A

large-scale

collection

of

phenotypic

data

describing

an

insertional

mutant

population

to

facilitate

functional

analysis

of

rice

genes

In

order

to

facilitate

the

functional

analysis

of

rice

genes

we

produced

about

insertion

lines

with

the

endogenous

retrotransposon

Phenotypes

of

these

lines

in

the

generation

were

observed

in

the

field

and

characterized

based

on

phenotype

descriptors

Nearly

half

of

the

lines

showed

more

than

one

mutant

phenotype

The

most

frequently

observed

phenotype

was

low

fertility

followed

by

dwarfism

Phenotype

data

with

photographs

of

each

line

are

stored

in

the

mutant

panel

web-based

database

with

a

dataset

of

sequences

flanking

insertion

points

in

the

rice

genome

-LRB-

-RRB-

This

combination

of

phenotypic

and

flanking

sequence

data

will

stimulate

the

functional

analysis

of

rice

genes

Introduction

Rice

is

the

most

important

staple

crop

for

half

the

world

's

population

Improvements

in

rice

yield

and

quality

beyond

the

benefits

of

the

green

revolution

of

years

ago

are

required

to

meet

the

demands

of

an

increasing

global

population

At

the

beginning

of

the

century

with

the

hope

of

finding

creative

solutions

to

the

problems

of

food

production

nutrition

and

transportation

nearly

the

entire

nucleotide

sequence

of

the

rice

genome

Oryza

sativa

L.

cv

Nipponbare

was

determined

through

a

world-wide

collaborative

effort

-LRB-

International

Rice

Genome

Sequencing

Project

-RRB-

The

sequence

data

provide

an

important

resource

for

promoting

the

discovery

of

important

genes

for

crop

improvement

Currently

a

Rice

Annotation

Project

-LRB-

RAP

-RRB-

using

nucleotide

sequences

of

full-length

cDNAs

-LRB-

The

Rice

Full-Length

cDNA

Consortium

-RRB-

is

in

progress

to

position

functional

genes

on

the

rice

genome

map

-LRB-

Ohyanagi

et

al.

-RRB-

however

many

genes

remain

unknown

due

to

lack

of

experimental

evidence

or

sufficient

similarity

with

characterized

genes

from

other

organisms

The

next

great

challenge

after

completion

of

genome

sequencing

is

the

functional

characterization

of

these

genes

and

discovery

of

genes

that

affect

vital

developmental

agronomic

or

biochemical

plant

functions

-LRB-

Hirochika

et

al.

-RRB-

Gene

annotation

is

mostly

based

on

the

sequence

similarity

to

known

genes

from

other

species

The

limitations

of

this

method

are

that

every

organism

may

have

unique

genes

that

do

not

have

homologues

even

in

closely

related

species

and

assignment

of

a

protein

's

function

based

on

similarity

may

only

give

a

partial

description

For

example

a

gene

may

contain

a

domain

that

is

conserved

among

protein

kinases

but

the

actual

substrate

of

the

enzyme

would

be

difficult

or

impossible

to

determine

without

experimental

evidence

Categorization

using

cladistic

associations

-LRB-

i.e.

a

phylogenetic

tree

-RRB-

is

more

sensitive

and

is

able

to

detect

BLAST

mis-hits

or

false

positives

-LRB-

Sjolander

-RRB-

However

characteristics

supported

by

experimental

data

e.g.

correlation

between

the

phenotype

and

the

function

of

the

protein

kinase

are

indispensable

to

the

exact

annotation

because

all

of

the

associative

algorithms

ultimately

depend

on

experimental

data

Mutational

analysis

through

gene

disruption

is

one

of

the

most

efficient

methods

for

identifying

gene

function

The

related

approaches

of

interrupting

gene

expression

with

RNAi

-LRB-

Miki

and

Shimamoto

-RRB-

and

overexpression

are

also

effective

because

the

functions

of

genes

can

be

determined

by

the

correlation

of

disrupted

genes

and

their

associated

phenotypes

Currently

more

than

T-DNA

insertion

lines

of

Arabidopsis

thaliana

have

been

created

and

are

publicly

available

-LRB-

Alonso

et

al.

-RRB-

Phenotyping

of

Ds

insertion

lines

in

Arabidopsis

is

also

in

progress

-LRB-

Kuromori

et

al.

-RRB-

For

characterizing

monocot

genomes

we

have

produced

more

than

disruption

lines

of

Oryza

sativa

cv

Nipponbare

-LRB-

Japonica

-RRB-

using

the

endogenous

retrotransposon

which

has

a

copy

and

paste

type

of

transposition

activity

-LRB-

Miyao

et

al.

-RRB-

There

are

two

native

copies

of

in

the

Nipponbare

genome

that

are

activated

specifically

in

cultured

cells

-LRB-

Hirochika

et

al.

-RRB-

On

average

ten

new

copies

of

are

transposed

in

each

cell

during

months

in

culture

When

plants

have

been

regenerated

from

the

cultured

cells

retrotransposition

is

immediately

inactivated

and

copies

become

fixed

and

segregate

in

a

Mendelian

fashion

in

the

next

generation

There

are

many

advantages

to

the

disruption

system

for

mutational

analyses

Because

the

flanking

regions

of

insertion

points

are

easily

amplified

by

TAIL-PCR

or

a

suppression

PCR

method

using

a

end

primer

of

insertion

sites

in

the

rice

genome

can

be

easily

determined

Furthermore

mutants

can

be

screened

by

PCR

using

the

end

primer

and

a

primer

from

any

desired

genomic

sequence

The

distribution

of

transposed

is

not

random

and

a

large

number

of

transpositional

hot

spots

are

detected

throughout

the

rice

genome

The

insertion

frequency

of

into

genic

regions

is

three-fold

higher

than

that

into

non-coding

regions

Owing

to

this

polarization

insertion

lines

have

great

advantages

for

the

functional

analysis

of

rice

genes

-LRB-

Miyao

et

al.

-RRB-

Because

is

an

endogenous

retrotransposon

regenerated

lines

can

be

grown

in

the

field

and

seeds

can

be

exported

without

regulations

associated

with

genetically

modified

organisms

such

as

rice

lines

that

have

undergone

transgenic

insertion

of

T-DNA

-LRB-

Jeon

et

al.

Sallaud

et

al.

Wu

et

al.

-RRB-

Ds

-LRB-

Greco

et

al.

Kim

et

al.

-RRB-

or

En/Spm

-LRB-

Kumar

et

al.

-RRB-

Mutant

lines

generated

by

retrotransposition

have

already

been

used

for

the

functional

analysis

of

rice

genes

For

example

insertion

mutants

with

a

dwarf

or

viviparous

phenotype

were

used

to

identify

and

analyze

genes

for

gibberellin

and

abscisic

acid

metabolism

-LRB-

Sakamoto

et

al.

Agrawal

et

al.

