mutations in the adrenoleukodystrophy gene

500 DODD ET AL. HUMAN MUTATION 9:500�511 (1997)

© 1997 WILEY-LISS, INC.

HUMU 765

MUTATION UPDATE

Mutations in the Adrenoleukodystrophy Gene

Andrew Dodd,1 Shelley A. Rowland,1 Sheryl L.J. Hawkes,1 Martin A. Kennedy,2 and Donald R. Love1*1School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand; Fax: 61-93737-4142Cytogenetic and Molecular Oncology Unit, Department of Pathology, University of Otago School of Medicine, Christchurch,New Zealand

Communicated by R.G.H. Cotton

Adrenoleukodystrophy (ALD) is a peroxisomal disorder that commonly manifests as demyelination ofthe central nervous system (CNS). The isolation of the ALD gene by positional cloning has led to theidentification of a variety of mutations in the ALD gene. One hundred and ten mutations have beenidentified to date, of which approximately 50% are missense mutations. While rapid DNA-based diag-noses of ALD is now possible, there appears to be no simple correlation between genotype and pheno-type. Hum. Mutat. 9:500–511, 1997. © 1997 Wiley-Liss, Inc.

KEY WORDS: adrenoleukodystrophy; missense mutations; DNA-based diagnosis

INTRODUCTION

Adrenoleukodystrophy (ALD) is an X chromo-some-linked disorder that is characterised in mostaffected males (hemizygotes) by progressive multifo-cal demyelination of the central nervous system(CNS) and by adrenocortical insufficiency (Moseret al., 1995a). X-Linked ALD is manifested as a rangeof clinical phenotypes, often found within the samekindred. This paper and a recent review use a classi-fication scheme to clarify some of the confusion con-cerning nomenclature with respect to the range ofX-linked ALD phenotypes (Moser et al., 1995a).

At the biochemical level, all ALD phenotypes areassociated with the accumulation of very-long-chainfatty acids (VLCFAs) that occurs mainly in neuralwhite matter, adrenal glands, cultured fibroblasts, andplasma. This accumulation is due to an impairmentof the Ä-oxidation of fatty acids, which comprises partof the degradation pathway of VLCFAs in peroxi-somes (Menkes and Corbo, 1977). Peroxisomes ofALD patients lack the ability to activate VLCFAs totheir coenzyme A (CoA) derivatives for subsequentÄ-oxidation (Singh et al., 1981). This activation stepis thought to be carried out by very-long-chain acyl-CoA synthetase (VLCFA-CoA synthetase).

The gene that encodes the primary biochemicaldefect in ALD maps to Xq28 (Migeon et al., 1981).The ALD gene comprises 10 exons that encode for a3.7-kb transcript and a predicted protein of 745 aminoacids (ALDP). This protein shows no homology toVLCFA-CoA synthetase but exhibits significant ho-mology to a peroxisomal membrane protein, PMP70,

a member of the ATP-binding cassette (ABC) mem-brane transporter superfamily of proteins (Mosser etal., 1993). In humans, the ABC transporter familyalso includes the cystic fibrosis transmembrane con-ductance regulator (CFTR), the multidrug resistance(MDR) gene product, and the TAP1 and TAP2 pep-tide transporters encoded in the MHC cluster (Fanenet al., 1994). Immunohistochemical studies haveshown that ALDP is a peroxisomal membrane pro-tein orientated toward the cytosol (Mosser et al.,1994; Watkins et al., 1995). ALDP is predicted tocontain six membrane-spanning segments (compris-ing a transmembrane domain) and a putative hydro-philic ATP-binding domain, designated the nucleotidebinding fold (NBF) (Mosser et al., 1993). The func-tion of ALDP is unknown, but it has been proposedto act in the importation of VLCFA-CoA synthetaseinto peroxisomes (Moser et al., 1995a; Contreras etal., 1994).

This report documents all the mutations that havebeen found in the ALD gene to date, together with adiscussion of their proposed effect on ALDP function.

ALD GENE MUTATIONS

The first mutations documented in the ALD genewere large intragenic deletions (Mosser et al., 1993).Subsequent studies have also documented large de-letions ranging in size from 0.5 to 19.2 kb, as deter-mined by Southern blot analysis of genomic DNA

Received 2 February 1996; accepted 21 October 1996.

*Correspondence to: Donald R. Love.

MUTATIONS IN THE ALD GENE 501

(Mosser et al., 1994; Cartier et al., 1993; Kok et al.,1995; Koike et al., 1994; Ligtenberg et al., 1995;Watkins et al., 1995). Together, these data indicatethe frequency of ALD patients with genomic dele-tions to be in the range of 3–7%. Cartier et al. (1993)identified the first missense mutation, leading to manyreports identifying a wide range of mutations in theALD gene, of which 20 mutations have been reviewedpreviously by Ligtenberg et al. (1995). Thus far nowhole gene deletions or promoter mutations havebeen identified (Ligtenberg et al., 1995; van Oost etal., 1994). Point mutations, microdeletions and in-sertion events are thought to account for the mani-festation of the disorder in most patients. Table 1summarises the current literature of ALD gene mu-tations and indicates molecular and clinical informa-tion about each mutation.

