 |
|
Biochemical-Clinical Correlation in Patients With Different Loads of the Mitochondrial DNA T8993G Mutation
Valerio Carelli, MD, PhD;
Alessandra Baracca, PhD;
Silvia Barogi, PhD;
Francesco Pallotti, MD, PhD;
Maria Lucia Valentino, MD;
Pasquale Montagna, MD;
Massimo Zeviani, MD;
Antonella Pini, MD;
Giorgio Lenaz, MD;
Agostino Baruzzi, MD;
Giancarlo Solaini, PhD
Arch Neurol. 2002;59:264-270.
ABSTRACT
| |
Objective To investigate the correlation between biochemical and clinical phenotype
in 6 patients from 3 unrelated families with different mutation loads (heteroplasmy)
of the T8993G mitochondrial DNA mutation associated with neuropathy, ataxia,
and retinitis pigmentosa–Leigh syndrome.
Methods We studied adenosine triphosphate (ATP) synthase activity (synthesis
and hydrolysis) in platelet-derived submitochondrial particles and assessed
mutant loads both in platelets used for biochemical analysis and in other
available tissues. Biochemical and molecular results were correlated with
clinical features.
Results The rate of ATP hydrolysis was normal, but ATP synthesis was severely
impaired (30% to 4% of residual activity) in patients harboring 34% to 90%
mutant mitochondrial DNA, without any evidence of a threshold for the expression
of this defect. There was little variation in heteroplasmy among tissues from
each patient, but wider variability was detected in 2 mothers. Correlation
of heteroplasmy and clinical and biochemical features suggested that ATP synthesis
is defective at mutant loads as low as 34% and is extremely reduced at mutant
loads above 80% when the phenotype is neuropathy, ataxia, and retinitis pigmentosa–Leigh
syndrome.
Conclusions This study indicates a close relationship between tissue heteroplasmy,
expression of the biochemical defect in platelets, and clinical involvement.
The biochemical defect was greater than previously reported, and we found
no evidence of a biochemical threshold. The uniform distribution of high mutant
loads among our patients' tissues suggests a differential tissue-specific
reliance on mitochondrial ATP synthesis.
INTRODUCTION
SINCE THE INITIAL description of the T8993G mitochondrial DNA (mtDNA)
point mutation in the adenosine triphosphate (ATP) synthase 6 gene in a family
with neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome,1 variable clinical expression within families has been
reported.2 Two main phenotypes were identified:
the typical NARP syndrome and maternally inherited Leigh syndrome, distinguished
by different degrees of heteroplasmy (coexistence of normal and mutant mtDNA)
of the T8993G mutation.1-6
Symptoms usually appear when mutant mtDNA exceeds 60%; full-blown NARP syndrome
characteristically occurs between 75% and 90% heteroplasmy, whereas the more
severe phenotype of maternally inherited Leigh syndrome usually occurs at
mutant mtDNA levels above 90%.3-6
However, retinal dystrophy–related visual loss seems to be the prevalent
symptom in the 60% to 75% range of mutant mtDNA, and in a few cases retinal
dysfunction occurred at mutant loads even lower than 60% and manifested in
an age-related fashion.4
Pedigree analysis indicated that mutant loads tend to increase from
mother to child, most frequently with a very rapid segregation "leap" toward
mutant homoplasmy.3 However, ascertainment
bias could partly account for this feature,3, 6
and cases of slow segregation or even regression of the mutant mtDNA load
have also been reported.1-2,5, 7-9
Moreover, the distribution of mutant load among tissues seemed to be generally
uniform in these patients, lacking the skewed segregation seen for other mtDNA
mutations.7, 10-13
This good genotype-phenotype correlation makes it possible to assess recurrence
risk and provide reliable prenatal diagnosis and genetic counseling.3, 7-8,10
The T8993G mutation changes leucine 156 to arginine in the ATP synthase
6 subunit.1-2 Defective catalytic
properties of the enzyme complex may result either from an impairment of proton
transport or from impaired coupling of proton translocation with ATP synthesis.14-17 Biochemical
studies aimed at clarifying the pathophysiologic mechanism of this mutation
have shown a clear-cut reduction in the rate of ATP synthesis in patient-derived
tissues and in cells containing very high load of mutant mtDNA.2, 14-16,18-23
However, the degree of ATP reduction differed among studies, and no attempt
was made to correlate mutant load and ATP synthesis.
