kber’s Heredtary Optic Neuropathy and Complex I Deficiency in Muscle Nils-Goran Larsson, MD,’ Oluf Andersen, MD, PhD,i Elisabeth Holme, MD, PhD,’ Anders Oldfors, MD, PhDJ and Jan Wahlstrom, MD, PhDg
We investigated a family with Leber’s hereditary optic neuropathy in which affected individuals were homoplasmic for the point mutation of the NADH-dehydrogenase 4 gene of mitochondrial DNA, described by Wallace and colleagues in 1988. The proband had bilateral optic atrophy, tremor, dystonia, and sharply defined lesions in the putamen on magnetic resonance images. Optic atrophy was found in another 3 of 13 investigated relatives on the maternal side. Additional neurological signs were found but only in patients with optic neuropathy. The morphological appearance and the respiratory chain function of muscle tissue were investigated in the proband, his mother, and 3 siblings. Polarographic measurements revealed complex I deficiency in the 5 investigated subjects. Morphological changes of mitochondria were found in 4 of these subjects. There was no decrease in complex I activity measured as NADH ferricyanide reductase or rotenone-sensitive NADH cytochrome c reductase activities. In other cases with complex I deficiency, good agreement between polarographic and spectrophotornetric measurements was found. This study showed that there is decreased activity of complex I of the respiratory chain in muscle and that cerebral striatal lesions occur in Leber’s hereditary optic neuropathy with the NADH-dehydrogenase 4 gene point mutation. Larsson NG, Andersen 0, Holme E, Oldfors A, Wahlstrom J. Leber’s hereditary optic neuropathy and complex I deficiency in muscle. Ann Neurol 1991;30:701-708
Leber’s hereditary optic neuropathy (LHON) (McKusick 308900) is a maternally inherited disease causing blindness predominantly in young men 11, 21. A point mutation (LHON mutation) that changes a highly conserved amino acid from arginine to histidine in subunit 4 of complex I (NADII ubiquinone oxidoreductase, EC 1.6.5.3)of the respiratory chain was found by Wallace and colleagues in patients with LHON { 3 } . The LHON mutation results in loss of a cleavage site for the restriction endonuclease SfaNI and is found in about half of the families with the disease 14-63. Some families with the LHON mutation are heteroplasmic; that is, mutated mitochondrial DNA (mtDNA) is found together with normal mtDNA in proportions that can vary both between individuals and between tissues of the same individual [5-8). In the early phase of the disease, tortuous and swollen retinal vessels are found. This peripapillary microangiopathy often precedes the visual loss {9, 101. Additional findings, that is, cardiac conduction defects [2, 11, 12) and neurological signs 113-161, have been described. The accumulated knowledge of LHON is based mostly on investigations of families where information concerning the LHON mutation is lacking. It is therefore important to do thorough investigations of
Patients A Swedish family with LHON was investigated (Fig 1). Fifteen members of the family (Cases III:9 and 15,IV:6 to 11 and 13, V:l to 5 and 13) were examined by an experienced neurologist (0.A,). For the other individuals, data were obtained from interviewswith relatives. Visual acuity was examined with a Hedin chart [17}. Color vision was investigated with Bostrom-Kugelberg pseudoisochromatic plates in persons below the age of 50 years [18) and with the Ishihara
From the Departments of +Clinical Chemistry, $Neurology, and Spathology, Gothenburg University, Sahlgren’s Hospital, and §Departrnent of Clinical Genetics, East Hospital, Gothenburg, Sweden.
Address correspondence to Dr N.-G. Iarsson, Department of Clinical Chemistry, Guthenburg University, Sahlgren’s Hospital, 5-413 45 Gothenburg, Sweden.
families that have the LHON mutation to delineate further the phenotype linked to this mutation. The LHON mutation affects a complex I gene, which implies deficient respiratory chain function. Most information on deficiencies in the respiratory chain function in mitochondrial disorders emanates from investigations of skeletal muscle, which is affected in the majority of patients. It is therefore of special interest to study muscle tissue from patients with LHON for comparison with findings in patients with other mitochondrial disorders. We report the clinical findings and the results of morphological and biochemical investigations of skeletal muscle in a family that is homoplasmic for the LHON mutation. Materials and Methods
Received Dec 17, 1990, and in revised form Mar 4 and Apr 29, 1991. Accepted for publication May 4, 1991.