-RRB-

Phenotypic

characteristics

of

most

of

the

insertion

lines

however

remain

to

be

described

A

large

number

of

plant

scientists

working

with

rice

or

other

monocotyledonous

species

could

benefit

from

a

systematic

phenotypic

analysis

of

many

insertion

lines

and

the

creation

of

a

public

database

To

promote

the

functional

analysis

of

rice

genes

the

phenotypes

of

all

of

our

mutant

lines

have

been

observed

in

rice

fields

through

the

collaboration

of

seven

laboratories

Collected

phenotypic

data

are

useful

for

predicting

the

function

of

a

disrupted

gene

In

this

paper

we

report

the

phenotypic

statistics

of

a

insertion

mutant

population

for

the

discovery

of

agronomically

important

genes

Materials

and

methods

Plant

materials

Nipponbare

calli

derived

from

embryos

were

grown

in

liquid

medium

-LRB-

Otsuki

-RRB-

containing

mg/l

for

months

In

total

about

plants

were

regenerated

Seeds

of

the

generation

were

harvested

from

each

plant

To

check

the

activity

of

under

various

hormone

conditions

liquid

media

containing

or

mg/l

with

or

without

mg/l

BA

were

used

Phenotyping

Ten

to

twenty-five

seeds

were

planted

per

line

Germination

rates

and

seedling

phenotypes

were

observed

in

the

nursery

After

month

from

seeding

the

seedlings

were

transferred

to

the

paddy

field

Phenotypes

in

the

field

were

observed

at

the

vegetative

stage

near

the

heading

stage

at

the

seed

maturation

stage

and

at

harvest

Lines

segregating

abnormal

plants

at

about

frequency

were

digitally

photographed

and

assigned

a

phenotype

ID

Database

All

phenotype

data

were

stored

into

a

relational

database

on

the

PostgreSQL

relational

database

managing

system

-LRB-

-RRB-

with

a

FreeBSD

operating

system

-LRB-

-RRB-

FreeBSD

was

used

for

the

statistical

analysis

Tabular

structure

for

phenotypic

description

consists

of

the

line

name

plant

serial

number

observation

date

observing

person

a

link

to

the

photograph

file

phenotype

ID

and

detailed

descriptions

Additional

data

for

each

line

are

inserted

as

a

new

row

Data

can

be

modified

only

by

the

observing

person

With

this

table

structure

observation

logs

for

each

line

by

many

persons

over

the

course

of

many

years

can

be

stored

without

conflict

For

counting

lines

of

each

mutant

phenotype

Perl

script

which

can

connect

with

the

PostgreSQL

server

with

a

Pg.pm

module

reads

the

phenotype

table

stores

line

names

and

phenotype

IDs

into

an

associative

array

-LRB-

hash

-RRB-

to

convert

from

a

redundant

number

of

phenotype

IDs

to

a

unique

number

for

each

line

The

number

of

lines

showing

each

phenotype

ID

in

the

hash

was

counted

Flanking

sequence

data

of

insertion

sites

are

also

stored

in

the

same

database

Loci

of

insertions

are

determined

by

BLASTN

searches

against

rice

genome

sequences

of

the

International

Rice

Genome

Sequencing

Project

-LRB-

IRGSP

-RRB-

Loci

of

annotated

genes

are

also

stored

in

the

relational

database

SOM

analysis

Software

for

SOM

analysis

was

downloaded

from

the

Neural

Networks

Research

Center

of

Helsinki

University

of

Technology

-LRB-

-RRB-

For

the

initialization

program

randinit

parameters

for

x

dimension

for

y

dimension

hexa

for

topology

bubble

for

neighborhood

function

and

for

seeds

were

used

For

the

map

training

program

vsom

parameters

for

first

learning

for

learning

length

in

training

for

initial

learning

rate

for

initial

radius

of

the

training

area

in

som-algorithm

parameters

for

second

learning

for

learning

length

for

initial

learning

rate

for

initial

radius

The

analyzed

data

were

visualized

using

the

umat

program

Results

Flow

of

phenotypic

analysis

Nipponbare

calli

from

seeds

were

independently

cultured

for

months

and

about

plants

were

regenerated

Seeds

-LRB-

generation

-RRB-

from

the

regenerated

plants

were

independently

harvested

and

labeled

with

a

line

name

Ten

to

twenty-five

plants

of

each

line

were

observed

over

a

full

developmental

cycle

in

the

nursery

and

paddy

Each

line

was

designated

by

N

for

Nipponbare

followed

by

a

letter

A-G

that

indicates

the

yearly

lot

and

four

figures

e.g.

Lines

named

with

and

NB

were

used

in

a

small-scale

pilot

study

and

NC

to

NG

were

used

in

large-scale

studies

To

evaluate

the

effect

of

hormone

-LRB-

auxin

and

cytokinin

-RRB-

concentration

in

the

medium

on

retrotransposition

activity

the

NC

line

was

subcultured

in

media

containing

or

mg/l

of

acid

-LRB-

-RRB-

with

or

without

mg/l

benzyl

adenine

-LRB-

BA

-RRB-

There

was

no

significant

difference

in

observed

transposition

events

among

lines

derived

from

the

subcultures

In

lots

ND

NE

NF

and

NG

calli

were

induced

in

a

medium

containing

mg/l

and

maintained

in

a

medium

supplemented

with

mg/l

for

months

The

cultural

conditions

for

each

line

are

available

with

the

phenotypic

description

list

from

the

mutant

panel

database

Co-segregation

of

mutant

phenotypes

with

insertion

can

be

detected

by

DNA

blot

hybridization

which

will

help

the

further

analysis

of

gene

function

If

co-segregation

is

detected

the

flanking

region

of

co-segregated

can

be

isolated

by

TAIL-PCR

or

suppression

PCR

-LRB-

Miyao

et

al.

-RRB-

Classification

of

phenotypes

To

provide

a

classification

system

and

a

database

useful

for

most

rice

scientists

phenotype

scoring

was

limited

to

phenotype

descriptors

belonging

to

classes

Because

environmental

conditions

such

as

day

length

temperature

and

soil

conditions

differ

significantly

among

the

seven

fields

in

which

this

project

was

conducted

some

variability

in

traits

such

as

heading

date

or

leaf

color

may

have

occurred

between

different

fields

Thus

some

of

the

present

data

may

vary

with

environmental

conditions

although

most

of

mutant

phenotypes

are

stably

expressed

Phenotype

classifications

and

a

summary

of

observations

are

shown

in

Table

Each

subclass

has

a

phenotype

ID

code

to

enable

data

entry

as

a

barcode

with

a

portable

recording

device

and

to

enable

data

compilation

from

all

seven

laboratories.Germination

This

trait

was

evaluated

by

measuring

germination

rate

under

defined

conditions

Since

wild

type

-LRB-

cv

Nipponbare

-RRB-

showed

a

germination

rate

higher

than

lines

that

showed

germination

rates

less

than

were

recorded

in

the

present

project

Since

several

laboratories

measured

germination

rates

of

all

lines

all

primary

data

are

also

stored

in

the

database

In

total

lines

showed

a

low

germination

rate

and

lines

showed

germination

rates

less

than

Some

of

the

lines

with

low

germination

rates

may

be

embryo

mutants.Growth

Growth

was

observed

at

the

seedling

stage

in

the

rice

nursery

or

at

an

early

stage

after

transfer

to

the

field

Weak

refers

to

mutants

that

formed

slim

seedlings

with

retarded

growth

probably

caused

by

a

deficiency

in

some

housekeeping

gene

product

-LRB-

Fig.

-RRB-

Some

of

the

Weak

lines

were

reclassified

eventually

to

Lethal

An

example

of

Abnormal

shoot

is

shown

in

Fig.

-LRB-

-RRB-

Another

example

is

represented

by

This

seedling

is

smaller

than

wild

type

and

its

leaves

are

short

and

wide

resembling

mutants

defective

in

genes

associated

with

gibberellin

biosynthesis

or

signaling

pathways

-LRB-

Sakamoto

et

al.

Uegchi-Tanaka

et

al.

-RRB-

Leaf

color

Frequently

appearing

pigmentation

phenotypes

are

Albino

-LRB-

Fig.

-RRB-

and

Virescent

-LRB-

Fig.

-RRB-

Completely

white

or

yellow

-LRB-

Fig.

-RRB-

seedlings

died

within

weeks

after

seeding

If

green

and

white

segments

coexisted

on

leaves

a

condition

called

virescent

the

seedlings

survived

in

the

field

Zebra

mutants

that

show

repetition

of

a

white

or

a

pale

green

band

and

a

green

band

in

the

longitudinal

direction

were

also

observed

-LRB-

Fig.

-RRB-

However

in

most

lines

the

Zebra

phenotype

was

limited

to

young

stages

The

Stripe

phenotype

-LRB-

Fig.

-RRB-

often

showed

extremely

biased

segregation

e.g.

only

one

plant

in

and

often

was

not

stably

inherited.Leaf

shape

Many

different

kinds

of

abnormally

shaped

leaves

were

observed

Short

Leaf

and

some

Wide

leaf

phenotypes

were

eventually

reclassified

as

Dwarf

or

Severely

dwarf

phenotypes

-LRB-

Fig.

-RRB-

The

Short

leaf

phenotype

is

similar

to

mutants

defective

in

gibberellin

biosynthesis

or

signal

transduction

The

line

could

not

develop

a

normal

flat

leaf

blade

and

eventually

died

-LRB-

Fig.