In the present study, we have expanded on a previous investigation of
the biochemical phenotype of the T8993G mutation15
by assaying ATP synthesis and hydrolysis in patient-derived platelets. We
studied a total of 6 individuals from 3 unrelated Italian families harboring
different amounts of mutant mtDNA. Biochemical results and degrees of heteroplasmy
were correlated in the same platelet preparations. In 2 families, we also
assessed mutant loads in multiple tissues. Finally, we correlated our results
with the clinical phenotypes of the patients.
SUBJECTS AND METHODS
PATIENTS AND CONTROLS
We investigated 6 patients from 3 unrelated Italian families (Figure 1A) carrying different loads of the
T8993G mutation. Clinical descriptions of these patients have been reported
by Puddu et al24 and Lodi et al25
(family 1), by Uziel et al6 (family 2), and
Pini et al26 (family 3).
|
|
|
|
Figure 1. Pedigrees and mitochondrial DNA
heteroplasmy in the 3 families. A, Pedigrees of the 3 Italian families investigated.
White symbol indicates unaffected individuals without the T8993G mutation;
gray symbol, carrier of the T8993G mutation with nonspecific symptoms; dark
gray symbol, individual with a partial syndrome (retinitis pigmentosa and
ataxia); black symbols, individuals with full-blown neuropathy, ataxia, and
retinitis pigmentosa (NARP)–Leigh syndrome. B, Gel autoradiogram showing
a polymerase chain reaction–restriction fragment length polymorphism
analysis of DNA isolated from the same platelets used for the biochemical
studies. C, Gels with mitochondrial DNA heteroplasmy assessed in different
tissues from the same individual (B indicates blood; U, urinary epithelium;
H, hair; and F, fibroblast).
|
|
|
We also investigated 12 control subjects chosen randomly from the general
population. Informed consent was obtained in all cases.
MITOCHONDRIAL DNA ANALYSIS
Total DNA was extracted from the same platelets used for biochemical
assays and from whole blood, leukocyte- or platelet-enriched pellets, skeletal
muscle, fibroblasts, hair follicles, and urinary epithelium, by means of the
standard phenol-chloroform method. To detect the T8993G mutation, a 551–base
pair (bp) segment of mtDNA was amplified by polymerase chain reaction, as
described previously.15 The mutation was detected
by restriction fragment length polymorphism analysis after digestion of the
polymerase chain reaction product with the restriction endonuclease AvaI. The copresence of 3 fragments, 1 uncut wild-type
(551 bp) and 2 cut mutant fragments (345 and 206 bp) indicated heteroplasmy.
To evaluate the ratio of mutant to wild-type mtDNA, we ran a last hot-cycle
polymerase chain reaction in the presence of deoxyadenosine 5'-triphosphate
[ -32P], and electrophoresed the digestion products through a 12% nondenaturing
polyacrylamide gel.27 The AvaI-digested fragments were quantified by scanning the gel with an
image analyzing system (PhosphorImager, model GS-363; Bio-Rad, Hercules, Calif).
BIOCHEMICAL ASSAYS
Platelets were isolated and purified from 50 to 100 mL of venous blood
under standardized conditions, as reported previously.28
To isolate mitochondria, platelets were suspended in a hypotonic medium (10mM
Tris hydrochloride, pH 7.6), and 4 minutes later the suspension was centrifuged
at 1500g for 10 minutes. The supernatant was then
centrifuged at 10 000g for 20 minutes to precipitate
mitochondria. The above procedure was performed twice. The mitochondria were
suspended at 4 to 8 mg/mL in 0.25M sucrose and 2mM EDTA, pH 8. Coupled submitochondrial
particles were prepared according to Baracca et al15
by exposing mitochondria to sonic oscillation on a sonicator (model Labsonic
U; B. Braun, Melsungen AG, Germany) for 20 seconds at the minimum output.
The particles were suspended in 0.25M sucrose to give a protein concentration
of 6 to 8 mg/mL and were assayed immediately for the ATP synthase activities.
The ATP synthesis rate was assayed by incubating 20 to 40 µg of
submitochondrial particles in 25 µL of 0.25M sucrose, 50mM Hepes, 0.5mM
EDTA, 2mM magnesium sulfate, 2mM potassium phosphate, and 20mM succinate,
pH 7.4, to which 0.2mM adenosine diphosphate was added to start the reaction.
