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Epilepsia partialis continua associated with a homoplasmic mitochondrial tRNASer(UCN) mutation.

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Epilepsia Partialis Continua
Associated with a
Homo lasmic Mitochondrial
tRNAs!r(UCN) Mutation
Markus Schuelke, MD,*S Melan Bakker,?
Gisela Stoltenburg, MD,$ Jurgen Sperner, MD,S
and Arpad von Moers, MD*
Epilepsia partialis continua (EPC) is a rare epileptic syndrome characterized by continuous focal seizures. We report on a 16-year-old girl who died of prolonged pharmacoresistant EPC in whom we identified a 7472insC
mutation within the mitochondrial transfer ribonucleic
acid (tRNA)Ser(UCN).
Additional symptoms included
ataxia, lactic acidosis, myopathy, sensorineural hearing
loss, severe headaches, and mental retardation. Quantification revealed 100% mutant mitochondrial DNA
(mtDNA) in the patient, 4% in her mother, and none in
her half-sister. This highly skewed mtDNA distribution is
most improbable (-3 x
if only explained by random genetic drift. Clustering of dysfunctional mitochondria and replicatory advantage of mutant mtDNA may
play a role in the rapid segregation towards homoplasmy
within one generation.
Schuelke M, Bakker M , Stoltenburg G, Sperner J,
von Moers A. Epilepsia partialis continua
associated with a homoplasmic
mitochondrial tRNASer(UCN)
Ann Neurol 1998;44:700-704
Progressive myoclonus epilepsies are a heterogeneous
group of diseases defined by a syndrome of frequent
myoclonic jerks and myoclonic or tonic-clonic seizures,
which lead to progressive ataxia and mental deterioration.' Two different mitochondrial transfer ribonucleic
acid (tRNA)Ly" mutations (8344A-+G, 8356T-C)
cause the MERRF syndrome in which myoclonic epilepsy is associated with ragged-red fibers (RRFs) in
Beyond that, single families were re-
From the Virchow University Hospital, Departments of *Neuropediatrics and *Neuropathology, Berlin, Germany; tuniversity Hospital Nijrnegen, Department of Human Genetics, Nijmegen, The
Netherlands; and $Medical University Lubeck, Department of Neuropediatrics, Lubeck, Germany.
SPresent address: University Hospital Nijmegen, Department of
Human Genetics, Nijmegen, The Netherlands.
Received Feb 2, 1998, and in revised form May 11. Accepted for
publication May 1 1 , 1998.
Address correspondence to Dr Schuelke, Virchow University Hospital, Department of Neuropediatrics, Augustenburger Plarz 1,
D-13353 Berlin, Germany.
ported who exhibit a MERRF syndrome-like phenoand the
type and carry mutations in the tRNALeu(UUR)
t ~ A S e r ( U C N )4-6 Th
e severity of the MERRF syndrome correlates with the degree of heteroplasmy.'
Heteroplasmy is thought to be a hallmark for pathogenic mitochondrial DNA (mtDNA) m ~ t a t i o n s . ~
Because respiratory chain complexes I, 111, and IV
are partially encoded by mtDNA, mitochondrial tRNA
defects may impair adenosine triphosphate (ATP) production by affecting transcription of these proteins. Although the exact pathogenesis of the various symptoms
remains unknown, Graham and colleagues' demonstrated in adenine nucleotide translocator knockout
mice that muscular ATP deficiency alone can cause mitochondrial disease with myopathy and RRFs. Increased damage by free radicals might be another
pathogenetic mechanism.' We present a clinical and
molecular study of a patient with a tRNASer(UCN)
mutation who died of prolonged status epilepticus at the
age of 16 years.
Patients and Methods
Case Report
This girl was born after an uneventful
pregnancy to healthy nonconsanguineous parents. After normal early childhood development, the parents noticed progressive learning difficulties in this child at the age of 8 years.