-RRB-

Several

lines

showed

pleiotropic

phenotypes

of

the

shoot

The

line

did

not

develop

tillers

showed

dwarfism

and

formed

rolled

or

twisted

leaves

-LRB-

Fig.

-RRB-

Since

these

phenotypes

are

difficult

to

characterize

exactly

by

only

phenotype

codes

additional

remarks

were

presented

in

the

comment

column

of

the

database

Frequency

distribution

of

the

leaf

width

mutants

was

biased

toward

the

narrow

type

-LRB-

Fig.

-RRB-

rather

than

the

wide

type.Culm

shape

Dwarfism

is

the

most

abundant

mutant

phenotype

along

with

sterility

The

Semi-dwarf

condition

is

characterized

by

plant

heights

that

are

of

wild

type

values

Dwarf

describes

plants

with

heights

smaller

than

Semi-dwarf

but

larger

than

Severely

dwarf

Plants

are

classified

Severely

dwarf

when

the

plant

height

is

smaller

than

cm

at

maturity

Some

dwarf

phenotypes

often

co-segregated

with

other

abnormal

phenotypes

such

as

Fine

leaf

Wide

leaf

Spiral

leaf

or

Abnormal

panicle

shape

The

dwarf

mutants

accompanying

other

shoot/panicle

abnormalities

are

expected

to

be

involved

in

hormonal

signaling

or

synthesis

pathways

Other

culm

phenotypes

such

as

Thick

culm

-LRB-

Fig.

-RRB-

appeared

infrequently.Spotted

leaf/lesion

mimic

Various

types

of

Spotted

leaf/lesion

mimic

phenotypes

e.g.

small

and

scattered

spots

large

and

dispersed

spots

were

observed

Heavy

lesions

caused

the

early

death

of

leaves

-LRB-

Fig.

-RRB-

Mutants

with

large

white

lesions

were

also

observed

Brown

spots

were

more

frequently

observed

than

white

ones.Tillering

In

the

tillering

class

Low

tillering

mutants

were

abundant

The

High

tillering

phenotype

tends

to

co-segregate

with

the

Fine

leaf

phenotype

The

Lazy

-LRB-

Fig.

-RRB-

mutants

have

open

and

recumbent

tillers

A

quite

unusual

phenotype

among

High

tillering

lines

is

that

produced

tillers

reiteratively

from

upper

internodes

and

failed

to

produce

panicles.Heading

date

In

this

category

lines

whose

heading

date

deviated

more

than

days

from

normal

were

classified

as

heading

date

mutants

This

category

includes

Early

heading

Late

heading

and

Non-heading

mutants

A

small

number

of

lines

segregated

as

Non-heading

mutants

that

did

not

form

panicles

even

at

harvest

-LRB-

five

to

months

after

sowing

-RRB-

The

number

of

Early

heading

lines

-LRB-

-RRB-

is

a

little

larger

than

that

of

Late

heading

lines

-LRB-

-RRB-

Spikelet

Each

spikelet

of

rice

is

composed

of

two

rudimentary

glumes

two

empty

glumes

and

one

floret

comprised

of

one

lemma

one

palea

and

three

kinds

of

floral

organs

-LRB-

two

lodicules

six

stamens

and

one

pistil

-RRB-

Abnormal

hull

refers

to

any

glume

abnormality

and

Abnormal

floral

organ

refers

to

any

abnormality

in

floral

organs

-LRB-

e.g.

an

abnormal

pistil

-RRB-

In

the

Abnormal

hull

class

there

is

a

mutant

that

failed

to

close

hulls

after

flower

opening

On

the

other

hand

mutants

that

could

not

open

flowers

due

to

underdeveloped

stamens

were

often

re-classified

as

Completely

sterile

mutants

Another

mutant

produced

an

extra

glume

Abnormal

flower

phenotypes

showing

shoots

growing

from

floral

organs

as

in

Fig.

-LRB-

-RRB-

were

also

detected

at

relatively

low

frequency.Panicle

The

most

frequently

observed

abnormal

panicle

phenotype

was

the

precocious

germination

of

seeds

while

still

attached

to

the

maturing

panicle

or

Viviparous

As

for

panicle

shape

Dense

panicle

and

Short

panicle

mutants

-LRB-

Fig.

-RRB-

were

also

frequently

observed

and

Long

panicle

and

Lax

panicle

were

somewhat

rare

Incomplete

emergence

of

the

panicle

from

the

flag

leaf

sheath

was

categorized

as

Neck

leaf

Sterility

Sterile

is

the

third

most

frequent

mutant

in

this

insertion

mutant

population

after

low

seed

fertility

and

dwarf

mutants

The

Sterile

and

Low

fertility

conditions

correspond

to

lines

with

less

than

and

ca.

seed

fertility

respectively.Seed

Only

Large

grain

Small

grain

and

Slender

grain

were

distinguished

in

this

category

The

frequency

of

Small

grain

was

two

and

four

times

higher

than

the

frequency

of

Large

grain

and

Slender

grain

respectively

Among

the

Others

white

or

dull

kernel

phenotypes

were

often

observed.Table

of

phenotype

dataClassPhenotypeID

germination

green

green

shapeWide

lamina

joint

leaf/lesion

mimicSpotted

leaf/lesion

dateEarly

floral

panicle

NC

through

NG

were

harvested

in

through

respectively

Numbers

of

mutant

lines

with

the

corresponding

phenotypes

are

listedFig

phenotypes

of

insertional

mutants

Phenotype

and

line

designation

are

indicated

on

each

photograph

Correlation

between

phenotypes

To

understand

how

the

phenotypic

descriptors

are

related

lines

showing

two

or

more

abnormal

phenotypes

were

selected

and

a

matrix

with

rows

and

columns

of

phenotypes

with

a

number

of

lines

showing

each

pair

of

phenotypes

was

devised

Values

for

the

number

of

respective

phenotypes

were

changed

to

x

in

the

matrix

-LRB-

x

is

ignored

by

the

SOM

program

-RRB-

and

data

were

subjected

to

self-organizing

map

-LRB-

SOM

-RRB-

analysis

-LRB-

Kohonen

-RRB-

The

SOM

algorithm

is

used

for

visualization

of

multidimensional

complex

data

using

an

unsupervised

learning

method

based

on

a

grid

of

artificial

neurons

The

correlation

data

of

phenotypes

was

reflected

in

a

two-dimensional

map

-LRB-

Fig.

-RRB-

Fig.

map

for

correlation

of

phenotypes

Lines

that

showed

two

or

more

abnormal

phenotypes

were

analyzed

by

SOM

Distances

between

phenotypes

indicate

the

magnitude

of

correlation

of

phenotypes

Color

Others

Long

leaf

Abnormal

lamina

joint

angle

Long

panicle

Abnormal

floral

organ

Slender

grain

Thick

culm

Grayscale

levels

of

each

node

represents

the

distance

between

references

Topologies

-LRB-

not

distances

-RRB-

of

phenotypes

on

the

SOM

coordinates

coincide

with

the

correlation

between

phenotypes

Distance

between

two

phenotypes

is

indicated

by

grayscale

color

on

the

SOM

For

example

assume

that

phenotype

A

B

and

C

appeared

in

and

lines

respectively

Seven

lines

showed

phenotype

A

and

C.