Incubation was carried out for 10 minutes at 30°C and 5 µL of 50%
trichloroacetic acid was added to stop the reaction. The mixture was centrifuged
to remove precipitated protein, and the resulting extract was assayed for
ATP by the luciferin-luciferase chemiluminescent method.29
The ATP hydrolysis rate was assayed as follows: 10 µg of submitochondrial
particles were incubated for 10 minutes at 30°C in 25 µL of buffer
containing 0.25M sucrose, 50mM Hepes, and 2mM magnesium chloride, pH 8, and
1mM ATP was added to start the reaction. To stop the reaction, trichloroacetic
acid was added and nonhydrolyzed ATP was determined by the luciferin-luciferase
method as above.
The ATP, adenosine diphosphate, Hepes, Tris, and trichloroacetic acid
were obtained from Sigma-Aldrich Corp (St Louis, Mo); 1243-102 ATP monitoring
reagent, a mixture of luciferin and luciferase, was a product of BioOrbit
(Turku, Finland).
RESULTS
MITOCHONDRIAL DNA ANALYSIS
Table 1 shows the results
of mtDNA analysis in different tissues. Identification of high mutant loads
(85%-91%) of the T8993G mutation in whole blood cells from all 4 probands
in the 3 families (Table 1 and Figure 1A) led to the diagnosis of NARP-Leigh
syndrome. Lower amounts of mutant mtDNA (19%-55%) were found in whole blood
cells from the mothers in families 1 and 2, whereas the mother in family 3
had no detectable mutant mtDNA (Table 1 and Figure 1A). To correlate
biochemical data and mutant loads in the same tissue, we extracted mtDNA from
the same platelets used to obtain submitochondrial particles. Figure 1B shows the mutation loads in platelets ("biochemical study"
column in Table 1) for each individual
investigated.
|
|
|
|
Table 1. Tissue Distribution of Heteroplasmy*
|
|
|
We also investigated the mtDNA heteroplasmy in different tissues from
5 individuals in families 1 and 3 (Table
1); 2 examples are shown in Figure
1C. In family 2, we investigated only platelet mtDNA ("biochemical
study" column in Table 1), but
degrees of heteroplasmy in whole blood cells (Table 1) were derived from published data.6
All individuals with NARP-Leigh syndrome had very high loads of mutant mtDNA,
and these loads were fairly homogeneous in different tissues (Table 1 and Figure 1C,
right panel). However, the mother from family 1, who harbored the lowest amounts
of mutant mtDNA, displayed more scattered values (Table 1 and Figure 1C,
left panel), and a similar trend was suggested by the 2 available evaluations
from the mother in family 2 (Table 1).
An extended investigation of multiple tissues from the mother in family 3
(Table 1) confirmed the absence
of the T8993G mutation in this individual, compatible with a de novo mutation
in her son.
BIOCHEMICAL STUDIES
We previously reported a more than 20-fold decrease of ATP synthase
activity in platelet-derived submitochondrial particles from 3 patients carrying
more than 80% mutant mtDNA (probands from families 1 and 2), whereas ATP hydrolysis
was essentially unaffected.15 In the current
work, we extended our biochemical investigation to a new case of NARP-Leigh
syndrome (family 3) and to the mothers from all 3 families. Figure 2 shows ATP hydrolysis and synthesis results obtained after
the whole data set is considered. The values in the mother from family 3,
who did not carry any mutant DNA, were pooled with those of the control group.
The ATP hydrolysis rate was essentially unaffected in all individuals carrying
the mutation as compared with controls (Figure
2A). However, the values in the mutant group were near the lower
end of the control range (and never below 20 nmol · min-1· mg-1of protein) (Figure 2A). The ATP synthesis rate, on the contrary, was clearly
reduced in individuals with mutant mtDNA, with residual enzymatic activities
ranging from 4% to 30% of normal (individuals 2-6 in Figure 2B). The ATP synthesis rate in individual 1 (Figure 2B) was at the lower end of the range of control subjects.
|
|
|
|
Figure 2. Biochemical investigation of adenosine
triphosphate (ATP) hydrolysis (A) and ATP synthesis (B) activities in platelet
submitochondrial particles. The data are presented as mean ± SEM. C
indicates control group (n = 12); 1, patient I:1 from family 1; 2, patient
II:1 from family 1; 3, patient II:2 from family 1; 4, patient I:1 from family
2; 5, patient II:1 from family 2; and 6, patient II:1 from family 3.
|
|
|
GENETIC, BIOCHEMICAL, AND CLINICAL CORRELATIONS
Figure 3 correlates the defect
of ATP synthesis with the T8993G mutant load evaluated in the same tissue.
