Variation

analysis

and

gene

annotation

of

eight

MHC

haplotypes

The

MHC

Haplotype

Project

The

human

major

histocompatibility

complex

-LRB-

MHC

-RRB-

is

contained

within

about

Mb

on

the

short

arm

of

chromosome

and

is

recognised

as

the

most

variable

region

in

the

human

genome

The

primary

aim

of

the

MHC

Haplotype

Project

was

to

provide

a

comprehensively

annotated

reference

sequence

of

a

single

human

leukocyte

antigen-homozygous

MHC

haplotype

and

to

use

it

as

a

basis

against

which

variations

could

be

assessed

from

seven

other

similarly

homozygous

cell

lines

representative

of

the

most

common

MHC

haplotypes

in

the

European

population

Comparison

of

the

haplotype

sequences

including

four

haplotypes

not

previously

analysed

resulted

in

the

identification

of

variations

both

substitutions

and

indels

-LRB-

insertions

and

deletions

-RRB-

which

have

been

submitted

to

the

dbSNP

database

The

gene

annotation

uncovered

haplotype-specific

differences

and

confirmed

the

presence

of

more

than

loci

including

over

protein-coding

genes

Combined

analysis

of

the

variation

and

annotation

datasets

revealed

gene

loci

with

coding

substitutions

of

which

were

non-synonymous

The

haplotype

-LRB-

PGF

cell

line

-RRB-

designated

as

the

new

MHC

reference

sequence

has

been

incorporated

into

the

human

genome

assembly

-LRB-

and

subsequent

builds

-RRB-

and

constitutes

the

largest

single-haplotype

sequence

of

the

human

genome

to

date

The

extensive

variation

and

annotation

data

derived

from

the

analysis

of

seven

further

haplotypes

have

been

made

publicly

available

and

provide

a

framework

and

resource

for

future

association

studies

of

all

MHC-associated

diseases

and

transplant

medicine

Introduction

The

MHC

has

long

been

believed

to

be

the

most

important

region

in

the

human

genome

with

respect

to

infection

inflammation

autoimmunity

and

transplant

medicine

-LRB-

Lechler

and

Warrens

-RRB-

This

was

recently

confirmed

by

the

largest

genome-wide

association

study

carried

out

to

date

for

seven

common

diseases

including

two

autoimmune

diseases

-LRB-

type

diabetes

and

rheumatoid

arthritis

-RRB-

and

one

inflammatory

disease

-LRB-

Crohn

's

disease

-RRB-

The

highest

associations

were

found

between

the

MHC

and

these

two

autoimmune

diseases

-LRB-

The

Wellcome

Trust

Case

Control

Consortium

-RRB-

The

complex

aetiology

of

MHC-associated

disease

coupled

with

high

density

polymorphism

linkage

disequilibrium

-LRB-

LD

-RRB-

and

frequent

non-Mendelian

inheritance

of

gene

loci

have

made

it

challenging

to

identify

variations

that

cause

or

contribute

to

disease

phenotypes

Additional

limiting

factors

have

been

our

incomplete

knowledge

of

the

allelic

variation

of

genes

and

regions

flanking

the

nine

classical

human

leukocyte

antigen

-LRB-

HLA

-RRB-

loci

and

the

lack

of

a

single

haplotype

reference

sequence

the

original

reference

sequence

being

a

composite

of

multiple

MHC

haplotypes

-LRB-

Mungall

et

al.

The

MHC

Sequencing

Consortium

-RRB-

Recognizing

that

the

future

identification

of

variants

conferring

susceptibility

to

common

disease

is

critically

dependent

on

fully

informative

polymorphism

and

haplotype

maps

the

MHC

Haplotype

Consortium

formed

in

with

the

aim

to

generate

these

critical

data

and

to

make

them

publicly

available

as

a

general

resource

for

MHC-linked

disease

studies

Similar

efforts

but

with

different

experimental

approaches

were

also

carried

out

in

Japan

-LRB-

Shiina

et

al.

-RRB-

and

the

USA

-LRB-

Smith

et

al.

-RRB-

To

develop

the

resource

eight

HLA-homozygous

MHC

haplotypes

were

selected

on

the

basis

of

conferring

either

protection

against

or

susceptibility

to

two

autoimmune

diseases

type

diabetes

and

multiple

sclerosis

and

that

represented

common

haplotypes

in

European

populations

In

the

subsequent

years

incremental

data

materials

and

tools

comprising

this

resource

have

been

released

-LRB-

Allcock

et

al.

Horton

et

al.

Stewart

et

al.

Traherne

et

al.

-RRB-

and

have

contributed

towards

the

construction

of

a

high-resolution

LD

map

and

a

first

generation

of

HLA

tag

single

nucleotide

polymorphisms

-LRB-

SNPs

de

Bakker

et

al.

Miretti

et

al.

-RRB-

and

the

identification

of

a

second

MHC

susceptibility

locus

for

multiple

sclerosis

-LRB-

The

International

Multiple

Sclerosis

Genetics

Consortium

Yeo

et

al.

-RRB-

In

this

paper

we

report

the

final

account

of

this

international

effort

including

analysis

of

the

last

four

of

the

eight

haplotypes

up-to-date

variation

statistics

gene

annotation

population-specific

aspects

and

a

detailed

description

of

the

databases

and

tools

for

viewing

and

accessing

the

data

in

the

context

of

existing

genome

annotation

Materials

and

methods

Variation

analysis

The

method

previously

reported

for

comparison

of

MHC

haplotype

sequences

-LRB-

Stewart

et

al.

Traherne

et

al.

-RRB-

was

extended

to

cover

all

eight

haplotypes

Briefly

the

most

suitable

method

proved

to

be

a

clone

by

clone

comparison

using

the

discrepancy-list

option

of

the

program

-LRB-

Green

unpublished

-RRB-

an

implementation

of

the

Smith

Waterman

sequence

alignment

algorithm

-LRB-

Smith

and

Waterman

-RRB-

using

the

alignment

of

a

haplotype

clone

sequence

with

the

appropriate

overlapping

reference

sequence

from

a

PGF

clone

or

clones

All

variations

were

submitted

to

dbSNP

using

the

submitter

handle

and

user

identifiers

of

the

form

-LSB-

PGF

BAC

clone

sequence

version

-RSB-

-LSB-

position

in

PGF

BAC

clone

sequence

-RSB-

-LSB-

variation

change

-RSB-

Thus

indicates

a

substitution

in

which

the

base

T

at

base

position

in

-LRB-

PGF

BAC

-RRB-

was

substituted

by

C

in

the

other

haplotype

In

the

case

of

indels

the

variation

change

consists

of

i

or

d'

-LRB-

for

insertion

or

deletion

-RRB-

followed

by

a

numerical

value

for

the

length

of

the

indel

in

turn

followed

by

the

inserted

or

deleted

sequence

if

this

were

of

or

fewer

bases

For

longer

indels

an

X

value

is

given

which

refers

to

a

look-up

table

-LRB-

-RRB-

Thus

indicates

a

deletion

in

after

base

of

the

eight

bases

TACACACA

Further

indicates

an

insertion

in

starting

after

base

of

the

five

bases

ATATT

indicates

a

deletion

after

base

in

of

a

sequence

coded

as

which

is

ATACACACACACAC

Major

indel

sequences

appearing

as

breaks

in

the

discrepancy

lists

between

two

clones

from

difference

haplotypes

were

extracted

and

subjected

to

analysis

by

RepeatMasker

to

detect

the

presence

of

retrotransposible

elements

Gene

annotation

The

finished

genomic

sequence

for

each

of

the

eight

haplotypes

was

analysed

using

a

modified

Ensembl

pipeline

-LRB-

Searle

et

al.

-RRB-

CpG

islands

were

predicted

on

unmasked

sequence

Interspersed

and

tandem

repeats

were

masked

out

by

RepeatMasker

-LRB-

Smit

AFA

Hubley

R

Green

P.

RepeatMasker

-RRB-

and

Tandem

Repeats

Finder

-LRB-

TRF

Benson

-RRB-

respectively

The

sequence

was

then

BLAST

searched

-LRB-

BLAST

basic

local

alignment

search

tool

Altschul

et

al.

-RRB-

using

a

vertebrate

set

of

complementary

DNAs

-LRB-

cDNAs

-RRB-

and

expressed

sequence

tags

-LRB-

ESTs

-RRB-

from

the

European

Molecular

Biology

Laboratory

-LRB-

EMBL

-RRB-

nucleotide

database

-LRB-

Kulikova

et

al.

-RRB-

followed

by

the

re-alignment

of

significant

hits

Non-redundant

proteins

were

aligned

similarly

Protein

domain

matches

were

provided

through

alignment

of

Pfam

to

the

genomic

sequence

using

Genewise

-LRB-

Birney

et

al.