Copyright 0 1991 by the American Neurological Association
701
I
Restriction Enzyme Analysis
m
Total DNA was isolated from blood, muscle, and cultivated fibroblasts and cleaved with the restriction endonuclease SfaNI (New England Biolabs, Beverly, MA) [31. The human mtDNA fragment from nucleotides 10254 to 11922 (fragment 13) 1251was used as aprobe. The methods for isolation of DNA, blotting, hybridization, and labeling of probes were as previously described {25].
II
V
Polymerase Chain Reaction and DNA Sequencing
VI
Fig 1. Pedigree of the family with Leber’s hereditary optic neuropathy. Key to the pedigree: healthy by history (0, healthy by examination blind by history @I, and optic neuropathy by examination ( .,a). The a m indicates the proband. Case V:3 had bilateral peripapillary microangiopatby.
(m, a),
o),
(a,
pseudoisochromatic plates for those above the age of 50 years, as well as with Sahlgren’s saturation test 117). Nystagmus was diagnosed only when phasic gaze-directed eye movements were recorded, and in no instance were pendular movements of iunaurotic eyes recorded as nystagmus. The vibration sensibility on fingers and toes was examined with a newly calibrated Bio-Thesiometer (Bio-Medical Company, Chagrin Falls, OH) at a vibration level appropriate for the age of the person 120). Muscle biopsy specimens were obtained from Cases IV:b and V:2 to 5. The control biopsy specimens were from 6 adults investigated for suspected neuromuscular disease without evidence of mitochondrial disease. M e have investigated another 151 patients for suspected mitochondrial disease. Fourteen had isolated complex I deficiency on polarographic measurements of the respiratory chain function in isolated muscle mitochondria. The biochemicd findings in these 14 patients were used for comparison with the patients with LHON. Three were adults (mean age, 37 years; range, 20 to 52 years) and 11 were children (mean age, 8 years; range, 1 to 15 years). All biopsy specimens were obtained after informed consent was obtained from the patients or the parents.
Muscle Biopsies Skeletal muscle specimens were obtained from the vastus lateralis muscle by open biopsy as described 1211. Local anesthesia of the skin was used in adults. In children general anesthesia was used.
A DNA fragment, 2 15 bp, was amplified by the polymerase chain reaction (PCR) [26], with the primers corresponding to nucleotides 11646 to 11665 on the heavy strand of mtDNA (NG12) and nucleotides 11841 to 11860 on the light strand of rntDNA (NG13). Total DNA (0.1 pg) was amplified in Tris-hydrochloric acid (HCI) buffer (10 mrnoVliter, p H 8.3) containing potassium chloride (KCI) (50 mmoliL), magnesium chloride (MgCl,) (1.5 mmol/L), gelatin (0.01 gm/L), 2-mercaptoethanol (50 mmol/L), deoxyribo-nucleotides (0.25 mrnol/L) (Pharmacia, Uppsala, Sweden), primers (50 pmol each), and TaqDNA-polymerase (5 units) (Perkin Elmer Cetus, Norwalk, CT), in a total volume of 50 p1. A DNA thermal cycler (Perkin Elmer Cetus) was used to perform 40 cycles of amplification with denaturation at 74°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 90 seconds. Samples of the amplification mixture (5 ~ 1 were ) digested with the restriction endonuclease SfaNI (1 unit) and separated in a gel with NuSieve GTG agarose (2.5%) (FMC BioProducts, Rockland, ME) and ultra pure agarose (0.7%). To generate single-stranded DNA for sequencing, the asymmetric primer method was used 1271. The singlestranded DNA was purified with microconcentrators, Centricon 30 (Amincon, Danvers, MA), according to the manufacturer. Sequencing was performed with the Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH). Singlestranded DNA corresponding to the light strand of mtDNA was obtained with primer NG12 (4 pmol) and primer NG13 (50 pmol). An oligonucleotide corresponding to nucleotides 11691 to 11710 of the heavy strand of mtDNA (NG15) was used as the internal sequencing primer. Single-stranded DNA corresponding to the heavy strand of mtDNA was obtained with primer NG12 (50 pmol) and primer NG13 (4 pmol). An oligonucleotide corresponding to nucleotides 11821 to 11840 of the light strand of mtDNA (NG20) was used as the internal sequencing primer.
