The

SPINK

gene

family

and

celiac

disease

susceptibility

The

gene

family

of

serine

protease

inhibitors

of

the

Kazal

type

-LRB-

SPINK

-RRB-

are

functional

and

positional

candidate

genes

for

celiac

disease

-LRB-

CD

-RRB-

Our

aim

was

to

assess

the

gut

mucosal

gene

expression

and

genetic

association

of

and

in

the

Dutch

CD

population

Gene

expression

was

determined

for

all

four

SPINK

genes

by

quantitative

reverse-transcription

polymerase

chain

reaction

in

duodenal

biopsy

samples

from

untreated

-LRB-

n

-RRB-

and

diet-treated

patients

-LRB-

n

-RRB-

and

controls

-LRB-

n

-RRB-

Genetic

association

of

the

four

SPINK

genes

was

tested

within

a

total

of

haplotype

tagging

SNPs

one

coding

SNP

patients

and

controls

The

study

cohort

was

further

expanded

to

include

CD

cases

and

controls

DNA

sequence

analysis

was

performed

on

six

members

of

a

multigeneration

CD

family

to

detect

possible

point

mutations

or

deletions

showed

differential

gene

expression

which

was

at

its

highest

in

untreated

patients

and

dropped

sharply

upon

commencement

of

a

gluten-free

diet

Genetic

association

tests

for

all

four

SPINK

genes

were

negative

including

in

the

extended

case/control

cohort

No

mutations

or

deletions

were

observed

in

the

multigeneration

CD

family

with

linkage

to

chromosome

nor

was

the

coding

SNP

disease-specific

exhibits

CD

pathology-related

differential

gene

expression

likely

derived

from

altered

goblet

cell

activity

All

of

the

four

SPINK

genes

tested

do

not

contribute

to

the

genetic

risk

for

CD

in

the

Dutch

population

Introduction

Celiac

disease

-LRB-

CD

-RRB-

is

a

chronic

inflammatory

condition

of

the

small

intestine

because

of

an

immunological

intolerance

for

the

food

protein

gluten

Patients

have

to

adhere

to

a

life-long

diet

devoid

of

gluten

to

prevent

the

detrimental

effects

of

a

prolonged

nutrient

and

mineral

deficiency

-LRB-

Green

and

Jabri

-RRB-

Susceptibility

for

CD

is

predominantly

determined

by

genetic

factors

and

the

complex

inheritance

patterns

suggest

the

interaction

of

multiple

genes

-LRB-

van

Heel

et

al.

-RRB-

It

is

well

established

that

the

adaptive

immune

response

to

gluten

plays

a

pivotal

role

in

the

pathogenesis

of

CD

activation

of

T

cells

follows

gluten-peptide

presentation

by

or

molecules

expressed

on

antigen-presenting

cells

-LRB-

Sollid

-RRB-

The

HLA-DQA

and

DQB

gene

variants

coding

for

these

molecules

are

the

major

genetic

determinants

for

CD

susceptibility

-LRB-

Koning

et

al.

-RRB-

Recently

the

importance

of

innate

immunity

in

CD

pathogenesis

was

also

underscored

by

the

observation

of

induced

expression

and

NKT

cell

chemotaxis

through

the

MICA

and

molecules

-LRB-

Hue

et

al.

Meresse

et

al.

-RRB-

However

no

genetic

contribution

of

the

cognate

genes

has

been

demonstrated

The

notion

of

crosstalk

between

the

adaptive

and

innate

immune

systems

is

not

limited

to

CD

and

gets

much

attention

in

studies

of

the

inflammatory

process

-LRB-

Hoebe

et

al.

-RRB-

This

raises

the

question

whether

some

aspects

of

innate

immunity

may

contribute

to

the

genetic

susceptibility

for

CD

The

innate

immune

system

uses

a

wide

array

of

defense

mechanisms

against

the

invasion

of

pathogens

These

encompass

the

expression

of

pattern

recognition

receptors

release

of

antimicrobial

molecules

and

preservation

of

epithelial

barrier

and

tissue

integrity

by

e.g.

serine

protease

inhibitors

-LRB-

Kimbrell

and

Beutler

-RRB-

One

branch

of

the

family

of

serine

protease

inhibitors

is

that

of

the

Kazal

type

-LRB-

SPINK

-RRB-

that

originally

consisted

of

four

members

in

humans

-LRB-

and

-RRB-

Recently

as

part

of

a

cluster

of

SPINK

genes

on

chromosome

that

already

included

and

five

new

SPINK

-LRB-

like

-RRB-

members

were

identified

that

were

located

more

distally

and

respectively

-LRB-

NCBI

Map

Viewer

build

-RRB-

However

these

new

members

lack

functional

annotation

and

were

therefore

not

included

in

this

study

SPINK

family

members

and

have

a

comparable

size

and

structure

coded

for

by

exons

with

a

single

Kazal

type

serine

protease

inhibitor

domain

in

contrast

contains

exons

that

encode

inhibitory

domains

All

four

SPINK

members

are

thought

to

be

involved

in

the

protection

against

proteolytic

degradation

of

epithelial

and

mucosal

tissues

although

their

major

site

of

expression

may

differ

is

expressed

in

the

pancreas

and

the

gastrointestinal

tract

and

mutations

in

this

gene

are

reported

in

various

forms

of

pancreatitis

-LRB-

Pfutzer

and

Whitcomb

-RRB-

-LRB-

located

on

-RRB-

is

expressed

in

the

testis

epididymis

and

seminal

vesicle

where

its

antimicrobial

function

may

be

involved

in

protection

of

fertility

-LRB-

Rockett

et

al.

-RRB-

was

originally

isolated

from

pig

intestine

-LRB-

Agerberth

et

al.

-RRB-

and

is

abundantly

expressed

in

human

and

porcine

goblet

cells

in

the

crypts

of

Lieberkühn

but

was

also

found

in

monocytes

and

in

the

central

nervous

system

-LRB-

Metsis

et

al.

Norberg

et

al.

-RRB-

is

expressed

in

the

thymus

vaginal

epithelium

Bartolin

's

glands

oral

mucosa

tonsils

and

the

parathyroid

glands

-LRB-

Magert

et

al.

-RRB-

Mutations

in

are

responsible

for

the

Netherton

syndrome

a

lethal

skin

disorder

characterized

by

ichthyosis

hair

shaft

defects

atopy

skin

barrier

defects

and

recurrent

bacterial

infections

-LRB-

Bitoun

et

al.

-RRB-

Mouse

models

of

the

Netherton

syndrome

have

shown

enhanced

proteolysis

of

desmoglein

and

filaggrin

in

mutants

-LRB-

Descargues

et

al.

Hewett

et

al.

-RRB-

Moreover

has

also

been

associated

with

asthma

and

atopic

dermatitis

-LRB-

Blumenthal

-RRB-

Interestingly

both

and

are

located

on

chromosome

This

region

contains

the

susceptibility

locus

that

emerged

repeatedly

in

linkage

studies

-LRB-

Babron

et

al.

-RRB-

Despite

the

fact

that

this

region

is

rich

in

candidate

cytokine

genes

and

intense

mapping

efforts

were

made

no

closely

associated

genes

were

identified

-LRB-

Ryan

et

al.

-RRB-

Likewise

is

located

on

chromosome

and

resides

within

a

linkage

region

-LRB-

-RRB-

where

we

previously

identified

a

novel

CD

locus

that

segregated

within

a

four-generation

Dutch

family

-LRB-

van

Belzen

et

al.

-RRB-

Taken

together

the

role

of

SPINK

genes

in

epithelial

and

mucosal

protection

and

the

important

genetic

locations

of

and

prompted

us

to

subject

the

four

conventional

members

of

the

SPINK

family

to

gene

expression

and

genetic

association

analyses

to

ascertain

their

possible

role

in

CD

pathogenesis

Materials

and

methods

Patient

material

Duodenal

biopsy

samples

were

collected

by

endoscopy

as

part

of

a

routine

CD

diagnostic

procedure

or

to

monitor

the

response

to

a

gluten-free

diet

in

previously

diagnosed

patients

All

patients

were

classified

using

the

Marsh

nomenclature

according

to

the

UEGW

criteria

-LRB-

Report

of

a

working

group

of

the

United

European

Gastroenterology

Week

in

Amsterdam

-RRB-

Two

biopsy

samples

taken

in

parallel

to

those

used

for

histological

examination

were

pooled

and

used

for

determination

of

gene

expression

In

total

individuals

were

examined

with

quantitative

reverse

transcription

polymerase

chain

reaction

-LRB-

qRT-PCR

-RRB-

of

which

were

normal

controls

and

were

CD

patients

-LRB-

Fig.

