Multiple populations of deleted mitochondrial DNA detected by a novel gene amplification method

Vol. 162, No. 2, 1989 July 31, 1989

AND BIOPHYSKAL

BIOCHEMICAL

RESEAFICH COMMUNKATIONS Pages 664-672

MULTIPLE POPULATIONSOF DELETEDMITOCHONDkIALDNA DETECTEDBY A NOVELGENEAMPLIFICATION METHOD Wataru Sato, Tomoko Yamamoto, Department

Akita Received

June

of Biomedical Chemistry, Faculty of Medicine, University of Nagoya, Nagoya 466, Japan *Department School

University

9,

Masashi Tanaka, Kinji Ohno, Gore Takada*, and Takayuki Ozawa

of Pediatrics, of Medicine, Akita

010,

Japan

1989

method for detecting small populations of SUMMARY: A gene amplification deleted mitochondrial DNA was used in analysis of skeletal muscle from a patient with ocular myopathy. Multiple populations of differently deleted mtDNA were detected in the patient muscle. The presence of deleted mtDNAs was further confirmed by comparison of the shift in the sizes of the amplified fragments with the shift in the positions of the primers used for the amplification (the primer shift PCR method). Other methods, namely Southern enzymic activity measurement, and Western blotting, were inefficient blotting, These findings suggest that the at detecting the mitochondrial abnormality. primer shift PCR method could be valuable for accurate diagnosis of ocular myopathy associated with mtDNA deletion. 0 1989 Academic Press, Inc.

Mitochondrial which

play

enzyme

a central

complexes

system

mtDNA are

first

inheritance The

(51. hereditary

process

mtDNA mutations

with

myopathy

several bIot

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

and is

human

(5,9-ll),

has

an

the

has

has

been

important

used

664

for

phosphorylation

inherited for from

been

our

both

maternal laboratory

with

proposed

cytoplasmic

(3), diseases

the

families

contributor

diseases

encephalomyopathy

Inc. reserved.

also

subsequent

degenerative

in

multisubunit

transmitted

presented

reported

It

four

responsible

been

also

polypeptides,

oxidative

of maternally

mtDNA is

(6,7).

life

analysis

was

the

of

maternally

cause

mutant

hydrophobic

functions

exclusively as the

inheritance

during

thirteen

and V) of

myopathy

of

and to

is

the

neuropathy

mutations

Southern

that

optic

accumulation

for

IV,

implicated

mitochondrial

maternal

III,

I, mtDNA

report

of

encodes

in energy-transducing

Because

of The

(4).

role

(Complexes

(1,2).

mutations

these

DNA (mtDNA)

Leber's that

the

segregation

of

to

the

aging

(8). analysis

(12),

of and

mtDNA

pancytopenia

in

patients (13).

Vol. 162, No. 2, 1989

Heteroplasmy with

of

the

normal-sized

mitochondrial

myopathy, in

BIOCHEMICAL

some

with the

different

in etiology

at

suggested

the

be an effect with

order

genetic

level,

reaction

(PCR),

small

a novel

which

named

positions method

undetectable

of of

of the

shift

primers for

deleted

used

in for

detecting

by the

conventional

it

primer This

has

Zeviani

possible

blot

based

et

blot

myopathy the

fragments

of

study,

deleted

method,

(11) may

are

too

at

the

method.

polymerase

confirms

this

are

some patients

PCR method,

method

been

al,

mtDNA which

on

shift

not

syndrome

that

mitochondrial

In

populations Southern

it

method

mtDNA deletions

deleted

amplified

amplification. small

is of

method

of

mitochondrial

mt.DNA in Kearns-Sayre

of

mtDNA. sizes

Thus,

levels.

Southern

the

in patients

conventional

discrete

populations

we developed we have

(9,ll).

However,

etiology

with

by the

without

of

conventional the

patients

and genetic

deletions

have

establish

some

undetectable

and

a cause.

