Mitochondrial DNA mutation in a Chinese family with myoclonic epilepsy and ragged-red fiber disease

Mitochondrial DNA mutation in a Chinese family with myoclonic epilepsy and ragged-red fiber disease

Vol. 174, No. February 3, 1991 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 14, 1991 Pages Mitochondrial DNA Mutation Epilepsy and...

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Vol.

174,

No.

February

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

14, 1991

Pages

Mitochondrial

DNA Mutation Epilepsy and

Kwang-Dar

Shih,

Department

Received

Tzu-Chen

of

Yen,

Biochemistry, Taipei

December

17,

in a Chinese Family with Ragged-Red Fiber Disease Cheng-Yoong

Pang,

and

National Yang-Ming 11221, Taiwan, R.O.C.

1109-1116

Myoclonic

Yau-Huei

Medical

Weil

College

1990

We analyzed the mitochondrial DNA of blood cells of 5 patients from a Chinese family with myoclonic epilepsy and ragged-red fiber disease. The results showed that in all the affected individuals there was a point mutation from A to G at the 8344th nucleotide pair, which was located in the tRNALys No such a mutation was found in mtDNA of either unaffected gene. members of that family or other healthy Chinese subjects. These findings are consistent with the recent report of Shoffner et a. (Cell 1990, 61:931-937), and confirm that the 0 1991ACademlc Press,I point muta?ion is indeed the cause of this disease.

Myoclonic maternally

epilepsy transmitted

mitochondrial maternal the

inheritance

simple In disease

'To

at

disease(3). and

of

creates

the

molecular

diagnostic

clinical

aspects,

with

alters

for

MERRF is

the

(1,2).

The

origin

site, the

of

of

the

that

an

associated

TYC loop

of

which

the

a rare

tRNALys

provides

involves

abnormalities

in the

whom reprint

requests

and correspondence

should

maternally central

inherited

nervous

A

with

disease.

that

Abbreviations : mtDNA, mitochondrial reaction; MERRF, myoclonic epilepsy

in

IV

has indicated

a

defects

sequence

8344 is

restriction test

I and

nucleotide

position

(MERRF) is

mitochondrial

patients

nucleotide

a CviJI

by the

from

The mutation

disease

Complexes

explained

DNA (mtDNA)

G transition

gene

is

fiber

associated

transfer

Analysis

mitochondrial

this

disease

electron

disease.

to

and ragged-red

system

be addressed.

chain DNA: PCR, polymerase and ragged-red fiber disease.

a

Vol.

174,

No.

and

skeletal

was

made

3, 1991

on the

microscopic

defects

The in

ataxia,

suggestive

though only

and present

of

the

3,

4L,

inherited

three

(cytochrome

mtDNA

c

subunits

oxidase),

(COI,

and two

a large

(13,14).

All

the

assigned

to

specific

numerous

of

these

According

the

characteristics

a hypothesis

mutation

in

this

group

the

8344th

one has

of

a small

mtDNA

matrix

of

for I

genes, of

the

in

1988

that

the

rRNA

or

III)

pair

In

and

in

the

the

I

1110

and

of

of

and

the

the

22

tRNAs

have

been

and

most

IV(14,15). et --

a1.(2)

result

of

mtDNA. G

8)

addition,

be the

an A to

c

6

Wallace

in

of

of

IV

largest

above,

mtDNA

b)

Complex

products

genes

(NDl,

of

a set

MERRF could

identified

subunits

cytochrome

Complexes

tRNA

of

mitochondria

(ATPase

and

mentioned

thousands

(cytochrome

and

rRNA

genome

(rotenone-sensitive

subunit

II,

highly

human

seven

translation

subunits

successfully

nucleotide

the

contains

subunits

mitochondrial

are

of

ATP synthase). and

including

patients(4).

ubiquinol:

V (oligomycin-sensitive encodes

proposed

one

(antimycin-sensitive

III

the

Complex

oxidoreductase),

oxidoreductase),

Complex

respiratory

made up

considered

cell

codes

is

clonic-tonic

are

the

certain

associated

generalized

Each

DNA in

of

with

symptoms,

half

in maternal

and

component .

and and

(3)

severity

and

than

DNA(mtDNA)

6)

5,

and

variable

atrophy

less

signs

associated

signs

DNA(lO,ll)

NADH:ubiquinone Complex

of

optic

mitochondrial

4,

is

MERRF

abnormalities

;

and

in

maternally

clinical

phosphorylation

epilepsy

(12) * The mitochondrial 2,

syndrome

of

biochemical,

skin

dysrthria,

mitochondrial

copies

the

neurological

Deafness

of

mitochondrial

oxidative

other

seizures.

the

or

clinical

dementia,

The

of

COMMUNICATIONS

diagnosis

constellation

patients

myoclonus

various

RESEARCH

blr histochemical,

mitochondrial

progressive

with

(1)

muscle

inheritence.

is

of

confirmation

skeletal

BIOPHYSICAL

The conventional

basis

(2)

electron the

AND

muscle(l,4-9).

symptoms:

of

BIOCHEMICAL

Recently,

transition patients

a

at from

Vol.