Morphological Examination of Muscle
Biochemist y
Enzyme histochemical and ultrastructural analysis of muscle was performed as described [21].
Polarographk measurtlments of the respiratory chain function were performed on isolated muscle mitochondria from fresh muscle tissue essentially as described by Scholte and colleagues [22, 231. The respiratory rate with ascorbate as substrate was measured in the presence of TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine) (500 pmol/L). A concentration of TMPD (600 FmoliL) was used by Scholte (personal commurilcation, 1788). Spectrophotometric measurements were done on freeze-thawed mitochondrial preparations as previously described 1241.
Results Clinical Findings Bilateral severe optic atrophy was found in 4 subjects. These all had gaze-evoked nystagmus and 3 had decreased vibratioa sensibility in the feet. The 3 males suffered severe visual loss at the age of 18 (Case IV:7), 26 (Case V:l), and 36 years (Case V:2). The female (Case II1:9) became blind at the age of 72 years. Bilat-
702 Annals of Neurology
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N o 5 November 1971
Fig 3. Restriction fragment analyJis of mtDNA in djfferent tissues showing homoplasmyfor the Leber’s hweditaty optic neuropathy malation. The polymerase chain reaction was used to amplifv a 215-bp mtDNA fragment sarrounding the point matation. The amplifiedfragment was cieaved with the restriction endonuclease SfaNI and separated in NuSiPve agarose with ethidiumbromide. The analysis of D N A from blood, muscle, and fibroblasts (FIBRO)from Cases IV:8 and V:2 to j (lanes 1 t o 5, respectivelyi and D N A from blood from a control (lane C) are shown. A size marker is shown in lane M. Fig 2. M R I of the brain of the proband (Case V:2). T l weighted seqaences show symmetrical, sharply defined low-signal areas in the putamen. The lesions are indicated with arrouir.
eral peripapillary microangiopathy was found in 1 female, aged 39 years (Case V:3). She had no additional neurological signs. There were no symptoms or signs of abnormal ophthalmological or neurological function in the other examined subjects. The proband, Case V:2 (see Fig l), was a farm worker with a history of alcohol abuse and smoking. He was healthy until the age of 36 years when he experienced progressive bilateral loss of vision. He was virtually blind within 6 months. From the age of 37 years, he noted a disturbance of equilibrium and a tremor in the left hand. At the age of 38 years, vision was hand movements. Bilateral severe optic atrophy without specific diagnostic clues was found. H e presented a cerebellar-extrapyramidal tremor of his left arm and moderate left-side rigidity. He had bilateral ankle clonus and extensor plantar response. H e had bilateral gaze-evoked nystagmus. Magnetic resonance imaging (MRI) of the brain showed bilateral sharply defined lesions in the putamen (Fig 2). After diagnostic workup and advice, he totally abstained from alcohol. At an investigation at the age of 39 years, the left-side tremor and the rigidity were reduced, but ophthalmological findings were unchanged. MRIs of the brain of his mother (Case IV:8) and a brother (Case V:4) appeared normal. Electrocardiograms in Cases IV:8 and V:2 to 5 were essentially normal. Preexcitation was not found.