-RRB-

The

patient

group

consisted

of

untreated

cases

with

villous

atrophy

-LRB-

MIII

-RRB-

and

patients

treated

with

a

gluten-free

diet

who

were

in

various

stages

of

mucosal

recovery

MII

-LRB-

crypt

hyperplasia

n

-RRB-

MI

-LRB-

lymphocyte

infiltration

n

-RRB-

and

-LRB-

complete

remission

n

-RRB-

The

biopsies

of

all

participating

patients

were

reevaluated

by

an

experienced

pathologist

and

only

CD

patients

with

a

proven

original

Marsh

III

lesion

were

included

in

this

study

The

genetic

association

study

on

all

four

SPINK

genes

was

initially

conducted

on

a

cohort

of

independent

CD

patients

and

independent

age

and

sex-matched

controls

all

of

which

were

from

Dutch

Caucasian

decent

In

a

second

stage

exclusively

focused

on

all

SNPs

we

added

controls

to

a

total

of

In

case

of

three

variants

with

suggestive

P

values

the

power

of

the

study

was

further

enhanced

by

adding

extra

CD

cases

to

a

total

of

In

parallel

we

also

examined

the

gene

in

a

previously

described

four-generation

Dutch

CD

family

-LRB-

van

Belzen

et

al.

-RRB-

Six

family

members

and

a

CEPH

control

were

subjected

to

DNA

sequence

analysis

of

the

coding

regions

and

splice

sites

boundaries

Additionally

we

performed

SNP

haplotype

analysis

in

this

family

All

patients

and

family

members

that

volunteered

for

this

study

signed

an

informed

consent

The

study

was

approved

by

the

Medical

Ethics

Committee

of

the

University

Medical

Center

Utrecht

Fig.

of

qRT-PCR

of

SPINK

genes

in

normal

controls

-LRB-

NC

-RRB-

and

CD

patients

either

untreated

-LRB-

MIII

-RRB-

or

on

a

gluten-free

diet

-LRB-

MII

-RRB-

The

Marsh

stages

refer

to

the

pathological

conditions

of

the

mucosa

characterized

by

atrophy

of

the

villi

-LRB-

MIII

-RRB-

hyperplastic

crypts

between

the

villi

-LRB-

MIII

MII

-RRB-

and

enhanced

lymphocyte

infiltration

-LRB-

MIII

MII

MI

-RRB-

Stage

indicates

complete

remission

comparable

to

controls

The

genes

tested

were

as

follows

-LRB-

a

-RRB-

-LRB-

b

-RRB-

-LRB-

c

-RRB-

and

-LRB-

d

-RRB-

Measurements

were

made

in

triplicate

on

pools

of

separately

prepared

cDNA

samples

Expression

data

were

normalized

to

the

normal

control

pool

-LRB-

e

-RRB-

Relative

expression

of

all

four

SPINK

genes

with

respect

to

in

the

healthy

duodenal

mucosa

Note

the

logarithmic

scale

here

The

GUSB

gene

was

used

as

an

endogenous

control

in

all

tests

Errorbars

indicate

standard

deviations

Expression

study

The

isolation

of

total

RNA

from

biopsy

samples

and

the

analysis

of

gene

expression

by

real-time

qRT-PCR

on

an

ABI

Prism

was

performed

as

described

before

-LRB-

Wapenaar

et

al.

-RRB-

We

used

the

commercially

available

Assay-on-Demand

test

for

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

and

the

endogenous

control

gene

GUSB

-LRB-

PDAR

Applied

Biosystems

Foster

City

CA

-RRB-

All

samples

were

tested

in

triplicate

on

pooled

cDNA

samples

representing

each

Marsh

class

The

results

were

confirmed

with

cDNA

from

individual

samples

tested

in

duplicate

Relative

levels

of

gene

expression

were

obtained

using

the

software

-LRB-

Applied

Biosystems

-RRB-

Genetic

association

and

data

analysis

Haplotype

tagging

SNPs

were

selected

for

and

based

on

HapMap

-LRB-

Phase

I

-RRB-

data

using

Haploview

-LRB-

Barrett

et

al.

-RRB-

For

each

haploblock

containing

SNPs

in

high

linkage

disequilibrium

one

or

more

representative

SNPs

were

selected

that

should

capture

the

genetic

variation

within

that

block

For

the

four

SPINK

genes

tested

this

resulted

in

a

set

of

haplotype

tagging

SNPs

and

one

coding

SNP

-LRB-

see

Table

and

Fig.

-RRB-

SNP

assays

were

obtained

from

Applied

Biosystems

and

analyzed

on

an

ABI

Prism

Hardy

Weinberg

Equilibrium

-LRB-

HWE

-RRB-

was

evaluated

separately

in

cases

and

controls

for

all

SNPs

tested

Allele

frequencies

were

compared

between

cases

and

controls

and

P

values

were

obtained

by

χ2

analysis

Table

distribution

of

SPINK

haplotype

tagging

SNPs

in

a

Dutch

CD

case-control

cohort

Cases

-LRB-

n

-RRB-

Controls

-LRB-

n

-RRB-

AlleleAllele

countsAllele

frequency

-LRB-

-RRB-

Allele

countsAllele

frequency

-LRB-

-RRB-

Gene

nameSNP

i.d.Position

Minor/majorMinorMajorMinorMajorMinorMajorMinorMajorχ2P

Basepair

position

according

to

NCBI

build

Coding

SNP

nonsynonymous

change

-LRB-

-RRB-

Fig.

organization

of

the

four

SPINK

genes

The

upperhorizontalline

indicates

exon

locations

-LRB-

verticalbars

-RRB-

and

SNP

positions

-LRB-

numberedasterisks

-RRB-

The

SNPs

are

numbered

for

each

gene

consecutively

as

they

appear

in

Table

SNP

no.

represents

the

nonsynonymous

-LRB-

-RRB-

coding

SNP

The

arrowpoints

indicate

the

orientation

of

transcription

The

lowerportion

of

the

figure

shows

the

pairwise

linkage-disequilibrium

structure

between

indicated

SNPs

given

by

D

statistics

based

on

the

European

population

in

the

HapMap

database

-LRB-

Phase

II

-RRB-

Darkerred

intensities

indicate

higher

D

values

-LRB-

numbers

indicate

D'

value

whereas

SNP

pairs

without

number

have

a

D

-RRB-

DNA

sequence

analysis

DNA

sequence

analysis

was

performed

on

in

six

members

of

a

four-generation

Dutch

CD

family

and

one

CEPH

control

-LRB-

family

individual

-RRB-

Of

these

six

family

members

four

were

affected

-LRB-

index

and

-RRB-

and

carried

the

disease-linked

haplotype

and

two

were

nonaffected

-LRB-

index

and

-RRB-

without

this

haplotype

-LRB-

Fig.