The biochemical defect became manifest at mutation loads between 10% and 34%.
Surprisingly, a marked decrease of ATP synthesis occurred at a relatively
low mutant load (34%) in individual I:1 from family 2. The correlation coefficient
(0.95) was significant (P<.001).
|
|
|
|
Figure 3. Correlation of adenosine triphosphate
(ATP) synthesis defect with mutant heteroplasmy in platelets. The asterisk
indicates the value (1.78 nmol · min-1· mg-1) of the mother from family 3 (patient I:1 in Figure 1A) who
was assigned to the control group because no mutant mitochondrial DNA (mtDNA)
could be detected (Figure 1B). NARP indicates neuropathy, ataxia, and retinitis
pigmentosa.
|
|
|
On the basis of the fairly uniform distribution of the T8993G mutation
in the tissues investigated (Table 1),
as previously reported by others,3, 7, 10-13,24
we assume that the defect of ATP synthesis identified in platelets occurs
in most other tissues. We then correlated the biochemical phenotype and clinical
symptoms in Table 2. Both patients
with NARP syndrome and those with NARP-Leigh syndrome seemed to have similarly
severe impairments of ATP synthesis, which is not consistent with the clinical
differences between the 2 syndromes, or with the fact that bilateral basal
ganglia and brainstem lesions are seen only in maternally inherited Leigh
syndrome. The late-onset clinical manifestations of individual I:1 from family
2 are consistent with the defect of ATP synthesis in her platelets, although
she had relatively low percentages of mutant mtDNA (34%-55%).
|
|
|
|
Table 2. Biochemical-Clinical Correlation*
|
|
|
COMMENT
This study indicates that high percentages of T8993G mutation (>80%)
induce a marked decrease in ATP synthesis (4%-9% of control values) but essentially
unaltered ATP hydrolysis. The preservation of ATP hydrolytic activity, together
with our previous finding of normal ATP-driven proton translocation, suggests
that the F1F0-ATP synthase complex is essentially fully
assembled in platelets.15 Other investigators
recently reached similar conclusions by studying fibroblast cell lines.16 However, it has also been reported that, in T8993G
homoplasmic mutant cybrid cell lines, the assembly of ATP synthase may be
impaired.23 The ATP synthesis defect we observed
agrees with previous studies, but the residual activities we found were strikingly
lower than those previously reported.14, 16, 18-23
Moreover, when we correlated ATP synthesis and mutant load in the same tissue,
we saw no complementation by wild-type mtDNA, and no threshold expression
of the biochemical defect (Figure 3).
However, 60% to 75% mutant mtDNA is required for clinical expression of typical
central nervous system symptoms.3-6
To our knowledge, this is the first study correlating mutant load and biochemical
defect in patient-derived tissues.
Several reasons may account for our findings. Our assays were performed
in submitochondrial particles derived from circulating platelets (ex vivo
tissue), whereas previous studies were carried out in patient-derived cell
lines (mainly lymphoblasts and fibroblasts or cybrid cell lines).14, 16, 18-23
Differences between biochemical studies in cell cultures and in vivo tissues
were found in 2 studies of patients with the A3243G mtDNA mutation (transfer
RNALeu) associated with mitochondrial encephalomyopathy, lactic
acidosis, and strokelike episodes syndrome.30-31
The first study30 demonstrated a linear relationship
between brain lactate level and the proportion of mutant mtDNA, irrespective
of clinical symptoms. The second study31 found
a significant decrease of muscle ATP production despite low levels of mutant
heteroplasmy, normal histochemistry findings, and normal respiratory chain
activity in vitro. A similar situation may apply to our patient I:1 from family
1. This woman had low levels of mutant mtDNA in different tissues and did
not have any typical symptoms (Table 1
and Table 2), although she did
have minor complaints compatible with her very low mutant load.4-6
Her platelet ATP synthesis was within the control range in the presence of
10% mutant heteroplasmy (Figure 3), but brain phosphorus P 31 magnetic resonance spectroscopy in this subject,
performed by Lodi et al,25 showed defective
energy metabolism. Brain heteroplasmy in this patient is unknown, but the
mutant load could be higher than in other tissues investigated. It is unlikely,
however, that her mutant load in the brain exceeds a critical level (about
60%) needed for central nervous system symptoms to manifest. This suggests
that in this patient ATP synthesis is already impaired in her brain at a mutant
heteroplasmy level that does not cause typical symptoms (<60%). This is
in accordance with the findings in the other mother from family 2, who had
defective platelet ATP synthesis in the presence of a relatively low mutant
percentage (34%).