mtDNA Analysis To detect the LHON mutation, D N A from blood was digested with the endonuclease SfuNI. The restriction enzyme site around nucleotide 11778 was lost in the proband and all maternally related relatives (Cases IIk9 and 15; IV:6 to 8, 11, and 13; V:l to 5 and 13). Cases IV:9 and 10 were paternally related to the proband and did not have the point mutation. No heteroplasmy was found after prolonged exposure of the blots. These results were confirmed by PCR amplification of blood DNA and SfaNI digestion of the 215-bp fragment surrounding the point mutation. Muscle D N A from the proband (Case V:2) and muscle and fibroblast DNA from the mother of the proband (Case IV:8) and his 3 siblings (Cases V:3-5) were also examined. All examined tissues were homoplasmic for the point mutation. N o normal mtDNA could be detected after SfaNI digestion of total D N A or the 215-bp PCR fragment (Fig 3 ) obtained from these tissues. By using PCR and direct sequencing of both mtDNA strands of the region surrounding the point mutation, the previously described guanine to adenine conversion at nucleotide 11778 {3] was identified in the proband. Biochemistry
A muscle biopsy specimen was taken from the proband, his 3 siblings, and his mother. Polarographic measurements of the respiratory chain function in muscle mitochondria showed reduced oxidation rates for complex I-linked substrates (pyruvate plus malate and glutamate plus malate) whereas normal oxidation rates were found for substrates dependent on complex 11, Iarsson et al: LHON and Complex 1 Deficiency
703
*
Table I . PolavographicMeasurements of the Respiratovy Chain Function in Isolated Muscle Mitochondria f+om Fi%deMembws ofthe Affected Family Age at Biopsy
C0.X IV:8 v:2 V.3 V:4 V.5 Controls (n = 6 ) Mean t SD hnge
Isolated Miruchondna
Pyruvare
+ Malate
Glut-a~e * Malare
Succinate
+
Ascorbace
Rorcnonc
+ TMPD
(mgpmtrmlgm
!yr)
nu.ir/ri
64 39 39 38 29
3.29 1.65 3.70 3.40
35 t 17 21-61
2 76 t 1.89 1.26-6.28
3.69
RCI
P:O
v
RCI
P:O
v
3.2 35 37 5.4 30
61 4 73.0 58.7 45.6 77.7
4.5 27 1.8
3.2 39
1.8
69.9
5.6 4.8 4.3 1.8 5.3
2.4
5.3 5.0
117 139 105 121 134
107 ? 9 95.4-118
6.6 f 2.3 3.2-9.2
3.7 0.49 3.3-4.7
114 t 14 95.4-128
3.6 ? 0.93 2.6-5 2
4 5 f 0.81 3 7-5.7
122 t 18 98.1-151
55.4 72.5 55.9 36.7
*
59
RCI
P:O
v
RC.1
3.7
23 I 322 249 246 285
16
1.5
15 1.5 1.6 1.5
1.7
2.9 3.4
2.2 2.3 2.6 2.4 2.4
1.8 2 1.2 2.0-5.0
2.4 t 0.30 2.1-2.9
290 44 214-339
15
2.5
26
P.0
1.8 19 1.7
*
0.17 1.2-1.7
1 8 t 0 16 1.5-2.0
v = respiratory rate in the presence of ADP, expressed in natoms O/mg proteinimin; RCI = respiratory control index, the stimulation of respiratory rate by ADP; P : O = moles ADP phosphory1ated:atoms 0 consumed.
111, and IV activities in all investigated subjects (Table 1). There was no correlation between the degree of complex I deficiency in muscle and the occurrence of blindness or other neurological symptoms. The blind proband (Case V:2) had the highest complex I activity, whereas his clinically unaffected brother (Case V:4) had the lowest activity. Among other patients investigated for mitochondrial disease, we found 14 with deficient complex I activity in muscle in the polarographic assay. In this group, there is a good correlation between the respiratory rates in the presence of the complex I-linked substrates pyruvate plus malate (r = 0.86) (Fig 4) or glutamate plus malate (r = 0.89) and spectrophotometric measurements of complex I activity (NADH ferricyanide reductase). Such correlation was not found in the investigated members of the LHON family (see Fig 4). The enzyme activities of complex I (NADH ferricyanide reductase and rotenone-sensitive N A D H cytochrome c reductase), complex I1 and I11 (succinate cytochrome c reductase), and complex IV (cytochrome c oxidase), as well as the activity of the citric acid cycle enzyme, citrate synthase, were normal (Table 2). The activities of fumarase, glutamate dehydrogenase, and malate dehydrogenase in muscle were also normal. The resting plasma lactate concentration (reference interval, 0.8 to 1.8 mmol/L) was slightly elevated to 1.9 mmol/L in Case V:2 and normal, 1.5, 1.O, 1.8, and 1.4 mmol/L in Cases IV:8 and V:3 to 5. Lactate in cerebrospinal Auid was measured in the proband (V:2), and the concentration was slightly elevated to 1.9 mmol/L (reference interval, 0.8 to 1.8 mmol/L).
