Human oculocutaneous albinism caused by single base insertion in the tyrosinase gene

BIOCHEMICAL

Vol. 164, No. 3, 1989 November

AND BIOPHYSICAL

15, 1989

HUMAN

OCULOCUTANEOUS

ALBINISM IN

Yasushi

Tomital

‘Department

’2 ,

THE

Atsushi

of Applied University

Received

September

, Shoji Shibahara’*

and Medicine,

comprises

formation

and

as

autosomal


20,000

(1).

The

O(‘A. the

life

the of

melanin

of

2Department Sendai,

two latter

the

also

type,

formation

and

Tagami2,

and

Dermatology, 980, Japan

tyrosine

in t.hr

of

and

Tohoku

tyrosin:-lscA

heritnble

to t-he

its

patients

to

pigments

[EC

catalyzes

two

and

oxidation

hydrosylase

of acti\

is

ity

to arid

:iholit

1 in

k~auserl catlr’c>l’.

tly

Ot’i\

is

tv~osinasc~-tle~~~ti\,~~

an

OC~IIL

t hroughc,llt

essential

h.vdvoz>-lation dopa

gr~tl~t~;atizc~d is

skin

nel-er

1.1.4.18.11

f~eactions:

irlht~r.itt~~i

disturhnnres

and

melanin

:-I

incidencr:

\-isual

rni~latlln

((f(‘-l), 1~)

tyrosinase-positi\-e of

of

:tlbirrism

and

leads

~:+~II(~.

disorders

c,hal*ac.trrized hairs,

tyrosinase

(dopa)

mutation

predisposes (2):

since (3)

as

Hachiro

of Miyagi

Octtlocut~irlf~~)i~s

pigments

types

dihydroxyphenylalanine to

,

such a tl.uncated t>-rosinasts I;~r~l,ing c:atal)-ticall>inacti\ rt. k‘r t hc-l,efolY~ is a conse
is

skin

major

biosynthesis

referred

eyes,

and

patients,

(1,2). trait,

melanin

defects

into In

the

lack

Okinaga’

grol~p

races

recessiv-e

nrurologic:

classified

in all

in

INSERTION

Hlhinism (OCX) is ;in iriboL.n rr’r0t’ of lack of melanin pigments iti thy 6‘~ es n~ld gene of onca affrackd r.hiid the t> posinase analysis reveals ii single-t):i:;r Seqlteric.tl reading fl.ame an&d introduces a prrn1al.ul.r P’lll~~:tiO~l~-LI the amino acid rrsidue 298.

heterogenous

found

hy-popigmentation

BASE

1989

a

is

SINGLE

GENE

t.ermination signal (TGA codon) after analysis of the mutated gene indicates that is one potential copper-binding region conclude that the albino phenotype of the inacLi\-r% tylosinase c:aused b>- the nonsense lil 198’J Academic errs*, 1°C. Albinism

BY

TYROSINASE

Takedal Shigeki

Physiology School of

26,

CAUSED

Tyrosinase-negati\-e oculocutaneous metabolism, characterized by a complete skin. Ke have isolated and characterized (S.S.) witkl tyrosirlase-negative OCA. insertion in the exon 2 that shifts the

optic

RESEARCH COMMUNICATIONS Pages 990-996

erlz? of

dopacluinontl, dopa

mr

*If

t.) rc,sinc which

oxidasr~

to :~IY

at:ti\-it

y,

~cspective1.v. Lrl

this

tgrosinase

*To

whom

Abbreviations: polymerase

study, gene

we of

have one

determined

patient

correspondence

should

OCA, oculocutaneous chain reaction.

0006-291X/89 $1.50 Copyright 0 1989 by Academic Press, All rights of reproduction in any form

Inc. reserved.

S.S.

be

the affected

nucleotide with

sequence t.y~osinase-negat.i\,r

of

the

crloncad i)(‘

\

arid

addressed. albinism;

990

dopa,

dihyd~osyphenylalanine;

I’C‘H,

Vol.

164, No. 3, 1969

found

a

BIOCHEMICAL

single-base

Moreover,

we

are

insertion able

to

in

show

AND BIOPHYSICAL

the

that

exon

such

2,

RESEARCH COMMUNICATIONS

causing

a truncated

a

nonsense

tyrosinase

mutation. is

catalytically

inactive.

MATERIALS

AND

METHODS

Preparation

ofgenomic

DNA

Peripheral lymphocytes, affected with tyrosinase-negative (4). These patients are M.T., and F.S.), and their (5,6).

