig.

-RRB-

condition

This

last

condition

where

the

masker

was

a

cathodic-first

symmetric

biphasic

pulse

with

no

phase

separation

was

performed

for

two

reasons

First

because

this

protocol

is

routinely

used

in

clinics

the

morphology

of

the

resulting

ECAP

response

is

well

documented

It

usually

consists

of

an

complex

-LRB-

Brown

et

al.

-RRB-

similar

to

the

response

obtained

in

experiment

for

the

anodic

phase

Second

it

allowed

us

to

compare

the

latencies

of

the

probes

responses

If

the

anodic

phase

is

the

most

excitatory

phase

of

a

biphasic

pulse

we

would

expect

the

response

to

show

a

longer

latency

when

the

anodic

phase

is

the

second

phase

of

the

probe

than

when

it

is

the

first

FIG.

Combinations

of

maskers

-LRB-

thick

lines

-RRB-

and

probes

-LRB-

thin

lines

-RRB-

used

in

experiment

A

Anodic

masking

condition

B

Cathodic

masking

condition

C

Biphasic

masking

condition

For

each

masking

condition

two

different

symmetric

biphasic

probes

were

used

an

anodic-first

and

a

cathodic-first

Results

For

all

masking

conditions

a

large

uncancelled

artifact

was

present

at

the

probe

onsets

Figure

shows

examples

of

these

artifacts

obtained

with

using

biphasic

cathodic-first

-LRB-

A

-RRB-

and

anodic-first

-LRB-

B

-RRB-

probes

in

the

anodic

masking

condition

The

size

of

this

artifact

did

not

depend

on

the

probe

level

and

was

still

present

at

subthreshold

levels

-LRB-

thick

lines

of

Fig.

-RRB-

For

means

of

clarity

and

because

this

uncancelled

artifact

was

clearly

distinct

from

the

neural

response

subsequent

figures

will

only

show

the

neural

response

part

of

the

recordings

Note

that

an

advantage

of

the

present

approach

is

that

it

compares

the

effects

of

different

maskers

whereas

the

artifact

occurred

near

the

onset

of

the

probe

FIG.

Examples

of

uncancelled

artifacts

obtained

in

experiment

for

ECAP

responses

to

cathodic-first

-LRB-

A

-RRB-

and

anodic-first

-LRB-

B

-RRB-

biphasic

probes

in

the

anodic-masking

condition

at

subthreshold

-LRB-

thick

line

-RRB-

and

suprathreshold

-LRB-

thin

line

-RRB-

levels

Figure

shows

the

anodic-first

-LRB-

thick

line

-RRB-

and

cathodic-first

-LRB-

thin

line

-RRB-

probe

responses

in

the

biphasic

masking

condition

for

The

response

latency

of

the

peak

was

shorter

for

the

anodic-first

probe

consistent

with

the

neural

activation

being

elicited

primarily

by

the

anodic

phase

All

subjects

demonstrated

this

trend

with

a

latency

difference

ranging

from

to

μs

-LRB-

Fig.

-RRB-

opposite

to

results

obtained

in

the

guinea

pig

-LRB-

Klop

et

al.

-RRB-

A

t

test

performed

on

the

data

from

the

six

subjects

revealed

that

this

difference

was

significant

-LRB-

df

p

-RRB-

Apart

from

this

latency

difference

the

responses

to

anodic-first

and

cathodic-first

probes

had

similar

amplitudes

To

further

improve

artifact

cancellation

the

two

responses

were

averaged

for

data

analysis

FIG.

Masking

effectiveness

of

anodic

and

cathodic

phases

A

Example

of

ECAP

recordings

obtained

with

subject

for

an

anodic-first

-LRB-

thick

line

-RRB-

and

a

cathodic-first

-LRB-

thin

line

-RRB-

biphasic

probe

in

the

biphasic

masking

condition

The

response

latency

is

shorter

for

the

anodic-first

probe

B

Latency

of

the

cathodic-first

probe

response

vs.

latency

of

the

anodic-first

probe

response

For

each

subject

the

two

probes

were

presented

at

an

identical

level

-LRB-

comfortably

loud

-RRB-

and

also

at

the

same

level

as

the

biphasic

masker

C

D

and

E

Example

of

ECAPs

evoked

by

a

symmetric

biphasic

probe

with

no

phase

separation

in

the

three

masker

conditions

anodic

masking

-LRB-

C

-RRB-

cathodic

masking

-LRB-

D

-RRB-

and

biphasic

masking

-LRB-

E

-RRB-

for

subject

Each

trace

corresponds

to

the

average

of

the

anodic-first

and

cathodic-first

biphasic

probe

responses

In

the

cathodic

masking

condition

the

neural

response

was

sometimes

not

clearly

measurable

In

D

for

example

there

is

no

excursion

of

the

trace

in

the

negative

portion

of

the

graph

Nevertheless

the

recording

still

shows

a

voltage

increase

in

the

same

region

where

the

probe

neural

response

occurs

The

amplitude

of

the

probe

response

was

in

this

case

assumed

to

be

the

magnitude

of

this

increase

F

Summary

of

ECAP

amplitudes

obtained

in

the

anodic-masking

-LRB-

black

bars

-RRB-

cathodic-masking

-LRB-

white

bars

-RRB-

and

biphasic-masking

-LRB-

gray

bars

-RRB-

conditions

An

example

of

the

averaged

responses

of

is

illustrated

in

Figure

for

anodic

masking

-LRB-

C

-RRB-

cathodic

masking

-LRB-

D

-RRB-

and

biphasic

masking

-LRB-

E

-RRB-

For

the

six

subjects

tested

the

amplitude

of

the

response

-LRB-

difference

between

and

-RRB-

was

greater

when

the

masking

phase

was

anodic

than

when

it

was

cathodic

-LRB-

Fig.

-RRB-

This

confirmed

the

previous

finding

that

the

anodic

phase

is

more

effective

in

driving

auditory

nerve

fibers

in

their

refractory

state

Furthermore

the

probe

responses

in

the

anodic

masking

and

biphasic

masking

conditions

were

strikingly

similar

suggesting

that

the

anodic

phase

is

the

effective

phase

of

a

symmetric

biphasic

pulse

This

last

observation

also

argues

strongly

against

the

responses

being

influenced

by

an

uncancelled

masker

artifact

Note

that

although

the

anodic

and

cathodic

maskers

had

the

same

level

and

differed

only

in

their

leading

polarity

the

level

of

the

biphasic

masker

was

presented

on

average

dB

higher

to

have

a

loudness

similar

to

that

of

the

other

two

maskers

This

increase

in

level

reflects

the

influence

of

phase

separation

on

loudness

reported

in

previous

publications

-LRB-

McKay

and

Henshall

Carlyon

et

al.

van

Wieringen

et

al.

Macherey

et

al.

-RRB-

For

and

the

amplitude

is

negative

in

the

cathodic

masking

case

because

the

subjects

exhibited

an

inverted

response

-LRB-

with

a

positive

peak

-RRB-

This

may

mean

that

the

cathodic

phase

acts

as

a

facilitator

and

not

as

a

masker

for

these

specific

subjects

possibly

because

of

residual

potential

summation

-LRB-

Miller

et

al.

De

Balthasar

et

al.

-RRB-

Experiment

masking

effectiveness

of

anodic

and

cathodic

phases

psychophysical

measures

Rationale

and

methods

Because

ECAPs

in

CIs

can

only

be

measured

intracochlearly

and

not

directly

on

the

nerve

trunk

the

recordings

potentially

contain

the

neural

response

traveling

not

only

toward

the

central

auditory

system

-LRB-

orthodromically

-RRB-

but

also

in

the

opposite

direction

toward

the

fibers

periphery

-LRB-

antidromically

Miller

et

al.