-RRB-

thereby

providing

protein

domain

data

to

the

annotator

Ab

initio

gene

predictions

were

performed

by

Genscan

-LRB-

Burge

and

Karlin

-RRB-

and

Fgenesh

-LRB-

Salamov

and

Solovyev

-RRB-

and

potential

transcriptional

start

sites

were

predicted

by

Eponine

-LRB-

Down

and

Hubbard

-RRB-

Analysis

results

were

displayed

and

annotation

was

performed

through

an

in-house

annotation

software

system

Genes

were

manually

annotated

according

to

the

human

and

vertebrate

analysis

and

annotation

-LRB-

HAVANA

-RRB-

guidelines

-LRB-

-RRB-

using

evidence

based

on

comparison

with

external

databases

as

of

August

All

gene

structures

are

supported

by

transcriptional

evidence

either

from

cDNA

EST

or

protein

In

general

annotations

are

supported

by

best-in-genome

evidence

Haplotype-specific

evidence

is

assigned

where

possible

As

with

previous

MHC

annotation

-LRB-

Stewart

et

al.

Traherne

et

al.

-RRB-

some

olfactory

receptors

have

been

built

upon

protein

homology

alone

because

of

their

restricted

expression

Locus

and

variant

types

were

annotated

according

to

established

standards

-LRB-

Harrow

et

al.

-RRB-

with

the

modification

that

within

the

MHC

region

the

artefact

locus

has

been

used

to

tag

historically

annotated

structures

that

are

no

longer

deemed

valid

HLA-A

HLA-B

HLA-C

and

allele

types

were

assessed

by

comparison

against

the

IMGT/HLA

database

-LRB-

Marsh

et

al.

-RRB-

Annotation

status

of

haplotypes

The

PGF

COX

and

QBL

haplotypes

have

already

been

annotated

in

detail

-LRB-

Stewart

et

al.

Traherne

et

al.

-RRB-

It

was

decided

however

to

re-annotate

and

update

this

annotation

to

maintain

consistency

between

all

eight

haplotypes

with

the

current

supporting

evidence

and

pipeline

analyses

The

SSTO

haplotype

was

manually

annotated

de

novo

The

new

annotation

from

the

PGF

haplotype

was

projected

through

a

DNA

DNA

alignment

to

each

of

the

remaining

haplotypes

-LRB-

APD

DBB

MANN

and

MCF

-RRB-

where

possible

This

projection

was

checked

thoroughly

and

non-alignable

regions

were

manually

adjusted

-LRB-

including

the

and

hypervariable

regions

-RRB-

Polyadenylation

sites

and

signals

were

not

annotated

for

haplotypes

APD

DBB

MANN

and

MCF

because

of

time

constraints

In

the

main

however

these

features

may

be

assumed

to

correspond

to

the

same

positions

as

in

the

first

four

haplotypes

Combination

of

variation

and

annotation

data

By

employing

a

series

of

Perl

scripts

the

array

of

haplotype

variation

was

combined

with

the

annotation

of

gene

loci

repeat

elements

and

microsatellites

extracted

from

the

Vertebrate

Genome

Annotation

-LRB-

VEGA

-RRB-

database

in

general

feature

format

-LRB-

GFF

-RRB-

to

determine

the

variation

status

of

all

loci

Distribution

of

sequenced

HLA

haplotypes

in

Europeans

To

assess

the

distribution

of

sequenced

haplotypes

at

the

population

level

founder

haplotypes

were

reconstructed

using

genotypic

data

from

Centre

d'Etude

Polymorphisme

Humain

-LRB-

CEPH

-RRB-

trios

-LRB-

de

Bakker

et

al.

-RRB-

A

kb

segment

spanning

the

HLA

genes

was

selected

for

the

analyses

This

segment

represented

by

SNPs

is

delimited

by

and

with

NCBI

build

chromosome

coordinates

and

respectively

Phased

haplotypes

with

known

HLA

alleles

were

then

used

to

construct

a

neighbor-joining

tree

-LRB-

Kumar

et

al.

-RRB-

and

a

phylogenetic

network

-LRB-

Bandelt

et

al.

-RRB-

Resources

All

sequences

presented

in

this

paper

have

been

submitted

to

the

EMBL/GenBank/DNA

Data

Bank

of

Japan

-LRB-

DDBJ

-RRB-

database

and

allocated

accession

numbers

For

clarity

all

bacterial

artificial

chromosome

-LRB-

BAC

-RRB-

clones

are

referred

to

using

their

accession

numbers

The

annotation

of

each

haplotype

has

been

entered

in

the

VEGA

database

and

is

accessible

through

its

browser

-LRB-

-RRB-

All

variations

from

the

study

were

submitted

to

dbSNP

-LRB-

-RRB-

using

the

submitter

handle

BAC

clones

from

the

-LRB-

PGF

-RRB-

and

-LRB-

COX

-RRB-

libraries

can

be

requested

from

BACPAC

resources

-LRB-

-RRB-

Clones

from

the

other

libraries

can

be

requested

from

john.elliott@ualberta.ca

The

web

site

for

the

MHC

Haplotype

Project

provides

links

to

various

data

resources

-LRB-

-RRB-

DAS

sources

for

all

substitutions

and

indels

are

available

from

as

follows

These

can

be

accessed

via

the

VEGA

browser

Results

and

discussion

Variation

analysis

One

of

the

main

aims

of

the

MHC

Haplotype

Project

was

to

generate

a

comprehensive

variation

map

of

this

most

variable

region

of

the

human

genome

To

achieve

this

eight

haplotypes

were

sequenced

and

subjected

to

variation

analysis

Table

details

the

lengths

of

the

sequence

contigs

the

number

of

sequence

gaps

and

the

allelic

types

of

major

HLA

loci

for

each

haplotype

Of

the

eight

haplotypes

sequenced

three

have

already

been

described

PGF

and

Cox

-LRB-

Stewart

et

al.

-RRB-

both

of

which

formed

single

contigs

of

approximately

Mb

and

QBL

-LRB-

Traherne

et

al.

-RRB-

of

approximately

Mb

but

with

five

gaps

The

remaining

haplotypes

sequenced

all

contained

gaps

their

coverage

ranging

from

Mb

-LRB-

DBB

with

gaps

-RRB-

to

Mb

-LRB-

MANN

with

gaps

-RRB-

Table

sequence

contig

length

number

of

gaps

and

HLA

allele

typesHaplotypeLength

-LRB-

bp

-RRB-

Cw

-LSB-

A

-RSB-

B

length

-LRB-

bp

-RRB-

and

number

of

gaps

in

each

haplotype

sequence

together

with

the

HLA

gene

types

obtained

by

BLAST

against

the

IMGT/HLA

database

Dashes

or

data

in

square

brackets

indicate

the

absence

or

the

partial

presence

respectively

of

a

gene

owing

to

a

sequence

gap

For

the

variation

analysis

each

of

the

above

haplotypes

was

compared

with

the

PGF

reference

sequence

resulting

in

the

identification

of

variations

-LRB-

substitutions

and

indels

Table

-RRB-

which

have

all

been

submitted

to

dbSNP

The

success

of

this

exercise

is

illustrated

by

the

fact

that

examination

of

this

public

database

-LRB-

NCBI

dbSNP

build

March

-RRB-

showed

that

there

were

only

a

further

variations

submitted

by

other

laboratories

in

this

region

which

were

not

identified

by

this

project

In

accordance

with

the

annotation

that

we

also

generated

for

each

haplotype

-LRB-

see

below

-RRB-

the

variations

shown

in

Table

were

further

classified

as

untranslated

region

-LRB-

UTR

-RRB-

exonic

intronic

intergenic

and

eight

more

sub-categories

-LRB-

Table

-RRB-

Coding

substitutions

which

are

of

particular

interest

with

respect

to

altered

functionality

were

further

classified

as

synonymous

non-synonymous

conservative

or

non-synonymous

non-conservative

and

grouped

depending

on

whether

they

affected

HLA

or

other

genes

-LRB-

Table

-RRB-

The

actual

variations

and

affected

amino

acids

can

be

viewed

using

the

VEGA

browser

as

illustrated

in

Fig.

and

described

in

the

corresponding

section

later

on

In

addition

we

have

analysed

all

haplotype

sequences

for

inversions

which

represent

another

important

variation

category

that

has

been

linked

to

genomic

disorders

-LRB-

Shaw

and

Lupski

-RRB-

Using

-LRB-

Ning

et

al.