The biochemical effect of T8993G mutation is still under investigation.15-16,23 The structural defect
due to the Leu156-to-Arg amino acid change in ATP synthase 6 subunit (F0) may have 2 main consequences. The first is a complete or almost complete
block of proton flow through the proton channel (F0) during ATP
synthesis.14 The second is a still viable proton
translocation but uncoupled from ATP synthesis, perhaps due to loss of the
rotational mechanism powered by proton flow.15
We previously demonstrated that the reverse, ATP-driven, proton flow through
the channel, from the matrix side to the mitochondrial intermembrane space,
is not affected.15 The residual ATP synthesis
may be accounted for by F1F0-ATP synthase complexes
containing a subunit 6 encoded by wild-type mtDNA. At a mutation level greater
than 80%, as seen in our patients with NARP-Leigh syndrome, the expected number
of active F1F0complexes would be less than one fifth
of normal. Moreover, partial inactivation of ATP synthase should have increased
the turnover rate of the remaining active F1F0-ATP synthase
molecules for ATP synthesis.32 The mean decrease
of ATP synthesis was in fact about 1/20th that of the controls. A possible
explanation for the remarkably low values of ATP synthesis found in this study
may depend on the preparation of our samples. We used submitochondrial particles,
which did not present enzymatic activities, such as myokinases or those related
to glycolysis. These activities may induce an overestimation of net ATP synthesis.
Previous studies used mainly crude mitochondrial preparations or cells, and
these interfering activities may not have been carefully inhibited. Moreover,
the low values of ATP synthesis and the absent complementation of wild-type
mtDNA might also depend on an additional deleterious factor operating in vivo:
part of the ATP synthesized by residual active F1F0complexes
might be hydrolyzed by the mutated F1F0complexes themselves,
which are capable of ATP hydrolytic activity. This phenomenon could paradoxically
enhance the depletion of cellular energy.
Genetic analysis in our 3 families showed most of the typical features
of the T8993G mutation, such as rapid segregation toward homoplasmy in 1 generation,
occurrence of de novo mutations, and similar levels of heteroplasmy in different
tissues of probands with high mutant loads.3
Tissue segregation of mutant mtDNA in patients with low to intermediate heteroplasmy
has not been reported before, to our knowledge, and our findings in 2 cases
(mothers from families 1 and 2) indicate greater variability. Similar observations
have been reported in Leber hereditary optic neuropathy and mitochondrial
encephalomyopathy, lactic acidosis, and strokelike episodes syndrome, with
an inverse correlation between variance of mutant mtDNA segregation among
tissues and mean mutant load.33-34
Our study suggests a close relationship of biochemical defect, tissue
heteroplasmy, and clinical expression. In particular, the 2 individuals with
lowest tissue heteroplasmy (the 2 mothers from families 1 and 2) showed either
absence or late onset of typical symptoms (retinitis pigmentosa and cerebellar
ataxia). The latter patient had clearly defective ATP synthesis in platelets,
but not as severe as in probands with NARP-Leigh phenotype (Figure 3). An inverse relationship between heteroplasmy and age
at onset has already been shown, and a generally good genotype-phenotype correlation
is well established.3-6
However, our biochemical-heteroplasmy correlation shows that a defect of ATP
synthesis may become evident even at low levels of mutant heteroplasmy (somewhere
between 10% and 34%) and in the absence of clinical symptoms. Increasing amounts
of mutant DNA (between 60% and 90%) presumably induce greater defects in ATP
synthesis, and these correspond to increasingly severe clinical manifestations.3 At high levels of mutation (>80%), ATP synthesis is
extremely reduced (down to 4% in platelets with 90% mutant mtDNA). At these
mutation loads, the clinical phenotype may be drastically changed from a slowly
progressive degenerative disease, such as NARP syndrome, to a much more devastating
phenotype, such as Leigh syndrome, even by relatively small increases of mutational
load.5 If the T8993G mutation induces a block
in proton flow, it is conceivable that a backup effect on the respiratory
chain would also occur, especially when nearly homoplasmic levels of mutant
DNA are reached. Thus, substantial overproduction of reactive oxygen species
may be an additional biochemical effect superimposed on an already extremely
depressed ATP synthesis; this may account for the shift to the devastating
Leigh disease.