-RRB-

It

is

possible

that

this

phenomenon

occurred

and

that

the

larger

ECAP

amplitude

of

the

anodic

response

obtained

in

experiment

did

not

reflect

a

larger

response

propagating

toward

the

central

auditory

system

-LRB-

Cartee

et

al.

-RRB-

Although

the

results

of

experiment

argue

against

this

hypothesis

a

perhaps

more

direct

way

to

tease

out

this

effect

is

to

perform

psychophysical

measures

which

require

perceptual

judgments

from

the

subjects

thereby

reflecting

neural

responses

propagating

centrally

The

aim

of

experiment

was

to

compare

the

masking

effectiveness

of

each

polarity

phase

psychophysically

and

also

to

measure

the

most

effective

phase

of

an

asymmetric

pulse

Is

it

the

short

high-amplitude

or

the

long

low-amplitude

one

We

used

two

asymmetric

pulse

train

stimuli

-LRB-

anodic

and

cathodic-first

-RRB-

with

a

phase

separation

as

maskers

of

a

biphasic

pulse

train

probe

presented

ms

either

after

the

first

or

after

the

second

phase

of

the

masker

-LRB-

Fig.

-RRB-

Contrary

to

experiments

and

where

we

were

constrained

by

the

limited

duration

of

the

recording

window

we

used

a

longer

phase

separation

to

minimize

refractory

effects

of

one

phase

of

the

masker

over

the

other

The

masker

was

a

per

second

pulse

train

either

anodic-first

-LRB-

conditions

and

b

-RRB-

or

cathodic-first

-LRB-

conditions

and

b

-RRB-

with

a

short

phase

of

μs

and

a

long

phase

eight

times

longer

and

lower

in

amplitude

Masker

and

probe

had

a

total

duration

of

ms

and

were

temporally

interleaved

the

masker

being

presented

on

electrode

and

the

probe

on

electrode

or

in

an

additional

set

of

measures

on

electrode

Four

subjects

-LRB-

and

-RRB-

were

asked

to

detect

the

anodic-first

biphasic

probe

in

the

presence

of

one

of

the

two

maskers

in

a

three-interval

two-alternative

forced-choice

task

where

only

one

interval

contained

the

probe

Feedback

was

provided

and

the

level

of

the

probe

was

adaptively

varied

in

a

two-down

one-up

adaptive

procedure

that

stopped

after

eight

reversals

For

subjects

the

step

size

was

μA

for

the

first

two

and

μA

for

the

last

six

reversals

For

subject

the

phase

duration

of

the

biphasic

probe

had

to

be

increased

to

μs

to

be

able

to

reach

a

comfortably

loud

level

and

the

step

sizes

were

and

μA

respectively

for

the

first

two

and

the

last

six

reversals

Each

estimate

was

the

average

value

of

the

last

six

reversals

We

also

performed

the

same

four

measures

with

the

polarity

of

the

probe

reversed

-LRB-

cathodic-first

instead

of

anodic-first

-RRB-

We

did

this

because

in

a

previous

study

-LRB-

Macherey

et

al.

-RRB-

we

reported

the

presence

of

a

low-amplitude

current

flow

between

the

two

phases

of

stimuli

with

relatively

long

phase

separations

-LRB-

similar

to

the

separation

used

here

-RRB-

The

polarity

of

this

current

flow

is

opposite

to

the

polarity

of

the

preceding

stimulus

phase

and

is

due

to

the

passive

discharge

of

the

implant

's

DC-blocking

capacitors

This

current

may

influence

the

detection

of

the

probe

by

predepolarizing

or

prehyperpolarizing

the

neural

membrane

after

each

phase

The

results

obtained

in

this

condition

were

similar

to

the

ones

obtained

with

the

initial

stimulus

polarity

and

will

therefore

not

be

shown

here

FIG.

Psychophysical

masking

patterns

A

Description

of

the

four

experimental

conditions

Anodic-first

asymmetric

masker

-LRB-

thick

line

-RRB-

with

a

phase

separation

of

ms

followed

by

a

biphasic

probe

presented

ms

after

the

end

of

the

short

high-amplitude

phase

-LRB-

-RRB-

or

ms

after

the

end

of

the

long

low-amplitude

phase

-LRB-

-RRB-

of

the

masker

Conditions

and

are

identical

to

and

except

that

the

masker

is

reversed

in

polarity

B

Difference

between

masked

and

unmasked

threshold

for

the

four

conditions

-LRB-

and

-RRB-

and

the

four

subjects

-LRB-

and

-RRB-

for

a

probe

on

electrode

The

probe

dynamic

ranges

were

and

dB

for

and

respectively

C

Same

as

B

with

the

probe

on

electrode

Results

The

four

CI

subjects

-LRB-

-RRB-

were

first

asked

to

balance

in

loudness

the

two

opposite-polarity

maskers

at

their

MCL

following

a

procedure

described

in

Macherey

et

al.

-LRB-

-RRB-

On

average

the

level

of

the

cathodic

masker

was

adjusted

higher

than

the

anodic

one

consistent

with

the

anodic-first

pulse

being

more

effective

than

the

cathodic-first

one

These

levels

were

used

in

the

following

part

of

the

experiment

where

the

subjects

had

to

detect

the

presence

of

the

biphasic

probe

in

each

of

the

four

conditions

and

also

in

the

unmasked

case

Figure

shows

the

threshold

shifts

-LRB-

difference

between

masked

and

unmasked

thresholds

-RRB-

obtained

with

the

probe

on

electrode

For

an

anodic

masker

the

short

high-amplitude

-LRB-

anodic

-RRB-

phase

produced

more

masking

than

the

long

low-amplitude

-LRB-

cathodic

-RRB-

phase

for

all

subjects

-LRB-

conditions

vs.

-RRB-

No

clear

pattern

was

however

obtained

in

the

case

of

a

cathodic

masker

-LRB-

vs.

-RRB-

showing

across-subject

variability

More

interestingly

although

presented

at

a

lower

level

the

short

high-amplitude

anodic

phase

-LRB-

-RRB-

was

a

more

effective

masker

than

the

short

high-amplitude

cathodic

phase

-LRB-

-RRB-

Similar

results

were

obtained

with

the

probe

on

electrode

-LRB-

Fig.

-RRB-

These

observations

were

corroborated

by

a

two-way

repeated

measures

ANOVA

performed

on

the

mean

data

of

all

subjects

The

treatment

factors

were

the

probe

channel

-LRB-

or

-RRB-

and

the

probe

condition

First

there

was

a

significant

effect

of

the

probe

condition

-LRB-

F

-LRB-

-RRB-

p

-RRB-

but

no

effect

of

the

probe

channel

-LRB-

F

-LRB-

-RRB-

p

-RRB-

or

of

the

interaction

factor

-LRB-

F

-LRB-

-RRB-

p

-RRB-

Second

pairwise

comparisons

revealed

that

the

short

high-amplitude

anodic

phase

was

a

better

masker

than

the

long

low-amplitude

cathodic

phase

-LRB-

p

-RRB-

and

also

than

the

short

high-amplitude

cathodic

phase

-LRB-

p

-RRB-

No

significant

difference

was

however

found

between

the

short

cathodic

and

the

long

anodic

phases

-LRB-

p

-RRB-

These

results

demonstrate

that

the

larger

ECAP

response

previously

measured

for

anodic

stimulation

was

not

solely

caused

by

antidromic

propagation

and

strengthens

the

finding

that

auditory

nerve

fibers

are

more

sensitive

to

anodic

stimuli

Discussion

Main

findings

We

have

reported

the

results

of

three

electrophysiological

and

psychophysical

masking

experiments

designed

to

compare

the

responses

of

the

human

auditory

nerve

to

anodic

and

cathodic

current

flows

In

experiment

the

anodic

phase

produced

the

usual

ECAP

morphology

for

all

subjects

whereas

the

cathodic

phase

showed

no

measurable

response

except

for

one

subject

This

observation

led

us

to

conclude

that

the

anodic

phase

was

more

effective

than

the

cathodic

phase

There

are

however

at

least

three

alternative

explanations

for

this

trend

First

the

cathodic

phase

response

of

experiment

may

have

had

a

shorter

latency

than

the

anodic

phase

response

and

may

have

consequently

been

mixed

within

the

uncancelled

stimulus

artifact

Although

this

hypothesis

is

not

supported

by

animal

data

usually

showing

longer

latencies

for

cathodic

stimuli

-LRB-

Miller

et

al.