-RRB-

we

found

no

evidence

of

any

inversion

polymorphism

within

the

generated

sequences

but

could

not

exclude

large-scale

-LRB-

e.g.

involving

entire

MHC

-RRB-

inversions

with

breakpoints

outside

the

MHC

regions

sequenced

here

Fig.

and

variation

data

in

VEGA

VEGA

overview

-LRB-

a

-RRB-

detailed

view

-LRB-

b

-RRB-

and

basepair

view

-LRB-

c

-RRB-

example

of

the

variation

in

the

locus

in

which

a

STOP

codon

is

present

in

all

haplotypes

except

MCFTable

of

substitutions

and

indels

amongst

Number

of

variations

found

by

comparing

the

PGF

haplotype

sequence

with

each

of

the

other

haplotype

sequences

in

turn.Table

of

substitutions

and

indels

within

different

sequence

regions

amongst

haplotypesSequence

regionBase

pairsCOXQBLSSTOAPDDBBMANNMCFSIDSIDSIDSIDSIDSIDSIDCoding247

,283,4723,1415713,1355902,6585056021472,8975092,1853932,126404

Total

,686,9373,8766134,0766683,3695427141563,5745542,8894372,783437

Pseudogenic

Transcript

Transcript

,7051,2431971,1862161,05315585291,2451921,08116126853

REPEATS

,4292,1102212,0152402,388255755932,0972172,0841931,530164

,5671,3814281,3164011,3113853461341,229318928241936271

Other

,8632,6052072,5182292,514207925562,7481992,1981772,170169

Total

in

,524,8596,0968565,8498706,2138472,0262836,0747345,2106114,636604

All

,297,59012,3331,93311,8591,92011,4181,8013,16953311,5381,6059,5361,3158,1391,200

Other

,7203,6344603,4234403,5644991,0611502,7173702,5663392,651345

,754,82915,9672,39315,2822,36014,9822,3004,23068314,2551,97512,1021,65410,7901,545

Variations

shown

in

Table

ascribed

to

sequence

regions

identified

during

annotation

These

included

exonic

UTR

and

intronic

regions

of

coding

pseudogenic

and

transcript

loci

repeat

elements

microsatellites

and

other

intergenic

regionsS

Substitution

ID

indelTable

variation

caused

by

substitutions

in

HLA

and

other

gene

lociCodons

variation

by

virtue

of

substitutions

analysed

for

their

effects

on

protein

sequences

and

listed

in

by

haplotype

for

HLA

genes

-LRB-

HLA-A

HLA-B

HLA-C

HLA-DRA

-RRB-

and

for

all

other

genes

according

to

the

changes

they

induced

in

codons

as

either

synonymous

non-synonymous

conservative

or

non-synonymous

non-conservative

changes

Gene

annotation

There

have

been

several

previous

annotations

of

the

gene

content

of

the

MHC

-LRB-

Horton

et

al.

Mungall

et

al.

Stewart

et

al.

The

MHC

Sequencing

Consortium

Traherne

et

al.

-RRB-

The

maximum

region

annotated

in

this

study

extends

from

the

telomeric

gene

in

the

MHC

extended

class

I

region

-LRB-

COX

haplotype

-RRB-

to

the

centromeric

gene

just

telomeric

of

the

MHC

extended

class

II

region

-LRB-

PGF

and

SSTO

haplotypes

-RRB-

The

PGF

haplotype

-LRB-

Stewart

et

al.

-RRB-

remains

the

longest

complete

MHC

haplotype

encompassing

annotated

loci

with

variants

The

number

of

variants

ascribed

to

each

locus-type

is

listed

in

Table

A

comparison

of

the

statistics

for

loci

in

each

haplotype

is

shown

in

Table

Table

statistics

for

PGF

annotationTypeNo.Total

splice

variants

annotated

in

the

PGF

haplotype.Table

annotation

statistics

for

eight

MHC

haplotypesLocus

Annotation

statistics

for

loci

in

each

haplotype

For

definitions

of

locus

types

see

Materials

and

methods

VEGA

database

and

browser

The

VEGA

database

provides

access

to

gene

annotation

of

the

eight

MHC

haplotype

sequences

a

valuable

public

resource

and

a

means

of

integrating

annotation

and

variation

data

The

VEGA

database

also

provides

the

facility

to

download

nucleotide

or

peptide

sequences

for

genes

of

interest

by

selecting

export

cDNA

or

export

peptide

from

the

menu

obtained

by

clicking

on

gene

cartoons

in

the

VEGA

detailed

view

or

basepair

view

window

From

these

any

desired

alignments

can

be

made

Variation

data

may

be

viewed

in

the

browser

linked

to

a

distributed

annotation

system

-LRB-

DAS

-RRB-

source

of

any

given

variation

-LRB-

see

Materials

and

methods

-RRB-

This

is

illustrated

An

example

of

the

use

of

this

browser

to

view

a

C

to

T

substitution

is

illustrated

for

the

locus

-LRB-

Fig.

-RRB-

An

overview

of

the

genomic

environment

is

given

in

Fig.

showing

the

gene

within

a

cluster

of

olfactory

gene

loci

on

chromosome

The

detailed

view

-LRB-

Fig.

-RRB-

shows

with

associated

variations

in

all

haplotypes

The

basepair

view

-LRB-

Fig.

-RRB-

illustrates

the

presence

of

the

C/T

substitution

in

all

haplotypes

except

MCF

and

its

positioning

above

the

translated

sequence

at

the

first

position

of

a

CAG

codon

indicating

the

presence

of

a

stop

codon

instead

of

glutamine

Annotation

changes

In

addition

to

loci

annotated

in

the

previous

studies

newly

recognised

with

official

Hugo

Gene

Nomenclature

Committee

-LRB-

HGNC

-RRB-

symbols

have

also

been

annotated

These

have

included

the

mitochondrial

coiled

coil

domain

protein

gene

-LRB-

Semple

et

al.

-RRB-

and

the

related

unprocessed

pseudogenes

and

as

well

as

the

zinc-finger

and

BTB

domain-containing

protein

gene

annotated

at

the

very

centromeric

boundary

of

the

sequenced

region

The

gene

-LRB-

De

Vet

et

al.

XXbac

-RRB-

of

the

MHC

class

III

region

was

annotated

as

a

separate

locus

from

the

adjacent

centromeric

locus

-LRB-

splice

variants

XXbac

and

XXbac

-RRB-

There

was

however

a

further

coding

splice

variant

of

-LRB-

XXbac

-RRB-

which

spanned

not

only

the

other

splice

variants

but

also

suggesting

that

this

is

a

possible

so-called

chimeric

transcript

-LRB-

Parra

et

al.

-RRB-

hypervariable

region

Of

the

five

newly

annotated

MHC

haplotypes

APD

alone

exhibited

the

HLA

antigenic

specificity

found

on

-LRB-

and

-RRB-

and

-LRB-

and

-RRB-

haplotypes

and

encoded

by

HLA

whereas

the

remainder

-LRB-

SSTO

DBB

MANN

and

MCF

-RRB-

exhibited

the

specificity

encoded

by

HLA

here

annotated

for

the

first

time

in

genomic

sequence

The

HLA

sequences

included

three

known

loci

-LRB-

HLA

HLA

and

HLA

-RRB-

as

well

as

three

novel

pseudogenes

-LRB-

DASS

DASS

and

DASS

-RRB-

DASS

corresponds

to

a

pseudogene

derived

from

the

gene

for

the

protein

kinase

interferon-inducible

double-stranded

RNA

dependent

activator

-LRB-

Chida

et

al.

-RRB-

for

which

the

symbol

has

now

been

recognised

A

further

processed

pseudogene

-LRB-

Jamain

et

al.

-RRB-

was

also

annotated

in

the

specificity

HLA-V

and

HLA-P

Our

analysis

showed

that

the

two

unprocessed

class

I

pseudogenes

HLA-V

and

HLA-P

-LRB-

previously

and

Geraghty

et

al.

-RRB-

should

in

fact

be

merged

together

individually

they

merely

represented

the

and

portions

of

a

single

unprocessed

pseudogene

separated

by

repeat

elements

According

to

our

annotation

guidelines

-LRB-

see

Materials

and

methods

-RRB-

the

newly

merged

locus

was

assigned

the

symbol

from

the

component

in

this

case

HLA-P

Best-in-genome

nucleotide

evidence

was

found

to

support

five

transcript

variants

at

the

end

which

together

with

evidence

for

continued

locus-transcription

led

us

to

designate

the

locus

as

a

transcribed

pseudogene

Because

transcription

appears

to

still

occur

at

this

locus

it

was

therefore

designated

as

a

transcribed

pseudogene

A

further

six

expressed

pseudogenes

were

identified

in

the

MHC

region

-LRB-

HLA

HLA-J

HLA

HLA

L

and

-RRB-

RCCX

hypervariable

region

This

module

within

the

MHC

class

III

region

named

for

its

gene

content

-LRB-

-RRB-

may

be

duplicated

or

triplicated

-LRB-

Chung

et

al.

-RRB-

and

the

pseudogenes

TNXA

and

contain

the

complement

component

gene

in

either

or

both

of

the

two

versions

and

-LRB-

Awdeh

and

Alper

-RRB-

This

gene

may

also

be

present

in

either

long

-LRB-

-RRB-

or

short

-LRB-

-RRB-

forms

depending

on

the

presence

or

absence

of

an

inserted

element

in

intron

Contrary

to

our

previous

annotation

-LRB-

Stewart

et

al.

-RRB-

see

also

legend

to

-LRB-

Fig.

-RRB-

the

PGF

haplotype

now

appears

to

possess

an

arrangement

in

which

precedes

whereas

COX

has

a

single

module

with

and

QBL

has

a

single

module

with

-LRB-

Traherne

et

al.