Approximately 50% of mutations described to dateare missense mutations, with the remainder nonsensemutations leading to premature termination of trans-lation (12/110) (Ligtenberg et al., 1995; Uchiyamaet al., 1994; Fanen et al., 1994; Fuchs et al., 1994;Braun et al., 1995; Kok et al., 1995; Watkins et al.,1995; Rowland et al., 1996; Krasemann et al., 1996;Feigenbaum et al., 1996), microdeletions (18/110)(Ligtenberg et al., 1995; Kemp et al., 1994; Barcelóet al., 1994; Fuchs et al., 1994; Kok et al., 1994; Fanenet al., 1994; Song et al., 1995; Braun et al., 1995;Krasemann et al., 1996; Feigenbaum et al., 1996),microinsertions (3/110) (Krasemann et al., 1996;Feigenbaum et al., 1996), amino acid deletions (4/110) (Koike et al., 1994; Ligtenberg et al., 1995; Braunet al., 1995; Watkins et al., 1995; Krasemann et al.,1996), amino acid insertions (2/110) (Krasemann etal., 1996; Feigenbaum et al., 1996), and missense mu-tations leading to RNA splice site defects (5/110)(Ligtenberg et al., 1995; Fanen et al., 1994; Kemp etal., 1994, 1995; Feigenbaum et al., 1996). One “same-sense” mutation has been documented (Fanen et al.,1994; Fuchs et al., 1994), which has been suggestedas an alternate sequence or silent polymorphism.

The mutations documented here are distributedthroughout the ALD gene from exon 1 to exon 9(Fig. 1). A large number of mutations lie within ex-ons 1, 5, 6, and 8 (Fanen et al., 1994; Fuchs et al.,1994; Koike et al., 1994; Krasemann et al., 1996). Ex-ons 6 and 8 encode for highly conserved regions withinthe NBF, in particular, the conserved Walker motifs anda 12 amino acid segment (Mosser et al., 1993).

Mutations in exon 1 appear to cluster mainly be-tween the putative third and fourth transmembranesegments. The majority of mutations in the ALD geneappear to be unique to each of the kindreds that havebeen investigated. Two exceptions to this rule have

been found. First, the E609K mutation has been de-tected in two kindreds (Ligtenberg et al., 1995). Sec-ond, a dinucleotide deletion at nucleotide position1801–1802 in exon 5 has been observed in sevenseparate studies and in a total of 14 different kindreds(Table 1). This microdeletion is the most frequent ofall mutations found in the ALD gene and appears torepresent a mutation hotspot (Kok et al., 1995;Barceló et al., 1994; Kemp et al., 1994; Fuchs et al.,1994; Ligtenberg et al., 1995; Krasemann et al., 1996;Feigenbaum et al., 1996). Analysis of these kindredshas revealed differing haplotypes, which suggests theabsence of a founder effect (Kok et al., 1995). Atpresent, all but one study of ALD patients have iden-tified mutations in the ALD gene, confirming thatthis gene encodes for the primary biochemical defectresponsible for ALD. Feigenbaum et al. (1996) foundno mutation in 7 of 44 patients but attributed this toexperimental strategy or promoter mutations. Re-cently an ALD pseudogene has been identified thatcontains sequences homologous to exons 7–10(Braun et al., 1996). The presence of this pseudo-gene, and possibly other ALD-related sequences,could interfere with some strategies for identifyingmutations based on the amplification of genomic se-quences (Cartier et al., 1993; Sarde et al., 1994; Braunet al., 1996). This difficulty can be circumvented bythe analysis of cDNA or using amplification primersdesigned to regions of maximum mismatch betweenthe pseudogene and the functional ALD gene, as sug-gested by Braun et al. (1996).

MUTATIONAL MECHANISMS

Approximately 64% of the missense mutationsfound in the ALD gene can be explained by deami-nation of methylcytosine in CpG dinucleotides(Barker et al., 1984), even though CpG dinucleotidesrepresent less than 6% of the nucleotides in the trans-lated portion of the gene. This class of mutation ac-counts for approximately 35% of all single base pairsubstitutions that cause genetic disease (Cooper andYoussoufian, 1988). The most frequent amino acidsubstitution found in the ALD gene involves argin-ine. This amino acid is charged, hence mutationsresulting in the replacement of arginine may resultin a charge change, which may affect protein func-tion and/or conformation. All but one of the substi-tutions at arginine residues can be explained byC-to-T or G-to-A transitions.

Several groups have identified a small number ofnucleotides within exon 5 that are particularly sus-ceptible to small deletions and insertions, in particu-lar the 1801–1802delAG microdeletion mutation(Kemp et al., 1994; Fuchs et al., 1994; Barceló et al.,