-RRB-

a

potentially

important

confounding

variable

is

the

location

of

the

recording

electrode

Most

animal

ECAP

data

are

recorded

using

an

electrode

positioned

on

the

nerve

trunk

Consequently

the

shorter

the

response

latency

the

more

central

the

excitation

site

is

This

may

however

not

be

the

case

for

intracochlear

recordings

where

the

recording

and

the

stimulating

electrodes

are

close

to

each

other

-LRB-

Miller

et

al.

-RRB-

Here

we

used

a

recording

electrode

mm

apical

to

the

stimulating

one

similar

to

what

is

used

clinically

and

this

choice

may

have

affected

the

morphology

and

the

latency

of

the

ECAP

responses

We

note

that

our

second

experiment

controlled

for

any

possible

effects

of

response

latency

to

the

probe

by

studying

the

effects

of

masker

polarity

for

a

fixed

probe

and

by

showing

that

although

the

leading

polarity

of

a

biphasic

probe

did

affect

the

response

latency

it

was

always

the

case

that

responses

were

larger

with

an

anodic

than

with

a

cathodic

masker

Second

as

previously

stated

the

response

to

the

anodic

phase

obtained

in

experiment

may

not

only

represent

the

neural

response

traveling

toward

the

central

auditory

system

As

the

recording

electrode

is

located

at

the

periphery

of

the

nerve

fibers

the

recorded

response

possibly

contains

the

antidromic

response

of

the

fibers

Although

this

is

possible

this

would

necessarily

imply

that

the

nerve

fibers

still

have

remaining

peripheral

processes

to

allow

the

propagation

of

antidromic

spikes

In

such

a

case

this

phenomenon

may

not

occur

in

subjects

with

a

long

duration

of

deafness

and

presumably

a

poor

peripheral

processes

survival

Furthermore

the

psychophysical

report

of

experiment

demonstrates

that

the

orthodromic

response

-LRB-

propagating

centrally

-RRB-

is

also

larger

for

the

anodic

than

for

the

cathodic

phase

Antidromic

propagation

can

not

therefore

be

the

only

account

for

our

finding

Third

the

cathodic

phase

may

excite

a

different

subpopulation

of

nerve

fibers

than

a

biphasic

pulse

with

no

phase

separation

In

such

a

case

the

masker-probe

paradigm

that

we

used

in

Experiments

and

would

not

be

effective

in

the

measure

of

the

cathodic

phase

response

i.e.

if

the

masker

does

not

effectively

mask

the

probe

the

subtraction

method

would

not

allow

the

neural

response

to

appear

Although

the

present

data

can

not

rule

out

this

possibility

it

is

worth

noting

that

the

masker-probe

paradigm

was

successfully

used

in

the

measure

of

the

anodic

phase

response

This

would

mean

that

a

biphasic

pulse

-LRB-

whether

anodic

or

cathodic-first

-RRB-

with

no

phase

separation

excites

the

same

subpopulation

as

a

single

anodic

phase

but

a

different

subpopulation

than

a

single

cathodic

phase

Consequently

this

would

not

change

the

conclusion

that

the

anodic

phase

is

the

most

effective

phase

of

a

biphasic

pulse

and

that

the

ECAPs

recorded

clinically

most

likely

represent

the

response

to

the

anodic

phase

of

biphasic

pulses

Although

the

observation

of

a

higher

sensitivity

to

anodic

current

flows

is

surprising

some

animal

data

have

already

shown

such

a

result

Miller

et

al.

-LRB-

-RRB-

recorded

ECAPs

to

monophasic

anodic

and

cathodic

pulses

from

the

cat

and

the

guinea

pig

They

observed

lower

cathodic

thresholds

for

cats

but

lower

anodic

thresholds

for

guinea

pigs

The

reason

for

this

species

discrepancy

was

however

unclear

and

the

authors

proposed

that

it

may

relate

to

differences

in

anatomy

or

in

electrode

size

and

position

Other

stimulus

parameters

have

shown

to

affect

human

and

animal

subjects

in

different

ways

-LRB-

Shepherd

and

Javel

Carlyon

et

al.

-RRB-

and

several

factors

may

underlie

those

species

differences

including

differences

in

anatomy

neural

properties

neural

survival

and

electrode

geometry

-LRB-

reviewed

by

Miller

et

al.

-RRB-

Factors

affecting

polarity

sensitivity

The

simplest

model

of

extracellular

stimulation

that

can

account

for

a

lower

cathodic

threshold

is

the

activating

function

of

Rattay

-LRB-

-RRB-

This

model

describes

the

spatial

pattern

of

polarization

of

an

infinite

homogeneous

fiber

subjected

to

an

extracellular

electrical

current

A

cathodic

stimulus

produces

a

large

depolarization

of

the

fiber

proximal

to

the

electrode

and

a

smaller

hyperpolarization

at

both

sides

of

the

depolarized

region

whereas

an

anodic

stimulus

produces

the

exact

opposite

pattern

of

polarization

This

model

is

however

too

restrictive

in

the

case

of

auditory

nerve

fibers

which

are

made

of

several

excitable

elements

-LRB-

peripheral

process

unmyelinated

cell

body

and

central

axon

-RRB-

that

presumably

have

different

biophysical

properties

and

hence

different

activation

thresholds

Moreover

auditory

nerve

fibers

are

curved

and

the

spatial

pattern

of

polarization

produced

by

an

electrical

pulse

may

show

multiple

regions

of

depolarization

and

hyperpolarization

-LRB-

Rattay

Rattay

et

al.

-RRB-

There

are

at

least

three

factors

that

may

influence

polarity

sensitivity

the

electrode

position

the

nerve

parameters

and

the

stimulus

current

level

They

are

discussed

in

the

following

paragraphs

Ranck

-LRB-

-RRB-

reviewed

the

different

mechanisms

of

electrical

excitation

of

the

central

nervous

system

and

showed

that

the

electrode

position

relative

to

the

fiber

had

a

crucial

effect

on

its

sensitivity

to

polarity

He

reported

that

myelinated

fibers

were

more

easily

excited

by

cathodic

stimuli

when

the

electrode

was

placed

over

the

central

axon

However

for

an

electrode

located

near

the

cell

body

on

the

opposite

side

from

the

central

axon

the

lowest

threshold

was

often

obtained

with

an

anodic

stimulus

Rattay

-LRB-

-RRB-

and

McIntyre

and

Grill

-LRB-

-RRB-

found

similar

place-dependent

polarity

effects

on

their

modeled

central

neurons

In

another

modeling

study

Rubinstein

-LRB-

-RRB-

focused

on

the

excitation

process

near

the

axon

termination

He

also

demonstrated

the

extreme

dependence

of

polarity

sensitivity

on

the

exact

electrode

position

As

a

CI

electrode

presumably

excites

a

large

number

of

nerve

fibers

with

different

orientations

and

locations

it

is

possible

that

each

fiber

is

excited

differently

by

anodic

and

cathodic

phases

-LRB-

Rattay

et

al.