-RRB-

All

coding

sequences

of

the

gene

were

PCR-amplified

including

the

intron

exon

boundaries

-LRB-

for

primers

and

protocols

see

supplementary

data

Table

-RRB-

PCR

products

were

examined

on

a

agarose

gel

and

purified

with

a

Millipore

Vacuum

Manifold

-LRB-

Billerica

MA

-RRB-

Samples

were

prepared

with

the

BigDye

terminator

cycle

sequencing

ready

kit

-LRB-

Applied

Biosystems

-RRB-

according

to

the

manufacturer

's

protocol

PCR

and

sequencing

amplification

were

performed

on

a

GeneAmp

PCR

system

-LRB-

Perkin

Elmer

Foster

City

CA

-RRB-

Sequences

were

run

on

an

ABI

Prism

analyzer

-LRB-

Applied

Biosystems

-RRB-

Analysis

and

sequence

alignment

was

carried

out

with

Sequence

Navigator

-LRB-

Applied

Biosystems

-RRB-

and

Vector

NTI

-LRB-

InforMax

Massachusetts

-RRB-

software

packages

Fig.

of

the

Dutch

multigeneration

CD

family

Only

affected

descendents

are

depicted

-LRB-

out

of

siblings

in

the

second

generation

were

affected

-RRB-

The

grandparental

haplotypes

that

are

boxed

and

shaded

are

identical

to

the

grandmaternal

at-risk

haplotype

-LRB-

noninformative

-RRB-

The

SNPs

are

ordered

-LRB-

top-to-bottom

-RRB-

as

they

appear

in

Table

Genotype

numbers

and

refer

to

A

C

G

and

T

alleles

respectively

Sequence

analysis

was

performed

on

family

members

and

Family

member

index

numbers

are

indicated

in

bold

Results

SPINK

gene

expression

in

the

CD

mucosa

The

expression

of

all

four

conventional

members

of

the

human

SPINK

family

was

determined

by

real-time

qRT-PCR

on

duodenal

biopsy-derived

cDNA

pools

from

normal

controls

and

CD

patients

either

untreated

or

in

various

stages

of

remission

on

a

gluten-free

diet

The

results

shown

in

Fig.

indicate

that

only

-LRB-

Fig.

-RRB-

is

differentially

expressed

and

that

its

transcriptional

activity

which

is

at

its

highest

in

Marsh

III

-LRB-

compared

to

controls

-RRB-

decreases

sharply

-LRB-

fourfold

-RRB-

when

patients

improve

and

make

a

transition

to

Marsh

II

To

preclude

that

the

results

for

might

be

biased

by

fortuitous

differences

in

individual

expression

levels

within

the

generated

pools

we

also

examined

the

control

and

case

samples

each

separately

This

did

not

change

the

observed

drop

in

expression

during

tissue

recovery

-LRB-

see

supplementary

Fig.

-RRB-

Likewise

we

performed

the

same

analysis

for

the

other

three

SPINK

genes

without

affecting

the

profile

already

observed

in

the

pools

-LRB-

results

not

shown

-RRB-

We

also

examined

the

relative

expression

of

the

four

SPINK

genes

with

respect

to

each

other

in

the

normal

intestinal

mucosa

This

showed

that

both

and

have

the

highest

expression

which

is

respectively

and

higher

compared

to

whereas

is

in

the

same

order

of

magnitude

-LRB-

fivefold

-RRB-

as

-LRB-

Fig.

-RRB-

In

conclusion

only

the

gene

appears

to

be

differentially

regulated

in

the

intestinal

mucosa

during

recovery

from

the

gluten-evoked

CD

lesion

This

observation

prompted

us

to

examine

whether

or

any

of

the

other

SPINK

genes

could

also

be

causally

related

to

the

CD

pathogenesis

Genetic

association

analysis

of

SPINK

genes

We

designed

a

haplotype

tagging

SNP

strategy

to

capture

all

genetic

variation

in

and

An

overview

of

these

four

SPINK

genes

with

their

genomic

organization

linkage-disequilibrium

structure

and

the

position

of

the

haplotype

tagging

SNPs

used

is

depicted

in

Fig.

Initially

these

haplotype

tagging

SNPs

were

tested

in

CD

cases

and

controls

-LRB-

Table

-RRB-

and

showed

no

significant

association

for

any

of

the

haplotype

tagging

SNPs

in

the

four

SPINK

genes

Despite

the

initial

negative

result

we

decided

to

pursue

further

because

it

is

expressed

in

goblet

cells

-LRB-

Metsis

et

al.

-RRB-

displayed

a

CD

pathology-related

differential

expression

in

the

intestinal

mucosa

and

mapped

within

a

CD

linkage

region

-LRB-

van

Belzen

et

al.

-RRB-

Initially

we

expanded

the

control

group

with

samples

to

a

total

of

for

all

SNPs

tested

As

a

result

the

coding

variant

and

its

flanking

haplotype

tagging

SNPs

and

yielded

suggestive

but

nonsignificant

P

values

of

and

respectively

-LRB-

data

not

shown

-RRB-

To

increase

the

power

of

the

study

even

further

we

subsequently

added

CD

cases

to

a

total

of

The

effect

on

the

P

values

of

the

three

SNPs

tested

was

such

that

they

dropped

below

the

significance

threshold

-LRB-

see

Table

-RRB-

From

this

we

conclude

that

the

four

SPINK

genes

tested

do

not

contribute

to

the

genetic

susceptibility

in

the

Dutch

CD

population

Table

distribution

of

three

selected

SNPs

in

the

extended

Dutch

CD

case-control

cohort

Cases

-LRB-

n

-RRB-

Controls

-LRB-

n

-RRB-

AlleleAllele

countsAllele

frequency

-LRB-

-RRB-

Allele

countsAllele

frequency

-LRB-

-RRB-

Gene

nameSNP

i.d.Position

Basepair

position

according

to

NCBI

build

Coding

SNP

nonsynonymous

change

-LRB-

-RRB-

sequence

analysis

in

a

multigeneration

family

We

have

previously

described

a

four-generation

CD

family

with

an

extraordinary

high

incidence

of

affected

individuals

-LRB-

see

Fig.

-RRB-

The

disease

segregated

with

a

grandmaternal

haplotype

on

chromosome

-LRB-

van

Belzen

et

al.

-RRB-

a

region

that

encompasses

The

apparent

dominant

inheritance

pattern

could

be

caused

by

a

mutation

that

is

rare

in

the

general

CD

population

but

present

with

a

high

phenotypic

penetration

in

this

specific

family

To

assess

if

any

functional

variants

of

the

gene

were

present

in

this

family

we

sequenced

all

its

exons

and

intron

exon

boundaries

in

six

family

members

However

we

did

not

observe

mutations

in

any

of

the

samples

tested

-LRB-

results

not

shown

-RRB-

Neither

was

the

exon

coding

SNP

specific

for

affected

individuals

as

all

seven

individuals

tested

-LRB-

including

the

CEPH

control

-RRB-

carried

the

most

frequent

GG

genotype

-LRB-

Fig.

-RRB-

To

exclude

the

possibility

of

deletions

in

to

be

misinterpreted

from

the

sequence

data

as

homozygous

genotypes

we

also

performed

segregation

analysis

of

the

grandparental

haplotypes

within

the

entire

family

but

observed

no

suspect

inheritance

pattern

-LRB-

Fig.

-RRB-

In

conclusion

we

have

found

no

evidence

that

is

a

candidate

gene

for

the

chromosome

CD

locus

in

the

Dutch

population

in

general

or

in

the

multigeneration

Dutch

CD

family

specifically

Discussion

Chronic

inflammatory

conditions

and

autoimmune

disorders

are

typically

characterized

by

a

deregulated

adaptive

and

innate

immune

system

The

innate

defense

consists

of

multiple

components

that

include

physical

barriers

antimicrobial

molecules

pattern

recognition

receptors

circulating

phagocytes

and

the

complement

system

-LRB-

Hoebe

et

al.

-RRB-

A

breach

of

the

epithelial

barrier

and

loss

of

microbial

containment

is

often

the

first

of

a

series

of

events

that

trigger

or

sustain

chronic

inflammatory

diseases

-LRB-

Tlaskalova-Hogenova

et

al.

-RRB-

as

described

e.g.

in

Crohn

's

disease

atopic

eczema

asthma

and

psoriasis

-LRB-

Schreiber

et

al.

-RRB-

In

CD

the

gut

lumen

separation

is

undermined

by

dietary

gluten

that

evokes

a

combined

innate

and

adaptive

immune

response

-LRB-

Londei

et

al.

-RRB-

It

is

the

joined

action

of

gluten

peptides

environmental

factors

and

genetic

determinants

that

precipitates

this

enteropathy

The

human

leukocyte

antigen

locus

is

the

major

genetic

contribution

to

the

adaptive

reaction

-LRB-

Koning

et

al.

-RRB-

Recently

we

identified

as

a

susceptibility

gene

in

the

Dutch

population

that

possibly

has

an

effect

on

epithelial

barrier

integrity

-LRB-

Monsuur

et

al.

-RRB-

Several

other

studies

have

underscored

the

involvement

of

innate

immunity

in

CD

however

without

identification

of

underlying

causative

gene

variants

-LRB-

Londei

et

al.