174,

three

3, 1991

BIOCHEMICAL

independent

like of

No.

to

report

a Chinese

A to

our

molecular

family

G transition

with at

individuals.

Shoffner

et of

BIOPHYSICAL

MERRF pedigrees(3).

affected

result

AND

studies MERRF.

nucleotide These

d.(3)

and

deficiencies

in

In

support

this

The pair findings the

results

COMMUNICATIONS

paper,

on mtDNA's

mitochondrial

MATERIALS

RESEARCH

from

five

showed

8344

in

energy

an

mtDNA's

confirm notion

we

the

that

would members

identical of

all

report

MERRF

is

the of the

metabolism.

AND METHODS

Prewaration of patients mtDNA from blood cells Whole blood was collected in either sodium EDTA or acid citrate dextrose after obtaining informed consent from the patients. An aliquot of 50 ~1 whole blood was removed for immediate DNA extraction and the remainder of the sample was spotted onto a filter paper and allowed to air dry. The 50 1.11 whole blood was added with 500 ~1 of lx TE buffer and washed for three times before centrifugation at 5,000 g for 30 sec. After removal of the supernatant, the pellet was added with 100 ~1 of lx K buffer (50 mM KCl, 2.5 mM MgC12, 0.5% Tween 20, lo-20 mM Tris-HCl, pH 8.3, and 100 pg/ml of fresh proteinase K), and incubated at 56" C for 1 hr. These samples were then heated at 94°C for 10 min to inactivate proteinase K. The final product was frozen at -20°C until use. Amwlification of desired mtDNA by PCR techniuue Oliqonucleotides (sense 20 mer between 7,583 and 7,602 and anti-sense 20 mer between 10,006 and 10,025) were prepared by use of a DNA synthesizer (Applied Biosystems, Inc.). About 100 ng of the mtDNA segment encompassing the structural genes of cytochrome and ATPase subunits 6 and 8 was amplified by the PCR -Ib tRNALyS technique ;sing a DNA amplification system (Perkin-Elmer/Cetus). Ten ~1 of the 10x PCR buffer and 16 yl of the Lambda dNTP's stock solution (final concentration for each dNTP was 1.25 mM) were added to an eppendorf vial. An aliquot of 2 ~1 of mtDNA solution from whole blood of the patient was added in an 0.5 ml eppendorf vial and the synthetic primer was added to a final concentration of 50 pmol for each primer. Doubly distilled water was added to adjust the final volume to 100 ~1. One-half pl(2.5 units) of the AmpliTaq DNA polymerase (Perkin Elmer/Cetus AmpTM kit) was introduced and the solution was spun down at 3,000 rpm for 3 min so that Taq DNA polymerase was thoroughly mixed with the oligonucleotide and template DNA. An aliquot of 30 to 50 1.11 mineral oil was layered on the top of the aqueous phase to prevent evaporation. The vials were placed in a Perkin-Elmer Cetus DNA thermal cycler to carry out PCR. Each reaction cycle consisted of 2 min at 95"C, 1 min at the annealing temperature of 55"C, and 2 min at 72°C for extension. A total of 30 cycles were performed for each run that was usually completed within 4 hr. Analysis of the amplified mtDNA was immediately carried out after the PCR cycle had been completed. An aliquot of 3 ~1 of each reaction mixture was electrophoresed in a 0.8% agarose mini gel and stained with ethidium bromide to detect the efficiency and fidelity of amplification. Each of the PCR operations was repeated at least once for the confirmation of the PCR fidelity. 1111

Vol.

