Brain (2000), 123, 908–919

Clinical and molecular genetic characteristics ofpatients with cerebrotendinous xanthomatosisAad Verrips,1 Lies H. Hoefsloot,2 Gerry C. H. Steenbergen,3 Joop P. Theelen,2 Ron A. Wevers,3 FonsJ. M. Gabreels,1 Baziel G. M. van Engelen1 and Lambert P. W. J. van den Heuvel3

1Department of Neurology, 2Department of Human Correspondence to: L. P. W. J. van den Heuvel, LaboratoryGenetics and 3Laboratory of Pediatrics and Neurology, of Pediatrics and Neurology, University Hospital Nijmegen,University Hospital Nijmegen, Nijmegen, The Netherlands PO Box 9101, 6500 HB Nijmegen, The Netherlands

E-mail: B.vandeHeuvel@ckslkn.azn.nl

SummaryCerebrotendinous xanthomatosis (CTX) is a lipid storagedisease caused by a deficiency of the mitochondrial enzyme27-sterol hydroxylase (CYP 27), due to mutations in itsgene. In this study we report on mutations in 58 patientswith CTX out of 32 unrelated families. Eight of thesewere novel mutations, two of which were found togetherwith two already known pathogenic mutations. Twelvemutations found in this patient group have been describedin the literature. In the patients from 31 families,mutations were found in both alleles. In the literature, 28mutations in 67 patients with CTX out of 44 families havebeen described. Pooling our patient group and the patients

Keywords: cerebrotendinous xanthomatosis; mutations; genotype–phenotype correlation; pathogenesis

Abbreviations: CTX � cerebrotendinous xanthomatosis; CYP 27 � sterol 27-hydroxylase

IntroductionCerebrotendinous xanthomatosis (CTX) is a rare, autosomalrecessive, lipid storage disease caused by a deficiency ofthe mitochondrial enzyme 27-sterol hydroxylase (CYP 27).Because of this deficiency, large amounts of cholestanol andcholesterol are produced. These metabolites accumulate inmany tissues, especially eye lenses, the CNS and muscletendons. Besides the cholestanol and cholesterol production,large amounts of bile alcohols are produced in CTX, whichare excreted in urine. Clinical characteristics of CTX arepremature bilateral cataracts, formation of tendon xanthomas(most often in the Achilles tendons), neurological andneuropsychiatric abnormalities such as pyramidal andcerebellar signs, peripheral neuropathy and dementia(Bjorkhem and Boberg, 1995). Most patients have cerebellarsigns and dementia from the age of 20 years onwards. Inchildhood, the combination of bilateral cataracts and diarrhoeais almost pathognomonic for the disease (Cruysberg et al.,1991; van Heijst et al., 1996). The biochemical diagnosis ismade by determination of the serum cholestanol level and

© Oxford University Press 2000

from the literature together, 37 different mutations in125 patients out of 74 families were obtained. Identicalmutations have been found in families from differentethnic backgrounds. In 41% of all the patients, CYP 27gene mutations are found in the region of exons 6–8. Thisregion encodes for adrenodoxin and haem binding sites ofthe protein. Of these 125 patients, a genotype–phenotypeanalysis was done for 79 homozygous patients harbouring23 different mutations, out of 45 families. The patientswith compound heterozygous mutations were left out ofthe genotype–phenotype analysis. The genotype–phenotype analysis did not reveal any correlation.

by the determination of bile alcohol excretion in urine(Wolthers et al., 1983, 1991).

In 1989, Andersson and colleagues characterized the cDNAencoding rabbit mitochondrial CYP 27 starting with rabbitenzyme protein, which is a member of the mitochondrialcytochrome P-450 enzyme family (Andersson et al., 1989).In 1991, the cDNA for human CYP 27 was isolated byhybridizing rabbit cDNA to a liver cDNA library and itsgene was localized on the long arm of chromosome 2 (Caliand Russell, 1991). The genomic structure of the CYP 27gene was elucidated in 1993; the gene contains nine exonsand eight introns and spans 18.6 kb of DNA (Leitersdorfet al., 1993). The mature enzyme consists of 498 aminoacids and contains putative binding sites for adrenodoxin andhaem; these sites are encoded by the region between exons6 and 8 (Leitersdorf et al., 1993). The enzyme is expressedin the CNS, liver, lung, duodenum and endothelial cells(Reiss et al., 1997). In 1991, the first mutations in theCYP 27 gene were described (Cali et al., 1991).

Genotype and phenotype in CTX 909

In this paper we present the phenotypes and genotypes(including eight novel mutations) of 58 patients with CTXout of 32 families, of which 21 were Dutch families; this isthe largest series ever reported. We have reviewed theliterature and identified 67 additional CTX patients out of44 families in whom the genotype had been established.Finally, we have performed a genotype–phenotype analysisfor 79 homozygous patients harbouring 23 different muta-tions, out of 45 families.

