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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).

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).

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.

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.

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.

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.

Table 1. 
Tissue Distribution of Heteroplasmy*
Tissue Distribution of Heteroplasmy*6
Table 2. 
Biochemical-Clinical Correlation*
Biochemical-Clinical Correlation*
1.
Holt  IJHarding  AEPetty  RKHMorgan-Hughes  JA A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet.1990;46:428-433.
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Tatuch  YChristodoulou  JFeigenbaum  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.
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White  SLCollins  VRWolfe  R  et al Genetic counseling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993. Am J Hum Genet.1999;65:474-482.
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Tatuch  YPagon  RAVlcek  BRoberts  RKorson  MRobinson  BH The 8993 mtDNA mutation: heteroplasmy and clinical presentation in three families. Eur J Hum Genet.1994;2:35-43.
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Makela-Bengs  PSuomalainen  AMajander  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.
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Uziel  GMoroni  ILamantea  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.
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Ferlin  TLandrieu  PRambaud  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.
8.
Block  RBGook  DAThorburn  DRDahl  H-HM Skewed segregation of the mtDNA nt 8993 (T→G) mutation in human oocytes. Am J Hum Genet.1997;60:1495-1501.
9.
Martin  MACampos  YGarcia-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.
10.
Harding  AEHolt  IJSweeney  MGBrockington  MDavis  MB Prenatal diagnosis of mitochondrial DNA 8993 T→G disease. Am J Hum Genet.1992;50:629-633.
11.
Ciafaloni  ESantorelli  FMShanske  S  et al Maternally inherited Leigh syndrome. J Pediatr.1993;122:419-422.
12.
Nagashima  TMori  MKatayama  K  et al Adult Leigh syndrome with mitochondrial DNA mutation at 8993. Acta Neuropathol.1999;97:416-422.
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White  SLShanske  SMcGill  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.
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Tatuch  YRobinson  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.
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Baracca  ABarogi  SCarelli  VLenaz  GSolaini  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.
16.
Garcia  JJOgilvie  IRobinson  BHCapaldi  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.
17.
Schon  EA Mitochondrial genetics and disease. Trends Biochem Sci.2000;25:555-560.
18.
Trounce  INeill  SWallace  DC Cytoplasmatic transfer of the mtDNA nt 8993 T→G (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.
19.
Houstek  JKlement  PHermanska  J  et al Altered properties of mitochondrial ATP-synthase in patients with a T→G mutation in the ATPase6 (subunit a) gene at position 8993 of mtDNA. Biochim Biophys Acta.1995;1271:349-357.
20.
Vasquez-Memije  MEShanske  SSantorelli  FM  et al Comparative biochemical studies in fibroblasts from patients with different forms of Leigh syndrome. J Inherit Metab Dis.1996;19:43-50.
21.
Vasquez-Memije  MEShanske  SSantorelli  FMKranz-Eble  PDe Vivo  DCDiMauro  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.
22.
Manfredi  GGupta  NVasquesz-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.
23.
Nijtmans  LGJHenderson  NSAttardi  GHolt  IJ Impaired ATP synthase assembly associated with a mutation in the human ATP synthase subunit 6 gene. J Biol Chem.2001;276:6755-6762.
24.
Puddu  PBarboni  PMantovani  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.
25.
Lodi  RMontagna  PIotti  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.
26.
Pini  AGobbi  GCarelli  VBertani  GAntoniani  CMarcello  N Leigh/NARP phenotype in a sporadic case with de novo mtDNA 8993 mutation [abstract]. Neuromusc Disord.1998;8:256.
27.
Moraes  CTRicci  EBonilla  EDiMauro  SSchon  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.
28.
Baracca  ABucchi  LGhelli  ALenaz  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.
29.
Stanley  PEWilliams  SG Use of the liquid scintillation spectrometer for determining adenosine triphosphate by the luciferase enzyme. Anal Biochem.1969;29:381-392.
30.
Dubeau  FDe Stefano  NZifkin  BGArnold  DLSoubridge  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.
31.
Chinnery  PFTaylor  DJBrown  DTManners  DStyles  PLodi  R Very low levels of the mtDNA A3243G mutation associated with mitochondrial dysfunction in vivo. Ann Neurol.2000;47:381-384.
32.
Matsuno-Yagi  AHatefi  Y Kinetic modalities of ATP synthesis: regulation by the mitochondrial respiratory chain. J Biol Chem.1986;261:14031-14038.
33.
Juvonen  VNikoskelainen  ELamminen  TPenttinen  MAula  PSavontaus  M-L Tissue distribution of the ND4/11778 mutation in heteroplasmic lineages with Leber hereditary optic neuropathy. Hum Mutat.1997;9:412-417.
34.
Macmillan  CLach  BShoubridge  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.
35.
Geromel  VKadhom  NCebalos-Picot  I  et al Superoxide-induced massive apoptosis in cultured skin fibroblasts harboring the neurogenic ataxia retinitis pigmentosa (NARP) mutation in the ATPase-6 gene of the mitochondrial DNA. Hum Mol Genet.2001;10:1221-1228.
36.
Wallace  DC Mitochondrial disease in man and mouse. Science.1999;283:1482-1488.
Original Contribution
February 2002

Biochemical-Clinical Correlation in Patients With Different Loads of the Mitochondrial DNA T8993G Mutation

Author Affiliations

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).

Arch Neurol. 2002;59(2):264-270. doi:10.1001/archneur.59.2.264
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.

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.16 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%.36 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.1,2,5,79 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.7,1013 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.1417 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.2,1416,1823 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).

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 [α-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.

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.

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).

On the basis of the fairly uniform distribution of the T8993G mutation in the tissues investigated (Table 1), as previously reported by others,3,7,1013,24 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%).

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.14,16,1823 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.36 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).14,16,1823 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.46 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.36 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.

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Article 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@neuro.unibo.it).

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