-RRB-

For

the

new

haplotype

sequences

reported

in

this

paper

SSTO

was

bimodular

with

two

copies

of

whereas

DBB

was

bimodular

with

followed

by

Although

a

sequence

gap

was

present

in

MCF

this

haplotype

appeared

to

be

bimodular

in

that

although

the

telomeric

copy

of

the

gene

could

not

be

identified

there

was

evidence

for

the

pseudogenes

TNXA

and

in

a

telomeric

module

The

second

centromeric

module

in

MCF

contained

The

RCCX

region

in

the

APD

and

MANN

haplotypes

was

incomplete

because

of

sequence

gaps

Variability

in

the

gene

has

been

reported

to

consist

of

extension

of

the

minisatellite

in

exon

from

copies

in

PGF

and

COX

to

copies

in

QBL

-LRB-

Traherne

et

al.

-RRB-

In

the

newly

annotated

haplotypes

we

found

the

minisatellite

to

extend

to

in

MANN

The

APD

DBB

and

MCF

possessed

copies

There

was

a

sequence

gap

in

this

region

in

the

SSTO

haplotype

MICA

The

known

allelic

polymorphism

of

MICA

reported

for

the

QBL

cell

line

sequence

in

which

a

four-base

insertion

-LRB-

GCGT

-RRB-

extended

the

open

reading

frame

in

coding

exon

haplotype

-LRB-

Traherne

et

al.

-RRB-

was

also

present

in

the

MANN

haplotype

The

insertion

was

absent

from

PGF

COX

and

SSTO

No

sequence

was

available

in

APD

DBB

and

MCF

for

this

gene

The

intronless

pseudogene

reported

to

have

a

full-length

open

reading

frame

in

the

PGF

COX

and

QBL

haplotypes

-LRB-

Stewart

et

al.

Traherne

et

al.

-RRB-

was

found

to

have

a

similar

open

reading

frame

in

the

DBB

and

MANN

haplotypes

but

to

have

the

frameshift

mutation

seen

in

the

original

chromosome

reference

sequence

-LRB-

Mungall

et

al.

-RRB-

in

the

SSTO

APD

and

MCF

haplotypes

The

QBL

haplotype

remains

the

only

one

in

which

there

was

a

single

nucleotide

deletion

in

a

polyC

tract

of

exon

-LRB-

Traherne

et

al.

-RRB-

DBB

MANN

and

MCF

resembled

PGF

and

COX

No

sequence

was

available

for

this

gene

in

SSTO

or

APD

The

PGF

haplotype

has

been

reported

to

have

a

disrupted

start

codon

for

alternative

splice

variant

of

-LRB-

Traherne

et

al.

-RRB-

This

disruption

was

not

present

in

COX

or

QBL

nor

was

it

present

in

the

further

haplotypes

reported

in

this

paper

namely

SSTO

DBB

MANN

and

MCF

APD

had

no

sequence

in

this

region

This

olfactory

receptor

has

been

reported

to

have

both

functional

and

non-functional

alleles

-LRB-

Ehlers

et

al.

-RRB-

the

latter

the

result

of

a

premature

stop

codon

at

amino

acid

position

introduced

by

a

substitution

in

the

coding

sequence

In

our

annotation

we

found

the

PGF

and

MCF

haplotypes

to

contain

the

full-coding

sequence

whereas

the

COX

QBL

SSTO

APD

DBB

and

MANN

haplotypes

to

contain

the

truncated

sequence

as

an

unprocessed

pseudogene

-LRB-

see

above

and

Fig.

-RRB-

Other

annotation

differences

Other

loci

included

in

the

current

but

not

the

previous

PGF

annotation

were

and

the

loci

without

symbols

listed

in

Table

Previously

annotated

loci

not

annotated

in

this

study

or

considered

artefacts

because

they

did

not

reach

our

current

standards

of

annotation

included

HLA-X

and

Table

newly

annotated

lociLocusLocus

Processed

Processed

Unprocessed

Unprocessed

Processed

Processed

Unprocessed

Unprocessed

TranscriptNewly

annotated

loci

without

HGNC

symbols

Non-canonical

splice

sites

Eight

variants

within

six

loci

were

shown

to

exhibit

haplotypic

variation

at

their

splice

sites

-LRB-

canonical

to

non-canonical

motif

Table

-RRB-

These

variations

may

affect

the

gene

expression

at

the

post-transcriptional

level

Hoarau

et

al.

-LRB-

-RRB-

have

already

described

the

differential

splicing

within

the

HLA

locus

and

this

can

clearly

be

seen

by

comparing

the

new

HLA

annotation

through

the

VEGA

genome

browser

Table

variation

at

splice

sitesGeneVariantAffected

exonsDonor

Acceptor

dbSNP

cluster

IDBest

loci

and

variants

that

are

affected

by

disruptive

variations

at

splice

sites

C

Canonical

splice

site

-LRB-

donor

ngt

acceptor

nag

-RRB-

NC

non-canonical

and

ND

no

data

-LRB-

gene

absent

or

gap

-RRB-

Donor

and

acceptor

variable

nucleotides

in

bold

with

equivalent

dbSNP

cluster

ID

number

given

in

column

to

right

The

and

genes

are

for

these

purposes

effective

duplicates

of

each

other

The

two

variants

share

the

same

splice

site

-LRB-

but

differ

elsewhere

in

structure

-RRB-

The

two

HLA

DQA

variants

share

the

same

donor

but

have

alternative

acceptors

Note

the

mutually

exclusivity

of

these

variants

amongst

the

haplotypes

-LRB-

Hoarau

et

al.

Hoarau

et

al.

-RRB-

Combination

of

variation

and

annotation

data

The

data

for

sequence

contig

length

gaps

variation

rate

within

haplotypes

and

PGF

coding

gene

annotation

have

been

combined

in

the

map

in

Fig.

This

illustrates

the

concentration

of

variation

around

the

HLA

gene

loci

specifically

in

areas

around

HLA-F

HLA-G

and

HLA-A

around

HLA-C

and

HLA-B

and

around

and

The

variation

status

of

genes

of

the

PGF

haplotype

is

shown

in

Table

Fig.

and

annotation

map

of

eight

MHC

haplotypes

The

map

represents

the

complete

reference

sequence

-LRB-

orange

bar

split

into

three

Mb

sections

-RRB-

labelled

PGF

and

marked

with

a

scale

-LRB-

Mb

-RRB-

and

approximate

megabase

positions

on

the

build

of

chromosome

-LRB-

grey

milestones

-RRB-

Below

the

reference

sequence

are

arrows

representing

gene

positions

and

orientations

colour-coded

for

variation

status

-LRB-

invariable

black

with

synonymous

variation

only

green

with

non-synonymous

conservative

variation

red

with

non-synonymous

non-conservative

variation

purple

see

Table

-RRB-

and

their

symbols

on

a

band

denoting

MHC

class

-LRB-

extended

class

I

green

class

I

yellow

class

III

pale

orange

class

II

light

blue

extended

class

II

pink

outside

MHC

pale

grey

-RRB-

Above

the

reference

sequence

coloured

bands

represent

the

sequences

of

the

other

seven

haplotypes

-LRB-

COX

orange

QBL

mauve

APD

yellow

DBB

green

MANN

light

blue

SSTO

dark

blue

MCF

purple

-RRB-

with

sequence

gaps

in

dark

grey

the

RCCX

hyper-variable

region

shown

with

green

-LRB-

block

-RRB-

and/or

red

-LRB-

block

-RRB-

or

black

-LRB-

block

absent

-RRB-

and

the

HLA

DRB

hyper-variable

region

in

shades

of

blue-green

Above

each

haplotype

bar

a

bar-graph

represents

total

variation

between

the

haplotype

and

the

reference

sequence

-LRB-

total

kb

-RRB-

in

dark

red

Re-examination

of

the

sequence

from

the

PGF

haplotype

which

contains

the

RCCX

region

has

shown

that

the

original

assembly

was

erroneous

Correction

of

these

errors

leads

us

now

to

the

conclusion

that

the

gene

precedes

the

gene

in

this

clone

sequence

This

new

gene

order

is

reflected

in

Fig.

status

of

the

main

coding

variant

of

each

gene

in

the

PGF

haplotype

annotationInvariableSynonymous

variation

onlyNon-synonymous

variationConservative

variationNon-conservative

coding

sequences

may

be

invariable

-LRB-

no

recorded

variation

-RRB-

have

synonymous

variation

only

-LRB-

variation

at

the

nucleotide

but

not

the

peptide

level

-RRB-

or

have

non-synonymous

variation

-LRB-

variation

at

both

the

nucleotide

and

peptide

level

-RRB-

which

in

turn

may

be

conservative

or

non-conservative

variation

according

to

the

criteria

of

positive

or

negative

values

in

the

matrix

The

main

coding

variant

is

that

numbered

in

the

VEGA

database

except

for

and

where

the

main

variant

is

not

coding

and

were

excluded

from

calculation

of

variation

because

the

order

of

these

genes

in

the

PGF

sequence

precluded

alignment

with

other

haplotype

sequences

Nevertheless

alignment

of

the

coding

sequences

for

each

gene

separately

showed

that

there

were

non-synonymous

non-conservative

variations

is

present

in

this

study

only

in

the

PGF

haplotype

and

therefore

here

appears

invariableaCoding

genes

where

the

main

variant

does

not

harbour

non-conservative

non-synonymous

variation

but

other

variants

do

-LRB-

HLA-DOA

MOG

and

-RRB-

bSimilarly

coding

genes

where

the

main

variant

does

not

harbour

conservative

non-synonymous

variation

but

other

variants

do

-LRB-

-RRB-

As

well

as

the

variations

reported

above

major

indels

revealed

as

breaks

in

discrepancy

lists

and

analysed

by

RepeatMasker

are

given

in

Table

Many

of

these

have

been

previously

reported

-LRB-

Dangel

et

al.