MethodsPatientsBetween 1983 and 1998 the clinical and biochemicalinformation of 42 Dutch CTX patients, of which seven werechildren, out of 21 families in the Netherlands were collected.DNA from these 42 patients and from 16 CTX patients outof 11 families from the UK, Belgium, Spain, Tunisia,Germany and China was also analysed. All patients hadelevated serum cholestanol levels and an excessive urinaryexcretion of bile alcohols, measured according to Wolthersand colleagues using capillary gas chromatography (Woltherset al., 1983, 1991). Informed consent was obtained fromeach participating subject or the parents of younger children.The study was approved by the Ethics Committee of theUniversity Hospital Nijmegen, The Netherlands.

Mutation analysisThe CYP 27 gene was amplified in four fragments (exons 1,2, 3–5 and 6–9), by PCR (polymerase chain reaction) fromgenomic DNA of leucocytes. Exons 3–9 with their intronboundaries were subsequently amplified separately, with thetwo PCR fragments 3–5 and 6–9 as templates (Luytenet al., 1995). The oligonucleotides used as primers for PCRamplification and for sequence analysis are those describedby Leitersdorf and colleagues (Leitersdorf et al., 1993).Human genomic DNA from patients from these 32 familieswas screened for mutations in the CYP 27 gene by singlestrand conformation polymorphism analysis using thePharmacia Phast System (Amersham Pharmacia Biotech.Ruusendaal, The Netherlands), or were directly sequenced.Cycle sequencing of the coding and the non-coding strandswas carried out by the Taq Dye Deoxy Terminator method(Applied Biosystems Inc., Forster City, Calif., USA) usingan ABI 377 DNA sequencer.

To analyse the effects of the splice site mutations, RNAfrom cultured fibroblasts was reverse transcribed to cDNAaccording to established procedures (Ploos van Amstel et al.,1996; Verrips et al., 1997). The CTX cDNA, amplified byPCR, was used for agarose gel electrophoresis and for DNAsequence analysis, as described above. The segregation ofnovel mutations has been studied in members of families 6,14, 16, 21, 28, 31 and 32 (Table 1). In families 24 and 30no family members could be evaluated.

ResultsPatientsThe clinical characteristics of 54 patients are listed in Table 1.In 10 of the 32 families, three patients were present and insix families two patients. Sixteen patients were sporadiccases and accurate clinical information could not be obtainedin two families (19 and 29). The phenotypes of families 2–8, 10, 12–16, 24 and 31 have been described in previouspublications. In two families (16 and 18) consanguinity waspresent: in both families the parents are first cousins. Amongthe general signs, bilateral premature cataracts were presentin 90% of the patients, tendon xanthomas in 45% andintractable diarrhoea in 33%. Among the most frequentneurological signs were pyramidal (67%) and cerebellar signs(60%) and low intelligence (57%). Epilepsy and peripheralneuropathy were both present in 24% of the patients. Thehigh prevalence of diarrhoea in our patient group in contrastto reports in the literature may be due to the fact that it isgenerally not known that diarrhoea is an importantphenomenon in this mainly neurological disease.

In all patients, excessive amounts of bile alcohols werefound in the urine. In the patients in whom serum cholestanollevels were determined, elevated levels were found. CranialMRI findings were available in 34 patients. In two-thirds ofthe patients global atrophy and parenchymal lesions wereseen. In the majority of the patients in whom an EMG wasperformed, axonal neuropathy could be established. Evokedpotentials studies (visual evoked potential, brainstem auditoryevoked potential and somatosensory evoked potential)revealed delayed central conduction times. A diffuse slowingwith paroxysmal discharges were the main EEG findings.

MutationsMutations found in the 32 families, together with theirdistribution among these families and the mutations describedin the literature are listed in Table 2. The distribution of themutations over the CYP 27 gene is depicted in Fig. 1. In the32 families presented, 20 different mutations were found ofwhich 12 were discovered in the Dutch CTX patients.