At the same time, her gait became ataxic, and she complained of rapid exhaustion after exercise. The girl's first
myoclonic attacks occurred at the age of 12 years, leading to
generalized tonic-clonic seizures within 1 year. The electroencephalogram (EEG) revealed diffuse background slowing,
multifocal spikes, spike waves, and a photomyoclonus. The
initial valproate treatment had to be discontinued due to
nausea and a rise in liver transarninases. The generalized seizures were controlled by the combination of ethosuximide
and lamotrigine, but the focal myoclonic jerks persisted, especially during light sleep. Because of progressive sensorineural hearing loss (50 dB at the age of 13 years), this girl was
fitted with hearing aids. Since the age of 12 years, she had
been plagued by recurrent long-lasting headaches which were
unresponsive to therapy and severely affected her school attendance. Cranial magnetic resonance imaging (MRI) at that
time was normal. At the same time, ergometry (1 W/kg of
body weight for 3 minutes) detected a lactic acidosis (pH
7.23, lactate 10.8 mmol/L, pyruvate 280 pmol/L) and a
muscle biopsy specimen revealed a cytochrome c oxidase
(COX) deficiency. Cardiomyopathy and retinitis pigmentosa
were excluded. At the age of 16 years, the girl presented with
a 1-day aura of left-sided colorful hallucinations, which rapidly led to epilepsia partialis continua (EPC) with continuous
twitching of the left hand and foot and a changing state of
consciousness. There was a severe lactic acidosis (pH 7.05,
lactate 17.6 mmol/L). At this time, cranial MRI (Fig 1A)
showed an area of increased T2-weighted signal intensity in
the right occipital cortex which corresponded to an area of
increased blood flow velocity on magnetic resonance angiogINDEX PATIENT II:2.
Copyright 0 1998 by the American Neurological Association
SUBJECT 11:l. The half-sister of Patient 11:2 is of normal
intelligence and is completely healthy.
SUBJECT 12. The mother of the index patient is suffering
from recurrent migraine. There is, however, no neurological
or mental deficit.
DNA Analysis
DNA was prepared from lymphocytes and additionally from
skeletal muscular tissue of the patient. A mutation screening
was done by standard restriction fragment length polymorphism (RFLP) analysis for previously described mtDNA
point mutations known to be associated with deafness or
myoclonic epilepsy. Both strands of all three mitochondria1
COX genes were analyzed by direct automatic sequencing.
Larger deletions were screened for by long-template polymerase chain reaction (PCR).
Quantitative Analysis of Mutated mtDNA
Quantification of the tRNAS"'"""
mutation was done by
RFLP analysis. The reverse primer (nt7608-7588) was 5'end labeled with [y32]P-ATP by T4-kinase. A forward mismatch primer 5'-CAA AAA AGG AAG GAA T C G AAC
CCA C-3' (nt7446-7470) was used to create a new BsiYI
restriction site in the presence of the mutation. The fragments were separated on a 10% polyacrylamide gel, detected
on a phosphor imager, and analyzed quantitatively by densitometry and volume integration. Quantification of the
7270T-C COX mutation was done by PCR amplification
with the primer pair nt7070-7090 and 1x7516-7495 and
subsequent RFLP analysis after Pst I digestion. The fragments were separated on a 3.5% agarose gel and analyzed
Statistical Methods
We presume that the number of segregating mtDNA units is
approximately 200 and that some 6 X 10' primary oocytes
are generated by about 24 mitotic divisions during oogenesis." T o obtain an oocyte with 100% mutant mtDNA copies out of a orimordial germ cell with 4% mutant mtDNA.
the percentaie of mutait mtDNA has to increase by about
4% in each of the 24 or so mitotic cell divisions. The probability (H,) of an increase by 4% (eg, 36-40%) can be calculated from the total number (N = 200) and the number
of mutant (M = 72; 36%) mtDNA copies in one cell before
and the total (n = 100) and the number of mutant (m =
40; 40%) mtDNA copies in one of the two cells after equal
division according to":
Fia 1. (A) The T2-wei~hted(TR = 2500 msec; TE = 80
m& MRI of Patient y1.2 immediate4 afier Onset of epilepsia
partialis continua (EPC) shows an area of increased signal
intensity in the right occipital lobe. One day before, the patient had complained about colorfil hallucinations within the
lefi visualjeLd. (B) The abnormal MRI signal correpondc to
an area of increased bloodflow velocity on MRA within the
vascular territory of the right posterior cerebral artery.
- -
raphy (MRA). Correspondingly, the EEG revealed continuous right-sided spike-wave activity and generalized back-
ground slowing. Despite correction of the blood pH and
intensive antiepileptic therapy, the areas of increased T2weighted signal intensity enlarged progressively. The vascular
abnormalities on MRA, however, could no longer be found
2 weeks later. With increasing frequency, the EPC progressed to generalized pharmacoresistant status epilepticus to
which the patient succumbed 3 months after admission.