Preliminary evidence indicating reactive oxygen species overproduction
and massive apoptotic cellular death in patients with T8993G mutation has
recently been presented, and in a manganese superoxide dismutase knock-out
mouse model, the lack of the intramitochondrial superoxide scavenging enzyme
induces an encephalopathy with the histopathological hallmarks of Leigh disease.35-36 It is increasingly clear that the
pathophysiologic mechanism of mitochondrial diseases combines impairment of
cellular energy production and oxidative stress damage.36
Clarifying the interplay of ATP synthesis defect, tissue-specific energy threshold,
and tissue-specific antioxidant capabilities will be the next challenge in
understanding the exact pathophysiologic characteristics of these diseases
and in designing effective therapies.
AUTHOR INFORMATION
Accepted for publication September 24, 2001.
Author contributions: Study concept and design (Drs Carelli, Montagna, Zeviani, Pini, Lenaz, Baruzzi, and Solaini); acquisition of data (Drs Carelli, Baracca, Barogi,
Pallotti, and Valentino); analysis and interpretation of data (Drs Carelli, Baracca, Pallotti, Valentino, and Solaini);
drafting of the manuscript (Drs Carelli, Barogi, and Solaini); critical revision of the manuscript for important intellectual content (Drs Baracca, Pallotti, Valentino, Montagna, Zeviani, Pini, Lenaz,
Baruzzi, and Solaini); statistical expertise (Drs
Carelli, Baracca, Barogi, and Pallotti); obtaining funding (Dr Solaini); administrative, technical, or material support (Drs Baracca and Valentino); supervision (Drs Montagna, Zeviani, Pini, Lenaz, Baruzzi, and Solaini).
This study was supported by Telethon-Italy, Rome, Italy (project code
1048), and Fondazione Gino Galletti, Bologna, Italy, for the study of dementia
and other neurodegenerative diseases in Italy.
We are indebted to the families for their collaboration. We thank Eric
A. Schon, PhD, and Salvatore DiMauro, MD, for reviewing the manuscript and
helpful discussion.
Corresponding author and reprints: Valerio Carelli, MD, PhD, Istituto
di Clinica Neurologica, Universita' di Bologna, Via U Foscolo 7, 40123 Bologna,
Italy (e-mail: carelli{at}neuro.unibo.it).
From the Istituto di Clinica Neurologica (Drs Carelli, Valentino, Montagna,
and Baruzzi) and Dipartimento di Biochimica (Drs Baracca and Lenaz), Universita'
di Bologna, Bologna, Italy; Doheny Eye Institute, University of Southern California,
Los Angeles (Dr Carelli); Scuola Superiore di Studi Universitari e di Perfezionamento
S. Anna, Pisa, Italy (Drs Barogi and Solaini); College of Physicians and Surgeons,
Columbia University, New York, NY (Dr Pallotti); Istituto Nazionale Neurologico
"C. Besta," Milano, Italy (Dr Zeviani); and Ospedale Maggiore, Bologna (Dr
Pini).
REFERENCES
| |
1. Holt IJ, Harding AE, Petty RKH, Morgan-Hughes JA. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet. 1990;46:428-433.
ISI
| PUBMED
2. Tatuch Y, Christodoulou J, Feigenbaum A, et al. Heteroplasmic mtDNA mutation (T G) at 8993 can cause Leigh disease
when the percentage of abnormal mtDNA is high. Am J Hum Genet. 1992;50:852-858.
ISI
| PUBMED
3. White SL, Collins VR, Wolfe R, et al. Genetic counseling and prenatal diagnosis for the mitochondrial DNA
mutations at nucleotide 8993. Am J Hum Genet. 1999;65:474-482.