Eight novel mutations were found in the 32 familiespresented. A 1151C→T transition in exon 6, resulting in thesubstitution of proline by leucine in codon 384 (families 5,14, 16 and 26), and a 1213C→T transition in exon 7, resultingin replacement of arginine by tryptophan in codon 405(family 6), were observed. A missense mutation in exon 6was found in the same allele as a C insertion in exon 1 onposition 5–6, an already known mutation (Segev et al., 1995)(Table 2). We found these two mutations on the same allelein families 5, 14, 16 and 26. In one allele from the patientsof family 16 and in both alleles in their mother, no mutationin the CYP 27 gene was found, indicating a co-segregationof these two mutations. In families 24 and 32, a 776A→Gtransition in exon 4, resulting in substitution of lysine byarginine in codon 259, was found. On the other allele in

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din

the

lite

ratu

re

Mut

atio

nN

ucle

otid

ech

ange

Am

ino

acid

chan

geE

thni

cor

igin

Mut

atio

ndi

stri

butio

nR

efer

ence

num

ber

New

*O

ld†

New

‡O

ld§

Hom

ozyg

ous

Het

eroz

ygou

s

(1)

E1

5–6i

nsC

26/2

7ins

CM

et1�

178a

a→pt

cM

et–3

3�17

8aa→

ptc

Fran

ce,

Net

herl

ands

5¶14

¶ ,16¶ ,2

6¶Se

gev

etal

.,19

95(2

)E

235

5del

C37

6del

CM

et11

8�23

aa→

ptc

Met

85�

23aa

→pt

cIs

rael

iD

ruze

––

Lei

ters

dorf

etal

.,19

94(3

)E

237

9C→

T40

0C→

TR

127W

Arg

94T

rpN

ethe

rlan

ds,

Bel

gium

–25

,28

Ver

rips

etal

.,19

99a

(4)

E2

380G

→A

401G

→A

R12

7QA

rg94

Gln

UK

,Sp

ain

–21

Wat

tset

al.,

1996

(5)

E2

409C

→T

430C

→T

R13

7WA

rg10

4T

rpJa

pan

––

Nak

ashi

ma

etal

.,19

94(6

)E

243

5G→

T45

6G→

TG

145G

•Gly

112

Gly

––

––

•Tyr

111

dele

ted

––

––

•Exo

n2

skip

ped

Japa

n–

–C

hen

etal

.,19

98b

(7)

I244

6�1g

→a

467�

1g→

aA

berr

ant

splic

ing?

Abe

rran

tsp

licin

g?Sp

ain

–21

Thi

sst

udy

(8)

E3

475C

→T

496C

→T

Q15

9XG

ln12

6(p

tc)

Net

herl

ands

–10

Ver

rips

etal

.,19

96(9

)E

352

5/52

6del

G54

6/54

7del

GT

175

�5a

a→pt

cT

hr14

2�5a

a→pt

cSu

rina

mC

reol

e2

–V

erri

pset

al.,

1996

(10)

E3

646G

→C

667G

→C

A21

6PA

la18

3Pr

oIt

aly,

Tun

isia

20–

Gar

uti

etal

.,19

96b

(11)

E4

691C

→T

712C

→T

R23

1XA

rg19

8(p

tc)

Ital

y–

–G

arut

iet

al.,

1997

(12)

E4

745C

→T

766C

→T

Q24

9XG

ln21

6(p

tc)

UK

–31

Thi

sst

udy

(13)

E4

776A

→G

797A

→G

K25

9RLy

s22

6A

rgG

erm

any

–24

,32

Thi

sst

udy

(14)

E4

779G

→A

800G

→A

W26

0XT

rp22

7(p

tc)

Ger

man

y–

24T

his

stud

y(1

5)E

480

8C→

T82

9C→

TR

270X

Arg

237

(ptc

)Pa

kist

an,

Net

herl

ands

–14

Ahm

edet

al.,

1997

(16)

E4

819d

elT

840d

elT

L27

2�12

aa→

ptc

Leu

239�

12aa

→pt

cM

oroc

co,

Jew

s–

–L

eite

rsdo

rfet

al.,

1993

(17)

I484

4�1g

→a

865�

1g→

aD

elet

ion

ofex

on4

(66

aa)

Net

herl

ands

318

,22

Ver

rips

etal

.,19

97(1

8)I4

845-

1g→

a86

6-1g

→a

Abe

rran

tsp

licin

g?A

berr

ant

splic

ing?

Mor

occo

,Je

ws

––

Lei

ters

dorf

etal

.,19

93;

Mei

ner

etal

.,19

94(1

9)E

585

0A→

T87

1A→

TK

284X

Lys

251

(ptc

)S.

Afr

ica,

Net

herl

ands

–8

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ner

etal

.,19

94(2

0)E

510

16C

→T

1037

C→

TT

339M

Thr

306

Met

Seph

ardi

cJe

ws,

Chi

na,

4,9,

11,2

9#7,

8,13

,23,

25B

raut

bar

etal

.,19

83;

Net

herl

ands

Res

hef

etal

.,19

94(2

1)E

610

61A

→G

1082

A→

GD

354G

Asp

321

Gly

Tur

key

–30

Thi

sst

udy

(22)