H,(N, M; n> =
H40(200,72;mh;2q100) = 40
= 5.88 X
Brief Communication: Schuelke et al: EPC with a tRNper(UCN)
Mu tation
The binomial coefficient
is calculated as
The probability ( p ) that the percentage increases in each of
the 24 cell cycles by 4% is therefore
4- 163bp
p = (5.88 x 1 0 - ~ ) ~=*3 x
Histochemical analysis of the skeletal muscle biopsy
specimen revealed a patchy COX deficiency but neither RRFs nor abnormal mitochondria on electron microscopy. By RFLP analysis and subsequent sequencing
, we found a homoplasof the patient's tRNASe'(UCN)
mic C insertion at nt7472. Quantification of this mutation in the family members revealed only 4% mutant
mtDNA in the mother and none in the half-sister (Fig
2). An additional homoplasmic T+ C mutation was
detected in the COX1 gene at position nt7270, which
causes an exchange from valine to alanine. This mutation, however, was homoplasmic in unaffected family members as well (Fig 3). Frequent other pathogenic mitochondrial tRNA mutations (3243A+ G,
8356T-+C), the NAIU' (neuropathy,
8993T+C/G, the 1 5 5 5 A 4 G mutation, and larger deletions were excluded.
We describe the clinical, radiological, and molecular
investigations of a 16-year-old girl in whom we found
a mitochondrial tRNASrr(UCN)mutation. She acutely
presented with EPC progressing to generalized status
epilepticus. The initial epileptogenic focus in the EEG
corresponded to an area of cerebral hyperperfusion on
MRA (see Fig 1B). Quantification of the mutation revealed 4% mutant mtDNA in the mother and none in
the half-sister.
In contrast to progressive myoclonic epilepsy which
is well known to be associated with several mitochondrial tRNA mutations,2x5EPC is a rare feature of mitochondrial disease. It has only been described in 1 patient with Leigh syndrome and in another with
respiratory complex I deficiency.12.13 This is the first
report of a mitochondrial tRNA mutation associated
with EPC. The 7472insC mutation was previously described by Tiranti and colleagues6 in a large kindred
702 Annals of Neurology
Vol 44
No 4 October 1778
4- 138bp
contr 11:l
Fig 2. Restriction fragment length polymorphism (RFLP) analysis and quantification of radioactive 5' end labeled polymerase chain reaction (PCR) products. The 1G3bp product is cut
into two fiapzmts of 138 plas 25 bp by BsiYI in the presence of the 7472insC mutation. Patient IL2 exhibits 100%
mutant mitochondrial D N A (mtDNA) within lymphocytic
(lym) and muscular (mus) DNA preparations. The mother
(Patient I:2) shows 4% mutant mtDNA and the half-rister
(Patient I L l ) shows no mutant mtDNA in lymphocytic DNA
from Sicily whose affected members exhibited symptoms identical to those of our patient: ataxia, sensorineural hearing loss, and a myoclonus; however, the
individuals in that study did not demonstrate abnormalities on EEG. Similarly, no RRFs were detected in
muscle biopsy specimens. Sensorineural hearing loss
seems to be a common feature of all previously
described tRNASrr(UCN) mutations (7445T+C,'*
7472ins~,' 7 5 1 2 ~ - - + c ~ ) .
The most well-documented MRI changes in mitochondrial disease are those of stroke-like episodes in
MELAS patients. As in our patient, the areas of increased focal T2-weighted signal intensity were generally associated with focal hyperemia and vasodilatation.I5 We assume that the ongoing focal neuronal
hyperactivity causes a lowering of pH, resulting in vasodilatation. The arterial dilatation disappeared after
correction of the metabolic acidosis. Despite a normal
blood pH, however, the EPC even worsened. This sug-
500bp I
300bp1OObp =b
11 :2
gests an ongoing primary cellular pathology whose trigger mechanisms remain unknown.
Molecular genetic studies revealed a C insertion at
nt7472 in the mitochondrial tRNASe'(UCN)
. For the
following reasons, we assume this mutation to be
pathogenic: (1) it shows minor heteroplasmy in unaffected family members, (2) it has previously been described in patients who exhibited a similar phenotype,
and (3) it was absent in 381 unrelated controls.6 The
C insertion might cause a three-dimensional distortion
of the T W loop, thus interfering with the aminoacylation of the tRNASe'(UCN)and leading to premature
translation termination. A similar effect has been demonstrated for the 8344A-G mutation.I6 Because the
patient was COX deficient, we sequenced all three mitochondrial COX genes and only found a 7270T-+C
mutation in the COXI gene. This mutation causes an
exchange from valine to alanine and is most probably a
benign polymorphism because (1) it is homoplasmic in
all family members; (2) the amino acid residue is not
strictly conserved between the species; and (3) the two
amino acids are similar (uncharged, nonpolar).