Dunn

et

al.

Dunn

et

al.

Gaudieri

et

al.

Horton

et

al.

Kulski

and

Dunn

Stewart

et

al.

-RRB-

These

indels

were

most

frequently

but

not

exclusively

associated

with

AluY

elements

Table

indels

in

the

form

of

retrotransposible

pos

nFlanking

lociPresence

in

CCComplex

region

-LRB-

A

-RRB-

X

Within

X

HLA-F

XX

plus

simple

MICF

X

MICF

HLA-H

inserted

in

XX

XX

SVA

E/F

plus

simple

rpt

XX

XX?AluJb

and

parts

of

XX

X

AluY

-LRB-

whole

part

-RRB-

and

HLA-C

X

part

-LRB-

-RRB-

HLA-C

XXX?XXXSVA

F

plus

GC

rich

rpt

X?XXXAluY

HLA-B

XXXAluSp

plus

part

-LRB-

-RRB-

HLA-B

X

-LRB-

part

-RRB-

and

AluSg

-LRB-

only

cf

CX

DB

-RRB-

HLA-B

part

-LRB-

-RRB-

X

X?SVA

B/F

plus

simple

XX

XX

HLA-DRA

intron

xxX

within

more

or

less

partial

intron

XX/X

intron

CC?Complex

region

-LRB-

B

-RRB-

intron

X

X

X?AluSq

CC?Complex

region

-LRB-

C

-RRB-

X

-LRB-

parts

-RRB-

X

part

and

from

CX

and

CCCComplex

region

-LRB-

D

-RRB-

X?X

X

-LRB-

parts

-RRB-

X

around

-LRB-

part

-RRB-

X

-LRB-

Zombi

-RRB-

-LRB-

parts

-RRB-

and

X

X

XX

HLA-DOB

X

XX

HLA-DOBXX

HLA-DOB

X?XXX

and

parts

of

and

AluSc

within

XXX

XAluY

-LRB-

-RRB-

AluJb

-LRB-

-RRB-

Where

there

was

a

break

in

the

discrepancy

list

match

between

two

clones

the

inserted

sequence

was

extracted

and

subjected

to

analysis

by

RepeatMasker

to

assess

the

number

of

major

indels

that

were

a

result

of

retrotransposible

elements

Chromosome

position

-LRB-

-RRB-

of

the

inserted

sequence

was

that

of

the

midpoint

where

the

sequence

was

an

insertion

in

PGF

or

the

position

before

the

deletion

in

PGF

Flanking

loci

were

retrieved

from

the

annotation

Insertion

in

a

haplotypes

is

indicated

by

deletion

by

X

complex

regions

by

C

Where

there

is

a

sequence

gap

in

a

haplotype

corresponding

to

the

indel

this

is

shown

by

Four

complex

deletion/insertion

events

are

listed

A

B

C

and

D

for

details

see

text

Four

of

these

major

indels

were

complex

and

designated

as

complex

regions

A

B

C

and

D

in

Table

They

include

three

known

regions

from

the

comparison

of

the

PGF

and

COX

haplotypes

-LRB-

Stewart

et

al.

-RRB-

Complex

region

A

-LRB-

involving

MIR

AluSx

AluSg

AluY

AluSx

and

elements

-RRB-

maps

between

and

and

was

found

to

be

deleted

in

COX

but

present

in

PGF

QBL

SSTO

MANN

and

MCF

Complex

region

B

-LRB-

involving

and

AluY

elements

-RRB-

maps

to

intron

of

HLA

and

was

also

found

by

comparing

PGF

with

COX

QBL

DBB

MANN

and

SSTO

Complex

region

C

-LRB-

SVA

and

low-complexity

repeat

elements

-RRB-

maps

between

HLA

and

HLA

and

was

noted

in

COX

as

a

deletion

of

the

SVA

and

low-complexity

repeats

Whereas

DBB

MANN

and

SSTO

displayed

the

same

deletion

as

well

as

a

telomeric

deletion

of

AluSx/MIRb

QBL

had

both

deletions

plus

that

of

an

intervening

kb

sequence

containing

Alu

and

elements

Complex

region

D

maps

between

HLA

and

HLA

and

is

more

complicated

than

previously

reported

At

the

telomeric

end

PGF

lacks

an

fragment

of

bp

that

is

present

in

COX

QBL

SSTO

and

MCF

and

is

also

absent

in

DBB

and

MANN

where

it

is

interrupted

by

about

kb

of

SVA

sequence

Centromeric

to

this

PGF

contains

an

AluSx

an

AluY

and

an

flanked

by

long

interspersed

nuclear

element

repeats

all

deleted

in

the

other

haplotypes

Further

towards

the

centromere

there

is

an

fragment

into

which

in

PGF

alone

there

are

insertions

of

an

AluSx

followed

by

an

AluY

a

subsequent

AluSg

present

in

all

haplotypes

contains

an

insertion

of

bp

of

SVA

sequence

in

just

COX

and

QBL

Finally

at

the

centromeric

end

of

this

region

PGF

uniquely

contains

intact

and

elements

Representation

of

haplotypes

within

European

populations

The

eight

haplotypes

analysed

in

this

study

were

selected

on

the

basis

of

their

association

with

type

diabetes

and

multiple

sclerosis

and

their

high

population

frequencies

To

determine

how

representative

these

haplotypes

are

with

respect

to

SNP

haplotypic

diversity

in

a

population

we

determined

their

distribution

in

the

haplotypic

tree

space

in

the

European

population

For

this

analysis

we

selected

a

segment

of

kb

spanning

the

HLA

and

HLA

genes

in

a

population

of

European

ancestry

with

known

HLA

allelic

data

-LRB-

de

Bakker

et

al.

-RRB-

Phylogenetic

analysis

of

founder

haplotypes

derived

from

genotypic

data

-LRB-

substitutions

-RRB-

shows

that

the

eight

haplotypes

selected

as

part

of

the

MHC

Haplotype

Project

share

identical

HLA

alleles

over

most

of

the

tree

space

-LRB-

Fig.

-RRB-

representing

almost

the

entire

variation

observed

in

the

population

assayed

with

the

exception

of

two

branches

-LRB-

and

-RRB-

Fig.

of

haplotypes

in

the

European

haplotypic

diversity

Phylogenetic

relationship

of

founder

SNP

haplotypes

from

CEPH

trios

spanning

a

segment

of

the

MHC

class

II

region

including

the

and

genes

-LRB-

substitutions

from

to

-RRB-

a

Sequenced

haplotypes

are

widely

distributed

in

this

NJ

tree

and

represent

the

vast

majority

of

the

variation

in

the

population

sampled

Four-digit

alleles

are

indicated

for

the

corresponding

and

genes

in

each

haplotype

ID

label

to

highlight

the

HLA

haplotypic

distribution

based

on

the

underlying

nucleotide

variation

The

NJ

tree

was

constructed

using

pairwise

genetic

distances

considering

the

Kimura

model

without

correction

for

rate

variation

among

sites

as

implemented

in

the

software

-LRB-

Kumar

et

al.

-RRB-

b

Each

haplotype

sequenced

is

associated

to

a

single

haplotype

cluster

This

phylogenetic

network

-LRB-

Bandelt

et

al.

-RRB-

also

shows

that

clusters

-LRB-

shaded

area

-RRB-

are

constituted

by

one

central

haplotype

and

its

derivatives

Circles

represent

individual

haplotypes

and

the

size

of

the

circle

is

proportional

to

the

haplotype

frequency

The

length

of

the

lines

connecting

nodes

is

relative

to

the

distance

between

them

e.g.

distances

within

shaded

areas

-LRB-

clusters

-RRB-

never

exceed

three

mutation

steps

Cluster

of

haplotypes

sharing

HLA

alleles

with

sequenced

cell

lines

are

named

accordingly

COX

and

QBL

PGF

APD

MCF

DBB

SSTO

MANN

HLA

haplotypes

and

indicate

the

two

major

haplotype

clusters

not

represented

in

the

MHC

haplotype

project

data

Haplotype

diversity

in

this

sub-population

is

restricted

to

relatively

few

haplotype

clusters

-LRB-

Fig.