Three-lines

showed

phenotype

B

and

C

The

color

between

phenotype

A

and

C

should

be

darker

than

the

color

between

phenotype

B

and

C

because

the

line

number

of

phenotype

A

lines

is

much

greater

than

that

of

phenotype

B

lines

although

the

phenotype

correlation

of

C

with

A

is

stronger

than

that

with

B

It

is

difficult

to

explain

multidimensional

data

completely

on

a

two

dimensional

map

but

a

SOM

map

indicates

generally

that

neighboring

phenotypes

have

relatively

tight

correlations

The

SOM

analysis

in

Fig.

shows

that

there

are

many

pairs

of

phenotypes

that

are

apparently

correlated

That

is

among

phenotypes

several

phenotypes

have

a

high

probability

to

emerge

simultaneously

with

other

specific

phenotypes

This

phenotypic

correlation

could

be

caused

by

pleitropic

expression

of

a

single

gene

or

could

reflect

developmental

causality

of

the

two

abnormalities

For

example

Long

leaf

and

Long

panicle

-LRB-

-RRB-

located

in

the

upper

left

corner

on

the

SOM

map

in

Fig.

have

a

very

strong

correlation

This

result

suggests

that

the

gene

involved

in

leaf

length

also

affects

panicle

length

On

the

contrary

long

leaf

and

dwarfism

at

the

opposite

corner

of

the

map

do

not

have

any

correlation

Both

dwarf

and

semi-dwarf

phenotypes

have

a

relatively

strong

correlation

with

sterile

and

low

fertile

phenotypes

Since

this

correlation

is

not

so

strong

a

part

of

Dwarf

and

Semi-dwarf

mutants

do

not

affect

seed

fertility

but

other

Dwarf

and

Semi-dwarf

mutants

may

be

regulated

by

genes

associated

with

some

housekeeping

functions

Correlations

between

mutant

phenotype

and

insertion

If

allelic

insertion

lines

show

the

same

phenotype

it

is

highly

probable

that

the

mutant

phenotype

is

caused

by

disruption

of

the

target

gene

by

a

insertion

We

searched

for

genes

that

were

disrupted

by

in

at

least

two

lines

A

total

of

genes

were

detected

based

on

the

Rice

Annotation

Project

-LRB-

Ohyanagi

et

al.

-RRB-

Table

is

a

partial

list

of

such

loci

and

their

mutant

phenotypes

For

example

five

lines

have

insertions

of

in

the

gene

-LRB-

Fig.

-RRB-

of

which

two

lines

have

the

insertions

in

exons

and

show

an

early

heading

phenotype

On

the

other

hand

the

other

three

lines

have

insertions

in

introns

and

are

not

early

heading

-LRB-

see

-RRB-

The

gene

is

a

key

gene

for

determining

heading

date

-LRB-

Yano

et

al.

-RRB-

Another

example

is

the

magnesium

chelatase

subunit

gene

in

Fig.

-LRB-

Jung

et

al.

-RRB-

insertion

into

this

gene

was

correlated

with

an

albino

phenotype

because

mutants

defective

in

this

gene

lack

active

chlorophyll

and

eventually

become

albino

All

correlations

between

phenotypes

and

disrupted

genes

by

insertion

are

displayed

on

the

mutant

panel

database

website

-LRB-

-RRB-

Table

of

loci

that

have

insertions

in

exons

in

at

least

two

linesLocus

green

leaf

low

kinase

spotted

leaf/lesion

of

unknown

function

domain

containing

proteinPale

green

leaf

semi-dwarf

long

culm

short

panicle

sterile

low

kD

Microtubule

associated

proteinNarrow

leaf

DNA

dehydrogenase

-LRB-

EC

-RRB-

-LRB-

BADH

-RRB-

Low

-LRB-

EC

-RRB-

Dwarf

low

kinase

domain

containing

proteinNarrow

methyltransferaseEarly

RING

domain

containing

proteinEarly

-LRB-

II

-RRB-

oxygenase

domain

containing

proteinDark

green

leaf

dwarf

severely

early

receptor

precursor

-LRB-

Ligand-gated

ion

channel

-RRB-

-LRB-

-RRB-

Low

Zn-finger

domain

containing

proteinLate

IX

Mg-chelatase

subunit

precursorLethal

albino

family

proteinSemi-dwarf

dwarf

low

tillering

sterile

low

b

synthase

-LRB-

Fragment

-RRB-

Dark

green

leaf

pale

green

leaf

withering

semi-dwarf

severely

dwarf

early

heading

late

heading

low

transcription

factor

low

domain

containing

proteinSeverely

of

unknown

function

XH

domain

containing

proteinEarly

headingLocus

name

and

description

are

from

RAP

dataFig

of

genes

that

have

insertions

of

-LRB-

A

-RRB-

gene

-LRB-

B

-RRB-

Magnesium

chelatase

subunit

gene

The

structure

and

insertion

points

of

these

genes

are

drawn

on

demand

from

our

web

site

-LRB-

-RRB-

Coding

regions

are

indicated

with

broad

lines

Positions

of

insertions

are

indicated

by

flags

The

direction

of

the

flag

is

the

direction

of

the

insertion

Detailed

descriptions

of

insertion

points

sequence

names

accession

numbers

and

line

names

will

appear

on

the

web

page

When

a

sequence

name

is

clicked

the

figure

will

be

re-drawn

with

a

flag

labeling

the

sequence

name

to

distinguish

closely

inserted

A

large-scale

phenotypic

characterization

of

an

population

generated

by

retrotransposition

has

revealed

that

this

population

contains

a

large

number

of

mutants

covering

many

easily

scored

phenotypes

In

addition

linkage

between

the

mutant

phenotype

and

a

specific

insertion

facilitates

greatly

the

isolation

of

the

causal

genes

and

the

elucidation

of

the

gene

functions

Discussion

Three-years

of

collaboration

among

seven

laboratories

has

led

to

a

large-scale

phenotypic

characterization

of

about

plants

generated

by

retrotransposition

We

have

examined

kinds

of

abnormal

phenotypes

in

rice

from

the

seedling

to

harvest

stages

that

are

easily

evaluated

in

the

field

This

project

has

revealed

that

this

population

contains

a

large

number

of

mutants

covering

a

wide

range

of

phenotypes

Although

several

mutant

phenotypes

may

be

environment-sensitive

and

their

expression

may

be

unstable

most

of

the

mutant

phenotypes

were

stable

We

also

deposited

additional

information

about

certain

phenotypes

in

a

comment

field

within

the

phenotype

description

table

if

this

information

was

provided

A

text

search

box

on

the

phenotype

list

web

page

is

envisioned

to

facilitate

use

of

the

resource

Because

plants

were

observed

inserted

segregate

among

individuals

An

average

of

new

copies

of

are

inserted

into

each

regenerated

plant

If

the

phenotype

co-segregates

with

an

insertion

of

the

phenotype

is

likely

to

be

caused

by

disruption

of

the

target

gene

Of

course

there

are

lines

whose

mutant

phenotypes

are

not

correlated

with

insertion

These

mutants

might

be

caused

by

insertions

of

other

native

transposons

by

chromosomal

aberrations

or

by

other

mutations

during

tissue

culture

In

this

case

their

causal

genes

could

be

isolated

by

usual

positional

cloning

methods

There

were

some

difficulties

in

clustering

phenotypes

using

standard

relational

algorithms

because

each

of

the

phenotypes

has

as

many

as

kinds

of

frequencies

against

other

phenotypes

respectively

SOM

analysis

is

a

kind

of

clustering

method

suitable

for

such

multi-dimensional

non-linear

data

-LRB-

Kohonen

-RRB-

SOM

analysis

confirmed

some

associations

due

to

hormonal

or

developmental

constraint

but

some

additional

intriguing

correlations

were

also

detected

e.g.

between

leaf

color/shape

and

heading

date

between

dwarfism

and

sterility

In

Fig.

the

Long

leaf

phenotype

is

strongly

correlated

with

the

Long

panicle

phenotype

The

Weak

phenotype

at

the

seedling

stage

has

a

relatively

strong

correlation

with

Lethal

Abnormal

shoot

Pale

green

Leaf

and

Spotted

leaf/lesion

mimic

phenotypes

indicating

that

the

Weak

plants

at

the

seedling

stage

likely

show

Lethal

Abnormal

shoot

Pale

green

and/or

Spotted

leaf/lesion

mimic

phenotypes

at

later

stages

Dwarf

Semi-dwarf

Sterile

Low

fertile

and

Low

tillering

phenotypes

showed

relatively

high

correlations

The

SOM

map

is

quite

useful

for

the

overview

of

phenotype

correlations

This

method

of

analysis

will

be

useful

not

only

for

considering

the

major

and

side-effects

of

gene

disruption

but

also

for

re-categorization

of

phenotypes

The

advantage

of

SOM

analysis

is

the

ability

to

analyze

many

different

kinds

of

data

simultaneously

Currently

metabolome

data

have

been

obtained

from

Arabidopsis

-LRB-

Hirai

et

al.

-RRB-

tomato

-LRB-

Schauer

et

al.