-RRB-

Interestingly

it

was

also

reported

that

the

epithelial

glycocalyx

and

the

bacterial

composition

in

the

CD

gut

is

distinct

-LRB-

Forsberg

et

al.

Tjellstrom

et

al.

-RRB-

In

search

of

genes

that

may

have

a

primary

contribution

to

CD

pathogenesis

we

focused

our

attention

to

the

SPINK

family

of

serine

protease

inhibitors

that

play

an

important

role

in

tissue

preservation

through

the

containment

of

uncontrolled

proteolysis

and

bacterial

growth

In

this

study

we

demonstrated

differential

gene

expression

of

mucosal

in

CD

Crypt

hyperplasia

is

a

feature

of

the

Marsh

III

and

Marsh

II

stages

of

CD

and

the

concomitant

increase

in

the

number

of

goblet

cells

may

contribute

to

the

increased

expression

However

the

observed

sharp

decrease

in

gene

expression

sets

in

during

the

MIII/MII

transition

whereas

crypt

normalization

is

observed

only

later

at

the

MII/MI

recovery

phase

This

suggests

that

downregulation

sets

in

soon

after

commencement

of

the

gluten-free

diet

This

differential

expression

probably

reflects

altered

goblet

cell

activity

but

its

functional

significance

and

regulatory

mechanism

in

CD

pathology

remains

to

be

established

The

combination

of

functional

relevance

and

mapping

to

CD

linkage

intervals

pointed

to

the

SPINK

family

members

as

attractive

functional

and

positional

candidate

genes

We

have

chosen

a

robust

strategy

for

genetic

association

testing

based

on

haplotype

tagging

SNPs

and

linkage-disequilibrium

structure

of

the

SPINK

loci

applied

to

a

considerably

sized

Dutch

case-control

cohort

With

our

study

design

we

had

power

to

confirm

association

with

and

-LRB-

relative

risk

allele

frequency

confidence

interval

-RRB-

whereas

this

was

even

-LRB-

RR

-RRB-

and

-LRB-

RR

-RRB-

for

These

power

estimates

reflect

a

Type

I

error

rate

of

which

is

appropriate

for

testing

a

previously

reported

result

Initial

detection

of

a

new

genetic

association

would

require

much

more

stringent

criteria

to

assure

reproducibility

and

power

would

be

correspondingly

less

In

parallel

we

examined

the

extended

Dutch

CD

family

for

variants

and

deletions

in

We

hypothesized

that

a

specific

mutation

although

rare

in

the

general

population

could

have

a

dramatic

impact

on

mucus

composition

bacterial

containment

and

gluten

sensitivity

thereby

explaining

the

apparent

dominant

and

high

penetration

inheritance

pattern

in

our

extended

CD

family

With

both

approaches

we

were

not

able

to

establish

a

genetic

involvement

of

the

SPINK

genes

tested

However

we

can

not

completely

rule

out

the

possibility

of

a

rare

noncoding

mutation

in

-LRB-

outside

the

splice

donor

and

acceptor

regions

-RRB-

that

might

specifically

segregate

in

this

atypical

CD

family

characterized

by

an

exceptional

high

prevalence

of

affected

members

Despite

this

negative

result

in

the

Dutch

CD

population

we

can

not

formally

rule

out

the

possibility

of

genetic

contribution

of

SPINK

genes

to

CD

in

other

European

populations

like

the

Italian

in

whom

unlike

the

Dutch

-LRB-

van

Belzen

et

al.

-RRB-

chromosome

linkage

was

established

-LRB-

Greco

et

al.

Percopo

et

al.

-RRB-

Genuine

population

heterogeneity

has

been

reported

before

e.g.

between

and

Crohn

's

disease

-LRB-

Lesage

et

al.

Croucher

et

al.

-RRB-

and

between

and

asthma

-LRB-

Blumenthal

Jongepier

et

al.