TAB

LE 1

. M

utat

ions

in t

he A

dren

oleu

kody

stro

phy

Gen

e

Type

of

mut

atio

nN

ucle

otid

eE

ffect

on

codi

ngR

estr

ictio

nan

d lo

cati

onch

ange

ase

quen

cesi

teb

Exo

nP

rote

inc

Phe

noty

ped

Mis

sens

eS

98L

CÃ

T a

t 67

9S

erÃ

Leu

at 9

8N

/A121

Pre

sent

ChA

LD,

Asy

R10

4CC

ÃT

at

696

Arg

ÃC

ys a

t 10

4�T

aul

18,17

�A

MN

�Aci

lR

104H

GÃ

A a

t 69

7A

rgÃ

His

at

104

+C

viR

I114

�A

DO

�Tau

l�A

scil

+A

lul

T10

5IC

ÃT

at

700

TryÃ

Ile a

t 10

5N

/A121

Abs

ent

AD

OL1

07P

TÃ

C a

t 70

6Le

uÃP

ro a

t 10

7N

/A120

�C

hALD

, A

MN

. A

DO

, A

syS

108W

CÃ

G a

t 70

9S

erÃ

Trp

at 1

08N

/A121

Dec

reas

edC

hALD

, A

MN

G11

6RG

ÃA

at

732

Gly

ÃA

rg a

t 11

6N

/A121

Abs

ent

ChA

LDA

123V

eC

ÃT

at

754

Ala

ÃV

al a

t 12

3+

Hae

III

19�

�A

141T

GÃ

A a

t 80

7A

laÃ

Thr

e at

141

�117

�C

hALD

N14

8SA

ÃG

at

829

Asn

ÃS

er a

t 14

8�H

incI

I14,

21C

onse

rved

res

idue

, pr

esen

tA

DO

S14

9NG

ÃA

at

832

Ser

ÃA

sn a

t 14

9�

18�

AM

NR

152C

CÃ

T a

t 84

0A

rgÃ

Cys

at

152

�1

17,2

1P

rese

ntC

hALD

, A

DO

R15

2PG

ÃC

at

841

Arg

ÃP

ro a

t 15

2�M

aeII

I18

�C

hALD

R16

3HG

ÃA

at

874

Arg

ÃH

is a

t 16

3�A

ciI

18�

Sym

pCar

�Tse

IY

174S

fA

ÃC

at

907

TyrÃ

Ser

at

174

+B

saX

I119

Con

serv

ed r

esid

ueC

hALD

Y17

4DT

ÃG

at

906

TyrÃ

Asp

at

174

+Ta

qI14,

20C

onse

rved

res

idue

ALD

Q17

8EC

ÃG

at

918

Gln

ÃG

lu a

t 17

8+

Bsa

I114

Non

cons

erve

dA

MN

+B

saJI

+E

coR

II+

Scr

FI

Y18

1CA

ÃG

at

928

TyrÃ

Cys

at

181

N/A

121�

AM

NC

R18

2PG

ÃC

at

931

Arg

ÃP

ro a

t 18

2�B

sLI

117�

AD

OD

194H

GÃ

C a

t 96

6A

spÃ

His

at

194

+H

phI

18�

ChA

LD+

MnI

ID

200V

AÃ

T a

t 98

5A

spÃ

Val a

t 20

0N

/A121

�C

hALD

L211

PT

ÃC

at

1018

LeuÃ

Pro

at

211

N/A

120C

hALD

L220

PT

ÃC

at

1045

LeuÃ

Pro

at

220

�Xcm

I18

�A

MN

�Eco

RII

+M

spI

+N

ciI

D21

1GA

ÃG

at

1048

Asp

ÃG

ly a

t 22

1N

/A121

Abs

ent

ChA

LD,

AM

NT

254P

CÃ

T a

t 11

47Ty

rÃP

ro a

t 25

4N

/A120

AM

NP

263L

CÃ

T a

t 11

74P

roÃ

Leu

at 2

63N

/A121

Dec

reas

edC

hALD

, A

MN

, A

DO

G26

6RG

ÃA

at

1182

Gly

ÃA

rg a

t 26

6�B

sII

14,

6,8

Con

serv

ed r

esid

ueA

MN

K27

6EA

ÃG

at

1212

LysÃ

Glu

at

276

+M

nII

16P

rese

ntC

hALD

G27

7RG

ÃA

at

1215

Gly

ÃA

rg a

t 27

7N

/A120

�A

MN

G27

7WG

ÃT

at

1215

Gly

ÃTr

p at

277

�117

�C

hALD

E29

1KG

ÃA

at

1257

Glu

ÃLy

s at

291

+Ta

qI12

��

E29

1DG

ÃC

at

1259

Glu

ÃA

sp a

t 29

1�

116A

bsen

tC

hALD

A29

4TG

ÃA

at

1266

Ala

ÃT

hr a

t 29

4N

/A121

�A

MN

(con

tinu

ed)

TAB

LE 1

. M

utat

ions

in t

he A

dren

oleu

kody

stro

phy

Gen

e (c

onti

nued

)