RESEARCH COMMUNICATIONS

mtDNAs is observed

myopathy

with

that

by the

populations

comparison

this

to

are

molecular

myopathy

to be detected In

the

than

mitochondrial

small

cases

possibility rather

In

mitochondrial

whether

deleted

(5,9-11).

mtDNA deletions

patients

established

and the

myopathy

however,

AND BIOPHYSICAL

for the

with

chain detecting

deletion the

shift

we have mtDNA,

in a patient

by in

employed which

with

are ocular

myopathy. PATIENT

AND METHODS

A 55-year-old female had opbthalmoplegia from age 45. She had Patient. proximal muscle weakness but no other neurological abnormalities. Her mother Serum lactate and pyruvate levels were and matern,al grandmother had ptosis. normal. Histochemistry of the biopsied skeletal muscle showed no typical namely, type-2 fiber atrophy ragged-red fibers but only mild myopathic changes; and subsarcolemmal mitochondrial accumulation. Southern blot analysis. Total DNA was extracted from 17 mg of the frozen biopsied muscle. DNA (100 ng) was digested with 12 units of BamHI and PstI Japan and separated electrophoretically on 0.6% obtained from Toyobo, Osaka, phage DNA digested with agarose gels. Size standards employed were lambda Hind111 and phage Xl74 DNA digested with IfaeIII from Nippon Gene, Toyama, Japan. DNA in the gels was denatured and transferred onto GeneScreenPlus membranes from Du Pont-NEN. Hybridization was carried out as described previously (5). Enzymological analysis. Mitochondria were isolated from the biopsied frozen skeletal muscle according to the method of Hookelman et al. (14). NADH-ubiquinone oxidoreductase Knzymic activities of rotenone-sensitive succinate-ubiquinone oxidoreductase (Complex II), ubiquinol(Complex I), cytochrome c oxidase (Complex IV), cytochrome c oxidoreductase (Complex III),

665

Vol. 162, No. 2, 1989

BIOCHEMICAL Table

Primers*

1.

AND BIOPHYSICAL

Synthesized

Sequence

5'+

primers

RESEARCH COMMUNICATIONS

used for

3'

PCR

Complementary

L 790 L 820 L 853

TGAACCTACGAGTACACCGA TTCATGCCCATCGTCCTAGA ACGAAAATCTGTTCGCTTCA

Hl338 Hl363

TCTTGTTCATTGTTAAGGTT GGGGAAGCGAGGTTGACCTG

7901 to 8201 to 8531 to

site**

7920 8220 8550

13381 to 13400 13631 to 13650

*Primers L790, L820, and L853 were used for amplification of the light strand of mtDNA. Primers Hl338 and H1363 were used for amplification of the heavy strand of mtDNA. **Numbering of mtDNA is according to Anderson et al. (1).

and antimycin-sensitive ATPase (Complex V) were Protein was measured by the bicinchoninic (151. et

al.

measured as reported acid method according

previously to Smith

(16).

Uestern blot analysis. Subunits of Complexes I, III, IV, and V were analyzed by the Western blot method using the specific antibodies against bovine holoenzymes, essentially accordin52v the method of Tanaka et al. (17). Antibody binding was detected with [ Illabeled protein A (Amersham, Backingamshire, UK). Polymerase chain reaction. Fragments of mtDNA were amplified from 10 ng of the total DNA in 100 ul of reaction mixture containing 200 UM of each dNTP, 1 UM of each primer, 2.5 units of Taq DNA polymerase from Perkin Elmer Cetus, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgClz, and 0.01% gelatin. The reactions were carried out for a total of 35 cycles with the use of a Thermal Cycler supplied by Perkin Elmer Cetus. The cycle times were as follows: denaturation 15 set at 94OC; annealing, 15 set at 45'C; primer extension, 60 set at 12'C. The amplified fragments were precipitated with ethanol and separated by electrophoresis on 1% agarose gels and detected fluorographically after staining with ethidium bromide. Primers used for PCR are listed in Table 1. They were synthesized using a Shimadzu model NS-1 DNA synthesizer or an Applied Biosystems model 380B DNA synthesizer and purified with NENSORB PREP Cartridge from Du Pant-NEN. RESULTS The Southern showed

only

a single

(Fig.

1A).