-RRB-

The

new

SPINK

members

on

chromosome

-LRB-

and

-RRB-

were

not

part

of

this

study

Currently

no

functional

annotation

is

available

for

these

genes

that

are

located

near

and

in

a

chromosomal

region

that

appears

to

have

been

subjected

to

gene

duplication

during

evolution

Therefore

we

can

not

exclude

their

possible

involvement

in

CD

or

any

other

inflammatory

disorder

Electronic

Supplementary

Material

Below

is

the

link

to

the

electronic

supplementary

material

Supplementary

Fig

-LRB-

GIF

kb

-RRB-

Supplementary

Table

Primers

and

reaction

conditions

used

for

DNA

sequence

analysis

-LRB-

PDF

KB

-RRB-

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

spink genes
celiac disease
genetic association

quantitative reverse-transcription polymerase chain reaction


A comparison of echocardiographic and electron beam computed tomographic assessment of aortic valve area in patients with valvular aortic stenosis The purpose of this study was to compare electron beam computed tomography -LRB- EBT -RRB- with transthoracic echocardiography -LRB- TTE -RRB- in determining aortic valve area -LRB- AVA -RRB- . Thirty patients -LRB- 9 females , 21 males -RRB- underwent a contrast-enhanced EBT scan -LRB- e-Speed , GE , San Francisco , CA , USA -RRB- and TTE within 17 ± 12 days . In end-inspiratory breath hold , a prospectively ecg-triggered scan was acquired with a beam speed of 50 -- 100 ms , a collimation of 2 × 1.5 mm and an increment of 3.0 mm . The AVA was measured with planimetry . A complete TTE study was performed in all patients , and the AVA was computed using the continuity equation . There was close correlation between AVA measured with EBT and AVA assessed with TTE -LRB- r = 0.60 , P < 0.01 -RRB- . The AVA measured with EBT was 0.51 ± 0.46 cm2 larger than the AVA calculated with TTE measurements . EBT appeared to be a valuable non-invasive method to measure the AVA . EBT measures the anatomical AVA , while with TTE the functional AVA is calculated , which explains the difference in results between the methods . Introduction Aortic valve sclerosis is common in the elderly population , affecting about 25 % of adults over 65 years of age -LSB- 1 , 2 -RSB- . The presence of aortic sclerosis , with or without demonstrable haemodynamic obstruction , is associated with an increase of approximately 50 % in the risk of cardiovascular death -LSB- 3 -RSB- . The timing of aortic valve replacement surgery has also an effect on mortality risk -LSB- 4 -RSB- . The indication for aortic valve replacement generally is based on haemodynamic variables , such as transvalvular pressure drop and the presence of symptoms . Significant reduction in the aortic valve area -LRB- AVA -RRB- is also an important indicator -LSB- 4 -RSB- . Hence , exact evaluation of the aortic valve and the AVA is necessary to make the best treatment decision . Evaluation of aortic valve stenosis by means of transthoracic echocardiography -LRB- TTE -RRB- has gained widespread acceptance in clinical routine and pressure gradients across the aortic valve have shown to correlate well with invasive measurements -LSB- 5 , 6 -RSB- . Moreover , TTE is used to determine the functional AVA , using the continuity equation -LSB- 5 , 6 -RSB- . However , the reliability of TTE measurements depends heavily on image quality , which is influenced by aortic valve calcification and adequacy of the ultrasound window -LSB- 7 -- 9 -RSB- . The results also depend on the patient 's haemoglobin level , heart rate and left ventricular function -LSB- 7 -RSB- . Thus , invasive confirmation is often still necessary preceding valve replacement surgery . For this purpose , cardiac catheterization evaluating AVA by applying the Gorlin formula is available -LSB- 10 -RSB- . However , this method may not give reliable results , depending on cardiac function and aortic regurgitation -LSB- 11 -RSB- . Aforementioned techniques rely on and are affected by physiological parameters . Moreover , catheterization is an invasive procedure that may be associated with serious complications -LSB- 12 -RSB- . Therefore , new techniques are necessary to evaluate the severity and progression of aortic sclerosis . These new techniques could measure the anatomical AVA , which measurement is independent of physiological parameters . Contrast-enhanced computed tomography is a useful technique to evaluate cardiac structures , like coronary arteries and aortic valves -LSB- 13 -RSB- . EBT , a non-mechanical computed tomography scan with a high temporal resolution -LRB- 50 -- 100 ms -RRB- , is widely used to assess cardiac calcification , which is also an early marker of the aortic sclerotic process -LSB- 1 , 2 -RSB- . To assess cardiac calcification , administration of contrast agent to the patient is not necessary . Several studies have shown the usefulness of assessing the degree of aortic valve calcification -LRB- AVC -RRB- with EBT as a measure of the severity of aortic valve sclerosis -LSB- 14 -- 17 -RSB- . However , these studies did not use EBT to measure the AVA . MacMillan et al. were the first to use contrast-enhanced EBT to measure the AVA -LSB- 18 -RSB- . They were able to determine AVA in six patients with known aortic sclerosis . We wished to compare EBT and TTE in order to compare the assessment of aortic stenosis with both methods . We hypothesize that EBT is an useful method to evaluate aortic stenosis ; both in terms of AVA as well as AVC . Methods Between June 2004 and February 2006 , patients with a known peak gradient -LRB- ≥ 30 mmHg -RRB- across their native aortic valve were invited to participate in the study during their control visit to the outpatient clinic of cardiology . All patients were under control for valvular aortic stenosis . The study was in accordance with principles that have their origin in the Declaration of Helsinki , and was approved by the local review board and all patients gave their informed consent . All patients were over 18 years of age , and had to be able to lie flat and hold their breath for 20 s. Patients with congenital heart defects , renal insufficiency -LRB- serum creatinin > 120 μmol/l -RRB- , known contrast allergy or history of hyperthyroidism , thyroid cancer , Kahlers disease , myasthenia gravis , pheochromocytoma or mastocytosis were excluded . Patients with a body weight over 100 kg were also excluded because image quality of EBT is impaired in obese patients . Electron beam computed tomography Scans were performed with an EBT scanner -LRB- e-Speed , GE , San Francisco , CA , USA -RRB- , in the dual 1.5 mm slice mode with an image acquisition time of 50 or 100 ms depending on patient size . First , non-enhanced EBT data were collected during end-inspiratory breath hold . The non-enhanced scan was ecg-triggered at 42 % of the R -- R interval . A set of 40 continuous axial coupes were obtained from under the trachea bifurcation to the heart . An Agatston calcification score of the aortic valve was obtained -LSB- 19 -RSB- . Area and volume of calcifications were calculated and summated . The localization and severity of AVC was graded using a scale of 1 to 6 according to Bahler et al. -LSB- 20 -RSB- . Second , contrast-enhanced EBT data were collected during end-inspiratory breath hold . To achieve optimal contrast enhancement , bolus time was determined for each patient individually before EBT scanning . Bolus timing , using 20 ml contrast agent , was done with the region of interest placed in the ascending aorta . The ecg-triggered multiphase scan was carried out with 120 ml contrast agent -LRB- Iomeron 400 ® , Bracco , Italy -RRB- followed by a saline bolus chaser . Contrast and saline bolus chaser were injected through a 18G venflon , placed in a cubital vein , with a flow rate of 4 ml/s . Ecg-triggered multiphase scanning started at 5 % of the R -- R interval , the first phase , and continued during systole till 50 % of the R -- R interval , the last phase . Depending on the patient 's heart rate and beam speed , measurements were performed during 7 to 10 phases . The data were transferred to a workstation -LRB- GE Advanced Workstation , GE Medical , Waukesha , WI , USA -RRB- and reviewed by 1 observer -LRB- R D -RRB- , blinded from echocardiographic results . An axial view of the aorta valve was created with a double oblique view . One oblique axis was put trough the aortic valve in the coronal view of the heart , see Fig. 1 -LRB- A -RRB- . The second oblique axis was set in the created oblique view , see Fig. 1 -LRB- B -RRB- . The AVA was than measured in six planes at several levels in the created axial view of the aortic valve , see Fig. 1 -LRB- B -RRB- . The plane with the smallest AVA was selected and measured three times using planimetry , see Fig. 1 -LRB- C -RRB- . The mean AVA was assessed in this way for each phase . The phase with the maximal mean AVA was selected and was considered to be the AVA of concern . This AVA was compared to TTE findings . Fig. 1All patients underwent electron beam computed tomography . -LRB- A -RRB- One oblique axis was put trough the aortic valve in the coronal view of the heart . Based on this orientation an oblique view was created . -LRB- B -RRB- The second oblique axis was set in the created oblique view . Based on this orientation an axial view of the aortic valve was made . The aortic valve area -LRB- AVA -RRB- was than measured in six planes at several levels in the created axial view of the aortic valve . -LRB- C -RRB- The AVA was measured three times at each level in each phase using planimetry . The mean AVA was assessed for each phase . The phase with the maximal mean AVA was selected and was considered to be the AVA of concern Transthoracic echocardiography A complete TTE study was performed in all patients by an experienced sonographer , blinded for EBT , following a standard procedure -LRB- GE Vingmed Ultrasound Vivid Five , GE Medical Systems , Waukesha , WI -RRB- . The diameter of the left ventricular outflow tract was measured from the parasternal long-axis view . Flow velocity in the left ventricular outflow tract was assessed by pulse-wave Doppler from the apical 4 chamber approach . Flow velocity across the aortic valve was measured in the apical 5 chamber view . The AVA was computed using the continuity equation -LSB- 5 -RSB- . The presence of aortic regurgitation was determined from standard images , and quantified as the short-axis area of the regurgitant jet as a percentage to the short-axis area of the left ventricular outflow tract , moreover standard images of the left ventricle were obtained to evaluate left ventricular function and hypertrophy . Left ventricular function was based on estimated ejection fraction and wall motion , and was graded as being normal if the ejection fraction ≥ 60 % . Left ventricular dysfunction was mild if the ejection fraction was 45 -- 60 % , moderate if the ejection fraction 30 -- 45 % , or poor if the ejection fraction was < 30 % . To evaluate left ventricular hypertrophy , posterior wall and septal thickness were measured from the parasternal length axis . Statistical analysis For the measurement of the AVA by EBT , 3 manual tracings made by 1 observer -LRB- R D -RRB- were averaged . Results of continuous normally distributed variables