-RRB-

and

other

plants

It

is

reasonable

to

predict

that

the

combinatorial

analysis

of

phenotype

and

metabolome

data

of

rice

will

reveal

correlations

between

metabolic

pathways

and

phenotypes

without

additional

genetic

information

Phenotype

databases

of

rice

have

been

developed

in

many

countries

e.g.

Oryzabase

-LRB-

Kurata

and

Yamazaki

-RRB-

RMD

-LRB-

Zhang

et

al.

-RRB-

and

IRRS

-LRB-

Wu

et

al.

-RRB-

Our

system

includes

both

phenotypic

and

insertion

data

on

the

rice

genome

in

a

relational

database

The

number

of

photographs

on

file

is

more

than

and

the

number

of

phenotype

description

records

is

more

than

Our

database

structure

enables

direct

access

of

Perl

script

to

the

database

and

the

extraction

of

many

kinds

of

correlations

Flanking

sequences

of

insertion

points

and

phenotypes

of

the

insertion

lines

provide

a

provisional

assignment

of

the

function

of

a

disrupted

gene

We

have

already

sequenced

more

than

flanking

sequences

from

of

the

insertion

lines

and

the

number

of

flanking

sequences

is

still

increasing

Of

the

total

annotated

loci

based

on

loci

have

more

than

two

insertions

in

exons

When

two

or

more

lines

have

insertions

in

a

common

gene

they

usually

exhibited

similar

phenotypes

These

data

are

a

strong

indication

of

the

function

of

a

disrupted

gene

However

several

phenotypes

such

as

Dwarf

and

Low

fertile

were

observed

in

many

lines

It

might

be

difficult

to

assign

a

gene

function

based

solely

on

correlation

for

these

frequently

observed

phenotypes

and

a

complementation

test

would

be

required

to

confirm

the

correlation

If

flanking

sequence

data

from

all

insertion

lines

can

be

obtained

this

correlation

will

be

more

useful

for

the

annotation

of

genes

We

are

continuing

to

sequence

the

flanking

regions

of

all

our

mutant

lines

In

this

study

we

collected

a

large

amount

of

phenotypic

data

in

seven

fields

under

natural

conditions

If

phenotypes

are

observed

under

other

conditions

such

as

drought

or

temperature

stress

conditions

or

under

pathogen

pressure

phenotypic

description

of

this

population

would

be

much

more

enriched

In

addition

the

present

study

evaluated

only

a

limited

number

of

traits

easily

scored

on

above-ground

organs

Thus

other

traits

such

as

roots

and

seed

storage

composition

remain

to

be

investigated

in

the

future

Expansion

of

this

study

to

include

new

traits

would

enable

investigators

to

find

new

correlations

with

disrupted

genes

Furthermore

integration

of

phenotype

data

with

those

of

microarray

experiments

metabolic

profiling

and

other

approaches

will

be

a

powerful

tool

for

revealing

new

aspects

of

plant

physiology

All

phenotype

and

flanking

sequence

data

can

be

obtained

via

At

this

site

all

annotated

rice

genes

and

locations

of

insertions

are

shown

on

a

clickable

chromosome

map

Details

containing

illustrated

gene

structures

with

insertion

points

phenotypes

of

corresponding

lines

nucleotide

sequences

flanking

the

disrupted

region

candidate

primer

sequences

for

segregation

analysis

and

annotation

from

RAP

data

corresponding

to

the

selected

position

are

displayed

Mutant

seeds

are

available

for

scientific

use

from

the

Genome

Resource

Center

at

NIAS

-LRB-

-RRB-

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

oryza sativa
phenotyping
retrotransposon
database


Gadolinium

and

nephrogenic

systemic

fibrosis

time

to

tighten

practice

Nephrogenic

systemic

fibrosis

-LRB-

NSF

-RRB-

is

a

relatively

new

entity

first

described

in

Few

cases

have

been

reported

but

the

disease

has

high

morbidity

and

mortality

To

date

it

has

been

seen

exclusively

in

patients

with

renal

dysfunction

There

is

an

emerging

link

with

intravenous

injection

of

gadolinium

contrast

agents

which

has

been

suggested

as

a

main

triggering

factor

with

a

lag

time

of

days

to

weeks

Risk

factors

include

the

severity

of

renal

impairment

major

surgery

vascular

events

and

other

proinflammatory

conditions

There

is

no

reason

to

believe

that

children

have

an

altered

risk

compared

to

the

adult

population

It

is

important

that

the

paediatric

radiologist

acknowledges

emerging

information

on

NSF

but

at

the

same

time

considers

the

risk

benefit

ratio

prior

to

embarking

on

alternative

investigations

as

children

with

chronic

kidney

disease

require

high-quality

diagnostic

imaging

Introduction

Nephrogenic

systemic

fibrosis

-LRB-

NSF

formerly

named

nephrogenic

fibrosing

dermopathy

-RRB-

is

a

systemic

disease

that

was

first

described

in

and

recognized

as

a

separate

clinical

entity

in

-LSB-

-RSB-

Until

now

all

confirmed

cases

of

NSF

have

been

reported

in

patients

with

chronic

kidney

disease

-LRB-

CKD

-RRB-

with

estimated

glomerular

filtration

rates

-LRB-

eGFR

-RRB-

ml/min

per

or

end-stage

renal

failure

requiring

renal

replacement

therapy

NSF

affects

both

sexes

with

a

reported

age

range

of

years

and

has

no

race

predilection

-LSB-

-RSB-

Relatively

few

paediatric

cases

have

been

reported

which

may

reflect

the

lower

incidence

of

CKD

in

children

Patients

present

with

swelling

induration

and

tightening

of

the

skin

often

with

a

peau

d'orange

appearance

or

textured

plaques

papules

or

nodules

usually

limited

to

the

extremities

-LRB-

from

ankles

to

mid-thighs

and

from

wrists

to

mid-upper

arms

-RRB-

The

skin

lesions

sometimes

involve

the

trunk

but

spare

the

neck

and

face

The

condition

develops

over

days

to

weeks

and

may

ultimately

result

in

severe

contractures

and

disability

Patients

may

experience

burning

itching

or

sharp

pains

in

involved

areas

Deep

pain

has

also

been

described

in

the

hips

and

ribs

Radiography

may

reveal

calcifications

of

the

soft

tissue

-LSB-

-RSB-

Systemic

involvement

may

be

fatal

with

fibrosis

of

the

lungs

skeletal

muscles

pleura

pericardium

myocardium

kidneys

muscle

bone

testes

and

dura

-LSB-

-RSB-

Clinical

differential

diagnoses

include

scleroderma

scleromyxoedema

systemic

sclerosis/morphoea

eosinophilic

fasciitis

and

eosinophilia-myalgia

syndrome

and

the

condition

may

mimic

cellulitis

panniculitis

or

drug

reactions

-LSB-

-RSB-

The

severity

of

CKD

appears

to

be

the

main

determinant

of

risk

Also

several

contributing

risk

factors

have

been

suggested

hypercoagulability

and

thrombotic

events

cardiomyopathy

hepatic

disease

idiopathic

pulmonary

fibrosis

systemic

lupus

erythematosus

brain

tumours

recent

surgical

procedures

especially

those

with

a

major

vascular

component

and

other

proinflammatory

conditions

including

liver

transplantation

-LSB-

-RSB-

A

specific

aetiology

is

still

unknown

The

only

common

factor

in

all

patients

is

the

underlying

kidney

disease

with

most

patients

dialysis-dependent

However

NSF

has

also

been

reported

in

patients

who

have

never

been

dialysed

-LSB-

-RSB-

The

physiopathological

mechanism

for

fibrosis

is