14.5

kb and

could

be

blot

When 2.1

band PstI

kb

detected

of

the

patient

of

16.6

was used

fragments by

the

skeletal

muscle

kb fragment as the

were

produced

restriction

observed

conventional

DNA

digested

from

1B).

Southern

only

two

No abnormal

blot

BandI

normal-sized

enzyme,

(Fig.

with

method

in

mtDNA bands

of

fragments the

patient

muscle. The skeletal were

enzymic muscle

within

activities mitochondria

normal

ranges.

of are kSterh

the

oxidative

shown

phosphorylation

in Table

blot 666

analysis

2.

All for

the subunits

complexes activities of

in

the

measured Complexes

I,

Vol,

162, No. 2, 1969

BIOCHEMICAL c

IV,

and V demonstrated

the

patient

mitochondria

In

PCR,

extraneous due

to

principle

use

of

deletion

is

is

of

of

distance amplified

from

The in

between

the

shift positions

in

to

ascertain to

an

were

results

that

decreased

not

patient

in

in

amplification

an amplified

unexpected

a fragment

primers

L and

by the

of

fragment

position

deleted the of

the In

primers.

is

the

mtDNA by the

sizes

of

the

primers

amplified

of

primer

from

H surrounding

by subtracting the

the

suhunits

sometimes

mtDNA in the

experiment,

a pair

the

is

not

mtDNA,

shift

we

method,

the

as follows.

can be obtained

and H. shift

first

primers

primers

deletion

of which

In the

the

of

of

2).

In order

misannealing the

amounts

of

fragments.

identified

the

misannealing

P

of mtDNA. A: Digestion with L?a#dlI. This enzyme producing a single linear fragment of 16.6 kb. enzyme cleaves mt.DNA at positions 6,910 and in the control (C) or in the patient (P).

that (Fig.

RESEARCH COMMUNlCATlONS c

P

Fig. 1. Southern blot analysis cleaves q tDNA at position 14,258 B: Digestion with Pst.1. This 9,020. No extra bands were seen

III,

AND BIOPHYSICAL

the

size

of

second use

The

the

amplified

fragment

another fragments

from

L'.

667

mtDNA by

deletion.

experiment, of

deleted

the

amplified L to

the

If

another pair should

the

sizes

of

size

of from

fragment primers

parallel of

deletion

L' the

Vol.

BIOCHEMICAL

162, No. 2, 1989 Table

2.

Enzymic activities

Enzyme

in

Patient

Complex Complex Complex Complex Complex

I II III IV V

from

356

(95) (801

amplified

fragment

presence

fIl338

of

the

In the

first

(position

is

3A,

lane

lane

P).

I

316 244 477 285 80

-

570 408 963 592 174

of mitochondrial

it

identical, of

the

is

protein.

concluded

primers

but

that due

to

the the

mtDNA.

kb

also

Range

(6) (6) (6) (6) (6)

misannealing

the

(Fig.

3A,

to

13,381-13,400),

control

(Fig.

due

by using

a 5.2

hut

not

are

experiment,

only

fragment

in nmol/min/mg

experiments

deleted

amplify

kh

two

(n)

& 93 k 68 t 183 k 113 t 34

421 319 777 443 125

RESEARCH COMMUNICATIONS

muscle mitochondria

skeletal

Control*

(85) (72) (74)

229 571 419 99

the

the

(%)

Activities are expressed *Values are means t SD.

calcuLated

AND BIOPHYSICAL

fragment C).

In

primers distance

derived patient,

fragments

with

the

The

of

the

(position

between from

the

order

1820

which

normal-sized

we could

amplify 1.1,

of

1.3,

amounts

of

the

5.2

kb,

and we could

mtDNA

in

the

not

only

the

5.2

0.95,

and

amplified

0.75

fragment

V

IV

c

is

the

sizes

Ill

8,201-8,220)

P

c

P

Fig. 2. Western blot analysis of the subunits of Complexes I, III, IV, and V. C: control mitochondria (15 ug protein); P: patient mitochondria (15 ug protein). I: Complex I; Molecular masses of the detected subunits of Complex I are indicated in kDa. III: Complex III; Core, core proteins; ISP, ironsulfur protein. IV: Complex IV; IV and V, subunits IV and V. V: Complex V; a and 9, subunits a and 6. 668

kb was:

Vol.