Type

of

mut

atio

nN

ucle

otid

eE

ffect

on

codi

ngR

estr

ictio

nan

d lo

cati

onch

ange

ase

quen

cesi

teb

Exo

nP

rote

inc

Phe

noty

ped

S34

2PT

ÃC

at

1410

Ser

ÃP

ro a

t 34

2+

Bsa

JI216

Pre

sent

AM

N+

Msp

I�S

au96

I+

Sm

aI

�MnI

IR

389G

CÃ

G a

t 15

51A

rg-G

ly a

t 38

9N

/A320

AM

NR

389H

GÃ

A a

t 15

52A

rgÃ

His

at

389

�Aci

I38,

16,1

7P

rese

ntA

MN

�Cac

8I

�Fau

IR

401Q

GÃ

A a

t 15

88A

rgÃ

Gln

at

401

�Aci

I34,

16,2

0C

onse

rved

res

idue

, pr

esen

tC

hALD

, A

MN

�BsI

IR

418W

CÃ

T a

t 16

38A

rgÃ

Trp

at 4

18�S

maI

44,20

Con

serv

ed r

esid

ueA

MN

+E

coR

II�A

vaI

�Nci

l ×2

�Msp

I+

Scr

FI

P48

4RC

ÃG

at

1837

Pro

ÃA

rg a

t 48

4+

Tha

I511

�C

hALD

, A

MN

, A

DO

,+

Hha

IS

ympt

Car

�Bsu

36I

G50

7VG

ÃT

at

1906

Gly

ÃVa

l at

507

�Eco

0109

I66

Con

serv

ed r

esid

ueC

hALD

�Hae

III

�Nla

IV�S

au96

I�B

qII

�Mw

oIG

512S

GÃ

A a

t 19

20G

lyÃ

Ser

at

512

+P

stI

615

,17

Con

serv

ed,

abse

ntA

dALD

, C

hALD

S51

5FC

ÃT

at

1930

Ser

ÃP

he a

t 51

5�S

acI

64,12

Con

serv

ed r

esid

ueA

MN

�Bsp

128

6I�B

anII

R51

8WC

ÃT

at

1938

Arg

ÃTr

p at

518

�Bsp

EI

61C

onse

rved

res

idue

AM

NR

518Q

GÃ

A a

t 19

39A

rgÃ

Gln

at

518

+S

au3A

I66

Con

serv

ed r

esid

ueC

hALD

�Bsp

EI

�BsI

I�B

amH

IP

534L

CÃ

T a

t 19

87P

roÃ

Leu

at 5

34+

Alu

I615

Con

serv

ed,

abse

ntC

hALD

P56

0LC

ÃT

at

2065

Pro

ÃLe

u at

560

�Msp

I714

Con

serv

ed r

esid

ueC

hALD

�N

ciI

+E

coR

IIM

566K

TÃ

A a

t 20

83M

etÃ

Lys

at 5

66�C

viR

I717

�A

MN

�NIa

III

�Nsp

IR

591Q

GÃ

A a

t 21

58A

rgÃ

Glu

at

591

�AcI

I7

16,1

7P

rese

ntA

MN

+E

coN

I

(con

tinu

ed)

TAB

LE 1

. M

utat

ions

in t

he A

dren

oleu

kody

stro

phy

Gen

e (c

onti

nued

)

Type

of

mut

atio

nN

ucle

otid

eE

ffect

on

codi

ngR

estr

ictio

nan

d lo

cati

onch

ange

ase

quen

cesi

teb

Exo

nP

rote

inc

Phe

noty

ped

+C

viR

I+

Hae

IS

606L

CÃ

T a

t 22

03S

erÃ

Leu

at 6

06+

TaqI

I81,

16,1

7C

onse

rved

, pr

esen

tA

DO

E60

9KG

ÃA

at

2211

Glu

ÃLy

s at

609

�88,

20�

AM

NE

609G

AÃ

G a

t 22

12G

luÃ

Gly

at

609

+A

ciI

88�

ChA

LDR

617G

CÃ

G a

t 22

35A

rgÃ

Gly

at

617

N/A

820�

AD

O,

AM

NC

R61

7CC

ÃT

at

2235

Arg

ÃC

ys a

t 61

7�A

ciI

81,8,

20C

onse

rved

ChA

LD,

Asy

�Sau

96I

R61

7HG

ÃA

at

2236

Arg

ÃH

is a

t 61

7+

Bsp

LU11

I8

1,8,

10,1

6,17

Non

cons

erve

d, a

bsen

tA

MN

C,

ChA

LD,

AM

N+

AfII

II�M

woI

�Aci

IA

626T

GÃ

A a

t 22

62A

laÃ

Thr

e at

626

�9

16,1

7A

bsen

tC

hALD

, A

MN

D62

9HG

ÃC

at

2271

Asp

ÃH

is a

t 62

9�F

okI

916P

rese

nt�

+M

wo

I�E

coR

II�S

crF

IR

660W

CÃ

T a

t 23

64A

rgÃ

Trp

at 6

60�M

spI

98,

15,1

6,17

Con

serv

ed,

abse

ntA

MN

, A

dALD

�Bsr

FI

Non

sens

eW

10X

GÃ

A a

t 41

6Tr

pÃS

top

at 1

0`N

/A121

Abs

ent

ChA

LD,

AM

NQ

133X

CÃ

q ~í

TUP

Gln

ÃS

top

at 1

33�A

lwN

I18

Trun

cate

dC

hALD

�Bbv

I�M

woI

�Pvu

IIW

137X

GÃ

A a

t 79

7Tr

pÃS

top

at 1

37�N

laIV

18Tr

unca

ted

ChA

LD�M

woI

+H

infI

\+

Ple

IQ

157X

CÃ

T a

t 85

5G

lnÃ

Sto

p at

157

�Sau

96I

18Tr

unca

ted

AM

N�H

aeII

IY

181X

CÃ

A a

t 92

9Ty

rÃst

op a

t 18

1�M

spI

18Tr

unca

ted

ChA

LD�N

ciI

�Scr

FI

Y21

2XC

ÃG

at

1022

TyrÃ

stop

at

212

+B

faI

114Tr

unca

ted

AM

NW

242X

GÃ

A a

t 11

12Tr

pÃst

op a

t 24

2N

/A120

ChA

LDR

464X

CÃ

T a

t 17

76A

rgÃ

stop

at

464

+B

gIII

41,13

Trun

cate

dA

MN

, A

DO

�Bsa

jIQ

466X

CÃ

T a

t 17

82G

lnÃ

stop

at

466

�Hae

III

417Tr

unca

ted

AM

N�E

coR

II�H

aeI

E47

7XG

ÃT

at

1815

Glu

Ãst

op a

t 47

7�

54,20

Trun

cate

dA

MN

Q59

0XC

ÃT

at

2154

Gln

Ãst

op a

t 59

0�P

stI

77Tr

unca

ted

AM

N,

ChA

LDQ

645X

CÃ

T a

t 23

19G

lnÃ

stop

at

645

�Eco

RII

916

Abs

ent

ChA

LD�S

crF

I+

Bfa

I(c

onti

nued

)