-RRB-

The

present

study

was

restricted

to

electrodes

mid-way

along

the

electrode

array

Subsequent

studies

should

explore

other

electrode

positions

and

other

electrode

designs

Computational

models

have

provided

a

simple

way

to

investigate

the

influence

of

neural

parameters

In

two

different

modeling

studies

Rattay

et

al.

-LRB-

-RRB-

suggested

that

intact

human

auditory

nerve

fibers

would

be

more

sensitive

-LRB-

showing

lower

excitation

thresholds

-RRB-

to

cathodic

stimuli

whereas

fibers

with

degenerated

or

demyelinated

peripheral

processes

would

be

more

sensitive

to

anodic

stimuli

First

they

predicted

that

for

intact

fibers

the

depolarization

magnitude

would

be

maximal

at

the

level

of

the

peripheral

process

and

would

be

larger

for

a

cathodic

than

for

an

anodic

pulse

Second

for

fibers

without

peripheral

processes

their

model

showed

that

the

only

site

that

could

be

depolarized

by

a

cathodic

pulse

was

the

cell

body

whereas

an

anodic

pulse

could

depolarize

the

central

axon

Given

that

the

cell

body

was

modeled

using

a

channel

density

ten

times

smaller

than

for

the

central

axon

it

had

a

much

higher

threshold

than

the

central

axon

thereby

leading

to

a

lower

anodic

threshold

A

similar

effect

occurred

in

the

case

of

demyelination

of

the

peripheral

process

because

unmyelinated

fibers

need

much

stronger

currents

to

be

excited

than

myelinated

ones

Given

that

peripheral

processes

in

deafened

ears

are

believed

to

be

somewhat

degenerated

-LRB-

Fayad

and

Linthicum

-RRB-

and

that

this

degeneration

is

often

accompanied

by

demyelination

the

predictions

of

Rattay

et

al.

-LRB-

-RRB-

give

a

plausible

explanation

for

the

higher

sensitivity

to

anodic

stimulus

currents

observed

in

implanted

subjects

This

would

therefore

imply

that

the

excitation

site

is

at

the

level

of

the

central

axon

which

is

consistent

with

a

report

of

van

den

Honert

and

Stypulkowski

-LRB-

-RRB-

on

the

responses

of

cats

fibers

with

and

without

peripheral

processes

and

cell

bodies

They

found

the

responses

of

the

degenerated

fibers

to

closely

resemble

those

of

the

normal

fibers

stimulated

at

high

current

levels

Although

the

exact

location

of

the

excitation

site

is

still

a

matter

of

debate

and

probably

depends

on

the

electrode

configuration

and

on

the

stimulus

current

level

Miller

et

al.

-LRB-

-RRB-

demonstrated

that

cats

fibers

were

almost

always

excited

at

the

level

of

their

central

axon

with

monopolar

stimulation

The

response

latencies

obtained

in

experiment

for

anodic

stimulation

-LRB-

μs

on

average

-RRB-

also

argue

for

a

stimulation

at

the

level

of

the

central

axon

given

that

they

are

similar

to

the

latencies

commonly

observed

in

human

CI

subjects

for

symmetric

biphasic

waveforms

but

also

comparable

to

those

measured

intracochlearly

in

cats

for

anodic

pulses

-LRB-

Brown

et

al.

Miller

et

al.

-RRB-

Given

that

the

main

morphological

discrepancies

between

humans

and

cats

are

the

length

of

the

peripheral

processes

-LRB-

longer

in

humans

-RRB-

and

the

size

and

degree

of

myelination

of

the

spiral

ganglion

cells

-LRB-

larger

and

unmyelinated

in

humans

Liberman

and

Oliver

Spoendlin

and

Schrott

Rattay

et

al.

-RRB-

we

would

expect

significant

ECAP

latency

differences

if

the

auditory

nerve

fibers

were

excited

at

their

peripheral

process

or

cell

body

sites

but

not

if

they

were

excited

at

the

level

of

their

central

axon

To

test

whether

neural

degeneration

and

demyelination

are

the

causes

of

our

finding

additional

psychophysical

measures

may

be

performed

Specifically

in

ears

with

substantial

degeneration

the

dominance

of

the

anodic

phase

should

be

enhanced

the

effect

of

phase

separation

on

threshold

should

be

reduced

-LRB-

Prado-Guitierrez

et

al.

-RRB-

and

the

slopes

of

the

strength-duration

functions

should

be

shallower

-LRB-

Miller

et

al.

-RRB-

Finally

we

reported

in

a

previous

study

-LRB-

Macherey

et

al.

-RRB-

a

comparison

of

thresholds

and

MCLs

for

anodic

and

cathodic-first

asymmetric

pulses

Although

we

did

find

lower

MCLs

for

anodic-first

pulses

consistent

with

the

present

results

we

did

not

observe

any

difference

at

threshold

It

is

possible

that

the

higher

sensitivity

to

anodic

stimuli

is

specific

to

suprathreshold

levels

One

hypothesis

proposed

by

Macherey

et

al.

-LRB-

-RRB-

was

that

cathodic-first

asymmetric

pulses

may

produce

a

cathodal

block

at

high

levels

This

seems

however

unlikely

given

that

cathodal

blocking

should

occur

at

very

high

levels

above

threshold

-LRB-

approximately

eight

times

higher

Ranck

-RRB-

This

would

correspond

to

a

current

level

higher

than

threshold

therefore

exceeding

the

common

dynamic

range

values

of

CI

users

-LRB-

for

example

the

probe

dynamic

ranges

of

experiment

ranged

from

about

to

dB

-RRB-

Another

possibility

is

that

the

cluster

of

fibers

recruited

at

threshold

is

equally

sensitive

to

anodic

and

cathodic

phases

or

that

alternatively

approximately

the

same

number

of

fibers

is

excited

by

an

anodic

and

by

a

cathodic

phase

At

higher

levels

more

spatially

remote

fibers

may

be

recruited

and

it

is

possible

that

a

majority

of

these

fibers

are

more

sensitive

to

anodic

stimulation

Interestingly

in

their

spatial

modeling

study

of

the

human

cochlea

Rattay

et

al.

-LRB-

-RRB-

predicted

that

the

nerve

fibers

close

to

the

stimulating

electrode

would

demonstrate

lower

cathodic

than

anodic

thresholds

but

that

fibers

more

spatially

remote

would

in

some

cases

exhibit

the

opposite

trend

However

we

note

that

in

experiment

the

greater

masking

effectiveness

of

the

anodic

phase

was

observed

for

probes

presented

to

electrodes

adjacent

to

that

used

for

the

masker

Applications

to

CIs

Our

results

have

implications

not

only

for

theories

of

electrical

stimulation

of

the

human

auditory

nerve

but

also

for

the

development

of

future

CIs

Asymmetric

pulses

may

be

used

in

a

CI

speech

processing

strategy

for

two

reasons

to

reduce

power

consumption

or

to

increase

spatial

selectivity

First

we

previously

suggested

that

asymmetric

pulses

presented

in

monopolar

mode

would

achieve

some

worthwhile

power

savings

-LRB-

Macherey

et

al.