Genomic

Cloning

DNA

was

and sequencing

collected identified family prepared

from were using

OCA, by histories from

ofgenomic

the

three unrelated Japanese patients transformed with Epstein-Barr virus two letters randomly chosen (S.S., indicate no consanguineous marriages

transformed

DN.4 encodin

cells.

g thehuman

tyrosinasegene

The genomic DNA library of the patient S.S. was constructed DNA segments encoding tyrosinase as described previously sequences of all the exons were determined by the method (8), except for the 3 - -end of exon 4. The exon 4 sequence polymerase oligonucleotides (complementary cycles, each thermal cycler previously

Genotype

and screened for (7). Nucleotide of Maxam and Gilbert was amplified using

chain

reaction (PCR) (9). The primers used were two 22-mer corresponding to the 5 ’ -end (1267/1288) and 3 - -end to 1427/1448) of the exon 4. The reaction was repeated for 25 consisting of 94 “C for 1 min, 50 “C for 2 min, and 72°C for 3 min using (Perkin Elmer Cetus). The amplified exon 4 was cloned as described

(7).

analysis

The primers oligonucleotides

used derived

for from

amplification wild-type

of

the tyrosinase

exon

2 cDNA

were (10):

two 22-mer 5 ’ -

ATTGTCTGTAGCCGATTGGAGG-3 ’ (902/923 ) and 5’-CTTCCAGTGTATTTCTAAAGCT-3’ (complementary to 1097/1118). A fraction of the amplified DNA (l/10 of sample) was spotted in duplicate to a sheet of nitrocellulose filter; one set was hybridized to the 32P-end-labeled normal probe of 20 mer, 5 ’ -GGAGCCTTGGGGTTCTGGAT-3 ’ (complementary to 1000/1019), the other set to the mutant probe of 21 mer, 5’GGAGCCTTGGGGGTTCTGGAT-3’, containing the single base insertion. Following hybridization at 55 “C overnight, the filters were washed for 15 min with lx saline sodium citrate (SSC) and 0.1% SDS at room temperature, and then washed for 15 min

with

the

Functionalanalysis

same

solution

at

56°C

of the albino

for

normal

tyrosinase

probe

or

58°C

for

mutant

probe.

cDNA

The expression plasmid, pRHOHT2, contains the full-length tyrosinase cDNA under the rat heme oxygenase gene promoter (7). The pRHOHTM1, containing the additional C residue between 1011 and 1012, was constructed as follows. The Safi/AvaI fragment (59/960) was isolated from the pRHOHT2 and the AvaI/NcoI fragment (960/1075+1) containing a single base insertion was prepared from the subcloned DNA carrying the exon 2 of the S.S. tyrosinase gene. Both fragments were then ligated to the larger fragment of the pRHOHT2 linearized with SalI and NcoI. The mock construct, pRHOHT0, contains the truncated tyrosinase cDNA (7). These plasmids were introduced into mouse K1735 amelanotic melanoma cells (11,12) as described previously (13). Following a 20-h incubation of transfection, cells were treated for 3 h at 42 “C and incubated for additional 16 h at 37 “C (7). The assay of tyrosine hydroxylase and dopa staining of transfected cells (dopa oxidase activity) were carried out as described previously (7).

991

Vol.

BIOCHEMICAL

164, No. 3, 1989

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

RESULTSANDDISCUSSION Cloning

and

We

structural

have

cloned

and

protein-coding isolated represented

in

1

was

residue

tyrosinase

gene

as

shown We

have

melanoma albino the

in

therefore

gene

using to

cloned

genomic

the

be

more

tyrosinase

DNA

of

the

internal

the

of

35

gene patient

S.S.

the

size

Genomic,

retains

the normal

Ilot,

(nll~leotid~

confirmed

hh.

and

.i is cnntninirrg

sitre

The

technique.

the

c‘xon

segment

Sa~3A1

thts

nlthou
the

DNA

and

than

harbol,ing S.S.,

3 I -end

cloned PCR

segments patient,

since

at

We

the

The

(Fig.l),

DNA

expected the

the

clone

DNA of

(Fig.1).

10).

gene

genomic gene

phage

tyrosinase

that

tyr.osinnse

tyrosinase

the

thus

suggests

organization not

is

albino the

phage

7 and

S.S.

the

overlapping

into

refs. the

the

isolated

cloned

of

analysis

not

the

1412;

sequence

of

are

of

sequenced

region clones

exon

analysis

of

rsxon

t.ht*

I)N.\ same’

l

hlrmarl t.llott

itIC;

seqr~e-r~~.~~

indj\-iriblal

(data

1. then

confirmed

cell-free

promoter

transcriptiorl

phenotype

of

mutation

the

in

this

its

activit)

system

patient

gene.

of

(data

is a consequence Compal?son

of

thr

not of

the

S.S. shown),

inactil-e

riucleotide

tyrosinase

I;renr

suggesting

Itsing

that.

tyrosinasc

t II?