FULL TEXT
|
ISI
| PUBMED
4. Tatuch Y, Pagon RA, Vlcek B, Roberts R, Korson M, Robinson BH. The 8993 mtDNA mutation: heteroplasmy and clinical presentation in
three families. Eur J Hum Genet. 1994;2:35-43.
PUBMED
5. Makela-Bengs P, Suomalainen A, Majander A, et al. Correlation between the clinical symptoms and the proportion of mitochondrial
DNA carrying the 8993 point mutation in the NARP syndrome. Pediatr Res. 1995;37:634-639.
ISI
| PUBMED
6. Uziel G, Moroni I, Lamantea E, et al. Mitochondrial disease associated with the T8993G mutation of the mitochondrial
ATPase 6 gene: a clinical, biochemical, and molecular study in six families. J Neurol Neurosurg Psychiatry. 1997;63:16-22.
FREE FULL TEXT
7. Ferlin T, Landrieu P, Rambaud C, et al. Segregation of the G8993 mutant mitochondrial DNA through generations
and embryonic tissues in a family at risk of Leigh syndrome. J Pediatr. 1997;131:447-449.
ISI
| PUBMED
8. Block RB, Gook DA, Thorburn DR, Dahl H-HM. Skewed segregation of the mtDNA nt 8993 (TG) mutation in human
oocytes. Am J Hum Genet. 1997;60:1495-1501.
ISI
| PUBMED
9. Martin MA, Campos Y, Garcia-Silva MT, et al. Slow segregation and rapid shift to homoplasmy coexist in a family
with the T8993 > G mutation. J Inherit Metab Dis. 1999;22:939-940.
FULL TEXT
|
ISI
| PUBMED
10. Harding AE, Holt IJ, Sweeney MG, Brockington M, Davis MB. Prenatal diagnosis of mitochondrial DNA 8993 TG disease. Am J Hum Genet. 1992;50:629-633.
ISI
| PUBMED
11. Ciafaloni E, Santorelli FM, Shanske S, et al. Maternally inherited Leigh syndrome. J Pediatr. 1993;122:419-422.
ISI
| PUBMED
12. Nagashima T, Mori M, Katayama K, et al. Adult Leigh syndrome with mitochondrial DNA mutation at 8993. Acta Neuropathol. 1999;97:416-422.
FULL TEXT
| PUBMED
13. White SL, Shanske S, McGill JJ, et al. Mitochondrial DNA mutations at nucleotide 8993 show a lack of tissue-
or age-related variation. J Inherit Metab Dis. 1999;22:899-914.
FULL TEXT
|
ISI
| PUBMED
14. Tatuch Y, Robinson BH. The mitochondrial DNA mutation at 8993 associated with NARP slows the
rate of ATP synthesis in isolated lymphoblast mitochondria. Biochem Biophys Res Commun. 1993;192:124-128.
FULL TEXT
|
ISI
| PUBMED
15. Baracca A, Barogi S, Carelli V, Lenaz G, Solaini G. Catalytic activities of mitochondrial ATP synthase in patients with
T8993G mutation in the ATPase 6 gene encoding for subunit a. J Biol Chem. 2000;275:4177-4182.
FREE FULL TEXT
16. Garcia JJ, Ogilvie I, Robinson BH, Capaldi RA. Structure, functioning, and assembly of the ATP synthase in cells from
patients with the T8993G mitochondrial DNA mutation. J Biol Chem. 2000;275:11075-11081.
FREE FULL TEXT
17. Schon EA. Mitochondrial genetics and disease. Trends Biochem Sci. 2000;25:555-560.
FULL TEXT
|
ISI
| PUBMED
18. Trounce I, Neill S, Wallace DC. Cytoplasmatic transfer of the mtDNA nt 8993 TG (ATP6) point mutation
associated with Leigh syndrome into mtDNA-less cells demonstrates cosegregation
with a decrease in state III respiration and ADP/O ratio. Proc Natl Acad Sci U S A. 1994;91:8334-8338.
FREE FULL TEXT
19. Houstek J, Klement P, Hermanska J, et al. Altered properties of mitochondrial ATP-synthase in patients with a
TG mutation in the ATPase6 (subunit a) gene at position 8993 of mtDNA. Biochim Biophys Acta. 1995;1271:349-357.