-RRB-

Each

cluster

consists

of

a

founder

haplotype

depicted

by

the

most

frequent

and

centrally

located

haplotype

within

the

cluster

Recently

derived

haplotypes

show

lower

frequencies

and

are

connected

to

the

central

haplotype

by

relatively

few

mutation

steps

-LRB-

in

this

case

up

to

three

-RRB-

This

phylogenetic

network

clearly

shows

that

all

the

sequenced

haplotypes

occupy

central

positions

in

their

respective

haplotypic

groups

Inferences

about

phylogenetic

relationships

between

haplotype

clusters

are

however

only

approximate

as

a

consequence

of

recombination

events

It

should

also

be

noted

that

SNP

haploptypes

derived

from

CEPH

pedigrees

of

European

ancestry

by

no

means

represent

an

exhaustive

sampling

of

European

diversity

Nevertheless

the

sampling

has

been

shown

to

represent

the

European

population

in

the

UK

reasonably

well

-LRB-

Ke

et

al.

-RRB-

In

conclusion

our

analysis

demonstrates

that

the

HLA

haplotypes

selected

for

the

MHC

Haplotype

Project

are

ancestral

haplotypes

representative

of

MHC

diversity

in

the

European

population

Conclusion

and

outlook

The

MHC

Haplotype

Project

has

succeeded

in

providing

a

new

public

resource

for

immune-linked

disease

and

population

genetic

studies

First

reports

from

studies

using

the

resource

indicate

that

it

adds

significant

power

to

the

identification

and

fine-mapping

of

disease-associated

variations

-LRB-

Yeo

et

al.

-RRB-

The

data

have

also

contributed

to

the

recent

identification

of

a

first

set

of

HLA

tag

SNPs

which

hold

great

promise

for

future

applications

in

clinical

settings

e.g.

to

complement

or

replace

classical

HLA-typing

in

transplant

medicine

-LRB-

de

Bakker

et

al.