~,;r~~sed

sequenc:r

of

h,v

t ht,

5.5.

u Single base insertion in the tyrosinase gene of one patient with tyrosinase-negative OCA. A. Schematic representation of the albino tyrosinast. gene. The direction of transcription is from left to right. Only relevant sites for EcoRI (E) and Hind111 (H) are shown. Solid lines represent the ~enomi~~ I’I~‘A segments carried by the isolated phage clones and the closed boxes indic~ate lhe exons. The phage clone carrying exon 5 is indicated by a solid line and dotkd lines, since the Hind111 fragment containing exon 5 is not localized within its insert. The nucleotide residues shown are numbered ftum t hC> of about 16 lib. transcription sequence

initiation containing

the additional the functional assigned the containing An arrowhead

C residue tyrosinase accession

single base indicates

site single

of the base

between precursor number insertion. an additional

human insertion

tyrosinase gene is enlarged.

1011

(7). An

and 1012. The nucleotide cDNA have been deposited M27160. B. Sequence ladder The sequence of the message C residue.

992

A part arrowhead

of

the t.l;on indic.ates

sequence data of in GenBank and of the frxgmerrt strand is shor*rl.

2

Vol. 164, No. 3, 1969

tyrosinase

gene

in

the

C

residue

with

patient’s

gene.

in

of

the

TGA

(amino of

acid

1012

cDNA

residues

the

for

codon frame

residues

amino

acids,

343-3851,

may

be

reveals

only

contains

one

an

proline to

at

an

aberrant

after

the

amino

of

511

amino

154-220

and

343-385)

lacking

one

of

inactive

(Fig.2).

consisting acid

298

gene

(1030/1032)

RESEARCH COMMUNICATIONS

(7,lO)

tyrosinase

reading

tyrosinase,

tyrosinase (amino

S.S.

signal,

AND BIOPHYSICAL

wild-type

the

change

regions

Genot.r;pe

and

human

truncated

In

the

termination

copper-binding

the

Namely,

in

wild-type

regions

of

1011

resulting

a premature

a

that

between

(Fig.11,

Since

BIOCHEMICAL

insertion

the

of

position one

292

containing

acid

two

mutation

residue

acids,

298. has

(ref.

lo),

copper

order

to gene,

confirm

the

we

amplified

presence the

of exon

an 2

additional

C

sequence

and

-18 -18

binding

residue carried

in

the

S.S.

out

dot

blot

MLLALLYCLLWSFQTSAG ******************

1 HFPRACVSSKNLMEKECCPPWSGDRSPCGQLSGRGSCQNILLSNAPLGPQFPFTGVDDRE 1 ************************************************************ 61 SWPS~FYNRTCQCSGNFMGFNCGNCKFGFWGPNCTERRLLVRRNIFDLSAPEKDKFFAYL 61 **********************+*************************************** 121 TLAKHTISSDYVIPIGTYGQHKNCSTPMFNDINIYDLFVWMHYYVSMDALLGGSEIWRDI 121 ************************i********************~***#***********

Top

such

analysis

tyrosinase

w

two

181 181

DFAHEAPAFLPWHRLFLLRWEQEIQKLTGDENFTIPYWDWRDAEKCDICTDEYMGGQHPT ***t*******t*:*L**:t***$*$*******:$$$*:**~**$:*****$********

211 241

NPNLLSPASFFSSW$IVCSRLEEYNS"QSLCNGTPEGPLRRNP~NHDKSRTPRL~SSADV ****************************************************KA*LFS

301

EFCLSLTQYESCSMDKAANFSFRNTL~~FASPLTGIADASQSS""NAL"~Y~N~T"SQVQ

361

GSANDPIFLLH"AFVD~IFEQWLRRHRPLQRVYPEANAPI~HNR~SY"VPF~PLYRN~DF

421

FISSKDLGYDYSYLQDS6PDSFQDYIKSYLEQASRIWSWL~AA"V~AVLTALLA~LVSL

481

LCRHKRKQLPEEKQPLLMEKEDYHSLYQSHL

Deduced amino acid sequences indicates the entire amino

line

of wild-type acid

sequence

and of

albino tyrosinase

tyrosinase. precursor,

and

the

bottom line indicates that of truncated tyrosinase of patient S.S. The amino acids are numbered beginning with the amino-terminal residue of mature tyrosinase (10). The preceding residues of a putative signal peptide are indicated by negative numbers. Asterisks indicate the identical amino acids. Two potential copper-binding regions (10) are underlined and a putative transmembrane segment is double-underlined. Small vertical lines indicate the locations of introns: intron 1 between nucleotide residues 901 and 902; intron 2, 1118 and 1119; intron 3, 1266 and 1267; and intron 4, 1448 and 1449.