E6

1151

C→

T11

72C

→T

P384

LPr

o35

1L

euN

ethe

rlan

ds,

Bel

gium

5¶14

¶ ,16¶ ,2

6¶T

his

stud

y(2

3)E

611

83C

→T

1204

C→

TR

395C

Arg

362

Cys

USA

,B

elgi

um,

Net

herl

ands

,15

,17,

191,

7,10

,18,

26,3

2H

arla

nan

dG

erm

any

Still

,19

68;

Cal

iet

al.,

1991

(24)

E6

1183

C→

A12

04C

→A

R39

5S•A

rg36

2Se

rJa

pan

––

Che

net

al.,

1998

c•V

al33

2de

lete

d(F

rom

cDN

A11

16,

89bp

del

upst

ream

end

exon

6)(2

5)E

611

84G

→A

1205

G→

AR

395H

•Arg

362

His

(ful

lJa

pan

––

Che

net

al.,

1996

,19

98a

leng

th)

Abe

rran

tsp

licin

g•V

al33

2de

lete

d(F

rom

cDN

A11

16,

89bp

del

upst

ream

end

exon

6)•E

xon

6sk

ippe

d

Genotype and phenotype in CTX 913T

able

2co

ntin

ued

Mut

atio

nN

ucle

otid

ech

ange

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ino

acid

chan

geE

thni

cor

igin

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atio

ndi

stri

butio

nR

efer

ence

num

ber

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ld†

New

‡O

ld§

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ozyg

ous

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eroz

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s

(26)

I611

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1g→

a12

05�

1g→

aA

berr

ant

splic

ing

•Del

29aa

,co

ntin

ueIt

aly,

UK

,B

elgi

umw

ithV

al33

2�16

aa→

ptc

Tur

key

2728

,30

Gar

uti

etal

.,19

97•D

el56

aa,

cont

inue

with

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307�

16aa

→pt

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aa,

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307

cont

inue

with

Asn

388

(27)

I6D

el1.

9kb

Del

1.9k

bD

elex

on7–

9D

elex

on7–

9It

aly

––

Dot

tiet

al.,

1995

;G

arut

iet

al.,

1996

a,19

97(2

8)E

712

02C

→G

1223

C→

GP4

01R

Pro

368

Arg

Japa

n–

–O

kuya

ma

etal

.,19

96(2

9)E

712

13C

→T

1234

C→

TR

405W

Arg

372

Trp

Net

herl

ands

–6

Thi

sst

udy

(30)

E7

1214

G→

A12

35G

→A

R40

5QA

rg37

2G

lnJa

pan

––

Che

net

al.,

1997

a(3

1)I7

1263

�1g

→a

1284

�1g

→a

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rran

tsp

licin

gSk

ippi

ngex

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362�

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→pt

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aly,

UK

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ethe

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ds12

1,6,

13,2

3,31

,22

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uti

etal

.,19

96b,

1997

(32)

I712

63�

5g→

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84�

5g→

tA

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ant

splic

ing

Skip

ping

exon

7,A

rg36

2�28

aa→

ptc

Ital

y–

–G

arut

iet

al.,

1997

(33)

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64-1

g→a

1285

-1g→

aA

berr

ant

splic

ing

•Asn

388�

57aa

→pt

cIt

aly

––

Gar

uti

etal

.,19

97•D

elof

Thr

389

and

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390

(34)

E8

1420

C→

T14

41C

→T

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4WA

rg44

1T

rpJa

pan

Kur

iyam

aet

al.,

1991

;K

imet

al.,

1994

;N

agai

etal

.,19

96(3

5)E

814

15G

→C

1436

G→

CG

472A

Gly

439

Ala

Chi

na29

#T

his

stud

y(3

6)E

814

21G

→A

1442

G→

AR

474Q

Arg

441

Gln

Japa

n–

Kur

iyam

aet

al.,

1991

;K

imet

al.,

1994

;K

uwab

ara

etal

.,19

96;

Oku

yam

aet

al.,

1996

(37)

E8

1435

C→

T14

56C

→T

R47

9CA

rg44

6C

ysC

anad

a–

Past

ersh

ank

etal

.,19

74;

Cal

iet

al.,

1991

Poly

mor

phis

ms

(put

ativ

e)(3

8)E

236

6A→

C38

7A→

CG

122G

Gly

89G

lyJa

pan

––

Nak

ashi

ma

etal

.,19

94(3

9)E

585

2G→

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3G→

AK

284K

Lys

251

Lys

Mor

occo

,Je

ws

––

Lei

ters

dorf

etal

.,19

93

The

32fa

mili

esin

who

mth

ese

mut

atio

nsar

epr

esen

t(h

omoz

ygou

sor

com

poun

dhe

tero

zygo

us)

are

liste

dac

cord

ing

toth

enu

mbe

ring

inTa

ble

1.N

ucle

otid

esgi

ven

inca

pita

lle

tters

are

pres

ent

inex

ons

and

thos

ein

low

erca

sein

intr

ons.