-RRB-

If

such

pulses

were

implemented

in

a

speech

processing

strategy

then

the

results

of

experiment

suggest

that

the

short

high-amplitude

phase

of

the

pulses

should

be

anodic

relative

to

the

active

electrode

In

this

case

the

effect

of

the

long

low-amplitude

phase

on

neural

excitation

should

be

kept

minimal

Second

for

so-called

longitudinal

bipolar

stimulation

where

each

intracochlear

electrode

is

stimulated

with

reference

to

another

nearby

intracochlear

electrode

the

pattern

of

excitation

along

the

cochlea

can

sometimes

show

a

bimodal

shape

with

peaks

of

excitation

near

the

two

stimulating

electrodes

-LRB-

Kral

et

al.

Chatterjee

et

al.

-RRB-

Frijns

et

al.

-LRB-

-RRB-

suggested

that

this

bimodality

may

be

reduced

by

the

use

of

asymmetric

pulses

Their

computational

model

showed

that

the

nerve

fibers

proximal

to

the

electrode

for

which

the

short

high-amplitude

phase

is

cathodic

would

be

more

effectively

excited

than

the

fibers

proximal

to

the

other

electrode

Our

results

suggest

that

a

similar

reduced

bimodality

and

enhanced

spatial

selectivity

may

be

obtained

in

CIs

but

crucially

with

more

excitation

near

the

electrode

presenting

the

short

anodic

phase

Note

that

in

the

present

work

we

used

asymmetric

pulses

with

relatively

long

phase

separations

which

are

unlikely

to

be

useful

in

multichannel

CIs

because

they

would

require

an

extremely

low

repetition

rate

However

we

would

expect

this

enhanced

spatial

selectivity

to

also

hold

for

bipolar

asymmetric

stimuli

with

a

shorter

or

no

phase

separation

which

may

be

more

appropriate

candidates

for

implementation

in

a

CI

speech

processing

strategy

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

cochlear implants
electrical stimulation
auditory nerve
psychophysical masking
stimulus polarity
asymmetric pulses