TAB

LE 1

. M

utat

ions

in t

he A

dren

oleu

kody

stro

phy

Gen

e (c

onti

nued

)

Type

of

mut

atio

nN

ucle

otid

eE

ffect

on

codi

ngR

estr

ictio

nan

d lo

cati

onch

ange

ase

quen

cesi

teb

Exo

nP

rote

inc

Phe

noty

ped

134r

epR

epla

cem

ent

at 7

87 T

GC

TG

Fram

eshi

ft�P

stI

19Tr

unca

ted

ChA

LDw

ith A

GC

ATT

442d

elC

Del

etio

n of

C a

t 44

2Fr

ames

hift

�Hae

III

18�

ChA

LD+

Dra

III

+M

wo

I52

4ins

TIn

sert

ion

of T

at

524

Fram

eshi

ftN

/A121

�C

hALD

660�

97de

lD

elet

ion

of 6

60�6

97Fr

ames

hift

�114

Trun

cate

dC

hALD

663d

elC

Del

etio

n of

C a

t 66

3Fr

ames

hift

�18

�C

hALD

927�

8del

Del

ecti

on o

f TA

at

927

Fram

eshi

ftN

/A121

Abs

ent

AD

O10

04�1

6del

Del

etio

n of

13b

pFr

ames

hift

N/A

120Tr

unca

ted

ALD

1077

�8de

lD

elet

ion

of G

G a

t 10

77�8

Fram

eshi

ft�B

slI

×2

117�

�10

80-1

del

Del

etio

n of

GC

at

1080

Fram

eshi

ftN

/A121

Abs

ent

ChA

LD11

71-8

del

Del

etio

n of

CG

CC

CA

AG

Fram

eshi

ft�

18�

ChA

LD11

82de

lGD

elet

ion

of G

at

1182

Fram

eshi

ftN

/A1

Trun

cate

dC

hALD

1521

insC

Inse

rtio

n of

C a

t 15

21Fr

ames

hift

N/A

321A

bsen

tA

MN

C16

36de

lCD

elet

ion

of C

at

1636

Fram

eshi

ftN

/A421

Abs

ent

ChA

LD17

97in

sAIn

sert

ion

of A

at

1797

Fram

eshi

ft�

517�

AM

N17

98-9

del A

AD

elet

ion

of A

A a

t 17

98�9

Fram

eshi

ft�

�C

hALD

1801

-2de

lAG

Del

etio

n of

AG

at

1801

�2Fr

ames

hift

�53,

4,5,

8,17

,20

Trun

cate

dC

hALD

, A

MN

, A

DO

, A

sy19

37de

lCD

elet

ion

of C

at

1937

Fram

eshi

ft�B

sII

61Tr

unca

ted

ChA

LD�B

spE

Ial

t198

9�23

7719

88�2

368

dele

ted

574

bp o

fFr

ames

hift

�6�

98

�A

MN

intr

on 7

inse

rted

giv

ing

acr

yptic

spl

ice

dono

r si

te22

04de

lGD

elet

ion

of G

at

2204

Fram

eshi

ft+

BsI

I81

Trun

cate

dA

DO

2177

-8de

lTA

Del

etio

n of

TA

at

2177

�8Fr

ames

hift

�81

Trun

cate

dC

hALD

Am

ino

acid

del

etio

ns a

nd in

sert

ions

(no

fra

mes

hift

)79

8-80

9 de

lD

elet

ion

of 1

2 bp

Del

L13

8ÃA

141

120A

LD12

58de

lGA

GD

elet

ion

of G

AG

at

1258

Loss

of

Glu

291

�MnI

I16,

16,2

0C

onse

rved

, ab

sent

AC

ALD

1968

�197

3del

GG

TD

elet

ion

of G

GT

with

inD

el G

528

or

529

�6

14,2

0C

onse

rved

res

idue

ALD

1968

�197

312

15A

ATin

sIn

sert

ion

of A

AT a

t 12

15In

s B

at

277

N/A

120

�A

dd23

55�2

357d

elA

TCD

elet

ion

of A

TCLo

ss o

f Ile

at

657

+B

sII

98�

ChA

LD�H

phI

Larg

e de

letio

nsde

l ex3

�10

Del

etio

n of

exo

ns 3

�10

��

3�10

17�

ChA

LDde

l ex7

�10

Del

etio

n of

exo

ns 7

�10

��

7�10

16A

bsen

tA

MN

del e

x7�1

0D

elet

ion

of e

xons

7�1

0�

�7�

1017

�A

MN

del e

x8�1

0D

elet

ion

of e

xons

8�1

0�

�8�

1017

�A

MN

del e

x7�1

0D

elet

ion

of e

xons

7�1

0�

�7�

1017

�A

MN

(con

tinu

ed)