It remains intriguing that within one generation,
mutant mtDNA increases from 4 to loo%, although
the sibling's mutant mtDNA is below the limit of detection. Degoul and colleagues" published a similar
case in which a near-homoplasmic 8993T-G
mutation in a 2-year-old girl could not be demonstrated in
the mother's blood cells and fibroblasts. Possible explanations include the following:
The DNA extracted from blood cells might not
reflect the tissue distribution of the mutant
mtDNA, and its abundance might be substantially higher in the mother's ovarian tissue.
Jenuth and co-workers" showed that rapid segregation of mtDNA polymorphisms in mice is
caused by random genetic drift in the female
germ line. This phenomenon alone, however,
can only account for shifts of 10 to 30% between two generations. If the assumptions in the
statistical section are true, the probability of a 4
to 100% shift is only approximately 3 X
Fig 3. Restriction jagment length polymorphism (RFLP) analysis of the 7270T- C
mutation/polymorphismin the cytochrome c
oxidase I (COX) gene. In the presence of the
mutation, Pstl cuts the polymerase chain
reaction (PCR) product of 447 bp into j a g ments of 202 plus 245 bp. All examined
fimily members are homoplasmic f i r this
mutation. On the right, two unrelated controls are shown for compariton.
3. Our statistics presume an entirely random distribution of mtDNA copies. In RRFs, however,
dysfunctional mitochondria tend to cluster.l 8 In
this case, segregation would be much faster.
4. Positive selection of mutant mtDNA may additionally explain the quick segregation towards
homoplasmy. The replicative advantage of mutant mtDNA has been reported previously.'9220
The above mentioned findings make genetic counseling difficult if not impossible unless pre-implantation diagnostic tools are employed.21
This study was made possible by a research grant of the Deutsche
Forschungsgemeinschaft (Schu 1187/1-I).
The authors thank the patient's family for participation in this
study, Prof W. Schulke for helping with the statistics, Prof H. H.
Goebel for evaluating the electron microscopic slides, Done Gruber
and Gabriele Metzke for recording the EEGs, and Christine Gerstenfeld for critical discussions.
J, Andermann E, et al. Progressive myoclonus epilepsies: clinical and genetic aspects. Epilepsia 1993;
34(Suppl):S 19-S30
Schoffner JM, Lott MT, Lezza AMS, et al. Myoclonic epilepsy
and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNALYsmutation. Cell 1990;61:93 1-937
Silvestri G, Moraes C T , Shanske S, et al. A new mtDNA mutation in the tFU'JALysgene associated with myoclonic epilepsy
and ragged-red fibers (MERRF). Am J Hum Genet 1992;51:
1213-1 217
Campos Y, Martin MA, Lorenzo G, et al. Sporadic MERRFI
MELAS overlap syndrome associated with the 3234
[ ~ J , J A L ~ u ( U U)R mutation of mitochondrial DNA. Muscle Nerve
1996;19:187-1 90
Nakamura M, Nakano S, Goto Y, et al. A novel point mutation in the mitochondrial tRNASer(UCN)
gene detected in a
family with MERRF/MELAS overlap syndrome. Biochem Biophys Res Commun 1995;214:86-93