Variations in the FTO gene are associated with severe obesity in the Japanese Variations in the fat-mass and obesity-associated gene -LRB- FTO -RRB- are associated with the obesity phenotype in many Caucasian populations . This association with the obesity phenotype is not clear in the Japanese . To investigate the relationship between the FTO gene and obesity in the Japanese , we genotyped single nucleotide polymorphisms -LRB- SNPs -RRB- in the FTO genes from severely obese subjects -LSB- n = 927 , body mass index -LRB- BMI -RRB- ≥ 30 kg/m2 -RSB- and normal-weight control subjects -LRB- n = 1,527 , BMI < 25 kg/m2 -RRB- . A case-control association analysis revealed that 15 SNPs , including rs9939609 and rs1121980 , in a linkage disequilibrium -LRB- LD -RRB- block of approximately 50 kb demonstrated significant associations with obesity ; rs1558902 was most significantly associated with obesity . P value in additive mode was 0.0000041 , and odds ratio -LRB- OR -RRB- adjusted for age and gender was 1.41 -LSB- 95 % confidential interval -LRB- CI -RRB- = 1.22 -- 1.62 -RSB- . Obesity-associated phenotypes , which include the level of plasma glucose , hemoglobin A1c , total cholesterol , triglycerides , high-density lipoprotein -LRB- HDL -RRB- cholesterol , and blood pressure were not associated with the rs1558902 genotype . Thus , the SNPs in the FTO gene were found to be associated with obesity , i.e. , severe obesity , in the Japanese . Introduction Obesity is the most common nutritional disorder in developed countries , and it is a major risk factor for hypertension , cardiovascular disease , and type 2 diabetes -LRB- Kopelman 2000 ; Wilson et al. 2003 -RRB- . Genetic and environmental factors contribute to obesity development -LRB- Maes et al. 1997 ; Barsh et al. 2000 ; Rankinen et al. 2006 -RRB- . Recent progress in single nucleotide polymorphism -LRB- SNP -RRB- genotyping techniques has enabled genome-wide association studies on common diseases -LRB- Herbert et al. 2006 ; Frayling et al. 2007 ; Scuteri et al. 2007 ; The Wellcome Trust Case Control Consortium 2007 ; Hinney et al. 2007 -RRB- . Using a large-scale case-control association study , we found that secretogranin III -LRB- SCG3 -RRB- -LRB- Tanabe et al. 2007 -RRB- and myotubularin-related protein 9 -LRB- MTMR9 -RRB- -LRB- Yanagiya et al. 2007 -RRB- are involved in susceptibility to the obesity phenotype . Genome-wide association studies have shown that the fat-mass and obesity-associated gene -LRB- FTO -RRB- is also associated with the obesity phenotype -LRB- Frayling et al. 2007 ; Scuteri et al. 2007 ; Hinney et al. 2007 -RRB- . This association was also found in many Caucasian and Hispanic American populations -LRB- Frayling et al. 2007 ; Scuteri et al. 2007 ; Dina et al. 2007 ; Field et al. 2007 ; Andreasen et al. 2008 ; Wåhlén et al. 2008 ; Peeters et al. 2008 -RRB- , whereas it was not found in the Chinese Han population -LRB- Li et al. 2008 -RRB- . Among Japanese , body mass index -LRB- BMI -RRB- was higher in subjects who had the A allele of rs9939609 , similar to that observed in Caucasians ; however , this finding was not significant -LRB- Horikoshi et al. 2007 -RRB- . Another group reported that rs9939609 was associated with BMI in the Japanese -LRB- Omori et al. 2008 -RRB- . Thus , the association of SNPs in the FTO gene with obesity in the Japanese remains controversial . To investigate the relationship between the FTO gene and obesity in the Japanese , we performed a case-control association study using patients with severe adult obesity -LRB- BMI ≥ 30 kg/m2 -RRB- and normal-weight controls -LRB- BMI < 25 kg/m2 -RRB- ; we found that SNPs in intron 1 of the FTO gene were associated with severe adult obesity . Materials and methods Study subjects The sample size for severely obese Japanese subjects -LRB- BMI ≥ 30 kg/m2 -RRB- was 927 -LRB- male : female ratio 419:508 , age 48.7 ± 14.2 years , BMI 34.2 ± 5.4 kg/m2 -RRB- , whereas that for Japanese normal weight controls -LRB- BMI < 25 kg/m2 -RRB- was 1,527 -LRB- male : female ratio 685:842 , age 48.1 ± 16.5 years , BMI 21.7 ± 2.1 kg/m2 -RRB- . The severely obese subjects were recruited from among outpatients of medical institutes . Patients with secondary obesity and obesity-related hereditary disorders were not included , and neither were patients with medication-induced obesity . The normal-weight controls were recruited from among subjects who had undergone a medical examination for screening of common diseases . Clinical features of the subjects are illustrated in Table 1 . Additionally , 1,604 subjects were recruited -LRB- male : female ratio 803:801 , age 48.7 ± 16.9 years , BMI 22.66 ± 3.16 kg/m2 -RRB- from the Japanese general population . Each subject provided written informed consent , and the protocol was approved by the ethics committee of each institution and that of RIKEN . Table 1Clinical characterization of obese and control subjectsObeseControlP valueGender -LRB- M/F -RRB- 419/508658/842Age -LRB- year -RRB- 49.1 ± 14.248.2 ± 16.50.049 Body mass index -LRB- kg/m2 -RRB- 34.50 ± 5.3921.65 ± 2.08 < 0.000001 Glucose -LRB- mg/dl -RRB- 129.2 ± 49.697.7 ± 23.9 < 0.000001 HbA1c -LRB- % -RRB- 6.5 ± 1.85.1 ± 0.6 < 0.000001 Total cholesterol -LRB- mg/dl -RRB- 209.9 ± 37.9201.2 ± 36.4 < 0.000001 Triglycerides -LRB- mg/dl -RRB- 153.2 ± 99.5104.0 ± 73.2 < 0.000001High-density lipoprotein cholesterol -LRB- mg/dl -RRB- 53.1 ± 18.965.1 ± 15.7 < 0.000001 Systolic blood pressure -LRB- mmHg -RRB- 136.4 ± 18.1123.4 ± 17.8 < 0.000001 Diastolic blood pressure -LRB- mmHg -RRB- 83.8 ± 12.076.0 ± 11.1 < 0.000001 P values were analyzed using Mann -- Whitney U test . Data are mean ± standard deviation DNA preparation and SNP genotyping Genomic DNA was prepared from the blood sample of each subject by using the Genomix -LRB- Talent Srl , Trieste , Italy -RRB- . We searched for dbSNPs with minor allele frequencies -LRB- MAF -RRB- > 0.10 in the FTO gene of Japanese people . We selected 90 SNPs and were able to construct Invader probes -LRB- Third Wave Technologies , Madison , WI -RRB- for them -LRB- Supplementary Table 1 -RRB- . SNPs were genotyped using Invader assays as described previously -LRB- Ohnishi et al. 2001 ; Takei et al. 2002 -RRB- . Nine SNPs -LRB- rs9937053 , rs9939973 , rs9940128 , rs7193144 , rs8043757 , rs9923233 , rs9926289 , rs9939609 , and rs9930506 -RRB- reported in a previous genome-wide association study -LRB- Scuteri et al. 2007 -RRB- were genotyped using TaqMan probes -LRB- C __ 29910458_10 , C __ 11776771_10 , C __ 29621384_10 , C __ 29387650_10 , C __ 29387665_10 , C __ 29693738_10 , C __ 30270568_10 , C __ 30090620_10 , and C __ 29819994_10 ; Applied Biosystems , Foster City , CA , USA -RRB- . Statistical analysis Genotype or allele frequencies were compared between cases and controls in three different modes . In the first mode , i.e. , the additive mode , χ2 test was performed according to Sladek et al. -LRB- Sladek et al. 2007 -RRB- . In the second mode , i.e. , the minor allele recessive mode , frequencies of the homozygous genotype for the minor allele were compared using a 2 × 2 contingency table . In the third mode , i.e. , the minor allele dominant mode , frequencies of the homozygous genotype for the major allele were compared using a 2 × 2 contingency table . A test of independence was performed using Pearson 's χ2 method . P values were corrected by Bonferroni adjustment and P < 0.00017 -LSB- 0.05 / 99 -LRB- total SNP number -RRB- / 3 -LRB- number of modes -RRB- -RSB- was considered significant . The odds ratio -LRB- OR -RRB- and 95 % confidence interval -LRB- CI -RRB- were calculated by Woolf 's method . We coded genotypes as 0 , 1 , and 2 , depending on the number of copies of the risk alleles . OR adjusted for age and gender was calculated using multiple logistic regression with genotypes , age , and gender as independent variables . Hardy -- Weinberg equilibrium was assessed using the χ2 test -LRB- Nielsen et al. 1998 -RRB- . Haplotype blocks were determined using Haploview -LRB- Barrett et al. 2005 -RRB- . Simple comparison of