not

yet

known

Histopathologically

biopsies

from

the

skin

lesions

show

a

proliferation

of

fibrocytes

-LRB-

which

may

extend

into

subcutaneous

tissue

-RRB-

together

with

thick

collagen

bundles

and

surrounding

clefts

with

a

variable

amount

of

mucin

and

elastic

fibres

Fibrocytes

have

a

specific

immunophenotype

and

express

HLA-DR

which

implies

that

they

are

recruited

from

the

circulation

and

do

not

arise

as

a

result

of

proliferation

in

situ

Inflammatory

cells

are

not

usually

evident

although

small

clusters

of

perivascular

mononuclear

cells

may

be

seen

-LSB-

-RSB-

Therapeutic

options

in

NSF

are

limited

Improvements

have

been

reported

with

extracorporeal

photopheresis

plasmapheresis

ultraviolet

phototherapy

intravenous

immunoglobulin

and

renal

transplantation

-LSB-

-RSB-

Link

with

gadolinium-based

contrast

agents

The

main

concern

for

the

radiologist

is

a

growing

body

of

evidence

that

clinical

use

of

gadolinium-based

contrast

agents

may

trigger

the

disease

A

number

of

patients

with

NSF

seem

not

to

have

had

any

previous

gadolinium

exposure

but

these

are

still

under

investigation

Grobner

-LSB-

-RSB-

was

the

first

to

report

this

association

in

January

Grobner

described

nine

patients

with

end-stage

renal

failure

on

dialysis

who

underwent

MR

angiography

-LRB-

MRA

-RRB-

with

gadodiamide

-LRB-

Omniscan

GE

Healthcare

Chalfont

St

Giles

UK

-RRB-

five

-LRB-

mean

age

years

-RRB-

developed

NSF

after

weeks

All

patients

who

developed

NSF

had

confirmed

metabolic

acidosis

whilst

all

patients

who

did

not

develop

NSF

had

normal

pH

and

bicarbonate

level

at

the

time

of

the

MRA

The

average

contrast

agent

volume

used

was

ml

Marckmann

et

al.

-LSB-

-RSB-

reviewed

case

notes

from

their

own

nephrology

department

from

August

to

May

and

found

patients

with

confirmed

NSF

all

exposed

to

gadodiamide

All

patients

were

adults

-LRB-

years

of

age

mean

years

-RRB-

developed

NSF

days

-LRB-

mean

days

-RRB-

after

gadolinium

exposure

and

had

an

eGFR

ml/min

From

this

cohort

of

renal

patients

the

authors

estimated

an

odds

ratio

between

and

for

developing

NSF

following

gadolinium

exposure

The

link

between

gadolinium

exposure

and

NSF

has

been

further

strengthened

by

two

reports

of

gadolinium

deposits

in

tissue

samples

of

patients

with

NSF

High

et

al.

-LSB-

-RSB-

detected

gadolinium

in

out

of

skin

and

soft-tissue

biopsies

from

seven

patients

with

documented

NSF

All

patients

with

NSF

received

gadolinium-based

contrast

medium

prior

to

disease

and

the

interval

between

dosing

and

biopsy

could

be

determined

with

certainty

only

for

two

of

four

patients

-LRB-

age

range

months

-RRB-

The

same

group

reported

in

a

research

letter

following

quantitative

analysis

of

the

four

tissue

samples

in

which

gadolinium

was

detected

that

the

amount

of

gadolinium

in

the

affected

tissue

of

patients

with

NSF

was

approximately

to

the

level

of

gadolinium

in

the

bone

of

healthy

volunteers

with

normal

renal

function

In

a

case

report

Boyd

et

al.

-LSB-

-RSB-

found

gadolinium

deposits

in

areas

of

calcium

phosphate

deposition

in

blood

vessels

in

a

patient

with

NSF

who

had

been

previously

exposed

to

gadodiamide

Sadowski

et

al.

-LSB-

-RSB-

retrospectively

reviewed

data

from

patients

with

confirmed

NSF

-LRB-

eight

males

five

females

age

years

-RRB-

all

exposed

to

gadodiamide

The

median

time

between

onset

of

NSF

symptoms

and

last

contrast-enhanced

MRI

examination

was

days

The

dose

of

contrast

agent

administered

varied

from

to

mmol/kg

-LRB-

actual

-RRB-

body

weight

At

the

time

of

the

contrast-enhanced

MRI

all

patients

had

CKD

-LRB-

eGFR

ml/min

per

-RRB-

and

were

hospitalized

for

a

proinflammatory

event

-LRB-

major

surgery

infection

or

vascular

event

-RRB-

The

group

with

NSF

had

significantly

lower

eGFR

more

proinflammatory

events

and

more

contrast-enhanced

MR

examinations

per

patient

-LRB-

P

-RRB-

than

did

the

control

group

The

incidence

of

NSF

in

this

study

and

institution

was

per

year

A

review

of

six

further

NSF

cases

that

were

also

related

in

time

to

gadodiamide

administration

has

recently

been

published

-LSB-

-RSB-

Patients

were

aged

years

and

the

onset

of

symptoms

consistent

with

NSF

was

between

days

and

months

after

gadodiamide

exposure

All

patients

had

severe

renal

failure

and

were

exposed

to

a

gadodiamide

dose

ranging

from

to

mmol/kg

body

weight

Despite

having

normal

serum

bicarbonate

five

of

the

six

patients

had

an

elevated

anion

gap

metabolic

acidosis

Broome

et

al.

-LSB-

-RSB-

have

recently

reported

a

retrospective

study

of

patients

with

NSF

all

of

whom

received

a

double

dose

-LRB-

mmol/kg

body

weight

-RRB-

of

gadodiamide

weeks

before

the

development

of

skin

fibrosis

This

group

included

eight

men

and

four

women

-LRB-

age

years

-RRB-

and

all

had

renal

insufficiency

at

the

time

of

gadodiamide

administration

The

odds

ratio

for

development

of

NSF

in

the

gadodiamide-exposed

patients

compared

with

the

gadodiamide-unexposed

patients

was

and

the

prevalence

of

NSF

among

gadodiamide-exposed

dialysis

patients

was

The

odds

ratio

for

development

of

NSF

with

double-dose

compared

to

single-dose

gadodiamide

administration

was

Ten

patients

were

dialysed

within

days

of

gadodiamide

administration

but

this

failed

to

prevent

the

development

of

NSF

Although

the

overwhelming

majority

-LRB-

around

-RRB-

of

patients

with

gadolinium-associated

NSF

had

been

exposed

to

gadodiamide

there

are

now

several

reports

of

exposure

to

other

gadolinium

contrast

agents

In

a

recent

editorial

in

Radiology

Kuo

et

al.

-LSB-

-RSB-

reported

patients

with

NSF

investigated

by

the

United

States

Food

and

Drug

Administration

-LRB-

FDA

-RRB-

In

of

these

the

NSF

was

linked

to

gadodiamide

in

to

gadopentetate

dimeglumine

-LRB-

Magnevist

Berlex

Imaging

Montville

N.J.

-RRB-

in

to

gadoversetamide

-LRB-

OptiMARK

Mallinckrodt

St.

Louis

Mo.

-RRB-

and

in

to

gadodiamide

plus

gadoversetamide

in

other

patients

the

associated

specific

gadolinium-based

MR

contrast

agent

was

not

definitively

identified

Sadowski

et

al.

-LSB-

-RSB-

also

reported

one

patient

with

confirmed

NSF

associated

with

both

gadodiamide

and

gadobenate

dimeglumine

-LRB-

MultiHance

Bracco

Diagnostics

Milan

Italy

-RRB-

NSF

related

to

specific

gadolinium-based

contrast

agents

is

also

monitored

by

the

manufacturers

who

have

recently

published

several

reports

-LSB-

-RSB-

In

a

retrospective

study

in

Scotland

of

haemodialysis

patients

Collidge

et

al.

-LSB-

-RSB-

found

a

positive

association

between

the

cumulative

dose

of

gadodiamide

and

the

development

of

NSF

Patients

who

developed

NSF

had

received

a

higher

median

cumulative

dose

of

gadodiamide

-LRB-

mmol/kg

vs.

mmol/kg

in

patients

without

NSF

-RRB-

A

case-control

study

by

Marckmann

et

al.