are expressed as mean ± SD , results of continuous not normally distributed variables are expressed as median -LRB- range -RRB- . The data were analyzed with the use of standard software -LRB- SPSS version 12.0.1 , SPSS Institute , Chicago , IL , USA -RRB- on a PC . The Spearman 's and Pearson 's correlation coefficients -LRB- r -RRB- were used to establish the presence of linear relationships . To compare not normally distributed data , the Mann -- Whitney U test was used . A Bland -- Altman analysis was used to evaluate the agreement between the AVA assessed with EBT and TTE . A level of significance below 0,05 was defined as clinically significant . Results A total of 61 patients were screened for participation in the study . Nine females and 21 males -LRB- age 70.9 ± 10.0 years -RRB- were enrolled in the study . Eight patients were excluded because of renal insufficiency , 1 because of known contrast allergy , 1 because of body weight over 100 kg , and 4 patients declined to undergo an EBT . Another 17 patients were excluded because the AVA could not be assessed by TTE . In the study group , the mean peak pressure gradient across the valve was 64 ± 21 mmHg . Ten patients had symptoms according to NYHA class I , 12 patients class II and 8 patients class III . Left ventricular end-diastolic diameter was 4.9 ± 0.6 cm , interventricular septal thickness was 1.2 ± 0.3 cm and posterior wall thickness was 1.1 ± 0.1 cm . Ninety percent of the patients had signs of left ventricular hypertrophy on TTE . Twenty two patients -LRB- 73 % -RRB- had normal left ventricular function , 6 patients -LRB- 20 % -RRB- had mild and 2 patients -LRB- 7 % -RRB- moderate left ventricular dysfunction . Mild aortic regurgitation was present in 8 patients -LRB- 27 % -RRB- and moderate aortic regurgitation in 2 patients -LRB- 7 % -RRB- . The average time between TTE and EBT was 17 ± 12 days . Aortic valve area Mean AVA assessed with TTE was 0.99 ± 0.31 cm2 . The median AVA measured with EBT was 1.34 -LRB- 0.69 -- 2.84 -RRB- cm2 . There was a significant correlation between AVA measured with EBT and AVA assessed with TTE -LRB- r = 0.60 , P < 0.01 -RRB- , as shown in Fig. 2 . The AVA measured with EBT was on average 0.51 ± 0.46 cm2 larger than the AVA assessed with TTE , which was confirmed with Bland -- Altman analysis , see Fig. 2 . The difference in AVA between EBT and TTE showed a not statistically significant trend to be larger in patients with mild aortic regurgitation than without aortic regurgitation -LRB- 0.62 -LRB- 0.23 -- 1.22 -RRB- cm2 and 0.33 -LRB- − 0.14 -- 1.52 -RRB- cm2 , respectively , P = 0.08 -RRB- . The difference in AVA did not differ between patients with normal left ventricular function and left ventricular dysfunction -LRB- 0.45 -LRB- − 0.14 -- 1.52 -RRB- cm2 and 0.43 -LRB- − 0.03 -- 1.26 -RRB- cm2 , respectively , P = 0.78 -RRB- Fig. 2By using electron beam computed tomography -LRB- EBT -RRB- planimetry the aortic valve area -LRB- AVA -RRB- can be measured . -LRB- A -RRB- AVA measured with EBT correlate well with those assessed with transthoracic echocardiography -LRB- TTE -RRB- -LRB- r = 0.60 , P < 0.01 -RRB- . -LRB- B -RRB- AVA measured with EBT is larger on average than AVA assessed with TTE , as is confirmed with Bland -- Altman analysis , exhibiting a mean difference of 0.51 ± 0.46 cm2 . -- -- -- = 95 % confidence interval Aortic valve calcification The median Agatston score of the aortic valve was 3363 -LRB- 425 -- 10230 -RRB- . There was a moderate , but significant , inverse correlation between the Agatston score of the aortic valve measured with EBT and AVA assessed with TTE -LRB- r = − 0.38 , P = 0.04 -RRB- , as shown in Fig. 3 . In 17 patients with an AVA < 1.0 cm2 the aortic valve was more calcified than in 13 patients with an AVA ≥ 1.0 cm2 -LRB- Agatston score 3879 -LRB- 1192 -- 10230 -RRB- and 2447 -LRB- 425 -- 6378 -RRB- , respectively , P = 0.04 -RRB- . Fig. 3Aortic valve calcification can be quantified with electron beam computed tomography using the Agatston score . The Agatston score correlates well with the aortic valve area -LRB- AVA -RRB- measured with transthoracic echocardiography -LRB- TTE -RRB- -LRB- r = − 0.38 , P = 0.04 -RRB- . -- -- -- = 95 % confidence interval Discussion EBT is an useful non-invasive method to evaluate aortic stenosis , both in terms of AVA and AVC . However , on average a larger AVA was measured with EBT than was assessed AVA with TTE . Indeed , this is not surprising since EBT determines the anatomical AVA , which is expected to be greater than the functional AVA assessed with TTE . Because of calcifications the valvular leaflets are less flexible , as a result the AVA will be irregularly shaped , hence the AVA becomes haemodynamically less efficient . This results in a discrepancy between the functional and anatomical AVA . To our knowledge , the publication of MacMillan et al. is the first study investigating the usefulness of contrast-enhanced EBT to assess AVA -LSB- 18 -RSB- . In this study , 8 patients with known calcific aortic stenosis underwent EBT one day after cardiac catheterization . In six out of eight patients close agreement was found , within a 0.25 cm2 margin , between catheterization-derived AVA and AVA measured with EBT . In 1 patient the AVA could not be determined EBT and in 1 patient the AVA was overestimated by EBT with 0.60 cm2 . The results of our study are in line with those of MacMillan et al. . Our study also showed that EBT measures larger AVA compared to TTE . The AVA is measured planimetrically with EBT , so it can be considered as the anatomical AVA . Our study as well as the study of MacMillan et al. compared this anatomical AVA to the functional AVA . This functional AVA is derived from haemodynamic characteristics of the aortic valve , -LRB- pressure changes and flow velocity changes -RRB- across the aortic valve . The functional AVA is supposed to yield the haemodynamic relevant AVA . However , using the continuity equation , the AVA is assumed to be circular , which does not reflect the actual morphology of the orifice of the stenotic valve , especially not in case of heavy calcification . TTE is the principal diagnostic tool in routine clinical practice to assess the AVA , next to cardiac catheterization . To determine the AVA with TTE , the continuity equation is used -LSB- 5 , 6 -RSB- , which yields the functional AVA . However , this technique has its limitations . First , TTE is not suitable in every patient with aortic valve sclerosis . Poor sonographic windows and calcified deposits in the aortic valve and cardiac wall hamper the determination of the left ventricular outflow diameter . A small fault in the measurement of this diameter can seriously influence the calculated AVA , because this diameter is squared in the continuity equation . Moreover , if the ultrasound beam is not directed parallel to the velocity jet , peak transvalvular velocity may be underestimated . Hence the peak pressure gradient and AVA may be underestimated . Second , additional cardiac dysfunction can influence the outcome of TTE measurements . The flow velocity can be decreased in case of poor left ventricular function , even though there is an anatomical small AVA . This leads to an underestimation of the severity of the aortic stenosis . Furthermore , the severity of aortic stenosis can be overestimated in cases with concomitant aortic regurgitation . Consequently , the aortic valve stenosis is overestimated -LSB- 7 -- 9 -RSB- . Our study also showed a larger difference in AVA between EBT and TTE in patients with additional mild aortic regurgitation EBT is not influenced by aortic regurgitation , therefore EBT is a more reliable technique , in those patients , to conclude about the severity of aortic stenosis . The larger AVA measured by EBT compared to TTE and cardiac catheterization may be related to slice orientation . In order to get an axial view of the aortic valve , the image has to be rotated in two planes by the observer . If the selected axial view is not situated perpendicular to the aortic valve , the AVA is overestimated . Moreover , from this axial view the plane with the smallest AVA has to be selected . Although the temporal resolution of EBT is high , it is possible that the smallest AVA is situated between 2 planes . Hence , the AVA can be overestimated . Nevertheless , we addressed this problem by acquiring several slices at different levels of the aortic valve . This is necessary to minimize the potential of AVA overestimation because of imprecise localization . In addition to the measurement of AVA , AVC can be quantified with EBT . Many studies have made clear that EBT is also effective in the evaluation of AVC -LSB- 14 -- 17 , 21 , 22 -RSB- . The findings of these studies suggest that elevated AVC scores are a marker for the presence of aortic stenosis ; our study underlined these findings . A diagnostic threshold value of 1000 Agatson units has a sensitivity of 93 % to detect severe aortic stenosis -LRB- AVA < 1.0 cm2 -RRB- according to Messika-Zeitoun et al. -LSB- 16 -RSB- . Rosenhek et al. concluded that the extent of AVC was a strong independent predictor of outcome -LSB- 23 -RSB- . In a study population of 126 patients with asymptomatic severe aortic stenosis , four-year event-free survival was 75 % in patients with mild AVC , compared to 20 % in patients with moderate or severe AVC . As mentioned above , the grade of AVC is a predictor of outcome in patients with aortic stenosis . Although the echocardiographic severity of aortic stenosis is correlated to AVC score , the AVA can not be predicted from the AVC score . So , to evaluate the severity of aortic valve stenosis and to indicate whether aortic valve replacement surgery is necessary it is inevitable to measure AVA . In our study , we showed a significant correlation between the AVA measured with EBT and the severity of aortic stenosis assessed with TTE . However , further research is needed to validate EBT as a technique to evaluate the AVA . Next to EBT , there are other non-invasive imaging techniques being evaluated for their value in AVA assessment . Alkadhi et al. compared 16 detector row computed tomography to tranesophageal echocardiography in the evaluation of AVA in 20 patients with and 20 patients without aortic stenosis -LSB- 24 -RSB- . They reported a mean difference of 0.06 cm2 . Reant et al. performed a similar study using magnetic resonance imaging -LRB- MRI -RRB- -LSB- 25 -RSB- . They enrolled 39 patients with aortic stenosis and compared the AVA measured with MRI to tranesophageal echocardiography and cardiac angiography . Their results suggest good correlation between techniques ; MRI and transesophageal echocardiography differed 0.01 cm2 , MRI and cardiac angiography differed 0.06 cm2 . Both studies show that non-invasive imaging techniques are a good possible alternative for the evaluation of aortic valve stenosis . Conclusion EBT appears to be valuable as a non-invasive method to assess the severity of aortic valve sclerosis . EBT holds the qualifications for accurate assessment of the anatomy , morphology and physiology of the aortic valve . However , further investigations will have to be done to verify whether EBT is a suitable non-invasive imaging technique for evaluation of aortic valve disease .