the clinical data among the different genotypes was performed using one-way analysis of variance -LRB- ANOVA -RRB- . Simple comparison of the clinical data between case and control groups was analyzed using Mann -- Whitney U test . Difference in BMI between genotypes was analyzed using a multiple linear regression , with BMI as the dependent variable and genotype as the independent variable , and with gender and age as covariates for BMI . Statistical analyses were performed using StatView 5.0 -LRB- SAS Institute , Cary , NC , USA -RRB- . Power was calculated by the Monte Carlo method . Results Case-control association studies We searched for dbSNPs with MAF > 0.10 in the FTO gene . By using Invader and TaqMan assay , we successfully genotyped 99 SNPs spanning the FTO gene -LRB- Supplementary Table 1 -RRB- . Using these SNPs , we performed tests of independence between the phenotype and genotypes of obesity at each SNP by using severely obese subjects -LRB- BMI ≥ 30 kg/m2 -RRB- and normal weight controls -LRB- BMI < 25 kg/m2 -RRB- . For each SNP , the lowest P value among the three different modes was selected as the minimum P value . All SNPs , including rs1421084 , were in Hardy -- Weinberg equilibrium -LRB- P > 0.01 -RRB- -LRB- Supplementary Table 1 -RRB- . The power of the test was calculated by Monte Carlo method with different MAFs and different effect sizes . Effect of the risk allele on penetrance was assumed to be multiplicative ; i.e. , the penetrances for three genotypes were assumed to be a , ar , and ar2 , respectively , where a and r denote the lowest penetrance and genotype relative risk , respectively . Supplementary Table 2 shows the calculated values of the power of the test with different MAFs and different genotype relative risks -LRB- r -RRB- . The lowest penetrance -LRB- a -RRB- was calculated for each gender by assuming the affection rates of 2.3 % for men and 3.4 % for women -LRB- Yoshiike et al. 2002 -RRB- . Genotype relative risk -LRB- r -RRB- was assumed to be the same for both genders . Supplementary Table 2 shows that the test has significant power at relative high risk allele frequency when genotype relative risk is > 1.7 . As shown in Fig. 1 and Supplementary Table 1 , 15 SNPs demonstrated significant associations with the obesity phenotype ; the threshold of significance using Bonfferoni correction was P < 0.00017 . These SNPs included rs9939609 -LRB- Frayling et al. 2007 -RRB- and rs1121980 -LRB- Hinney et al. 2007 -RRB- that were reported to be significantly associated with the obesity phenotype in the Caucasian population , as determined by genome-wide association studies ; rs9930506 -LRB- Scuteri et al. 2007 -RRB- showed marginal association with obesity in the Japanese . Linkage disequilibrium -LRB- LD -RRB- analysis revealed that these 15 SNPs were in almost complete LD -LRB- D' > 0.98 , r2 > 0.80 -RRB- and were located within the same LD block of approximately 50 kb -LRB- Fig. 1 -RRB- . The most significant association was observed for rs1558902 -LSB- additive mode , P = 0.0000041 and allele-specific OR -LRB- 95 % CI -RRB- adjusted for age and gender was 1.41 -LRB- 1.22 -- 1.62 -RRB- -RSB- . The minor alleles of rs9939609 -LRB- MAF = 0.24 -RRB- and rs1121980 -LRB- MAF = 0.26 -RRB- were significantly more frequent in the obese group than in the normal-weight control group -LRB- additive mode , P = 0.000012 and P = 0.000051 , respectively -RRB- , and ORs were 1.38 -LRB- 95 % CI = 1.20 -- 1.59 -RRB- and 1.33 -LRB- 95 % CI = 1.16 -- 1.52 -RRB- , respectively -LRB- Table 2 , Supplementary Table 1 -RRB- . The MAF of both SNPs in the control group was 0.18 ; this was consistent with data obtained from the haplotype map of the human genome -LRB- HapMap -RRB- -LRB- Supplementary Table 1 -RRB- . Our data indicated that the SNPs in the FTO gene were associated with severe obesity in the Japanese . Fig. 1Linkage disequilibrium -LRB- LD -RRB- mapping , polymorphisms , and P values obtained in the test of independence between the phenotype and genotypes of obesity at various single nucleotide polymorphisms -LRB- SNPs -RRB- in the fat-mass and obesity-associated gene -LRB- FTO -RRB- gene . P values are expressed as negative logarithm of the minimum P values obtained in the three models -LRB- additive , minor allele dominant , and minor allele recessive modes -RRB- . LD coefficients -LRB- D' -RRB- between each pair of SNPs were calculated and are displayed as a strand in the LD blocks . Minor allele frequencies of all SNPs used in this analysis are ≥ 10 % . The genomic structure is shown in the upper . The gray bar marks the LD block associated with obesityTable 2Associations of single nucleotide polymorphisms -LRB- SNPs -RRB- in the fat-mass and obesity-associated gene -LRB- FTO -RRB- gene with obesity existing in the 50-kb linkage disequilibrium -LRB- LD -RRB- blockdbSNP IDAlleleGenotypeAdditive modeRecessive modeDominant modeCaseControl1/2111222Sum111222SumOR -LRB- 95 % CI -RRB- χ2P valueχ2P valueOR -LRB- 95 % CI -RRB- χ2P valueOR -LRB- 95 % CI -RRB- rs9937053A/G593604949136341477312501 .31 -LRB- 1.13 -- 1.51 -RRB- 12.30.00047 2.00.161.30 -LRB- 0.90 -- 1.88 -RRB- 13.00.000311.37 -LRB- 1.16 -- 1.63 -RRB- rs9939973A/G613674969247550494115201 .32 -LRB- 1.15 -- 1.51 -RRB- 15.70.000077 a3 .00.0811.36 -LRB- 0.96 -- 1.93 -RRB- 16.10.000061 a1 .40 -LRB- 1.19 -- 1.66 -RRB- rs9940128A/G603664989247550094115161 .31 -LRB- 1.15 -- 1.50 -RRB- 15.20.00010 a2 .60.111.33 -LRB- 0.94 -- 1.89 -RRB- 15.90.000068 a1 .40 -LRB- 1.19 -- 1.65 -RRB- rs1421085C/T4933853792457443101915191 .38 -LRB- 1.20 -- 1.59 -RRB- 19.60.000011 a3 .30.0681.44 -LRB- 0.97 -- 2.12 -RRB- 20.00.0000078 a1 .47 -LRB- 1.24 -- 1.74 -RRB- rs1558902A/T4834153692552449102115221 .41 -LRB- 1.22 -1.62 -RRB- 21.20.0000041 a4 .60.0321.55 -LRB- 1.04 -- 2.31 -RRB- 20.80.0000052 a1 .48 -LRB- 1.25 -- 1.75 -RRB- rs1121980A/G613674999277350494715241 .33 -LRB- 1.16 -- 1.52 -RRB- 16.50.000051 a3 .60.0591.40 -LRB- 0.99 -- 1.99 -RRB- 16.50.000050 a1 .41 -LRB- 1.19 -- 1.66 -RRB- rs7193144C/T4933953292055447101415161 .39 -LRB- 1.21 -- 1.61 -RRB- 20.40.0000067 a4 .00.0441.49 -LRB- 1.01 -- 2.22 -RRB- 20.30.0000067 a1 .47 -LRB- 1.24 -- 1.74 -RRB- rs8043757T/A4831954190854436102715171 .36 -LRB- 1.18 -- 1.57 -RRB- 17.40.000037 a4 .20.0401.51 -LRB- 1.02 -- 2.25 -RRB- 16.40.000052 a1 .42 -LRB- 1.20 -- 1.69 -RRB- rs8050136A/C5133653892556450101815241 .38 -LRB- 1.20 -- 1.59 -RRB- 19.40.000012 a4 .70.0311.53 -LRB- 1.04 -- 2.26 -RRB- 18.50.000017 a1 .45 -LRB- 1.22 -- 1.71 -RRB- rs3751812T/G5134053492555458101315261 .38 -LRB- 1.20 -- 1.59 -RRB- 19.60.0000098 a5 .10.0241.56 -LRB- 1.06 -- 2.31 -RRB- 18.50.000017 a1 .45 -LRB- 1.22 -- 1.71 -RRB- rs9923233C/G5133553391955449101015141 .38 -LRB- 1.20 -- 1.60 -RRB- 19.80.0000093 a5 .00.0251.56 -LRB- 1.06 -- 2.30 -RRB- 18.70.000015 a1 .45 -LRB- 1.23 -- 1.72 -RRB- rs9926289A/G503235319045642599314741 .37 -LRB- 1.19 -1.58 -RRB- 18.70.000020 a3 .90.0471.48 -LRB- 1.00 -- 2.19 -RRB- 18.10.000021 a1 .45 -LRB- 1.22 -- 1.72 -RRB- rs9939609A/T5133453491956443100515041 .38 -LRB- 1.20 -- 1.59 -RRB- 19.50.000012 a4 .50.0341.52 -LRB- 1.03 -- 2.24 -RRB- 18.70.000015 a1 .45 -LRB- 1.23 -- 1.72 -RRB- rs7185735G/A5134053692755455101415241 .38 -LRB- 1.20 -- 1.59 -RRB- 19.90.0000089 a5 .00.0251.55 -LRB- 1.05 -- 2.30 -RRB- 18.80.000014 a1 .45 -LRB- 1.23 -- 1.72 -RRB- rs9931494G/C643634949217150494215171 .35 -LRB- 1.18 -- 1.55 -RRB- 18.40.000018 a5 .60.0181.52 -LRB- 1.07 -- 2.15 -RRB- 16.90.000039 a1 .42 -LRB- 1.20 -- 1.67 -RRB- rs17817964T/C623615009236852493015221 .30 -LRB- 1.14 -- 1.49 -RRB- 13.50.00022 5.80.0161.54 -LRB- 1.08 -- 2.19 -RRB- 11.40.000751.33 -LRB- 1.13 -- 1.57 -RRB- rs9930506G/A673654889208252191315161 .28 -LRB- 1.12 -- 1.46 -RRB- 12.80.00038 3.50.0611.37 -LRB- 0.98 -- 1.92 -RRB- 12.10.000511.34 -LRB- 1.14 -- 1.58 -RRB- rs9932754C/T663684919257852591915221 .29 -LRB- 1.13 -- 1.48 -RRB- 13.60.00023 4.20.0401.42 -LRB- 1.01 -- 2.00 -RRB- 12.60.000401.35 -LRB- 1.14 -- 1.59 -RRB- rs9922619T/G663684899237852991915261 .29 -LRB- 1.13 -- 1.48 -RRB- 13.50.00024 4.30.0381.43 -LRB- 1.02 -- 2.01 -RRB- 12.30.000441.34 -LRB- 1.14 -- 1.58 -RRB- rs7204609C/T13441837392527371752915190 .83 -LRB- 0.73 -- 0.93 -RRB- 9.680.0022 5.00.0250.77 -LRB- 0.62 -- 0.97 -RRB- 7.50.00630.79 -LRB- 0.67 -- 0.94 -RRB- rs12149832A/G533495259276248098215241 .33 -LRB- 1.15 -- 1.53 -RRB- 15.20.000098 a3 .50.0611.43 -LRB- 0.98 -- 2.08 -RRB- 14.80.00012 a1 .39 -LRB- 1.17 -- 1.64 -RRB- The odds ratio -LRB- OR -RRB- for each SNP was adjusted simultaneously for age and gender using additive modelCI confidence interval , χ2 chi-squareaSignificant P value -LRB- P < 0.00017 -RRB- Analysis of various quantitative phenotypes with rs1558902 To investigate whether the genotypes of SNP rs1558902 are associated with the phenotypes of metabolic disorders , we compared the following among the different genotypes in the cases , controls , and combined groups : ANOVA results , BMI , levels of fasting plasma glucose , hemoglobin A1c -LRB- HbA1c -RRB- , total cholesterol , triglycerides , HDL cholesterol , and blood pressure . As rs1558902 showed the most significant association with obesity and its call rate was the highest , we analyzed various quantitative phenotypes by using this SNP . The quantitative phenotypes regarding BMI and the levels of fasting plasma glucose , HbA1c , total cholesterol , triglycerides , HDL cholesterol , and blood pressure were not found to be significantly associated with the genotypes at rs1558902 in either the case or control group -LRB- Table 3 -RRB- . Although there was no significant difference in BMI values among genotypes in either the control or case group , the direction of the difference -LRB- AA > AT > TT -RRB- was in accordance with the association between the qualitative obesity phenotype and the genotype shown . Table 3Comparison of various quantitative phenotypes among different genotypes at single nucleotide polymorphism -LRB- SNP -RRB- rs1558902 in obese and control subjectsObeseControlAA -LRB- n = 48 -RRB- AT -LRB- n = 341 -RRB- TT -LRB- n = 536 -RRB- AA -LRB- n = 52 -RRB- AT -LRB- n = 448 -RRB- TT -LRB- n = 1022 -RRB- Age -LRB- year -RRB- 49.8 ± 15.349.6 ± 14.348.8 ± 14.146.9 ± 15.446.9 ± 16.748.8 ± 16.5 P value0 .640.098 BMI -LRB- kg/m2 -RRB- 35.16 ± 5.7034.61 ± 5.4334.39 ± 5.3321.94 ± 2.23 21.62 ± 2.1021.65 ± 2.06 P value0 .580.56 Glucose -LRB- mg/dl -RRB- 142.8 ± 54.8125.4 ± 43.2130.8 ± 53.3101.7 ± 44.196.3 ± 18.198.2 ± 24.7 P value0 .0540.34 HbA1c -LRB- % -RRB- 6.9 ± 2.16.4 ± 1.76.5 ± 1.85.1 ± 1.25.0 ± 0.55.1 ± 0.7 P value0 .190.15 Total cholesterol -LRB- mg/dl -RRB- 215.1 ± 46.7211.3 ± 38.8208.6 ± 36.6195.6 ± 38.8201.4 ± 37.8201.4 ± 35.6 P value0 .370.53 Triglycerides -LRB- mg/dl -RRB- 171.7 ± 119.5151.3 ± 102.1153.2 ± 96.0111.7 ± 70.6102.0 ± 71.4104.4 ± 74.2 P value0 .420.63 HDL cholesterol -LRB- mg/dl -RRB- 53.2 ± 13.854.8 ± 24.052.0 ± 15.462.1 ± 14.265.1 ± 15.965.3 ± 15.6 P value0 .140.53 SBP -LRB- mmHg -RRB- 134.2 ± 20.4137.0 ± 17.8136.2 ± 18.2122.7 ± 17.3123.2 ± 18.8123.5 ± 17.5 P value0 .610.91 DBP -LRB- mmHg -RRB- 80.3 ± 11.784.1 ± 12.083.9 ± 12.075.5 ± 11.175.2 ± 11.776.3 ± 10.9 P value0 .140.22 Data of each quantitative phenotype were compared among different genotypes at the rs1558902 in obese and control subjects . P values were analyzed using analysis of variance in each group of obese and control subjects . Data are mean ± standard deviation.HDL high-density lipoprotein , SBP systolic blood pressure , DBP diastolic blood pressure . Finally , we examined the BMI distribution of rs1558902 in the Japanese general population and found that rs1558902 genotype was significantly associated with BMI -LRB- Table 4 -RRB- . This result would confirm the association of rs1558902 with obesity . Table 4Association of body mass index -LRB- BMI -RRB- with rs1558902 genotypes in the Japanese general populationAAATTTP value -LRB- additive model -RRB- aBMI -LRB- kg/m2 -RRB- -LRB- n -RRB- 23.17 ± 3.20 -LRB- 59 -RRB- 22.79 ± 3.26 -LRB- 482 -RRB- 22.57 ± 3.11 -LRB- 1063 -RRB- 0.041 aThe difference in BMI according to genotypes was analyzed using a multiple linear regression , with BMI as the dependent variable and genotype as the independent variable and with gender and age as covariates for BMI . Data are represented as mean ± standard deviation Discussion Recent genome-wide association studies have shown that the FTO gene is associated with obesity -LRB- Frayling et al. 2007 ; Scuteri et al. 2007 ; Hinney et al. 2007 -RRB- . The associations between variations in the FTO gene and the obesity phenotype have been observed in many Caucasian subjects -LRB- Frayling et al. 2007 ; Scuteri et al. 2007 ; Dina et al. 2007 ; Field et al. 2007 ; Andreasen et al. 2008 ; Wåhlén et al. 2008 ; Peeters et al. 2008 -RRB- . However , these associations were controversial with regard to Asian subjects -LRB- Horikoshi et al. 2007 ; Li et al. 2008 ; Omori et al. 2008 -RRB- . BMI values did not significantly differ among the genotypes in the general population of Chinese and Japanese -LRB- Horikoshi et al. 2007 ; Li et al. 2008 -RRB- . We performed a case-control association study with regard to severe obesity and found that the SNPs in the FTO gene were significantly associated with severe obesity . Although the SNPs demonstrated the most significant association in the Japanese , which was different from that in Caucasians , the significantly associated SNPs existed in a similar block as that in Caucasians . Therefore , the FTO gene could also contribute to the development of severe obesity in the Japanese . BMI was modestly different among rs1558902 genotypes in the general population in this study ; rs9939609 was not significantly associated with BMI in the general population -LRB- AA 23.22 ± 3.14 vs AT 22.79 ± 3.25 vs TT 22.58 ± 3.13 , P = 0.063 -RRB- . In the Japanese population , rs1558902 may be more tightly associated with BMI than rs9939609 . The National Nutrition Survey of Japan reported that the prevalence of subjects with a BMI of ≥ 30 kg/m2 is only 2.3 % in men and 3.4 % in women aged 20 years and older -LRB- Yoshiike et al. 2002 -RRB- , and the mean BMI was approximately 23 kg/m2 for ages 15 -- 84 years -LRB- Yoshiike et al. 1998 -RRB- . Inconsistency in the results of effects of variations in the FTO gene on BMI between Japanese and Europians may be due to the relatively small mean and variance of BMI in the former than the latter . The significant SNPs were located in intron 1 of the FTO gene . The rs1558902 and other significant SNPs , for example , rs9939609 and rs1121980 , would affect transcriptional activity of the FTO gene , although further investigation is necessary . The precise mechanism by which the FTO gene leads to obesity development is unclear -LRB- Gerken et al. 2007 ; Sanchez-Pulido et al. 2007 -RRB- . However , the FTO gene is expressed in the hypothalamus and regulated by fasting and leptin -LRB- Frayling et al. 2007 ; Gerken et al. 2007 -RRB- . Using large-scale case-control association studies , we determined that the SCG3 -LRB- Tanabe et al. 2007 -RRB- and MTMR9 -LRB- Yanagiya et al. 2007 -RRB- genes are involved in susceptibility to the obesity phenotype . These two genes are expressed in the hypothalamus . Genetic studies in mice have suggested that mutations in several genes , such as those encoding leptin , proopiomelanocortin , and melanocortin-4 receptor , are implicated in a monogenic form of inherited obesity -LRB- Barsh et al. 2000 ; Rankinen et al. 2006 -RRB- . Such mutations have also been reported in obese humans . As most such genes are expressed in the hypothalamus and have been indicated to play important roles in the regulation of food intake , genes expressed in the hypothalamus are likely to be good candidates for susceptibility to obesity . In summary , we have identified the genetic variations in the FTO gene that may influence the risk of severe obesity in the Japanese . Electronic supplementary material Below is the link to the electronic supplementary material . Summary of the association of SNPs between cases and controls -LRB- DOC 315 kb -RRB-

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

obesity
japanese population
association
snp