Nuc

leot

ide

and

amin

oac

idnu

mbe

ring

are

inth

eol

d(C

ali

and

Rus

sell,

1991

)an

dth

ere

cent

lypr

opos

edne

wno

men

clat

ure

(Ant

onar

akis

,19

98).

*Nuc

leot

ide

num

beri

ng:

the

Aof

the

first

AT

Gis

num

ber

1(A

nton

arak

is,

1998

);† nu

cleo

tide

num

beri

ng:

the

Gof

the

first

GC

Ais

num

ber

1(C

ali

and

Rus

sell,

1991

);‡ am

ino-

acid

num

beri

ng:

the

first

met

hion

ine

ofth

etr

ansl

ated

fram

eis

num

ber

1(A

nton

arak

is,

1998

);§ am

ino-

acid

num

beri

ng:

the

first

met

hion

ine

ofth

etr

ansl

ated

fram

eis

num

ber

33,

alan

ine

(num

ber

1)is

the

first

amin

oac

id(C

ali

and

Rus

sell,

1991

).¶ 5–

6ins

Can

d11

51C

→T

onon

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lele

.# 10

16C

→T

and

1415

G→

Con

one

alle

le.

aa�

amin

oac

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emat

ure

term

inat

ion

codo

n;in

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inse

rtio

n;de

l�

dele

tion.

914 A. Verrips et al.

Fig. 1 Schematic diagram of the mutations in the CYP 27 gene. The length of the exons is given proportional to their size. The numberswithin the mutation symbols correspond with those in Table 2. Ten of the 16 missense mutations are found in the region of exons 6–8.Deletions and insertions are found in exons 1, 2, 3 and 4. Nonsense mutations are found in exons 3 (one), 4 (four) and 5 (one). Seven ofthe 11 mutations affecting pre-mRNA splicing are found in the region of exons 6–8. Mutations 6, 24 and 25 are nucleotide substitutionsin exons affecting pre-mRNA splicing, resulting in aberrant splice products (Chen et al., 1996, 1998a, b, c).

family 24, a novel nonsense mutation was present: a 779G→Atransition in exon 4, changing codon 260 into an opaltermination codon. Another novel nonsense mutation wasfound in family 31: a 745C→T transition in exon 4 changingcodon 249 into an amber termination codon, together with asplice site mutation on the other allele were present. In family21, patients were compound heterozygotes for a novel splicesite mutation: a 446�1g→a transition in the splice donorsite in intron 2. On the other allele a missense mutation inexon 2 was found (already known) (Watts et al., 1996). Infamily 30, a 1061A→G transition leading to a replacementof asparagine by glycine in codon 354 was found, togetherwith an already known splice site mutation on the other allele(Garuti et al., 1997). A 1415G→A homozygous transition inexon 8, resulting in the substitution of glycine by alanine incodon 472 was found in family 29, together with the known1016C→T transition in exon 5 (Reshef et al., 1994). Noneof the novel mutations mentioned above were found in anyof the 50 controls (100 alleles).

Overview of mutations in the Dutch CTXpatients and in the world literatureIn the Dutch CTX patients, three mutations were mostfrequent, being found in almost two-thirds of the alleles.These were the 1016C→T transition in exon 5 (Reshef et al.,1994), a 1263�1g→ a transition in intron 7, leading to exonskipping and a frameshift (Garuti et al., 1996b), and finallythe 5–6 C insertion in exon 1 (Segev et al., 1995), togetherwith the exon 6 1151C→T missense mutation. Since theserecurrent mutations were identified in patients from differentgeographical origins, it is likely that they are ancient variantsoccurring frequently in CTX. An additional 42 families (67patients) in whom genotyping has been done were identifiedfrom the literature. These, taken together with our 32 families,gave a total of 45 families which were homozygous for onemutation, and 29 families which were compoundheterozygous.

Mutations 2, 5, 6, 9, 16, 18, 24, 25, 34, 35 and 37 wereonly found in homozygous patients. Mutations 1, 4, 6, 10,

Genotype and phenotype in CTX 915

Fig. 2 Allele frequencies in CTX mutations. In 74 families (a total of 148 alleles), 37 mutations were found. Except for one family inwhich only one mutant allele has been identified, mutations were found on both alleles. The mutation numbers on the horizontal axiscorrespond with those in Fig. 1 and Table 2. Cross-hatched columns � The Netherlands; black columns � abroad.

15, 17, 19, 20, 23, 26, 27, 30, 31 and 36 were found in bothhomozygous and compound heterozygous families. Mutations3, 7, 8, 11–14, 21, 22, 28–30, 32 and 33 were only found incompound heterozygous patients.