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

aortic valve stenosis
electron beam computed tomography
echocardiography


The effect of acute hyperglycemia on retinal thickness and ocular refraction in healthy subjects Purpose To quantify the retinal thickness and the refractive error of the healthy human eye during hyperglycemia by means of optical coherence tomography -LRB- OCT -RRB- and Hartmann -- Shack aberrometry . Patients with diabetes mellitus -LRB- DM -RRB- often experience subjective symptoms of blurred vision associated to hyperglycemia . The nature and origin of this phenomenon are still unclear . Blurred vision during hyperglycemia could be a result of transient refractive alterations due to changes in the lens -LSB- 5 , 12 , 15 , 25 , 27 , 36 , 37 -RSB- , but it could also be caused by changes in the retina . Macular edema , or retinal thickening due to abnormal fluid accumulation within the macula , is a common cause of visual loss -LSB- 1 , 14 , 22 -RSB- . The degree of retinal thickening has been found to be significantly correlated with visual acuity -LSB- 24 -RSB- . Furthermore , a change in retinal thickness , resulting in a change in axial eye length , could also induce a change in ocular refractive error . For instance , it can be calculated that with a 50 μm increase in retinal thickness , the ocular refractive error becomes 0.15 D more hyperopic -LSB- 30 -RSB- . Several studies have demonstrated that retinal thickness is affected by DM -LSB- 2 , 7 , 8 , 18 , 21 , 23 , 26 , 32 , 33 , 35 , 38 -RSB- . In general , an increase in retinal thickness has been reported in patients with long-term DM and advanced stages of retinopathy -LSB- 7 , 8 , 18 , 32 , 33 , 35 -RSB- . However , in diabetic patients with and without minimal diabetic retinopathy , a decrease in retinal thickness has been observed -LSB- 2 , 23 -RSB- . In healthy subjects , it has been shown that the average retinal thickness did not change during normo-insulinaemic hyperglycemia -LSB- 13 -RSB- . It is unclear whether the thickness of the different retinal areas , such as the foveal area , the pericentral foveal area , and the peripheral foveal area , changes during acute hyperglycemia and suppression of insulin . A change in retinal thickness and/or ocular refractive error could explain the subjective symptoms of blurred vision in patients with DM and hyperglycemia . Therefore , in the present study the effect of hyperglycemia on retinal thickness and ocular refractive error was investigated in healthy subjects during suppression of endogenous insulin . Retinal thickness was measured by means of optical coherence tomography -LRB- OCT -RRB- , which is a non-invasive technique that provides cross-sectional retinal images , and produces an objective measurement of the retinal thickness , independent of the refractive status of the eye -LSB- 10 , 11 , 29 -RSB- . Furthermore , aberrometry was used to measure the ocular refractive error . This technique makes it possible to detect small changes in ocular refraction -LSB- 19 -RSB- . Subjects and methods Five healthy subjects -LRB- two males and three females -RRB- participated in the study . The mean age of the subjects was 24.8 years -LRB- range 21.2 -- 32.6 -RRB- , and their mean body mass index -LRB- BMI -RRB- was 24.2 kg/m2 -LRB- range 21.4 -- 29.7 -RRB- . The subjects were screened during a first visit , which included medical history-taking , a physical examination -LRB- measurement of visual acuity , weight , height and blood pressure -RRB- and collecting a fasting blood sample . Exclusion criteria were a history of DM -LRB- or a fasting plasma glucose > 5.5 mmol/l -RRB- , a BMI of > 30 kg/m2 , elevated blood pressure -LRB- > 140 / 85 mmHg -RRB- , a visual acuity of < 0.5 -LRB- Snellen -RRB- or a history of ocular pathology . The investigators of the ocular parameters -LRB- NW and MD -RRB- were not informed about the blood glucose levels . Furthermore , the investigators who induced hyperglycemia -LRB- EE and SS -RRB- were not informed about the results of the ocular measurements . The study protocol was approved by the Medical Ethics Committee of the VU University Medical Center in Amsterdam , and written informed consent was obtained from all subjects after the purpose and nature of the study had been explained to them . Procedure to induce hyperglycemia After a 10-hour overnight fast , the subjects were given a subcutaneous injection of a low dose -LRB- 100 μg -RRB- of synthetic somatostatin -LRB- Sandostatin , Novartis , Basel , Switzerland -RRB- in order to suppress endogenous insulin secretion . Each subject underwent an oral glucose tolerance test -LRB- OGTT -RRB- -LRB- 75 g glucose -RRB- 30 minutes after the somatostatin injection , and blood glucose levels were measured with a blood glucose analyzer -LRB- HemoCue Diagnostics BV , Oisterwijk , the Netherlands -RRB- . Endogenous insulin levels were measured by means of immunometric assays -LRB- Luminescence , Bayer Diagnostics , Mijdrecht , the Netherlands -RRB- in the Endocrinology Laboratory at the Department of Clinical Chemistry of the VU University Medical Center . The subjects remained in fasting state during the entire procedure . Ocular measurements Retinal thickness was measured with the Stratus OCT -LRB- Model 3000 , Carl Zeiss Meditec , Dublin , CA , USA -RRB- , which combines a low-coherence scanning interferometer -LRB- wavelength 820 nm -RRB- with a video camera to visualize the fundus of the eye . The fast macular thickness OCT scan protocol was used to obtain six cross-sectional macular scans , 6 mm in length , which were positioned at equally spaced angular orientations -LRB- 30 ° -RRB- centred on the fovea . The cross-sectional images were analyzed with OCT3 mapping software that uses an edge-detection technique to locate the vitreoretinal interface and the anterior surface of the retinal pigment epithelium . Retinal thickness was defined as the distance between these two surfaces . Two OCT scans were made of each subject before , and every 30 minutes during the period of hyperglycemia . In order to quantify the retinal thickness , the foveal map constructed by the software was divided into nine Early Treatment Diabetic Retinopathy Study -LRB- ETDRS -RRB- areas -LSB- 6 -RSB- : the central fovea -LRB- central circle with a diameter of 1 mm -RRB- , and the pericentral area -LRB- donut-shaped ring with an inner diameter of 1 mm and an outer diameter of 3 mm -RRB- and peripheral area -LRB- donut-shaped ring with an inner diameter of 3 mm and an outer diameter of 6 mm -RRB- , both of which were divided into four quadrants . Retinal thickness was calculated for all separate areas , and for the average pericentral and peripheral regions . Ocular refractive error was determined with an IRX3 aberrometer -LRB- Imagine Eye Optics , Paris , France -RRB- , which performs wavefront analysis of the eye according to the Hartmann-Shack principle -LSB- 19 -RSB- . After pupil dilation and paralysis of accommodation with 1.0 % cyclopentolate and 5.0 % phenylephrine eye-drops , a series of three aberrometry measurements was made before , and every 30 minutes during the hyperglycemic condition . From these measurements , the equivalent refractive error was calculated as : equivalent refractive error -LRB- ERE -RRB- = sphere + -LRB- cylinder / 2 -RRB- . The measurements at baseline and during maximal hyperglycemia were analyzed , and any change was considered to be meaningful if the difference between the measurements was greater than the threshold of 50 μm for retinal thickness and 0.2 diopters -LRB- D -RRB- for ERE . The threshold of 50 μm exceeded the 95 % confidence interval for the detection of a change in retinal thickness , which has been reported to be approximately 40 μm -LSB- 4 , 20 , 28 -RSB- . A refractive change of more than 0.2 D also surpasses the precision -LRB- defined as 95 % confidence interval -RRB- of the aberrometer for measuring sphere , cylinder , and consequently ERE -LSB- 3 , 31 -RSB- . In each subject , the significance of a change was obtained from the precision of the measurement instruments and the difference in the ocular parameters at baseline and during hyperglycemia . In the whole group , the significance of a change could be determined by means of Wilcoxon matched pairs signed rank sum tests . P-values ≤ 0.05 were considered to be statistically significant . Results The changes in blood glucose after the administration of somatostatin and glucose are shown in Fig. 1 . Mean blood glucose levels rose from 4.0 mmol/l -LRB- range 3.4 -- 4.5 mmol/l -RRB- to 18.4 mmol/l -LRB- range 