-LSB-

-RSB-

conducted

in

patients

with

histologically

verified

NSF

and

matched

controls

found

that

increasing

cumulative

gadodiamide

exposure

high-dose

epoietin-b

treatment

and

higher

serum

concentrations

of

ionized

calcium

and

phosphate

increase

the

risk

of

gadodiamide-related

NSF

in

patients

with

renal

failure

Pathophysiology

Although

not

completely

understood

several

factors

are

known

to

contribute

to

the

physiopathological

mechanism

in

NSF

Gadolinium

chelates

are

mostly

eliminated

through

the

kidneys

Renal

impairment

will

therefore

prolong

the

presence

of

these

agents

in

the

body

facilitating

the

release

of

the

toxic

free

gadolinium

ion

-LRB-

-RRB-

by

dissociation

from

its

chelate

-LRB-

chemical

imbalance

acidosis

anion

gap

inflammation

-RRB-

and

its

subsequent

binding

to

endogenous

ions

This

chemical

process

known

as

transmetallation

may

be

promoted

by

acidosis

and

anion

gap

e.g.

under

inflammatory

conditions

-LSB-

-RSB-

It

has

therefore

been

suggested

that

both

the

chemical

stability

of

a

particular

chelate

-LRB-

cyclic

compounds

are

more

stable

than

linear

ones

in

vitro

-RRB-

and

the

dose

administered

are

risk

factors

for

triggering

NSF

-LSB-

-RSB-

Once

released

forms

hydroxides

and

phosphates

-LRB-

insoluble

at

a

pH

-RRB-

and

these

are

probably

engulfed

by

phagocytic

cells

-LSB-

-RSB-

This

might

affect

the

reticuloendothelial

system

inhibiting

the

activity

of

certain

enzymes

with

consequent

foreign

body

and

fibrous

reactions

-LSB-

-RSB-

Gadodiamide

the

agent

involved

in

NSF

in

most

patients

to

date

is

the

least

stable

in

terms

of

susceptibility

to

transmetallation

in

vitro

being

an

open-chain

compound

-LSB-

-RSB-

Free

gadolinium

ions

may

remain

in

the

body

for

weeks

to

months

allowing

more

time

for

toxic

effects

-LSB-

-RSB-

Gadolinium

is

a

well-known

inorganic

calcium

channel

blocker

and

its

acute

toxicity

may

also

partly

be

explained

via

this

mechanism

-LSB-

-RSB-

A

review

of

the

properties

of

commercial

gadolinium

contrast

agents

and

their

relation

to

NSF

is

presented

in

Table

Table

and

approval

status

of

extracellular

gadolinium

contrast

agents

-LRB-

CNS

central

nervous

system

MRA

magnetic

resonance

angiography

not

available

-RRB-

Chemical

nameTrade

nameManufacturerCharge

and

chemical

structureConditional

stability

at

pH

-LRB-

log

-RRB-

Excess

chelate

-LRB-

mg/ml

-RRB-

Thermodynamic

stability

constant

-LRB-

log

-RRB-

Kinetic

stabilityaBody

region

-LRB-

s

-RRB-

approvedApproval

statusApproved

doses

for

imaging

-LRB-

mmol/kg

-RRB-

Approved

doses

for

children

-LRB-

mmol/kg

-RRB-

NSF

related

to

gadoliniumGadodiamideOmniscanGE

HealthcareNonionic

sCNS

whole

bodyUSA

EU

JapanBody

CNS

MRA

From

months

Yes

-LRB-

cases

worldwide

-RRB-

Gadopentetate

dimeglumineMagnevistBayer

Schering

PharmaIonic

minCNS

whole

bodyUSA

EU

JapanBody

CNS

MRA

-LRB-

doses

of

may

be

used

if

necessary

for

children

older

than

years

-RRB-

Yes

-LRB-

cases

worldwide

-RRB-

Gadobenate

dimeglumineMultiHanceBraccoIonic

N/ACNS

liverUSA

EULiver

CNS

MRA

not

approvedNot

approved

yearsYes

-LRB-

case

in

a

patient

coadministered

Omniscan

-RRB-

GadoversetamideOptiMARKTycoNonionic

N/ACNS

liverUSABody

CNS

MRA

not

approvedNot

approved

yearsYesGadoterate

meglumineDotaremGuerbetIonic

monthCNS

whole

bodyEUBody

CNS

MRA

NoGadoteridolProHanceBraccoNonionic

hCNS

whole

bodyUSA

EU

JapanBody

CNS

MRA

not

from

years

caution

months

to

years

contraindicated

monthsNoGadobutrolGadovistBayer

Schering

PharmaNon-ionic

minCNS

MRAEU

CanadaBody

not

approved

CNS

MRA

-LRB-

imaging

FOV

-RRB-

MRA

-LRB-

imaging

FOV

-RRB-

Not

approved

yearsNoGadoxetic

acidPrimovistBayer

Schering

PharmaIonic

N/ALiverUSA

EUBody

μmol/kg

or

ml/kg

CNS

not

approved

MRA

not

approvedNot

approved

yearsNoGadofosvesetVasovistBayer

Schering

PharmaIonic

linearN/AN/AN

AN/AAbdominal

and

limb

vesselsEUMRA

Not

approved

yearsNoaKinetic

stability

dissociation

half-life

at

pH

Table

modified

from

Bellin

MF

-LRB-

-RRB-

Eur

J

Radiol

NSF

in

children

NSF

in

children

has

also

been

reported

Five

patients

under

years

of

age

have

thus

far

been

reported

-LRB-

discussed

below

-RRB-

out

of

a

total

of

ten

in

the

central

database

of

the

International

Center

for

Nephrogenic

Fibrosing

Dermopathy

Research

-LRB-

Dr.

S.

Cowper

personal

communication

-RRB-

However

possible

patients

have

been

reported

to

an

online

paediatric

nephrology

forum

in

June

-LRB-

-RRB-

and

these

are

still

being

investigated

in

relation

to

previous

gadolinium

exposure

In

Jan

et

al.

-LSB-

-RSB-

reported

two

paediatric

patients

with

nephrogenic

fibrosing

dermopathy

a

female

patient

with

a

life-long

history

of

kidney

failure

and

an

boy

with

a

history

of

membranoproliferative

glomerulonephritis

type

II

Both

patients

had

the

clinical

and

histopathological

features

of

NSF

and

no

contrast-enhanced

MRI

procedures

were

known

to

be

associated

with

these

patients

However

this

was

before

the

publication

of

a

possible

link

with

gadolinium

Jain

et

al.

-LSB-

-RSB-

reported

two

more

patients

with

NSF

in

a

boy

on

peritoneal

dialysis

and

a

male

on

haemodialysis

with

a

history

of

previous

failed

renal

transplants

both

diagnosed

with

NSF

on

skin

biopsies

The

boy

had

a

solitary

MRA

performed

in

June

approximately

months

prior

to

development

of

NFD

The

history

of

the

male

was

more

complicated

as

he

underwent

separate

MRA

procedures

all

with

gadolinium

contrast

agents

between

April

and

August

At

least

nine

of

the

MRAs

were

performed

prior

to

the

NFD

diagnosis

in

January

-LSB-

-RSB-

Also

both

patients

had

persistent

metabolic

acidosis

in

the

months

preceding

the

development

of

NSF

which

may

have

been

a

contributing

factor

-LSB-

-RSB-

DiCarlo

et

al.

-LSB-

-RSB-

described

another

patient

with

histopathologically

proven

NSF

This

patient

was

a

male

renal

transplant

recipient

-LRB-

years

following

transplant

-RRB-

who

had

recently

begun

peritoneal

dialysis

due

to

graft

failure

No

relationship

to

MRI

investigations

was

mentioned

Recently

Auron

et

al.

-LSB-

-RSB-

reported

two

patients

with

NSF

One

of

the

patients

was

a

boy

previously

reported

at

the

age

of

years

by

Jan

et

al.

-LSB-

-RSB-

in

-LRB-

discussed

above

-RRB-

The

second

patient

was

a

male

who

at

the

age

years

was

diagnosed

with

acute

monoblastic

leukaemia

and

years

later

underwent

bone

marrow

transplant

and

later

developed

kidney

failure

No

references

to

contrast-enhanced

MRI

are

available

from

these

reports

Our

own

hospital

has

a

large

paediatric

renal

unit

with

up

to

renal

transplants

per

year

We

have

actively

used

MRI

both

for

concurrent

conditions

and

complications

to

CKD

as

well

as

for

providing

a

presurgical

vascular

road-map

using

contrast-enhanced

MRA

-LRB-

Gd-DTPA

Magnevist

Schering

Germany

mmol/kg

i.v.