506 DODD ET AL.

TAB

LE 1

. M

utat

ions

in t

he A

dren

oleu

kody

stro

phy

Gen

e (c

oncl

uded

)

Type

of

mut

atio

nN

ucle

otid

eE

ffect

on

codi

ngR

estr

ictio

nan

d lo

cati

onch

ange

ase

quen

cesi

teb

Exo

nP

rote

inc

Phe

noty

ped

Spl

ice

defe

ct16

09 G

ÃA

GÃ

A a

t 16

09S

plic

e m

utat

ion

at 4

08N

/A317

�A

MN

2020

+1G

ÃA

GÃ

A a

t 20

20+

15´

spl

ice

sign

alN

/AIn

tron

61

�A

MN

C,

ChA

LD,

AM

N,

AD

Ode

l202

1�20

54D

elet

ion

of 2

021�

2054

Alte

ratio

n of

spl

ice

�78,

18�

ChA

LDac

cept

or s

itein

s225

1In

sert

ion

of 8

bp

at 2

251

Alte

ratio

n of

spl

ice

�98,

18�

AM

Nac

cept

or s

iteS

ame

sens

eG

1934

AG

ÃA

at

1934

No

effe

ct (

leuc

ine)

�61

,4�

�

a Posi

tion

of n

ucel

otid

e on

ALD

tra

nscr

ipt

(Mos

ser

et a

l., 1

993)

.b T

he a

ltera

tion

of r

estr

ictio

n en

zym

e si

tes

is li

mite

d to

tho

se w

idel

y av

aila

ble

and

thus

is n

ot a

com

plet

e co

nsid

erat

ion

of s

ites

tha

t ar

e af

fect

ed.

c The

mut

atio

nal e

ffect

s on

the

pro

tein

pro

duct

are

des

crib

ed a

s fo

llow

s: t

runc

ated

(tr

unca

ted

prot

ein

prod

uct)

; ab

sent

(no

pro

tein

iden

tifie

d);

pres

ent

(pro

tein

iden

tifie

d);

cons

erve

d/no

ncon

serv

ed(m

utat

ion

caus

ed a

con

serv

ed/n

onco

nser

ved

amin

o ac

id a

chan

ge);

con

serv

ed r

esid

ue (

mut

atio

n af

fect

ed a

res

idue

in a

con

serv

ed r

egio

n).

d Phe

noty

pe n

omen

clat

ure

mod

ified

from

Mos

er e

t al

. (19

95a)

: chi

ldho

od c

ereb

ral A

LD (

ChA

LD);

ado

lesc

ent

cere

bral

ALD

(A

dALD

); a

dult

cere

bral

ALD

(A

CA

LD);

adr

enom

yelo

neur

opat

hy (

AM

N);

adre

nom

yelo

neur

opat

hy c

ereb

ral (

AM

NC

) ad

rena

l ins

uffic

ienc

y�on

ly (

AD

O);

asy

mpt

omat

ic (

Asy

); s

ympt

omat

ic c

arri

er (

Sym

ptC

ar);

Add

ison

�s d

isea

se (

Add

); s

peci

fic p

heno

type

not

sta

ted

(ALD

).f Fi

rst

reor

ted

de n

ovo

mut

atio

n in

the

ALD

gen

e.N

/A, n

ot a

naly

sed;

�, N

o da

ta. 1

, Fan

en e

t al

., 19

94; 2

, Car

tier

et a

l., 1

993;

3, K

emp

et a

l., 1

994;

4, F

uchs

et

al.,

1994

; 5,

Bar

celó

et

al.,

1994

; 6,

Koi

ke e

t al

., 19

94;

7, U

chiy

ama

et a

l., 1

994;

8,

Ligt

enbe

rg e

t al.,

199

5; 9

, Son

g et

al.,

199

5; 1

0, M

atsu

mot

o et

al.,

199

4; 1

1, B

erge

r et

al.,

199

4; 1

2, V

orge

rd e

t al.,

199

5; 1

3, R

owla

nd e

t al.,

199

6; 1

4, B

raun

et a

l., 1

995;

15,

Yas

utak

e et

al.,

199

5;16

, Wat

kins

et

al.,

1995

; 17,

Kok

et

al.,

1995

; 18,

Kem

p et

al.,

199

5; 1

9, B

arce

ló e

t al

., 19

95; 2

0, K

rase

man

n et

al.,

199

6; 2

1, F

eige

nbau

m e

t al

., 19

96.


1994; Kok et al., 1994; Krasemann et al., 1996;Feigenbaum et al., 1996). The origin of themicrodeletion is unclear. Sequence motifs typicallyassociated with slipped mispairing, such as hairpinstem-and-loop formations or misalignment due todirect or inverted repeats, are present in the region,but not in configurations that can easily account forthe observed changes (Krawczak and Cooper, 1991).

BIOLOGICAL AND CLINICAL RELEVANCE

The majority of missense mutations identified inthe ALD gene result in nonconservative amino acidchanges within codons that are highly conservedamong ABC transporter proteins. The analysis ofthese mutations has the potential to reveal genotype–phenotype relationships, although the intrafamilialvariability of phenotype defies any simplistic model-ling (see later). To date, studies have primarily cor-related the location of mutations with those domainsthat are conserved in the ABC transporter superfam-ily of proteins. Four domains appear to be function-ally important: the nucleotide-binding fold in theC-terminal region of the protein; the domains con-cerned with dimerisation and peroxisomal signaling;and the transmembrane domain.