993

a

Vol.

164, No. 3, 1989

hybridization amplified

an

to

the

(Fig.3).

derived

from

control

insrvtiorr

of

homozygous and

analysis DNA

onl?;

obligate

rrormal

for

and

I)atient t> rosinase

gene

OC,I

indicating

‘To 292

0C.A

In

OCA

is

ensrrre

that

the

a molecular

the

of

gene.

Mouse

expression

plasmid

pRHOHT2

tyr-osinase

cDNA

with

amelanotic: (data

the

(7) insertion

melanoma not

shown)

and

Pedigree

coding of we

of

as

t.he

patitxnt

same

well

the

IrNA

t?f. ;.it’t’

1%it h tbo( h of

~IIII!

indic:ititr$

mrrtation

as indil

cor~r~esl~orrdin:;

a

trer,

r !I. 2

Lkr:+i

tt P

t tr:tt

itltr;+l


,Jositirjtr

t trr

(Eic.A),

of

tyrosinase

the

cells entire

no

4

the

()

region same

101 1 and

inse1.t 1012

levels C)nl>-

of

ixansfectrrl

protein-coding

(Fig.4B).

@

1012 in thtl c.c!tlorl analysis

weye

detect,able

activit,v

rind

1011 functional

between

contain

@Oe@

the

containing

residue

l

t.,t-osirtast’

tjet.ween

melanoma

C

used

albino

out.

pRHOHTM1

ceils

of cells

NORMAL

PROBE

MUTANT

PROBE

the

S.S.

wi1.h

t.lre

of :ts

(Fiq.4). tyr.osinase

hum:~n

pRHOH1

Analysis

of

Patients

Square albino

with

mRqA

t.~ansfr~<:trd

OCA.

r.epresents male and circles indicate female. Filled symbols indicate the phenotype and partially filled symbols indicate heterozygote car-met< for. OC.4 allele. Amplified DNAs were derived from father of S.S., 1; mother of S.S., 2; patient S.S., 3; sibling of S.S., -1; patient. M.T., 5; patient P.S., 6; and normal individual, 7.

994

2,

“lclrrse

234561 Genotype

1.

to

t-1~1, sit)lrng

hyblidizetl

normal

i.5

r~ot.mal

Ihrs

r~pt~.t(.d

as

pr‘obe,

the

S.S.

L?-:~J (1. I?..;

are

DNA

same

the

the

carried

containing

(I

and

fat

a C residue

OCA,

0

1 Fig.3

cDN4

amelanotic

01‘ with

in

the

~orttuin~?

of

parents

genotrric.

of

at

the

to

h~~;hr~idi:?t~i

indeed

segregation

mr~t.ant

gene

mutation

gene

S.S.

tire

only

probe

that,

her

ttre

contains

hybridize

heterogenorrs.

insertion

basis

tyrosinase

but

no

normal

genomic

the

tyrosinase

genetically of

with

the

pn~ents

their

Y.T.

contains

t r.rpt,ession

is

patient the

F.S.

patient

,Mor,eo\-et,,

only

contrast,

patient

).

to

the

krer

becarrse

able

and

notmal, Itrdeed,

allele,

1012

the

RESEARCH COMMUNICATIONS

ryvosinase

analysed

of

(Fig.3

the

S.S. and

nest

trait.

hybridized of

that

‘I‘t,attsiett

@CA

the

1011

family

was

w hereas

phenotypicall>-

probes

also

S.S..

thi1.d

the

the

S.S.,

We

the

is

mutant.

M.T.

patient.

in

probe

that

allele.

S.S.

of

patient

between

OCA

of

r,aYriers

hrtet~oz>--yous

the

AND BIOPHYSICAL

mutant

indicating

alleles

family-

The

residue

the

OCA

the

DNA, C

for

mutant

Sirrce

BIOCHEMICAL

xrth

Vol.

164, No. 3, 1989

BIOCHEMICAL

(PSI . . . . . .

HSE

I

Exon

I)

Aval

I

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Ecor?

(Ndel)

Ncol

Tyrosinase

ExonS pRHOHT2 pRHOHTM1 pRHOHT0

E3

*N.D.

.*,*.