-RRB-

While

costs

and

other

limitations

of

the

current

-LRB-

capillary

-RRB-

sequencing

technology

have

restricted

our

study

to

only

few

-LRB-

eight

-RRB-

MHC

haplotypes

the

number

of

new

variations

found

combined

with

the

fact

that

no

variation

plateau

has

yet

been

reached

indicates

that

there

are

many

more

variations

to

be

discovered

The

recent

introduction

of

several

new

and

massively

parallel

sequencing

platforms

-LRB-

for

review

see

Bentley

-RRB-

has

created

the

opportunity

to

do

just

that

by

re-sequencing

haplotypes

and

eventually

entire

genomes

at

the

population

level

and

as

integral

part

of

case

control

studies

Because

of

its

wide-ranging

medical

importance

the

MHC

can

be

expected

to

be

among

the

first

regions

of

the

human

genome

to

be

sequenced

in

this

way

Such

sequencing

will

provide

the

critical

and

until

now

missing

data

to

identify

causal

variations

and

their

underlying

mechanisms

on

an

unprecedented

scale

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

major histocompatibility complex
haplotype
polymorphism

retroelement
genetic predisposition to disease
population genetics


Pediatric delirium in critical illness : phenomenology , clinical correlates and treatment response in 40 cases in the pediatric intensive care unit Objective To study the phenomenology , clinical correlates , and response to treatment of delirium in critically ill children in the pediatric intensive care unit -LRB- PICU -RRB- . Introduction Delirium is a neuropsychiatric disorder secondary to a general medical condition , and must be considered a serious complication of the underlying disease or its treatment . In the revised fourth edition of the Diagnostic and Statistical Manual of Mental Disorders -LRB- DSM-IV-TR ® -RRB- , delirium is defined by four concurrent diagnostic criteria : -LRB- 1 -RRB- acute onset and fluctuation of -LRB- 2 -RRB- a disturbance of consciousness with reduced ability to focus , shift or maintain attention and -LRB- 3 -RRB- a change of cognition with memory deficit , disorientation , language disturbance , perceptual disturbances or hallucinations , -LRB- 4 -RRB- caused by the direct physiological consequences of a general medical condition -LSB- 1 -RSB- . It is frequently seen in critically ill adult and geriatric patients -LSB- 2 -- 5 -RSB- and is associated with a poor prognosis , reflected by longer hospital stay , worse functional and cognitive outcome , and a higher mortality rate after discharge from hospital -LSB- 3 -RSB- . In mechanically ventilated critically ill adults , delirium is an independent predictor of elevated 6-month mortality and a longer hospital stay -LSB- 6 -RSB- . If appropriate diagnostic tools validated for bedside use by non-psychiatrists -LSB- e.g. Delirium Rating Scale -LRB- DRS -RRB- , Confusion Assessment Instrument for the Intensive Care Unit -LRB- CAM-ICU -RRB- -RSB- are used , delirium is diagnosed in over 80 % of critically ill adult patients -LSB- 7 -RSB- . Thus , systematic monitoring for delirium and appropriate treatment with haloperidol in critically ill adult patients were included in the recently published clinical practice guidelines for sedatives and analgesia of the Society of Critical Care Medicine -LSB- 8 -RSB- . However , the optimal management of patients with delirium and the effects of the pharmacological treatment on the outcome are still key concerns for today -LSB- 9 -RSB- . Given lack of age-appropriate diagnostic criteria and assessment tools in children , even less is known about the incidence , clinical presentation , response to treatment and consequences of childhood delirium in general , and in critically ill children in particular -LSB- 10 -- 12 -RSB- . The few available published data on childhood delirium suggest that morbidity and mortality are higher in children with than in children without delirium -LSB- 13 -RSB- . Therefore , delirium in children should be considered a serious complication and be treated accordingly . Unfortunately , while there are comprehensive guidelines on the diagnosis and treatment of delirium in adults , clinical guidelines for delirium in children are nonexistent . The aim of this study was to investigate the incidence , patient and population characteristics , clinical presentation and response to treatment of delirium in a cohort of critically ill children admitted to a tertiary pediatric intensive care unit -LRB- PICU -RRB- . Given the necessarily multidisciplinary approach to assessment and treatment of these children , the input of four disciplines -- child psychiatry , pediatric intensive care medicine , child neurology and adult neuropsychiatry -- was used . Methods Design , setting and patients A descriptive study was carried out over a 4-year period -LRB- January 2002 to December 2005 -RRB- in an eight-bed tertiary PICU . This PICU is a tertiary referral center for both general and surgically critically ill children in the southeastern region of the Netherlands -LRB- population 1.4 million , 350 annual admissions -RRB- . Critically ill children , acutely , non-electively and consecutively admitted , were prospectively sampled . Both mechanically ventilated and non-ventilated patients were included . Diagnostic approach All children with -LRB- 1 -RRB- confusion , agitation , anxiety , moaning , discomfort , or behavioral disturbances with no acceptable medical explanation or -LRB- 2 -RRB- failure of standard analgosedative treatment were systematically assessed for the presence of delirium in a two-step diagnostic approach . The standard analgosedative treatment can be summarized as follows : children who required analgosedation because of obvious or expected pain or because of stress related to their underlying disease or treatment received adequate doses of opioids and/or benzodiazepines according to internationally published guidelines for analgesia and sedation in critically ill children -LSB- 14 -RSB- . Drug doses were individually tailored to achieve optimal patient comfort and were slowly reduced in order to avoid a withdrawal syndrome . In the event that a withdrawal syndrome was suspected , based on clinical observation or the revised Finnegan score , specific treatment with long-acting benzodiazepines -LRB- e.g. lorazepam -RRB- or opioids -LRB- e.g. methadone -RRB- was started according to internationally published guidelines -LSB- 15 , 16 -RSB- . At the time the assessment for delirium was initiated , none of the patients had signs of imminent life-threatening respiratory , circulatory or neurological failure , while ongoing asphyxia , respiratory acidosis , metabolic disturbances , fighting the ventilator due to inappropriate ventilator settings and withdrawal syndrome all had been excluded systematically as an explanation for the observed behavior . The first step of the diagnostic approach was a systematic assessment by a child neuropsychiatrist -LRB- J.S. -RRB- using DSM-IV criteria for delirium . Criteria were evaluated on the basis of -LRB- 1 -RRB- hetero-anamnestic information from parents , nurses , intensivists , and child neurologists about behavior and behavioral changes and -LRB- 2 -RRB- child psychiatric examination . Based on the findings , patients were categorized as having a -LRB- probable -RRB- delirium or not . In a second step , the provisional diagnosis of delirium was further tested in a daily multidisciplinary consensus meeting . The team consisted of the child neuropsychiatrist , the attending pediatric intensivist , and occasionally a geriatric neuropsychiatrist specialized in delirium in geriatric patients and/or a child neurologist . If this team agreed that alternative explanations for a child 's behavior were unlikely , the consensus diagnosis was delirium . Based on the dominant clinical presentation , cases of delirium were classified as `` hyperactive '' when psychomotor agitation was present , and as `` hypoactive '' when retardation and/or inhibition was present . A number of children presented with cognitive and/or attentional disturbances in the context of severe anxiety states , often accompanied with moaning and restlessness , but without clear agitation or retardation . This latter group was classified as `` emerging '' or `` veiled '' delirium -LSB- 17 -RSB- , in reference to the way delirious syndromes have been described as `` partial delirium '' in adult ICU patients or as `` subsyndromal delirium '' in elderly medical patients -LSB- 7 , 18 , 19 -RSB- . The different presentation forms were not always clear-cut , and some fluctuated dramatically over time . The severity of illness was scored according to the Pediatric Index of Mortality -LRB- PIM -RRB- and Pediatric Risk of Mortality -LRB- PRISM -RRB- -LSB- 20 -RSB- . Therapeutic approach Whenever delirium was identified or suspected , a two-track treatment approach consisting of both psychosocial and pharmacological interventions was implemented . Psychosocial interventions -- the parents ' presence and comforting throughout the day -LRB- and night -RRB- , familiar music , favorite toys , pictures of home and pets , friends , school , lighting schedules , sometimes even fragrances -- are standard in the PICU . The parents also received an information leaflet on childhood delirium -LSB- 21 -RSB- . All patients were also treated with antipsychotic medication after the referring pediatric intensivist had agreed and the parents , because of the off-label use , had given informed consent , which was never refused . In children with psychomotor agitation that was acutely threatening to their health status , haloperidol at a loading dosage of 0.15 -- 0.25 mg i.v. was used , given slowly over a period of 30 -- 45 min , followed by a maintenance dose of 0.05 -- 0.5 mg/kg/24 h i.v. -LSB- 11 , 22 , 23 -RSB- . In less acute situations , and when oral medication was possible , risperidone at a loading dose of 0.1 -- 0.2 mg p.o. was used , followed by a maintenance dose of 0.2 -- 2.0 mg/24 h p.o. as the treatment of choice . Clinical response and side effects were recorded by the child neuropsychiatrist and the pediatric intensivists . In order to tailor the treatment for delirium , daily discussions were held with the multidisciplinary team . Adjustment of treatment was based on the clinical observations and judgements of the parents , nurses , intensivists and child psychiatrist . Children were followed up for 6 weeks after discharge from the hospital either at the outpatient clinic or by contacting the parents by telephone . As the study solely involved the structured recording of routine clinical practice , under Dutch law no institutional review board approval was required . Results From January 2002 to December 2005 , there were 877 acute , non-elective admissions to the PICU . Distribution of age and gender are shown in Table 1 . In 61 cases -LRB- 7 % -RRB- , a systematic assessment by a child neuropsychiatrist was requested , usually for agitation , anxiety , moaning , discomfort , behavioral disturbance or problematic analgosedation . Table 1Number and incidence of delirium in the total sample * by age and genderAgeTotal sample * Patients with deliriumIncidence -LRB- % -RRB- 0 -- 2.99 years51314 2.7 Male310 9 2.9 Female203 5 2.53 -- 5.99 years106 4 3.8 Male 56 3 5.4 Female 50 1 2.06 -- 8.99 years 80 6 7.5 Male 46 1 2.2 Female 34 514.79 -- 11.99 years 77 3 3.9 Male 61 3 4.9 Female 16 0 012 -- 14.99 years 70 710Male 35 514.3 Female 35 2 5.715 -- 18 years 31 619.4 Male 13 430.8 Female 18 211.1 Total87740 4.6 Male52125 4.8 Female35615 4.2 * Critically ill children , acutely , non-electively and consecutively admitted during a 4-year period Of these 61 patients , 40 -LRB- 61 % -RRB- were diagnosed with delirium , yielding a cumulative incidence of 5 % -LRB- boys 5 % ; girls 4 % -RRB- . Age-specific incidences increased from 3 % in the 0 -- 3 years age group -LRB- boys 3 % ; girls 3 % -RRB- , to 19 % in the 16 -- 18 years age group -LRB- boys 31 % ; girls 11 % -RRB- -LRB- Table 1 , Fig . 