-RRB-

A

review

of

all

contrast-enhanced

scans

in

children

referred

via

our

nephrology

service

-LRB-

March

to

March

-RRB-

showed

that

nephrology

patients

-LRB-

neonate

to

years

of

age

median

years

-RRB-

had

had

MRI

scans

with

a

follow-up

of

at

least

months

There

were

no

patients

with

NSF

in

this

high-risk

cohort

Regulatory

advice

Regulatory

advice

can

be

obtained

from

the

websites

listed

in

Table

The

FDA

has

issued

a

general

warning

for

all

gadolinium-based

contrast

agents

and

advise

against

their

use

in

patients

with

eGFR

ml/min

per

and

in

all

patients

with

acute

renal

insufficiency

unless

the

diagnostic

information

is

essential

and

not

available

with

non-contrast

enhanced

magnetic

resonance

imaging

At

the

FDA

's

request

the

manufacturers

of

gadolinium-based

contrast

agents

have

revised

the

product

labels

which

now

include

a

new

boxed

warning

and

a

new

warnings

section

that

describes

the

risk

of

NSF

The

European

Medicines

Agency

has

so

far

not

published

any

update

of

general

contraindications

and

is

not

in

a

position

to

do

so

since

all

but

one

gadolinium-based

agent

are

licensed

by

individual

member

states

regulatory

bodies

However

the

issue

was

discussed

at

a

meeting

of

its

pharmacovigilance

group

in

June

and

May

and

issued

specific

advice

as

follows

-LRB-

-RRB-

The

use

of

Omniscan

and

Magnevist

is

contraindicated

in

patients

with

severe

renal

impairment

-LRB-

i.e.

GFR

or

eGFR

-RRB-

Omniscan

is

also

contraindicated

in

patients

with

renal

dysfunction

who

have

had

or

who

are

awaiting

liver

transplantation

For

patients

with

moderate

renal

impairment

-LRB-

GFR

or

eGFR

-RRB-

or

neonates

and

infants

up

to

year

of

age

Omniscan

and

Magnevist

should

be

used

only

after

careful

consideration

All

patients

should

be

screened

for

renal

dysfunction

by

obtaining

a

history

and/or

laboratory

tests

especially

before

Omniscan

or

Magnevist

are

used

Haemodialysis

shortly

after

administration

of

a

gadolinium-containing

MRI

contrast

agent

in

patients

currently

under

haemodialysis

may

be

useful

for

removal

of

contrast

agent

from

the

body

However

there

is

no

evidence

to

suggest

that

haemodialysis

can

prevent

or

treat

development

of

NSF

Careful

consideration

should

also

be

given

to

the

use

of

the

other

gadolinium-containing

MRI

contrast

agents

in

patients

with

severe

renal

impairment.Table

for

regulatory

updates

and

for

information

and

registration

of

cases

-LRB-

accessed

on

September

-RRB-

OrganizationInformation

providedURLEuropean

Medicines

AgencyRegulatory

informationhttp

Food

and

Drug

AdministrationRegulatory

informationhttp

for

healthcare

professionalshttp

Center

for

Nephrogenic

Fibrosing

Dermopathy

ResearchUpdated

information

and

contact

details

for

registration

of

caseshttp

Consequences

for

paediatric

practice

There

is

an

associated

high

morbidity

and

mortality

with

CKD

and

imaging

aids

clinical

management

such

as

assessment

of

secondary

vasculopathies

infections

etc

and

surgical

planning

prior

to

renal

transplantation

Patients

at

risk

also

include

children

with

known

or

suspected

CKD

as

well

as

newborns

and

infants

with

renal

immaturity

or

congenital

cardiopathy

It

seems

plausible

that

impaired

excretion

dissociation

of

the

gadolinium

chelate

and

prolonged

tissue

retention

of

gadolinium

is

a

trigger

for

NSF

in

patients

with

CKD

This

risk

increases

with

cumulative

dose

of

gadolinium

with

decreasing

GFR

ml/min

per

and

in

the

presence

of

acidosis

and

proinflammatory

conditions

The

scarcity

of

reported

children

with

NSF

may

well

be

due

to

the

lower

prevalence

of

CKD

and

secondary

conditions

in

children

It

is

therefore

imperative

that

paediatric

practitioners

are

prudent

and

immediately

tighten

their

local

policies

for

use

of

intravenous

gadolinium-based

contrast

agents

It

is

difficult

at

present

to

suggest

general

guidelines

because

The

absolute

risk

of

gadolinium

exposure

as

well

as

the

risk

associated

with

the

suspected

contributing

clinical

and

biochemical

factors

is

unknown

Each

case

must

therefore

be

assessed

individually.The

risk

attributable

to

the

kinetic

stability

of

different

gadolinium-based

agents

is

not

clear

The

majority

of

cases

have

been

reported

in

association

with

a

less

stable

agent

-LRB-

gadodiamide

-RRB-

however

we

are

still

in

an

early

stage

of

recording

cases

and

also

the

market

share

and

the

actual

use

of

different

agents

in

this

patient

population

is

not

fully

known

Thus

it

seems

reasonable

to

discontinue

the

use

of

gadodiamide

in

at-risk

patients.The

relationship

to

dose

is

not

completely

known

Since

there

is

no

indication

that

we

are

dealing

with

a

side

effect

not

related

to

dose

it

seems

reasonable

to

reduce

the

dose

of

gadolinium

if

possible

and

also

to

monitor

the

cumulative

dose

in

at-risk

patients.It

is

unclear

whether

acidosis

correction

before

gadolinium

administration

and

haemodialysis

immediately

after

gadolinium

administration

can

prevent

the

development

of

or

treat

NSF

but

such

actions

should

be

considered

in

children

already

on

haemodialysis

This

underlines

the

necessity

for

good

communication

between

radiologists

and

nephrologists

both

for

issuing

local

guidelines

and

for

management

of

the

individual

child

It

is

often

the

case

that

MRI

is

requested

after

an

inconclusive

US

examination

In

our

own

experience

more

meticulous

patient

preparation

and

dedicated

rescanning

of

the

patient

with

US

can

often

give

diagnostically

adequate

results

In

patients

with

CKD

not

yet

on

dialysis

CT

is

not

a

desirable

alternative

due

to

ionizing

radiation

and

the

nephrotoxicity

of

iodinated

contrast

agents

However

CT

may

provide

an

option

in

patients

who

are

already

on

dialysis

MRI

without

intravenous

contrast

agent

administration

has

not

yet

been

validated

for

vascular

anatomy

prior

to

renal

transplantation

but

several

techniques

may

be

valuable

such

as

steady-state

free

precession

imaging

and

time-of-flight

angiography

Patients

with

renal

impairment

may

be

referred

for

MRI

from

non-nephrology

units

and

a

crucial

question

is

how

to

identify

them

Our

approach

would

be

to

include

a

mandatory

tick

box

on

the

referral

form

requiring

the

referring

clinician

's

statement

about

renal

function

and

also

to

include

a

question

about

any

known

renal

problems

on

the

patient/guardian

prescan

checklist

This

is

a

pragmatic

approach

which

will

need

revision

after

an

initial

phase

Conclusion

We

would

advocate

prudence

and

in

practice

suggest

Information

collection

from

clinicians

and

patients/guardians

on

renal

dysfunction

and

active

collaboration

with

the

local

nephrology

unit

A

case-by-case

assessment

in

terms

of

the

necessity

for

gadolinium-enhanced

MRI

and

whether

an

alternative

test

-LRB-

repeat

US

noncontrast-enhanced

MRI

CT

-RRB-

would

be

acceptable

Cumulative

dose

recording

reduction

of

the

dose

of

any

gadolinium-based

contrast

agent

and

acting

accordingly

to

guidance

published

in

the

literature

Longer

follow-up

especially

in

high-risk

children

GFR

measurement

or

estimation

should

be

part

of

a

systematic

prescan

investigation

in

children

with

known

or

suspected

renal

dysfunction

however

a

general

screening

is

probably

not

justified

To

minimize

the

risks

we

should

take

advantage

of

the

current

knowledge

and

would

suggest

using

cyclic

nonionic

compounds

as

they

are

more

stable

in

vitro

and

therefore

theoretically

less

likely

to

undergo

transmetallation

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

nephrogenic systemic fibrosis
gadolinium
chronic kidney disease
children