Overall, five mutations were found in eight or more alleles(Fig. 2). These are mutations 2 (in Israeli Druze patientsonly), 20 (in Sephardic Jews, Chinese and Dutch patients),23 (USA, Belgium, The Netherlands, Germany), 31 (Italy,UK, the Netherlands), and 30, 34 and 36 (only in the Japanesepatients).

Of all point mutations identified in the CYP 27 gene, eightwere transversions and 26 were transitions. There were 13point mutations in CpG dinucleotides; eight C→T transitionstogether with four G→A transitions were found in the samecodon. These mutations led to a substitution of arginine byanother amino acid. Codon 395 was affected in three patientsby a point mutation. Several non-CpG point mutations werefound within 10 bp up- or downstream of mutation sitesharbouring tetra- and trinucleotide motifs. These genestructures are hypothesized to be hotspots for point mutationsor deletions (Cooper et al., 1995). Two CTTT tetranucleotidemotifs were present in exon 4 in the vicinity of mutations17 and 18. A TTTG motif was present in exon 3 (mutation10) and in exon 8 in the neighbourhood of mutation 35.Trinucleotide motifs were CTT (mutations 10) and TGA(mutation 21). Tetra- and trinucleotide motifs within a 10 bpregion of deletions were AAGT (mutation 2), CTTT (mutation9), TTGG and GAA (mutation 16).

Genotype–phenotype correlationsGenotypical and phenotypical characteristics of 125 patientsout of 74 families identified in the literature, together withthe presented cohort were determined. Forty-six patients out

of 29 families were compound heterozygous, 79 patients (45families) were homozygous for 23 different mutations. Inthese homozygous patients we examined possible differencesin sex, age of onset, diagnosis, biochemical characteristicsand the presence of signs and symptoms with respect tomutation site (exon 1–5 versus exon 6–9) and mutation type(missense versus other types of mutations, frameshift ormutations resulting in a premature termination codon versusother types of mutations). No specific genotype–phenotypecorrelation could be established.

Apart from the different phenotypes displayed betweenpatients from different families, there is also a strikingintrafamilial phenotypic variability in CTX (Dotti et al.,1996; Nagai et al., 1996) (Table 1).

DiscussionThe diagnosis of all the CTX patients was made on clinicalgrounds. The biochemical diagnosis was made by thedetermination of the bile alcohols in urine and of thecholestanol in serum, rather than by measurement of theCYP 27 enzyme activity. As mutations in the CYP 27 genewere found in 63 of the 64 alleles in all of these patients, itis highly unlikely that another gene is involved in thepathogenesis of CTX.

Including the novel mutations presented in the currentpatient cohort, 37 different mutations have been described inthe CYP 27 gene in CTX patients. They consist of 16missense mutations (resulting in amino acid replacements),three mutations in the last nucleotides of exons (resulting inboth amino acid replacements and affecting pre-mRNAsplicing), three deletions, one insertion, eight splice site andsix nonsense mutations (Cali et al., 1991; Leitersdorf et al.,1993, 1994; Kim et al., 1994; Meiner et al., 1994; Nakashima

916 A. Verrips et al.

Table 3 Evolutionary conservation of CYP 27 residues substituted in patients with CTX.

CYP 27 amino K259R D354G P384Lacid substitutionHuman mutated: R G LHuman wild-type: QNSLYATFLPKWTRPVLPFWK---LTWALYHLSKDPEIQEALHEE---VPQHKDFAHMPLLKAVLKETLRabbit wild-type: QNS.Y.TFLPKWTRP.LPFWK---LTWALYHLSK.PEIQ.AL..E---VPQHKDFAHMPLLKAVLKETLRat wild-type: .NS.Y.TFLPKW.RP.LPFWK---LTWALYHLSK.PEIQEALH.E---VPQ.KDFAHMPLLKAV.KETLMouse wild-type: .........PKWTR..L..W.---L.W.LY.LS..P..Q.ALH.E---..........PLLKA.LKETL

CYP 27 amino R405W G472Aacid substitutionHuman mutated: W AHuman wild-type: RLYPVVPTNSRIIEKEIEVDG---HPFGSVPFGYGVRACLGRRIARabbit wild-type: RLYPV.P.NSRI..KEIEV.G---HPFGSVPFGYGVRACLGRRIARat wild-type: RLYPVVPTNSRII........---HPFGSVPFGYGVR.CLGRRIAMouse wild-type: RLYPVVP.NSR.....I.V..---HPF.S.PFG.G.R.C.GRR.A

The amino acid sequence alignment of human CYP 27 with that from rabbit, rat and mouse. Points indicate the amino acid residues thatwere different from the human CYP 27 sequence. Evolutionarily conserved (wild-type) amino acids are indicated in bold.

et al., 1994; Reshef et al., 1994; Segev et al., 1995; Garutiet al., 1996a, b, 1997; Okuyama et al., 1996; Watts et al.,1996; Verrips et al., 1996, 1997, 1999a; Ahmed et al., 1997;Chen et al., 1996, 1997, 1998a, b, c).