PUBMED
20. Vasquez-Memije ME, Shanske S, Santorelli FM, et al. Comparative biochemical studies in fibroblasts from patients with different
forms of Leigh syndrome. J Inherit Metab Dis. 1996;19:43-50.
FULL TEXT
|
ISI
| PUBMED
21. Vasquez-Memije ME, Shanske S, Santorelli FM, Kranz-Eble P, De Vivo DC, DiMauro S. Comparative biochemical studies of ATPases in cells from patients with
T8993G or T8993C mitochondrial DNA mutations. J Inherit Metab Dis. 1998;21:829-836.
FULL TEXT
|
ISI
| PUBMED
22. Manfredi G, Gupta N, Vasquesz-Memije ME, et al. Oligomycin induces a decrease in the cellular content of a pathogenic
mutation in the human mitochondrial ATPase 6 gene. J Biol Chem. 1999;274:9386-9391.
FREE FULL TEXT
23. Nijtmans LGJ, Henderson NS, Attardi G, Holt IJ. Impaired ATP synthase assembly associated with a mutation in the human
ATP synthase subunit 6 gene. J Biol Chem. 2001;276:6755-6762.
FREE FULL TEXT
24. Puddu P, Barboni P, Mantovani V, et al. Retinitis pigmentosa, ataxia, and mental retardation associated with
mitochondrial DNA mutation in an Italian family. Br J Ophthalmol. 1993;77:84-88.
FREE FULL TEXT
25. Lodi R, Montagna P, Iotti S, et al. Brain and muscle energy metabolism studied in vivo by 31P-magnetic
resonance spectroscopy in NARP syndrome. J Neurol Neurosurg Psychiatry. 1994;57:1492-1496.
ABSTRACT
26. Pini A, Gobbi G, Carelli V, Bertani G, Antoniani C, Marcello N. Leigh/NARP phenotype in a sporadic case with de novo mtDNA 8993 mutation
[abstract]. Neuromusc Disord. 1998;8:256.
27. Moraes CT, Ricci E, Bonilla E, DiMauro S, Schon EA. The mitochondrial tRNALeu(UUR) mutation in mitochondrial
encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS): genetic,
biochemical, and morphological correlations in skeletal muscle. Am J Hum Genet. 1992;50:934-949.
ISI
| PUBMED
28. Baracca A, Bucchi L, Ghelli A, Lenaz G. Protonophoric activity of NADH coenzyme Q reductase and ATP synthase
in coupled submitochondrial particles from horse platelets. Biochem Biophys Res Commun. 1997;235:469-473.
FULL TEXT
|
ISI
| PUBMED
29. Stanley PE, Williams SG. Use of the liquid scintillation spectrometer for determining adenosine
triphosphate by the luciferase enzyme. Anal Biochem. 1969;29:381-392.
FULL TEXT
|
ISI
| PUBMED
30. Dubeau F, De Stefano N, Zifkin BG, Arnold DL, Soubridge EA. Oxidative phosphorylation defect in the brains of carriers of the tRNAleu(UUR) A3243G mutation in a MELAS pedigree. Ann Neurol. 2000;47:179-185.
FULL TEXT
|
ISI
| PUBMED
31. Chinnery PF, Taylor DJ, Brown DT, Manners D, Styles P, Lodi R. Very low levels of the mtDNA A3243G mutation associated with mitochondrial
dysfunction in vivo. Ann Neurol. 2000;47:381-384.
FULL TEXT
|
ISI
| PUBMED
32. Matsuno-Yagi A, Hatefi Y. Kinetic modalities of ATP synthesis: regulation by the mitochondrial
respiratory chain. J Biol Chem. 1986;261:14031-14038.
FREE FULL TEXT
33. Juvonen V, Nikoskelainen E, Lamminen T, Penttinen M, Aula P, Savontaus M-L. Tissue distribution of the ND4/11778 mutation in heteroplasmic lineages
with Leber hereditary optic neuropathy. Hum Mutat. 1997;9:412-417.
FULL TEXT
|
ISI
| PUBMED
34. Macmillan C, Lach B, Shoubridge EA. Variable distribution of mutant mitochondrial DNAs (tRNALeu(3243)) in tissues of symptomatic relatives with MELAS: the role of mitotic
segregation. Neurology. 1993;43:1586-1590.
FREE FULL TEXT
|