The putative NBF of ALDP is a critical functiondomain in ABC transporters. Mutations found in theNBF are predominantly located in regions that areconserved among ABC transporters; these regionsare important for the structure and function of theNBF. The conserved regions include the 12-amino

acid segment and the two Walker motifs, designatedA and B (Walker et al., 1982) (Fig. 1); the latter motifsare thought to be essential for ATP binding (Fanen etal., 1994). The Walker A motif corresponds to the Ploop or glycine-rich loop known to be involved in phos-phoryl transfer in many nucleotide binding proteins(Higgins, 1992). Therefore, mutations in the Walkermotif regions may affect the correct binding of ATP.

A number of structure motifs (~-helices and Ä-sheets) are predicted to form the core nucleotidebinding fold (Hyde et al., 1990), from which severalloops extend. Mutations in the structural motifs mayhave an indirect effect on ALDP function by alter-ing the structure of the ATP-binding domain, therebyleading to diminished ATP binding or hydrolysis. Theloop 3 region is of particular interest, as it may play arole in coupling the energy of ATP hydrolysis, throughconformational changes, to the transport processthrough direct interactions with other domains(Higgins, 1992). Mutations in the loop regions maytherefore have a direct effect on the transportfunction of ALDP.

Other explanations of functional changes in ALDPmay involve the way in which ALDP forms a func-tional transporter. The ALD gene product shows allthe characteristics of an ABC transporter, but withonly a single domain containing putative membranespanning segments and one ATP-binding domain(Fig. 1). The functional entity of a typical ABC trans-porter consists of two sets of these domains. In sometransporters, such as CFTR, both sets are assembled

FIGURE 1. Distribution of mutations within the ALD gene (up-dated and modified from Ligtenberg et al., 1995). The boxesrepresent the 10 exons of the ALD gene (drawn to scale,Sarde et al., 1994). The lines between these boxes representintrons (not to scale). Black boxes represent the six putativemembrane spanning segments. The location of these segmentsis in contrast to the earlier reports by Ligtenberg et al. (1995)and Kok et al. (1995). The membrane spanning segmentsindicated in this figure were predicted from hydropathy plotsgenerated using the peptide structure software in the Genet-ics Computer Group (GCG) software package (WisconsinPackage, Version 8, September 1994, Genetics Computer

Group, 575 Science Drive, Madison, WI, USA 53711). Thestippled area represents the conserved region encompassingthe ATP-binding domain termed the nucleotide binding fold(NBF), which includes the two Walker motifs (WA and WB)indicated in light grey. The striped box represents the 12 aminoacid segment (LSGGEKQRIGMA) that is highly conserved inother ABC proteins. L3 represents the loop 3 region pro-posed to be involved with coupling ATP hydrolysis to thetransport process (Higgins, 1992). Each vertical bar repre-sents a mutation documented in this report. In the case ofthe frameshift mutations, the last amino acid not altered bythe mutation is indicated.

508 DODD ET AL.

in a single polypeptide chain, while others, such asTAP1 and TAP2, are formed by dimerisation of twopolypeptides with structures analogous to that ofALDP. This functional requirement suggests thatALDP may exist as a homodimer or that it may forma heterodimer with an homologous protein. We can-not exclude the possibility that mutations in an au-tosomal gene encoding for a presumed partner ofALDP are responsible for the disease in some rarepatients. It has been proposed that PMP70 or ALDRP,a recently identified ALD-like gene, might be such apartner (Valle and Gartner, 1993; Lombard-Platet etal., 1996); however, there is no biochemical evidenceto support this proposition.

Peroxisomal proteins are synthesised by cytosolicribosomes and sorted into the peroxisome post-translationally (Lazarow and Fujiki, 1985). It is wellestablished that peroxisomal proteins have a target-ing signal that is localised in the C-terminal regionof the protein (Subramani, 1993). No such targetingsignal has been identified in ALDP. Therefore, somemutations may disrupt the unknown targeting signalto the peroxisome, and consequently the truncatedprotein may be sorted into other organelles or be re-tained in the cytoplasm, where ALDP is easily ac-cessed by proteinases and degraded. This has beenwell documented in primary hyperoxaluria type 1(PH1), where one-third of patients have point mu-tations in the PH1 gene that cause mistargeting ofthe protein from the peroxisome to the mitochon-drion (Purdue et al., 1990). Also, some mutations inthe NBF of the CFTR protein have been describedthat result in defective sorting of proteins to subcel-lular structures (Gregory et al., 1991).

The relative number of missense mutations in exon1 is high, especially between the third and fourthputative transmembrane segments. The reason forthis increased frequency can only be partially attrib-uted to the high CpG richness in this region. Muta-tions identified in these less conserved regions mayact by impairing the stability of the protein, or resultin aberrant substrate selection, transport, or homo/heterodimerisation (Valle and Gartner, 1993;Contreras et al., 1994; Mosser et al., 1994; Kamijo etal., 1994). Recent studies have also suggested a rolefor this region in targeting and inserting PMP70 intothe peroxisomal membrane (Liper et al., 1995).