Morphological Examination of Muscle All but 1 patient (IV:8) showed mitochondrial abnormalities. In 4, large subsarcolemmal accumulations of mitochondria were observed in the muscle fibers (Figs 5a and 5b). The subsarcolemmal mitochondria were frequently enlarged. In 1 patient paracrystalline inclusions were found in occasional muscle fibers (Fig 5c). In 2 of the patients several mitochondria with large
I
-1
0
20 40 60 Ox i da t i o n r a t e ip y r u v a t e/ma l a t el nmol O/min mg p r o t e i n
80
Fig 4. Corre(ation between polarographic and spectrophotometric measurements of complex I activity in isolated muscle mitocbondriu. The open circles s h w the activities in 14 patients with isolated complex I deficiency on pohrogruphic analysis. The regression line has been calcakated with the leust square method from the results in this group (r = 0.86). Thejlled circles s h w the activities in the jive investigated members of the family with Leber's hereditavy optic newrapatby (Cases IV:8 and V:2 to 51.
electron-dense inclusions were present (Fig 5d). The amount of lipid appeared to be slightly increased in scattered muscle fibers in all subjects. The intermyofibrillar network was slightly irregular in 2 subjects.
Discussion We found that in a family with homoplasmy for the LHON mutation there is a functional deficiency of complex I in intact muscle mitochondria. This deficiency was not only found in patients with optic neuropathy, but also in healthy individuals with the mutation. The deficiency was moderate and accompanied by morphological changes of mitochondria in 4 of the 5 investigated subjects. Similar morphological changes of muscle mitochondria in LH ON have been reported by others [28-30). Parker and associates reported de-
704 Annals of Neurology Vol 30 No 5 November 1991
Table 2. Spectrophotometric Measurements o f Respivatoty Chain Enzyme Actizlities in Isolated Muscle Mitochondria fmm Five Members of the A#ected Fami4 Rotenone-
Sensitive NADH
NADH
Ferricyanide Reductase
Cytochrome c
Succinate Cytochrome c Reductase
Cytochrome c Oxidase
Citrate Synthase (pnollmin mg
ikimg protein)
protein)
245 444 262 468 485
9.34 11.6 13.1 17.7 13.7
2.12 2.50 2.45 2.28
263 f 41 207-309
10.0 +- 1.37 7.70-11.8
2.03 2 0.44 1.26-2.54
(pmoNmin mg protein}
Reductase (nmoltmin tng protein)
(nmolimin mg protein)
6.08 5.02 7.51 7.41 6.74
24 3 150 248 25 1 289
6.39 ? 1.87 3.08-8.50
281 ? 149 139-523
Case 1V:B v:2 v:3 v:4 V:5
Controls (n Mean
Range
?
=
SD
6)
creased complex I activity in thrombocytes measured as the rotenone-sensitive N A D H ubiquinone reductase activity [3 I}. Uemura and colleagues reported normal complex I activity in muscle, measured as the rotenone-sensitive N A D H cytochrome c reductase activity [30], which was also found in our patients using the same method. This is at variance with our experience from other mitochondrial disorders, where good agreement is found between polarographic measurements of complex I activity in intact mitochondria and the spectrophotometric assays. Complex I consists of at least 25 different polypeptides and very little is known about the function of the different subunits and how they interact with each other and with other parts of the oxidative phosphorylation system 1321. Measurements of the respiratory rate in intact mitochondria are more likely to reflect the in vivo activity of complex I than are the different spectrophotometric assays. There is no coupling to ADP phosphorylation in these assays. The different electron acceptors used interact with the respiratory chain in different ways and may be more or less sensitive to defects in different parts of the complex. Another possibility is that the only effect of the mutation in complex I is an extraordinary sensitivity to endogenous or exogenous inhibitors resulting in a depressed in vivo activity. If there is a reversible inhibition of complex I, the preparation and assay procedures would be crucial for the detection of a depressed activity. Highly energydependent tissues (i.e., the central nervous system, the heart, and skeletal muscle) are preferentially affected in mitochondrial disorders [33]. These tissues, however, are not affected in a uniform pattern. In some disorders, for example, cytochrome c oxidase deficiency, the variable clinical pictures may be due to tissue-specific isoenzymes [34, 351. In the disorders due to mutations of mtDNA, heteroplasmy
1.95