In 19 of the 32 families in this study, mutations werelocated in the region of exons 6–8 of the CYP 27 gene.Although the mutations were distributed throughout the wholegene, 15 of the 37 mutations (41%) were found in this regionthat comprises 28.4% of the nucleotides of the CYP 27 gene.This finding may indicate that this conserved part of thegene, coding for adrenodoxin and haem binding sites, playsa pivotal role in the function of the enzyme. The pathogenicityof individual CTX mutations is based on their predictedeffect on the CYP 27 protein and on segregation in families.None of the novel mutations were found during the analysisof 50 control chromosomes. Among the CYP 27 genemutations identified in our CTX cohort are variants whichare likely to have deleterious effects on the function of theCYP 27 protein. Thus, the deletion/insertion (mutations 1and 9), and nonsense mutations (mutations 8, 12, 14) willcause premature termination of translation and result intruncated CYP 27 proteins. The different splice site mutations(mutations 7, 17, 26, 31) will lead to incorrect splicingand exon skipping, resulting in incorrect CYP 27 proteins.However, the majority of the mutations detected in the CTXcohort are amino acid substitutions. Our data, together withthe results of previous studies, indicate that 16 out of 37 aremissense mutations of which five are novel to the describedpatient cohort. These mutations are inferred to be pathogenicwhen they substitute amino acids, which, in view of theirconservation through evolution, are presumed to be offunctional importance. Except for the 1061A→G transition,which leads to a replacement of asparagine by glycine incodon 354, all novel missense mutations detected in ourCTX cohort are substitutions of strongly conserved aminoacids by non-conservative ones (Table 3). Three novelmissense mutations (mutations 3, 13, 22) were found in

unrelated CTX families. Except for the presence of twomutations together on the same allele in families 5, 14, 16and 26, screening of the remaining exons of the CYP 27gene in patients with missense mutations did not reveal othermutations. Therefore, it is likely that these missense mutationsare indeed pathogenic mutations and not innocuouspolymorphisms. However, the ultimate proof that these aminoacid substitutions can indeed result in impairment of CYP27 function are expression studies which are currently beingconducted in our laboratory. In one allele of the patientsfrom family 16, no mutations were found. In the mother ofthese patients the absence of mutations in the CYP 27 geneindicated co-segregation of mutations 1 and 22. It is possiblethat in this family, a mutation is present in the promoterregion of the CYP 27 gene or at a branch point within oneof the introns.

It is remarkable that the amino acid arginine is frequentlyinvolved in missense mutations. In 12 missense mutations,there is involvement of CpG dinucleotides leading to thereplacement of arginine by another amino acid. Arginine canform two hydrogen bonds which may be of major importancefor the conservation of tertiary structure or may play a rolein substrate binding. Both of these aspects could influencethe catalytic activity of the enzyme.

Mutations 1, 3, 4, 10, 15, 19, 20, 22, 23, 26 and 31 werefound in CTX patients from different ethnic backgrounds(Table 2). The mutations may be ancient variants frequentlyoccurring in CTX. In order to determine whether thesemutations are introduced into the population by a singlefounder, it is necessary to study the chromosome 2 haplotypesof the patients carrying these identical mutations.

In a large series of 58 CTX patients out of 32 unrelatedfamilies, we found 21 different mutations and a strikingphenotypic heterogeneity, even within families. No genotype–phenotype correlation could be established with these patientstaken together with all CTX patients reported in the literature.Several authors have stressed the marked phenotypic

Genotype and phenotype in CTX 917

heterogeneity between CTX patients, even between patientswith the same mutation (Dotti et al., 1996; Nagai et al., 1996).Since the phenotype varies between patients independent oftheir biochemical characteristics, other features must beresponsible for these clinical differences and it has beensuggested that environmental factors are responsible (Chenet al., 1996; Nagai et al., 1996; Garuti et al., 1997). In CTXthe same mutation may result in different phenotypes, ormutations at different sites of the CYP 27 gene may resultin the same or in different phenotypes. Recently, we describeda spinal variant of CTX, spinal xanthomatosis, that has arelatively mild clinical course compared with the classic formof CTX, which shows cerebellar involvement, dementia,tendon xanthoma formation and peripheral neuropathy earlyin the disease process. Mutation analysis in these patientsrevealed missense mutations, predominantly in exons 5 and6 of the gene, that were also found in the classical form ofCTX (Verrips et al., 1999a). This polyphenotypy in CTX isthe result of a complex pathophysiology. The CYP 27deficiency, caused by mutations in the CYP 27 gene, leadsto several, different cascades of metabolic derangement, suchas excessive production of cholestanol and bile alcohols(Batta et al., 1987; Bjorkhem and Boberg, 1995). Thecontribution of each of these pathological metabolic processesto the phenotype is poorly understood at the present time.The excessive cholestanol production and its accumulationwithin many tissues, particularly the CNS, play a major rolein the disease process. Recently it was shown that in ratsfed a cholestanol enriched diet, cholestanol accumulated inPurkinje cells, resulting in apoptosis (Inoue et al., 1999).However, in patients with sitosterolaemia, a rare lipid storagedisorder, serum hypercholestanolaemia is also found. Thesepatients do not develop neurological disease (Bjorkhem andBoberg, 1995).