Two studies so far have directly addressed the is-sue of mutational effects on ALDP. Watkins et al.(1995) looked for immunoreactive material in theperoxisomes of 35 ALD patients using antisera raisedagainst the C-terminal 18 amino acids of ALDP andfound no immunoreactive material in more than two-thirds of patients. Feigenbaum et al. (1996) studied

immunocytofluorescence and Western blotting of fi-broblasts and/or white blood cells with two anti-ALDP antibodies in 44 ALD patients. In this study,50% of patients with missense mutations were foundto lack immunoreactivity. These data provide addi-tional evidence that some missense mutations prob-ably result in the synthesis of an unstable protein thatis rapidly degraded in the cytosol.

Both studies demonstrated that all patients withnonsense mutations lacked detectable ALDP;Feigenbaum et al. (1996) demonstrated decreasedimmunoreactivity with some missense mutations.Watkins et al. (1995) found that three patients hadmissense mutations in the putative transmembranedomain. Only one of these three patients exhibitednegative immunofluorescence, with the mutationaltering a glutamic acid residue (amino acid position291) in the EAA-like motif (Saurin et al., 1994; Shaniet al., 1995). The EAA motif appears to play an im-portant role in the transport function of prokaryoticABC transporters (Koster and Bohm, 1992; Saurinet al., 1994). It has therefore been suggested that theEAA-like motif may play a similar role in eukaryotictransporters by maintaining the stability of ALDP, orin its interactions with a partner protein (Watkins etal., 1995; Shani et al., 1995).

Feigenbaum et al. (1996) found three mutationsthat resulted in the synthesis of stable but presumablynonfunctioning protein localised to the hydrophobicareas of the putative transmembrane domain. The ef-fect of these mutations on the charge and/or hydro-phobicity of the affected residue indicates that theseresidues are likely to be critical for ALDP function.

Two patients with missense mutations in the pu-tative hinge region between the transmembrane andATP-binding domains exhibited positive immunof-luorescence, in contrast to the one patient with amutation in the region downstream of the ATP-bind-ing domain (Watkins et al., 1995). Finally, eight pa-tients with missense mutations in the ATP-bindingdomain exhibited differing immunofluorescence pat-terns (Watkins et al., 1995; Feigenbaum et al., 1996).The conclusion from these studies is that the corre-lation of mutation analysis and immunofluorescencepatterns should lead to the identification of thoseresidues that are critical for ALDP function, althoughno correlation appears to exist between clinical phe-notype and immunofluorescence pattern.

FUTURE PROSPECTS

It is apparent from this report that further studiesneed to be undertaken before conclusions about theassociation of specific mutations with ALDP func-tion can be drawn. No clear-cut association of geno-


type with phenotype has been determined. Thisobservation is not surprising because of the knownintrafamilial phenotypic variability in ALD (Moserand Moser, 1989). This variability is not due to al-lelic variation or compound heterozygosity (Smithet al., 1991). It has therefore been suggested thatmodifier genes may be implicated in the variable ex-pression of the disease, or in some cases stochasticfactors (Sobue et al., 1994). Maestri and Beaty (1992)concluded that the most efficient method to identifythe autosomal modifier locus is to perform linkageanalysis with discordant affected sibs. This analysishas not been performed to date.

A possible cause of phenotypic diversity might bea consequence of the interaction of ALDP with otherproteins. Valle and Gartner (1993) speculated thatanother half-ABC transporter, namely PMP70, maybe a candidate partner protein. This protein is defec-tive in some patients with Zellweger syndrome, whichis another inborn error of peroxisome biogenesis. Bothheterozygosity at the PMP70 locus and mutations inthe ALD gene might lead to phenotypic variability(“digenic” ALD). A close relative of ALDP, ALDRP,has recently been identified (Lombard-Platet et al.,1996). This protein has been proposed as a candi-date partner for ALDP, or as a phenotypic modifier.However, ALDRP has a different expression patternfrom ALDP; hence its role as a putative partner couldbe facultative or nonequivalent in some tissues(Lombard-Platet et al., 1996). Although the partnerprotein for ALDP in humans has not been identi-fied, a recent report has suggested that the apparentsubunits of the ALDP-associated ABC transporterhave been identified in Saccharomyces cerevisiae(Shani et al., 1995). It remains to be determinedwhether the yeast partner protein has a homologuein humans that interacts with ALDP.

Regardless of the difficulties in explaining the phe-notypic variation in ALD, further elucidation of ge-netic defects in patients with ALD should improveour understanding of the biological interactions ofALDP. In this respect, studies to date have been lim-ited to the analysis of ALD patients. Importantly, thereconstruction of the full-length ALD transcript hasthe potential to address the effect of mutations onALDP function by in vitro reconstruction experi-ments. In addition, the development of a mousemodel for ALD should lead to the identification ofmodifier genes by breeding mice carrying a mutantALD gene to various inbred laboratory mouse strains,with subsequent linkage analysis in backcross mat-ings. This work may support the role of a gene linkedto the HLA class II region in disease manifestation.This gene has been shown in humans to be impli-

cated in the inflammatory demyelination of the CNS,which in characteristic of ChALD (Berger et al.,1995). A mouse model for ALD should also provepivotal in the development of therapies for ALD.

ACKNOWLEDGMENTS

We acknowledge the financial support of theAuckland Medical Research Foundation, the Can-terbury Medical Research Foundation (M.A.K.), theLottery Grants Board of New Zealand, the Univer-sity of Auckland Research Grants Committee, andthe Health Research Council of New Zealand, whichhave supported our mutational analysis studies andmouse modeling of ALD.

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