174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Preparation of asvmmetricallv amplified mtDNA template After the symmetrical PCR was carried out as described above, the PCR products containing desired mtDNA segment were removed and checked by agarose gel electrophoresis. A fraction of the amplified mtDNA segment was used as the template to carry out the asymmetric PCR. The protocols were the same as aforementioned except that 0.3 pmol sense primer and 30 pmol antisense primer were used instead, and a total of 35 cycles were performed. DNA seauencinq The mtDNA was sequenced directly using asymmetrically amplified mtDNA template, a synthetic primer (from 8,150 th to 8,166 th), 7-deaza-2'-deoxyguanosine 5'-triphosphate, and Taq DNA polymerase. The primer was annealed with the ssDNA template in an approximately 1:l molar ratio. For each set of four sequencing reactions, the following reagents were mixed with sterile H2 to a final volume of 25 ~1 in a microcentrifuge tube: 2 pmol ssDNA, 2 pm01 primer, 5 ~1 of 5x PCR buffer (50 mM KCl, 2.5 mM MgC12 ,lO-20 mM Tris-HCl, pH 8.3), and 2 11 extension/ labeling mixture. The mixture was incubated at 51°C for 10 min and then added with 2 1.11 of [cY-~~ S]dATP (1,000 Ci/mmol, approximately 10 pCi/pl). The extension/labeling reaction was carried out at 51°C for 3 min, and then an aliquot of 6 ~1 of the reaction mixture was added to each tube containing the d/ddNTP mixture to terminate the reaction. The mixture was briefly mixed by pipetting up and down, and then incubated at 70°C for 5 min. Then, 4 ~1 of stop solution was added to each tube and allowed to stand at room temperature. The mixture was heated at 90°C for 5 min and 2.5-3.0 ~1 of each reaction mixture was immediately loaded on the gel. After electrophoresis at 1,500 volts for 2 gel was placed onto a Whatman No.1 filter hr, the sequencing paper and dried over a gel dryer (Bio-Rad Laboratories) at 80°C for 2 hr. The gel was then subjected to exposure onto an X-ray film to obtain an autoradiogram. RESULTS The mtDNA's were

amplified

individuals

in

the

arm

point

of

the

patients tRNALys

was

examined.

the

tRNALys

wild

type

2 shows the

detected

of

the

was the

pedigree

from

four

we found

that

8344th

1).

This

(Fig.

mutation

sequence

of

in this

same as the

Cambridge

of the

members 1112

of

this

6

of

grandfather

ref.

there

was

pair

of

alters 3).

TVC loop family

the

No such

healthy

a

Chinese region

of

revealed

sequence(6). family.

PCR affected

nucleotide

3 in

in mtDNA's

MERRF disease asymmetric

by

the

structure

The nucletide

gene

A that

lineage,

(Fig.

with

directly mtDNA's

of A to G at

the

mutation

subjects

of a family

the

same maternal

transition

[email protected] of all TVC

members

examining

BY

common

five

by PCR and sequenced

technique.

a

from

It

a Fig.

indicates

Vol.

174,

No.

BIOCHEMICAL

3, 1991

disease

the

individuals

mtDNA's

Fig.

is

heteroplasmy

affected

region

COMMUNICATIONS

1. Autoradiogram of the sequencing gel used to locate the mtDNA's of a normal subject and site of mutation in the with myoclonic epilepsy three patients in the family An A to G transition at and ragged-red fiber disease. singlethe the 8344th nucleotide pair was detected in stranded mtDNA produced by the asymmetric PCR technique. The sequence was determined with the dideoxynucleotide DNA chain termination method using sequencing grade Taq polymerase (Promega).

Moreover,

of

RESEARCH

MERRF2

~RRFI

that

BIOPHYSICAL

AGCT -,_ T-4

AGCT

Fig.

AND

of

2.

were

tRNALys I

through

transmitted

was not of the

aligned

found

When the

family. with

respect

a homologous

in the

to

base could

maternal

mtDNA's

of

nucleotide the

all

the

sequences stem

conserved

be identified

The pedigree of the family members studied in MEFZRF disease is transmitted shows that the maternal lineage.

1113

lineage.

Fig.

in all

1. It through

Vol.

174,

the

No.

3, 1991

species

8344th

BIOCHEMICAL

examined

nucleotide

conserved mtDNA

from

pair

affect

translation

mutant

mtDNA

of

mtDNA-encoded

electron

functions

of the

appears

This

containing

the

T(YC loop

function.

tRNALys

to

molecules,

in

in

polypeptides

alters

are

oxidative

the

mitochondria

turn

that

human

causes

the

and

the

be functionally

position

of

and

Thus,

mutation

functions

transport

COMMUNICATIONS

Drosophila(3).

G at this

of

the

to

RESEARCH

of A with

impairment

biosynthesis

BIOPHYSICAL

human

of the

and substitution could

AND

the

involved

in

phosphorylation

mitochondria. DISCUSSION

The results there

exists

pair

in

the

is

we could

1.11) of blood

three

that

outside

transition

MERRF through

and

with

the

small

that

nucleotide

MERRF

patients

aforementined

of the

very

disease

just

quantities

but

in mtDNA

has

at

the

results

by

(50-100

found

human

to in

and once

in

Chinese,

Moreover,

mutation

human

the

point

1114

populations, both

was pointed

out

the

cases

patients is

is

once

history

mutation

a

that Third,

family

that

It

causes

pair

and in

the

subjects

Drosophila(3).

the

of the

in

the

of [email protected] from

lineage.

cause First,

healthy

analysis the

the

in

nucleotide

twice

suggest

maternal

been

8344th

same disease.

is

provide

disease.