Tiranti V, Chariot P, Carella F, et al. Maternally inherited
hearing loss, ataxia and myoclonus associated with a novel point
mutation in mitochondrial tRNAS"'UCN' gene. Hum Mol
Genet 1995;4:1421-1427
Zeviani M, Amati P, Savoia A. Mitochondria1 myopathies. Curr
Opin Rheumatol 1994;6:559-567
1 . Berkovic SF, Cochius
Brief Communication: Schuelke et al: EPC with a tRNASrr(UCN)Mutation
8. Graham BH, Waymire KG, Cottrell B, et al. A mouse model
for mitochondrial myopathy and cardiomyopathy resulting
from a deficiency in the heardmuscle isoform of the adenine
nucleotide translocator. Nat Genet 1997;16:226-234
9. Beal MF. Mitochondria, free radicals, and neurodegeneration.
Curr Opin Neurobiol 1996;6:661-666
10. Smirnow NW,Dunin-Barkowski WI. Mathematische Statistik
in der Wissenschaft. Berlin: DVW, 1973:39-40
11. Jenuth JP, Peterson AC, Fu K, et al. Random genetic drift in
the female germ line explains the rapid segregation of mammalian mitochondrial DNA. Nat Genet 1996;14:146-151
12. Antozzi C, Franceschetti S, Filippini G, et al. Epilepsia partialis
continua associated with NADH-coenzyme Q reductase deficiency. J Neurol Sci 1995;129:152-161
13. Elia M, Musumeci SA, Ferri R. Leigh syndrome and partial
deficit of cytochrome c oxidase associated with epilepsia partialis continua. Brain Dev 1996;18:207-21 l
14. Reid FM, Vernham GA, Jacobs HT. A novel mitochondrial
point mutation in a maternal pedigree with sensorineural deafness. Hum Mutat 1994;3:243-247
15. Ooiwa Y, Uematsu Y, Terada T, et al. Cerebral blood flow in
mitochondrial myopathy, encephalopathy, lactic acidosis, and
stroke-like episodes. Stroke 1993;24:304-309
16. Enriquez JA, Chomyn A, Attardi G. mtDNA mutation in
MERFF syndrome causes defective aminoacylation of tRNALYs
and premature translation termination. Nat Genet 1995;lO:
17. Degoul F, Franqois D, Diry M, et al. A near homoplasmic
T8993G mtDNA mutation in a patient with atypic Leigh syndrome not present in the mother’s tissues. J Inherit Metab Dis
1997;20:49 -53
18. Shoubridge FA. Mitochondria1 DNA diseases: histological and
cellular studies. J Bioenerg Biomembr 1994;26:301-310
19. Yoneda M, Chomyn A, Martinuzzi A, et al. Marked replicative
advantage of human mtDNA carrying a point mutation that
causes the MELAS encephalomyopathy. Proc Natl Acad Sci
USA 1992;89:11164-11168
20. Weber K, Wilson JN, Taylor L, et al. A new mtDNA mutation
showing accumulation with time and restriction to skeletal
muscle. Am J Hum Genet 1997;60:373-380
21. Lissens W, Sermon K. pre-implantation diagnosis: current status and new developments. Hum Reprod 1997;12:1756-1761
Central Nervous System
Nitric Oxide Formation in
Cerebral Systemic Lupus
L. Brundin, MD,* E. Svenungsson, MD,?
E. Morcos, MD,* M. Anderson, MD,*$
T. Olsson, MD,$ I. Lundberg, MD,?
and N. P. Wiklund, MD*
Systemic lupus erythematosus (SLE)is an inflammatory
disease in which up to two thirds of the patients present
neurological symptoms. The diagnosis of the disease is
based on clinical findings and the presence of autoantibodies, and the pathogenesis is unclear. The purpose of
this study was to determine if the pathogenesis was partly
mediated via nitric oxide (NO) formation. Cerebrospinal
fluid (CSF) samples from 15 patients with cerebral SLE
were analyzed for the NO metabolites nitrite and nitrate
using capillary electrophoresis. The severity of neurological symptoms was scored by dividing the patients into
two groups with either mild or moderatehevere CNS involvement. All patients with cerebral SLE showed increased levels of NO metabolites. In CSF, there was a
relationship between signs of NO production and clinical
results showing that increased levels of nitrite and nitrate
were associated with more severe neurological symptoms.
These findings may shed new light on the pathogenesis of
cerebral SLE, and analysis of nitrate and nitrate may
prove to be of value in monitoring the activity of the
Brundin L, Svenungsson E, Morcos E,
Anderson M, Olsson T, Lundberg I,
Wiklund NP. Central nervous system nitric
oxide formation in cerebral systemic lupus
erythernatosus. Ann Neurol 1998;44:704-706
Systemic lupus erythematosus (SLE) is an inflammatory disease mainly affecting women of fertile age. The
diagnosis is based on clinical findings of typical multiorgan involvement in combination with laboratory
tests showing increased production of defined autoantibodies. Up to two thirds of these patients experience
From the *Department of Clinical Neuroscience, Divisions of
TRheumatology and $Urology, and SNeuroimmunology Unit of the
Department of Medicine, Karolinska Institute, Karolinska Hospital,
Stockholm, Sweden.
Received Dec 2, 1997, and in revised form May 13, 1998. Accepted
for publication May 13, 1998.
Address correspondence to Dr Brundin, Department of Clinical
Neuroscience, Division of Neurology, Karolinska Institute, Karolinska Hospital, S 171 77 Stockholm, Sweden.
Copyright 0 1998 by the American Neurological Association
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associates, mutation, homoplasmic, partiality, epilepsies, continue, mitochondria, trnaser, ucn
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