NOlIt?

pRHOHT2

pRHOHTM

1

pRHOHT0

Fig.rl Transient Expression of the Tyrosinase Gene of Patient, S.S. A. Schematic representation of the expression plasmids. Solid lines, 5 . - and 3 ’ -flanking regions of the rat heme oxygenase genr (13); closed box, heat, s11c~cl~ element; arrowhead, TATA like sequence; stippled bo.xes, parts of exons 1 and 5 of t,he rat heme oxygenase gene; and open box, full-length tyrosinase clJNA. I<. Functional analysis of tyrosinase encoded by the wild-tj-pe or mlctant gene. Mouse Ii1735 amelanotic melanoma cells were tzansfected with the indical.ccl expression plasmids, and then assayed for tyrosine hydroxylase activity (oi~rl box) or dopa osidase activity (closed box). Shown is one of two indeprndtant experiments, which are of essentially identical results. Basal activity was measured in untransfected cells (indicated as None). Asterisks ind icwtc t hi> tyrosinase activity, that is not detectable (N.D.).

the

wild-type

construct

hydroxylase.

Moreover,

melanin-deposited of

transient

tyr.osine transfpcted

(pRHOHT2)

(dopa

about

w-ith

under activity

the

detectable

of

t.ransfected

oxidase-positive),

expression hydroxylase

1.4%

showed

construct

which

similar nor

cells

dopa

is

conditions osidase

carrying

the

995

activity with

consistent (14).

activity mutation

with In

was

of

tyrosinr

pRHOHT2 the

efficient?

contrast, detectable

(pRHOHTMI

:IP

neit.hrr in ) or

the

of c.ells

wit 1-1 t ht,

Vol. 164, No. 3, 1989

mock

construct

(pRHOHT0)

expression

of

showing

the

each

(data S.S.

found

all

in

the

similar

shown).

unable

clonsidering

to these

albino

patients

the

albino

trait

AND BIOPHYSICAL

the

untransfected

amount

for

the

(S.S. carrier

and and

for

transient nnalysii,

from the

the

plasmid

mutated

DNA

qcsrlfx

of

ttle

tyrosinase.

a molecular

screen

The

nuclease-mapping

that

conclude

that

basis

The

(None).

transcribed

functional

M.T.).

Sl

indicate

we

is

RNA

results

observations, gene

by

of

RESEARCH COMMUNICATIONS

cells

confirmed

These

code

tyrosinase

two

in was

of

not

is

or

construct

presence

introduced patient

BIOCHEMICAL

data

the

of

the

bastt

insertion

tyrosinase-negat.ive

presented

same

single

will

mutation

in

Of’A

enable

IIS

other

to

albino

in

drtret,t

patient:;.

ACKNOWLEDGMENTS We

thank

Drs.

introduction virus,

of and

and

K.

Dr.

Mr. H.

supported Clllture

K.

S,

patients Maeda

Yonekura in

of

the

Sonoda, to for for

part

by

C.J.,

Nance,

Kondo, us,

Dr.

technical

to

H.

Sato, Tohda

for

from

Iijima, the

and

N.

supply

of

thank

Prof.

We also

synthetic Y.T.

S.

assistance.

providing

grants

Y.

oligonucleotides. the

Ministry

lto

H. Okrzmot

Education,

world Science

Japan.

REFERENCES 1.

Witkop

2.

GeIlet* Witkop,

Jr,

22, 55-74. C.J., Quevedo,

W.E., W.C.

Rawls,

R.F.

& Fitzpatrick,

& White,

J.G.

T.13.

(1983)

Basis of Inherited Disease (Stanbury, J.B., Wyngaarden, Fredrickson, D.S., Goldstein, J.L. & Brown, M.S., eds) McGraw-Hill, New York. 3. Lrrner, 4.B., Fitzpatrick, T.R., Calkins, E. & Summerson, Biol. (‘hem. 178, 185-195. -1. Tohda, H., Oikawa, A., Katsuki, T., Hinuma, Y. & Seiji, Kes. 38, 253-256. 5. Sonoda, Ii., Nagao, S. & Iijima, S. (1978) Rinsho Hifuks Japanese). 6. Migamoto, L)ermatol. 7. ‘T‘aketia, 8. 9. 10. Il. 12. 13. 14.

C., Kawada, A., Mito, Y. & Ohtaki, 49, 260-265 (in Japanese ). A., Tomita, Y., Okinaga, S., Tagami,

N. ( 1987

(1970) in

Am. The

J. HI/~.

.MetahcG~.

J.B., pp. 301-346, W.H. M. 32,

.J.

Cancet

9-16

1 Nishi-Nihon

H. & Shibahara,

(1949)

(1978)

ttle

Einstein-Bar.1

This

of

for

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