1 -RRB- . Fig. 1Incidence of delirium in the sub-groups by age and gender The child psychiatric disorders diagnosed in the 61 referrals are summarized in Table 2 . Table 2Child psychiatric diagnosis at the first consultation -LRB- n = 61 -RRB- nDiagnosis40delirium 5adjustment disorders with anxiety and depressed mood , post operative 4psychological -- psychiatric factors affecting the medical condition 3anxiety disorder 3emotional and behavioral problems during chronic ventilation 2adjustment disorders with depressed mood 1mood disorder 1adjustment disorder with anxiety 1sleeping problem 1feeding problem Table 3 summarizes the population characteristics of the sample diagnosed with delirium , while Table 4 lists the underlying somatic disease or causative pharmacological treatment . Table 3Population characteristics of the 40 PICU cases with delirium , 2002 -- 2005CharacteristicsFrequency -LRB- total n = 40 -RRB- Age -LRB- mean ± SD -RRB- 7.6 ± 5.9 Malen25 age -LRB- mean ± SD -RRB- 7.6 ± 6.1 Femalen15 age -LRB- mean ± SD -RRB- 7.6 ± 5.8 Ethnicity White36 -LRB- 90 % -RRB- African 3 -LRB- 7.5 % -RRB- Asian 1 -LRB- 2.5 % -RRB- Mechanical ventilation34 -LRB- 85 % -RRB- PIM score -LRB- mean ± SD -RRB- 9.96 ± 16.20 PRISM score -LRB- mean ± SD -RRB- 23.54 ± 24.94 Major somatic pharmacological features Recent increase or decrease of analgosedative medication22 -LRB- 55 % -RRB- Neurological disorders21 -LRB- 52 % -RRB- Infectious disorders20 -LRB- 50 % -RRB- Respiratory disorders12 -LRB- 30 % -RRB- PIM , Pediatric Index of Mortality ; PRISM , Pediatric Risk of MortalityTable 4Patient characteristics of the 40 PICU cases with delirium 2002 -- 2005No . SexAgePrimary diagnosis on admission PICUMechanical ventilationDelirium typeTreatment 1M 3 monthsMultiple congenital malformations + EmergingHaloperidol 2F 4 monthsMeningococcal septic shock + EmergingRisperidone 3M 4.5 monthsSevere CLD+E mergingRisperidone 4F10 monthsNear drowning + EmergingHaloperidol 5F 1 yearPneumonia + EmergingHaloperidol 6M 1 yearSepsis due to perforated appendicitis + EmergingHaloperidol 7M 1 yearSubarachnoid bleeding + EmergingHaloperidol 8M 1 yearPericardial effusion with pretamponade + HyperactiveHaloperidol 9M 1 yearMultiple dysmorphia , upper airway obstruction -- EmergingHaloperidol10F 2 yearsMeningococcal septic shock + HyperactiveHaloperidol11M 2 yearsADEM + HyperactiveRisperidone12M 2 yearsCervical mass , upper airway obstruction + EmergingHaloperidol13M 2 yearsAspiration and pneumothorax + Hypoactive -- 14F 2 yearsMeningococcal meningitis with sepsis and DIC+H yperactiveHaloperidol15F 3 yearsCystic fibrosis and pneumonia + HypoactiveHaloperidol16M 4 yearsIntracerebral hemorrhage , Marfan syndrome + HypoactiveHaloperidol17M 5 yearsMedulloblastoma post surgery + EmergingRisperidone18M 5 yearsUpper respiratory tract infection + HyperactiveHaloperidol19M 6 yearsMultiple trauma + EmergingRisperidone20F 8 yearsMeningo-encephalitis + HyperactiveRisperidone21F 8 yearsViral encephalitis + Hyperactive -- 22F 9 yearsStatus asthmaticus + HyperactiveRisperidone23F 9 yearsTBI , gunshot wound + HypoactiveRisperidone24M 9 yearsStatus asthmaticus + HyperactiveHaloperidol25M 9 yearsNeural tube defect and drain dysfunction -- Emergingfirst Haloperidol , then Risperidone26M11 yearsHypovolemic shock , typhus abdominalis -- HyperactiveHaloperidol27F12 yearsTBI + HypoactiveHaloperidol28M12 yearsTBI and fracture of lower leg + EmergingHaloperidol29M13 yearsSepsis , paronychia -- HyperactiveHaloperidol30M13 yearsStatus epilepticus + EmergingHaloperidol31F14 yearsTBI + HyperactiveHaloperidol32F15 yearsPost TBI+H ypoactiveHaloperidol33M15 yearsPostoperative state + HypoactiveRisperidone34M15 yearsAcute lymphoblastic leukemia -- HypoactiveHaloperidol35M15 yearsTBI + EmergingHaloperidol36F15 yearsStatus asthmaticus + HyperactiveHaloperidol37M16 yearsMultiple trauma -- HypoactiveHaloperidol38F16 yearsBacterial meningitis + HyperactiveRisperidone39M16 yearsRespiratory failure , Duchenne disease + EmergingHaloperidol40M17 yearsSeptic shock + EmergingHaloperidolCLD , chronic lung disease ; ADEM , acute disseminated encephalomyelitis ; DIC , diffuse intravascular coagulation ; TBI , traumatic brain injury The underlying features were : recent increase or decrease in analgosedative medication -LRB- n = 22 -RRB- , neurological disorders -LRB- n = 21 -RRB- , infections -LRB- n = 20 -RRB- and respiratory disorders -LRB- n = 12 -RRB- . Usually , a combination of these existed . All but two patients were treated with an antipsychotic drug . Twenty-seven children were given haloperidol , 10 risperidone , and 1 child received both drugs in succession . In most cases , the beneficial results were observed rapidly , especially in the hyperactive forms , sometimes even after a single dose -LSB- 11 -RSB- . Sometimes it took a few hours or days . Two patients experienced acute dystonia as a likely side effect of the haloperidol , responding well to biperidene . Two children received no medication : one because of lack of consensus in our expert team , and one because of an endotracheal intubation at the time that medication was indicated . In most cases , the medication was stopped or tapered off successfully during hospitalization or afterwards in an outpatient setting . Five children -LRB- 12.5 % -RRB- with delirium died of their underlying disease ; the mean PIM was 10 % and the mean PRISM 24 % -LRB- Table 3 -RRB- . Discussion This is the first systematic multidisciplinary study of the phenomenology and treatment of delirium in 40 critically ill children in a PICU context . The low cumulative incidence of 5 % is mainly the result of the low incidence in the younger age groups -LRB- < 9 years old -RRB- , this segment constituting the majority of the total sample -LRB- 80 % -RRB- . A clearly higher incidence is seen in the older age groups . However , in critically ill adult patients substantially higher incidences have been reported , ranging from 10 -- 30 % in general hospital settings to 50 % in postoperative patients and up to 80 % in the terminally ill -LSB- 2 -RSB- . There are several possible explanations for this difference . First , the incidence of delirium in young critically ill children may be truly low : differences in age-related resilience and underlying conditions may contribute to true differences in the incidence between the very young and the very old . However this explanation seems unlikely , given the tendency in the very young to develop delirium under even much less severe circumstances -LSB- 1 , 11 , 24 -RSB- . A second explanation may relate to the fact that extensive psychosocial interventions are provided as a routine in Dutch PICU settings , with a possibly preventive effect on delirium in much the same way as observed in geriatric patients -LSB- 25 -RSB- . A third factor may be an anti-delirium effect of the routinely used analgosedative medication , although especially benzodiazepines may have excitatory side effects in children . A fourth factor may relate to reluctance on the part of the intensivists and/or child neurologists to request psychiatric evaluation in the case of behavioral changes for fear of adding stigmatization to an already burdened system . Perhaps the fifth , most likely and important explanation is that parents , nurses , pediatric intensivists and child neurologists do not easily recognize delirium , because the medical condition of these critically ill PICU children is so complex and changeable . It is possible that a psychiatric consultation was readily requested only in cases of anxiety and/or hyperactive delirium , not in the hypoactive and or veiled ones . The differential diagnosis of pediatric delirium consists of acute stress reactions , acute anxiety states , adjustment disorders with mixed emotions , dissociative and/or regressive states and childhood-onset psychosis -LRB- see also Table 2 -RRB- . However , differentiating delirium from extreme stress and agitation due to acute and life-threatening conditions is not only impossible , it is also unwanted , because it is usually irrelevant at that point of time in the process of medical care . Causative treatment , if possible , is always the first step to be taken . Furthermore , the diagnosis of delirium in children is complicated by the fact that the criteria for adult delirium are not always easily applicable to children because of important differences in age and developmental levels . The DSM-IV criteria for delirium are not always useful in the case of pediatric delirium , especially in a PICU context . This is a source of concern . Moreover , delirium is not mentioned in the DSM-IV section on child psychiatry . DSM-IV describes as an essential feature of delirium the `` disturbance of consciousness '' leading to `` impairment of the ability to focus , shift and sustain attention '' . This , however , is of little relevance in the critically ill in a PICU context , where disturbance of attention is routinely present due to the disorder -LRB- s -RRB- itself . In fact , attention is often the first `` to go '' -LSB- 17 , 26 -RSB- . In addition , patients almost always require treatment with opioids and benzodiazepines , which also have a strong impact on attention . Furthermore , it has been hypothesized that a disturbance of consciousness is not a discriminating characteristic of delirium in an ICU setting -LSB- 27 -RSB- . Our case series suggests that in addition to the hyper - and hypoactive forms of delirium , a third form may be characterized by anxiety , moaning , and/or restlessness . This was referred to as an `` emerging '' or veiled delirium , as described previously -LSB- 17 -RSB- . In the PICU population this state did not develop into a frank hyper - or hypoactive form , but appeared to exist in its own right , accompanied by disturbances of consciousness and cognition . Although atypical presentations of disorders are often not captured in classification systems , the high prevalence of `` emerging '' delirium in our sample -LRB- 17/40 -RRB- stresses the importance of further phenomenological study . Adhering too strongly to DSM-IV criteria for adult delirium , for clinical use in a PICU context , may result in persistent under-diagnosis . On the other hand , the incidence of delirium may be overestimated by using the CAM-ICU , so most important is the issue of what constitutes delirium in critical illness -LSB- 28 -RSB- . There are accumulating indications that pediatric delirium can be subtle and accompanied or even dominated by other neuropsychiatric signs such as : reduction of awareness of the caregiver and/or the surrounding environment , purposeless actions , restlessness , inconsolability , signs of autonomic dysregulation and other subtle higher cortical dysfunctions -LSB- 29 -- 32 -RSB- . Parents and nurses have a way of discriminating patterns in their children that may be diagnostically important . Thus , parents sometimes state : `` This is not my child anymore . '' Pediatric delirium therefore may have various subtle presentations and can be considered a spectrum disorder , which makes it sometimes difficult to diagnose -LSB- 33 -RSB- . Neither haloperidol nor risperidone is registered for the treatment of childhood delirium , although both are mentioned as the treatment of choice for adults -LSB- 1 -RSB- . Moreover , haloperidol is not registered for i.v. administration , even though it is used in this way in many places . We prefer risperidone in non-acute situations because of the theoretically lower risk of extrapyramidal side effects . Haloperidol and risperidone have been used for other indications in young children as well , such as childhood psychosis -LSB- 34 -RSB- or aggression in autism -LSB- 35 -RSB- . There are two limitations regarding our observations of treatment response . First , no severity scale for pediatric delirium was used , because none exists for this PICU population . Second , because no studies on the natural course of childhood delirium exist that have established the rate of spontaneous remission , spontaneous remissions may have been misclassified as response to treatment . In our opinion , however , the time frame of response points towards a medication effect . Most medications employed in pediatrics and child psychiatry are used off-label -LSB- 36 -RSB- , which means that special attention should be paid to information and informed consent procedures . Given the relatively high incidence of extrapyramidal symptoms with haloperidol , the `` off-label '' use needs further study -LSB- 37 -RSB- . This study has several limitations . First , it was a study of routine clinical practice . Observations were based on referrals emerging from care as usual . Although the focus on delirium may have altered referral paths and rates , we did not actively advocate any change , nor did we screen all admissions for delirium . In the absence of a hard clinical indication , no routine blood level measurements were performed to rule out persistent high levels of sedatives as a possible explanation for any neuropsychiatric symptom . Next , the lack of DSM-IV criteria for pediatric delirium and the disputed relevance of its main criterion in a -LRB- P -RRB- ICU setting make a standardized diagnosis difficult . Finally , treatment was provided in an open setting and based on consensus , especially among child psychiatrist , pediatric intensivist and child neurologist . In conclusion , critically ill children in a PICU can develop delirium , with a hyperactive , hypoactive or veiled presentation , despite adequate analgosedation and intensive psychosocial interventions . The approach using an algorithmic structuring and an intensifying of daily clinical care , including the use of multidisciplinary daily consensus meetings , appeared effective in assessing , diagnosing and treating childhood delirium at the PICU . The findings suggest that the incidence is much lower than in adults , but a likely explanation is that delirious states requiring child psychiatric referral are still frequently under-diagnosed . There is also still a great need for developing delirium criteria in critically ill patients , children and adults alike , in a -LRB- P -RRB- ICU setting . Treatment with haloperidol or risperidone was successful in all patients . Future research is necessary to identify the risk factors for pediatric delirium in a multivariate prospective approach , to develop `` easy to use '' bedside tools for non-psychiatric trained team members for the early detection of delirium in all pediatric PICU patients , and to study the effects of interventions in a double-blind and ideally placebo-controlled fashion . Disclosure Jim van Os is a speaker or member of the advisory board for Lilly , BMS , Janssen-Cilag and AstraZeneca . He received grant or research support from Lilly , GSK , BMS and AstraZeneca . Albert Leentjens participated in research for Boehringer Ingelheim and is on the advisory board of a study related to Parkinson 's disease by the same company . The other authors have no financial relationships to disclose .

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