162,

No.

2,

1969

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Fig. 3. Detection of multiple populations of deleted mtDNA by the primer shift PCR method. A: PCR amplification using primers L820 and Hl338. B: PCR amplification using primers LB53 and Hl336. Sizes of amplified fragments are indicated in kb. A single band derived from the normal-sized mtDNA is seen in the control (C). Four additional bands derived from the deleted mtDNAs are seen in the patient (P).

0.75

kb

> 1.3

(position

the

kb > 0.95

In

kb.

4.9

the

control,

fragment,

kb

amplified. kb > 0.8

populations

of

present

in

parallel

pairs

of

between 1.9,

to

1.5,

1.3

which

were

is

5.75

kb

amplify

fragments

indicates

that

from

for

kb,

we could

the

identified with three

sizes

of

1.0,

0.8,

of

the

resulLs

amplified

the

mtDNAs,

among these

mutant

mtDNA deletions using

abnormal

previous of

2.3,

populations 669

L790

of

addition

to

and

kb

0.45

was:

at

0.45

least 4.45

amplified

this

and

four kb are

fragments

with

a 4.45

patient,

Hl363,

fragments

with

of

3.9, In

experiments. and

normal-

kb

mtDNAs.

deletions

0.8,

the

and

mtDNA

in

primers

amplify

that

of

the

them

in

4,25,

amounts

between

fragment

4.1,

3.9,

L853

0.65,

demonstrate

of

primer

from

the

in the

additional

derived patient,

mtDNAs with

sizes

distance

the

mutant

the

the

In

deleted

other By

used.

HI33EI;

C).

that

population

experiment,

fragment

deletions

the

second

lane

These

kb.

of

kb

amounts

Assuming

search were

the

mtDNA with

largest

primers which

> 0.65

populations

order

with

the

primer

a 4.9 38,

of

patient.

had the In

kb

mutant

the

the

deletion

(Fig.

fragments

In

with

only

The order

kb > 1.0

kb.

was coupled

mtDNA was amplified

were

kb,

> 1.1

8,531-8,550)

was 4.9 sized

kb

0.65

mtDNA with

kb

the

distance

the

sizes

4.25,

addition, (Table deletions

various

of

and 4.45 we could 3). of

This 3.45,

Vol.

162, No. 2, 1989 Table

BIOCHEMICAL

3.

Distance between primers tkbl

Fragment

combination

size

in

Deletion

size

fkbl

Ckbl

L820 t H1338

5.2

5.2 1.3 1.1 0.95 0.75

3.9 4.1 4.25 4.45

L853 t Hl338

4.9

4.9 1.0 0.8 0.65 0.45

3.9 4.1 4.25 4.45

L790 t H1363

5.75

5.75 2.3 1.9 1.5 1.3 0.8 OS65

3.45 3.9 4.25 4.45 4.95 5.1

The PCR condition

and 5.1 the

gene

ended

within

the

detect

is described

kb were

within

not

RESEARCH COMMUNICATIONS

Fragment size as a function of primer amplification of deleted q tDNA

Primer combination

4.95,

AND BIOPHYSKAL

for

present

in

either

gene

deleted

the

CO2,

for

in Patient

and Methods.

patient

ATPase

tRNA-Lys,

ND5.

Using

These

t,is.sue.

other

6,

deletions

ATPase

8,

of

primers,

combinations

started

or

COl,

and

we could

mtDNA. DISCUSSION

The patient etiology

so far

methods muscle only

reported as the

(Figs. showed

1,

2 and Table

and

immunochemical

and

the

of

minimal

in

patient

primer

shift

deleted

mtDNA (Fig.

of

The (Fig. that

the

was able

3) which

could

of

deficiencies

detect

not

be detected

Fig.