In 1987 it was reported that a defect of the blood–brainbarrier was present in CTX, which was reflected by anelevation of the CSF/serum albumin quotient (Salen et al.,1987). In the CSF, high amounts of apolipoprotein B, theprotein component of low-density lipoproteins and a carrierof cholestanol, were present. This finding suggested anincreased influx of sterols from the blood into the CNS. Thedefect in the blood–brain barrier disappears after severalmonths of chenodeoxycholic acid therapy, so the deficiencyof CYP 27 itself cannot be responsible for this dysfunction.It can be hypothesized that the phenotype in CTX is theresult of a primary membrane dysfunction, followed by anincreased influx of sterols into the eye lens (blood–lensbarrier), the CNS (blood–brain barrier), peripheral nerves(blood–nerve barrier), and vessel wall (endothelial cellmembrane) leading to accelerated arteriosclerosis.

It is unlikely that use of an animal model will clarifythe complex genotype–phenotype relationship. Mice with adisrupted CYP 27 gene, which resulted in a markedly reducedsynthesis of bile acids, had normal plasma levels of cholesteroland cholestanol. In bile and in faeces of these CYP 27 –/–mice, only traces of bile alcohols were found. There was

no cholestanol accumulation or CTX-related pathologicalabnormalities (Rosen et al., 1998).

Since 1975, chenodeoxycholic acid has been commonlyused as a therapy for CTX (Salen et al., 1975) and hasproven to be effective (Berginer et al., 1984). With thistherapy there is a considerable decrease in the serumcholestanol level and a sharp decline in the excretion of bilealcohols in the urine (Wolthers et al., 1983; Batta et al.,1985). Perhaps the most effective inhibitor of cholestanolproduction is a combination of chenodeoxycholic acid witha β-HMG-CoA reductase inhibitor, resulting in a furtherlowering of an already normal serum cholestanol level(Verrips et al., 1999b) and facilitating the long-term washoutof cholestanol from the CNS. Finally, as therapy is available,the early recognition of CTX is important. Because of thephenotypic heterogeneity, in all siblings of novel CTX patientsdetermination of the genotype must be done to exclude orconfirm the diagnosis.

AcknowledgementsThe authors wish to thank F. Barkhof, Department ofDiagnostic Radiology, Free University Hospital, Amsterdam,The Netherlands, for the evaluation of the MRIs, andR. P. Kleyweg (family 25), Department of Neurology, HospitalDordrecht; M. de Visser (family 18), B. M. van Geel (family23) and J. J. P. Kastelein (family 2), Academic MedicalCentre, Amsterdam; J. W. B. Moll (family 10), UniversityHospital Rotterdam; J. H. J. Wokke (family 3), Departmentof Neurology, University Hospital Utrecht, Utrecht, TheNetherlands; S. Tam (family 29), Department of ClinicalBiochemistry, Queen Mary Hospital, Hong Kong, China;R. Denays (family 26), Department of Neurology, NewPaul Brien Centre, Brussels, Belgium; P. Hart (family 27),Neurology Department, Atkinson Morley’s Hospital, London,UK; R. Perez Moyano (family 21), Almeria, Spain; T. J. Walls(family 31), Regional Neurosciences Centre, Newcastle uponTyne, UK; J. Mebis (family 28), Algemeen ZiekenhuisMiddelheim, Department of Internal Medicine, Antwerpen,Belgium; N. Miladi (family 20), Institut National deNeurologie, Tunis, Tunisia; L. van Malderghem (family 19),Institute de Pathologie et de Genetique, Gerpinnes (Loverval),Belgium; R. Kimmelre (families 30 and 32), Heinrich HeineUniversitat Dusseldorf, Klinik fur Stoffwechselkrankheitenund Ernahrung, Dusseldorf, Germany; and S. Berndt (family24), Department of Neurology, Paderborn, Germany forreferring the patients.

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Received July 15, 1999. Revised October 12, 1999.Accepted November 11, 1999