Second,

from

G(3)

manifestations

least

of genetic

strongly

mutation

clinical

the

et

fiber

pedigrees.

conserved

in

the

A to G at

Americans

the

has never

these

arisen

and Shoffner

ragged-red

with

from

the

from

evidence

families

the

of mtDNA's

of

affected

resulted

that

8344th

the

all

diagnosis

study

correlates

white

out

clear-cut

at the

of

point

of this

mutation

mutation

cells

findings

epilepsy

in

to

obtain

myoclonic

highly

mutation

demonstrated

samples.

lines

persons

unequivocally

point

worthy

analysis

The

study

mtDNA of blood

It

molecular

this

an identical

examined. method

from

it and with

transmitted by

Shoffner

Vol. &

No. 3, 1991

174,

BIOCHEMICAL

that

d.(3)

tRNALys I

this

which

will

in

recognizing

capability These

molecular

clinical

features

cause

such

symptoms

usually

of

cascades

this

unclear

of more

disease

point

of

the

specificity

or

synthetsis.

protein cause

characteristic deficiencies

with

as to how the

alterations

in

polypeptide

synthesis

and

of

mitochondrial

respiratory

question

mutation

in

can lead

observed the further

that can

Provided

to the

in patients molecular

is mtDNA

we still

questions,

event

awaits

during

of a mitochondrion.

of

TYC loop

it's

associated

intriguing

important

a molecular

the

still

functions

the

finally are

RESEARCH COMMUNICATIONS

metabolism.

a

such

these

acid

impairment

An even

respiratory answered

is the

extent

alters affects

amino

assembly

complexes.

turn

of MERRF that

it

coordinated

what

in

energy

However,

mutation

abnormalities

in mitochondrial

tRNALys

point

AND BIOPHYSICAL

to

characteristic

mechanism

MERRF. of

how

and

to

damage that

have

with

enzyme

we

the have

explain

why

clinical The elucidation

pathogenesis

of

study. ACKNOWLEDGMENTS

This work was supported by grants (No. NSC 79-0412-BOlO-58 and NSC 80-0412-BOlO-10) from the National Science Council and partly by the Institute of Biomedical Sciences, Academic Sinica, Republic of China. One of the authors, Yau-Huei Wei, expresses his appreciation of the National Science Council for a distinguished research award received in the course of this study. REFERENCES 1. Rosing, H.S., Hopkins, L.C., Wallace, D,C,, Epstein, C-M., and Weidenheim, K. (1985) Ann. Neurol. 17, 228-237. Zheng, X., Lott, M-T., Hodge, J.A., Schurr 2. Wallace, D-C., Lezza, A.M.S., and Elsas, L.J.(1988) Cell 55, 601-610 T.G., Lott, M.T., Lezza, A.M., Seibel, P. 3. Shoffner, J.M., Ballinger, S.W., and Wallace, D.C. (1990) Cell 61, 931-937 K., Ohama, E., and Ikuta, F. (1988 4. Takeda, S., Wakabayashi, Acta Neuropathol. 75, 433-440. 5. Hopkins, L.C., and Rosing, H.S. (1986) Adv. Neurol. 43, 105 117. 6. Berkovic, S.F., Carpenter, S., Evans, A., Karpati, G., Tyler, J.L., Shoubridge, E-A., Andermann, F., Meyer, E., Diksic, M., and Arnold, D. (1989) Brain 112, 1231-1260. 1115

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174,

No.

3, 1991

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

7. DiMauro, S., Zeviani, M., Nakagawa, M., and Bonilla, E., Devivo, D.C. (1985) Ann. Neurol. 17, 521-538. 8. Fukuhara, N., Tokiguchi, S., Shirakawa, K., and Tsybaki, T. (1980) J. Neurol. Sci. 47, 117-133. 9. Wallace, D.C. (1987) Birth Defects 23, 137-190. 10. Giles, R.E., Blanc, H., Cann, H.M., and Wallace, D.C. (1980) Proc. Natl. Acad. Sci., USA. 77, 6715-6719. Cell Genet. 7, 11. Case, J.T., and Wallace, D.C. (1986) Somatic. 103-108. 12. Wallace, D.C. (1986) Somatic Cell Mol. Genet. 12, 41-49. 13. Clayton, D. A. (1984) Annu. Rev. Biochem. 53, 573-594. 14. Attrdi, G. (1981) Trends Biochem. Sci. 6, 86-89. 15. Tzagoloff, A. and Myers, A.M. (1986) Ann. Rev. Biochem. 55, 249-285. 16. Anderson, S.A., Bankier, A.T., Barrell, B.G. de Bruijn, M.H. L Coulson, A.R., Duorin, J., Eperon, I.C., Nierlich, D.P., Roe, B.A., Sanger, F. Schreier, P.H., Smith, A.J.H., Staden, R. and Young, I.G. (1981) Nature 290, 457-465.

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