multiple by the

2). small

patient

In

but no

enzymological

enzymic

phosphorylation 2 and

the

detected

the of

unknown

myopathy

analysis

Furthermore,

to

670

blot

of

conventional

mitochondrial

Southern

oxidative (Table

by the

examination of

1). the

myopathy

was analyzed

characteristic

mitochondria

PCR method

as ocular

The histological

tissue showed

subunits

patient

2).

tissue

changes.

analyses

defects the

the

muscle

fibers

myopathic

mtDNA in

had been diagnosed

biopsied

no ragged-red

nonspecific

abnormal

here

activities

complexes

were

contrast,

the

populations

conventional

of Southern

Vol. 162, No. 2, 1969

blot

method.

definite

The

novel

mitochondrial

both

significant

that

daughter.

In

disease

is

might of

study

led

deletion

to

demonstrated

mitochondrial daughter,

were

patient

has

RESEARCH COMMUNICATIONS

that

this

patient

has

a

abnormality.

deletions

maternally

have

not

but

while

identical

are

here,

inherited.

(5).

This

the

were

the

mtDNA,

family

and

found

in the

deletions

overlap

to

observation

is

first

transmitted

A small of

deletions these

genetically

presented

instability

myopathy,

from

history

discrete

suggests

mutation

and consequently

in

a higher

the

a

mother

to

that

the

the

mtDNA

frequency

mutations.

In conclusion, primer

and they

the the

method

of

mother

extent,

evidence

AND BIOPHYSICAL

gene

In a previous mtDNA of

BIOCHEMICAL

shift

the

PCR method

clinical

diagnosis

molecular

genetic

detection seems

of to

of

mitochondrial

basis

of maternal

small

populations

be a promising diseases inheritance

of novel

and

for

deleted

mtDNA by the

approach

for

accurate

elucidation

in mitochondrial

of

the

diseases.

ACENOWLEDGMENTS We thank Prof. Hiroko Yamamoto and Dr. Hiroshi Koga, Department of Neurology, Fujita-Gakuen Health University School of Medicine for permission to study the patient tissue and Prof. Matean Everson, Language Center, University of Nagoya for advice on language and style. This work was supported in part by the Grants-in-aid for General Scientific Research (62570128) to M.T. and for Scientific Research on Priority Areas (Bioenergetics, 01617002) to T-0. from and Culture of *Japan and by Grant 01-02-39 the Ministry of Education, Science, from the Ministry of Health and Welfare, Japan, to T.O. REFERENCES 1. Anderson, S., Bankier, A.T., Barrell, B.G., de Bruijn, M.H.L., Coulson, Nierlich, D.P., Roe, B.A., Sanger, F., A.R., Drouin, J., Eperon, I.C., A.J.H., Staden, R., and Young, I.G. (1981) Schreier, P.H., Smith, Nature 290, 457-465. 2. Chomyn, A., Mariottini, P., Cleeter, M.W.J., Ragan, C.I., Matsuno-Yagi, A., Hatefi, Y., Doolittle, R.F., and Attardi, G. (1985) Nature 314, 592-597. 3. Giles, R.E., Blanc, H., Cann, H.M., and Wallace, D.C. (1980) Proc. Natl. Acad. Sci. USA 77, 6715-6719. J. (1983) N. Engl. J. Med. 309, 142-146. 4. Egger, J., and Wilson, 5. Ozawa, T., Yoneda, M., Tanaka, M., Ohno, K., Sato, W., Suzuki, Il., Nishikimi, M., Yamamoto, M., Nonaka, I., and Hcrai, S. (1988) Biochem. Biophys. Res. Commun. 154, 1240-1247. 6. Wallace, D.C., Singh, G., Lott, M.T., Hodge, J.A., Schurr, T.G,, Elsas, L.J.11, and Nikoskelainen, E.K. (1988) Science 242, Lezza, A.M.S., 1427-1430. 7. Yoneda, M., Tsuji, S,, Yamauchi, T., Inuzuka, T., Miyatake, T., Horai, S., i, 1076-1077. and Ozawa, T. (1989) Lancet 8, Linnane, A.W,, Marzuki, S., Ozawa, T., and Tanaka, M. (1989) Lancet i, 642-645. 671

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162,

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1969

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

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