16.1 -- 22.0 mmol/l -RRB- after the OGTT . Endogenous insulin was suppressed by the subcutaneous injection of somatostatin during the glucose load to a mean value of 2.1 pmol/l -LRB- range 0.4 -- 4.5 pmol/l -RRB- , and remained below basal secretion level -LRB- < 110.0 pmol/l -RRB- for 147 minutes -LRB- range 75 -- 270 minutes -RRB- . Subject 01 had a delayed elevation of blood glucose level , compared to the other subjects . This person received a second 75 g oral glucose load after 30 minutes in order to induce a rise in the blood glucose level . In all subjects , the blood glucose and endogenous insulin levels normalized within 6 hours after the OGTT . Fig. 1Graph of normalized blood glucose levels in the five subjects after the administration of somatostatin and glucose . Data are normalized by subtracting the value at baseline from the measured value in each subject . The oral glucose load -LRB- 75 g -RRB- was administered at T 0 . Subject 01 received an extra 75 g oral glucose load at T 30 Figure 2 shows the normalized ERE of the five subjects during hyperglycemia . Mean ERE at baseline was 0.6 D -LRB- SD 0.6 -RRB- and 0.7 D -LRB- SD 0.6 -RRB- during maximal hyperglycemia ; no significant change was found in the group as a whole . A small , but significant hyperopic shift of 0.4 D -LRB- SD 0.2 -RRB- in ERE was measured in subject 01 during maximal hyperglycemia , compared to the start of the procedure -LRB- p < 0.001 -RRB- . No significant change in ERE was found in any of the other subjects . Normalized retinal thickness parameters are shown in Fig. 3a -LRB- central foveal area -RRB- , Fig. 3b -LRB- average pericentral foveal area -RRB- and Fig. 3c -LRB- average peripheral foveal area -RRB- . Average central foveal thickness , average pericentral foveal thickness , and average peripheral foveal thickness at baseline were 202 μm -LRB- SD 8 -RRB- , 277 μm -LRB- SD 5 -RRB- , and 243 μm -LRB- SD 8 -RRB- . During maximal hyperglycemia , average central foveal thickness , average pericentral foveal thickness , and average peripheral foveal thickness were 203 μm -LRB- SD 7 -RRB- , 275 μm -LRB- SD 3 -RRB- , and 242 μm -LRB- SD 9 -RRB- . No significant differences were found in the group as a whole . Furthermore , none of the subjects had any significant changes in the thickness of the central fovea , the pericentral fovea , or the peripheral fovea during maximal hyperglycemia , compared to baseline . The nine separate ETDRS areas were not affected by hyperglycemia . At baseline and during hyperglycemia , any change in retinal thickness that occurred in the various areas was less than 15 μm , which was within the previously defined threshold of 50 μm . Fig. 2Graph of the normalized equivalent refractive error -LRB- ERE -RRB- in diopters -LRB- D -RRB- of the five subjects . Data are presented as mean ± SD ; three measurements were made of each subject every 30 minutes during the procedure . Data are normalized by subtracting the value at baseline from the measured value in each subject . The oral glucose load was administered at T 0 . * Significant difference between ERE at T 0 and T 210 -LRB- maximal hyperglycemia -RRB- , p < 0.001 Fig. 3Graphs and maps of normalized retinal thickness parameters in the five subjects during hyperglycemia : -LRB- a -RRB- central fovea , -LRB- b -RRB- pericentral fovea , -LRB- c -RRB- peripheral fovea . Data are normalized by subtracting the value at baseline from the measured value in each subject . Each measured area has been indicated by a dark grey area on the retinal maps . No significant changes in retinal parameters were found in any of the subjects . The oral glucose load was administered at T 0 Discussion Blurred vision is a symptom that occurs frequently in patients with DM and hyperglycemia . The underlying mechanism is still unclear , and therefore the present study was carried out in an attempt to identify a possible cause of this symptom . The effect of reproducible hyperglycemia on retinal thickness and refractive error was studied in healthy young subjects who did not suffer from the systemic effects of DM . No changes in the thickness of the central , pericentral or peripheral foveal areas were found in any of the subjects during hyperglycemia . In addition , no significant change was measured in any of the nine different ETDRS areas of the macula . In their study , Jeppesen et al. -LSB- 13 -RSB- also found no significant difference in retinal thickness in healthy subjects during normo-insulinaemic hyperglycemia . Before and 180 minutes after the start of a hyperglycemic clamp they measured the average thickness of the retina , and found that retinal thickness was not affected by hyperglycemia . Although in the present study retinal thickness was measured under different circumstances than in the study of Jeppesen et al. -LRB- hypo-insulinaemic hyperglycemia instead of normo-insulinaemic hyperglycemia -RRB- , the results confirm those of Jeppesen et al. . Retinal thickness has been reported to change in patients with long-term DM and retinopathy . A morphological change in the retina may even occur in the early stages of diabetic retinopathy -LSB- 2 , 7 , 8 , 18 , 21 , 23 , 26 , 32 , 33 , 35 , 38 -RSB- . These changes in retinal thickness are usually due to abnormal fluid accumulation resulting from a breakdown of the blood-retinal barrier -LSB- 34 -RSB- . Goebel et al. -LSB- 8 -RSB- measured retinal thickness by means of OCT in 136 patients with different stages of diabetic retinopathy and with a mean DM duration of 16 years . Mean foveal thickness was 307 ± 136 μm in the diabetic subjects , compared to 153 ± 15 μm in healthy subjects . It seems that only long-term hyperglycemia and/or long-term fluctuations in blood glucose levels have any significant influence on the blood -- retina barrier and retinal thickness . From the findings of the present study , it appears that the blood -- retina barrier does not seem to be affected by a single episode of acute hyperglycemia . Nevertheless , the fact that no change in retinal thickness could be determined does not exclude the possibility that there could be early dysfunction of the blood retina barrier . Other means of examination could evidence such a dysfunction of the blood retina barrier following acute hyperglycemia . A factor that could have biased the results of this study is the administration of a synthetic somatostatin analogue to the subjects . Somatostatin is a peptide hormone that inhibits several hormones , including IGF-1 and insulin . IGF-1 is a growth factor that is produced by the hypoxic retina to mediate angiogenesis , resulting in neovascularisation . Somatostatin analogues not only inhibit neovascularisation in patients with advanced diabetic retinopathy , but also stabilize the blood -- retinal barrier in patients with diabetic macular edema -LSB- 16 , 17 -RSB- . It could have been possible that in the present study an increase in retinal thickness during hyperglycemia was prevented by somatostatin . Nevertheless , the efficacy of synthetic somatostatin in the treatment of advanced diabetic retinopathy was investigated by Grant et al. -LSB- 9 -RSB- . With maximally tolerated doses of somatostatin -LRB- ranging from 200 to 5000 μg/day -RRB- , after a period of 15 months one out of 22 eyes required panretinal photocoagulation , compared to nine of 24 eyes that were not treated with somatostatin . From the results of the Grant et al. study , it seems that only frequent , large doses of somatostatin over a long period of time have any significant effect on the progression of diabetic retinopathy . Although the effect of somatostatin on the healthy retina has not been investigated yet , it seems to be unlikely that the results of the present study were biased by the administration of one single , low dose -LRB- 100 μg -RRB- of somatostatin . In conclusion , the results of this study indicate that in healthy subjects , hyperglycemia does not cause any change in retinal thickness . Furthermore , ocular refraction in general was not affected by hyperglycemia . However , there were interindividual variations , as illustrated by subject 01 , who had a hyperopic shift of refraction during hyperglycemia . Therefore , it seems that a refractive change during hyperglycemia can not be explained by a change in retinal thickness . It could well be that other refractive components , such as the lens , are involved in causing blurred vision and refractive alterations during hyperglycemia .

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

hyperglycemia
diabetes mellitus
optical coherence tomography