occurs with a different percentage of mutated D NA in different organs [S, 25, 361. This may determine whether symptoms from a certain organ occur or not. From work with transmitochondrial cell lines, it has been shown that mitochondria with the same genotype have different rates of respiration in different nuclear backgrounds [37). This suggests that nuclear genes influence to what extent a certain mtDNA mutation affects the respiratory rate in an organ. The occurrence of optic neuropathy in six Finnish families with the LHON mutation is possibly linked to a locus on the X chromosome {38]. hnkage to the X chromosome would explain a higher vulnerability in men. In our family, which is homoplasmic for the W O N mutation, the optic nerve is obviously the most susceptible tissue. There were additional neurological signs, but these were only found together with optic neuropathy. In several large pedigrees with LHON, no associated neurological symptoms have been described [1, 21. Discrete signs of central nervous system involvement may not have been diagnosed since no neurological examination was performed. Minor neurological signs, including reflex abnormalities, have been described in some LHON families [ 151. In other famtlies major neurological signs occurred in childhood. Both an acute encephalopathy with respiratory difficulties [ 131 and an extrapyramidal-pyramidal syndrome [16}, similar to familial striatal degeneration { 391, have been described. In the family of Novotny and coworkers 1161 the adult manifestation of W O N and the childhood manifestation of striatal degeneration occurred both isolated and in combination. This family had a maternal pattern of inheritance, but the LHON mutation was not found IS]. The most consistent computed tomography (CT) findings in this family were symmetrical low-attenuation areas in the putamen [40). These findings are similar to the MRI findings in our most Larsson et al: LHON and Complex I Deficiency
705
Fig 5 . Morphological changes in muscle. (a) rncubation for succinate dehydrogenase shows subsarcolemmal accumulation of stained material in the majority of the muscle fibers (Case V:4). (b) Large subsarcolemmal accumulations of mitochondria (Case V:3). (c) Paracrystalline inclusions in mitochondria (Case V:Sj. (d) Large electron-dense inclusion in a mitochondrion (Case V:2).
706 Annals of Neurology Vol 30 No 5
November 1991
affected individual (Case V:2). The history of this patient agrees with the notion that the clinical manifestation of LHON not only is governed by genetic factors but also is influenced by exogenous factors. The acute or subacute occurrence of blindness indicates that it is precipitated by a sudden event that compromises the metabolism in the affected tissue. Signs of sudden metabolic incompensation are also seen in other mitochondrial disorders, most marked in the stroke-like episodes of MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), where a deficiency of complex I is often found {411. Exogenous factors, for example, cyanide in cigarette smoke, have been suggested to be of impotrance for the development of LHON 129, 42, 431. Cyanide, however, is most effective as a cytochrome c oxidase inhibitor. A better candidate would be a complex I inhibitor, for example, nitric oxide. Nitric oxide, which has been identified as the active principle of endothelid-derived relaxation factor (EDRF) 144, 451 is released not only from endothelial cells but also by other cells (e.g., neutrophils and activated macrophages) 146, 47). Activated macrophages were shown to inhibit the first complexes of the respiratory chain in target cells by Granger and Lehninger 1481. In these cells nitric oxide was convincingly shown to be the effector molecule, whch binds to iron-sulphur ciusters and inhibits complex I and I1 activity of the respiratory chain and the citric acid cycle enzyme aconitase 148-5 I}. Heavy exercise increases the urinary excretion of nittic oxide-derived nitrate, and in infection the excretion is increased several hundred-fold 1521. The effects of nitric oxide could also be mimicked by cigarette smoke, which contains 500 to 1,000ppm of nitric okide in the filtered gaseous phase 1537. It is possible that increased nitric oxide exposure or production may be the trigger that precipitates an acute metabolic incompensation and subsequent tissue damage in susceptible individuals.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
15. 16.
7. 8.
9. 20. 21.
This study was supported by grants from the Swedish Medical Research Council (project numbers 585 and 7122), the Kronprinsessan Margaretas Arbetsnhnd for synskadade, First of May Flower Annual Campaign, The Goteborg Medical Society, Linnea and Josef Carlsson foundation, SvenJerring foundation, and Wilhelm and Martina Lundgren foundation.
22.
23.
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Larsson e t al: LHON and Complex I
Deficiency 707
27. Gyllensten UB, Ehrlich HA. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci USA 1988;85:7652-7656 28. Federico A, Manneschi L, Meloni M, et al. Histochemical, ultrastructural and biochemical study of muscle mitochondria in Leber’s hereditary optic atrophy. J Inherited Metab Dis 1988; ll(SUpp1 2):193-197 29. Nikoskelainen E, Hassinen IE, Paljirvi L, et al. Leber’s hereditary optic neuroretinoparhy, a mitochondrial disease? Lancet 1984;2:1474 30. Uemura A, Osame M, Nakagawa M, et al. Leber’s hereditary optic neuropathy: mitochondrial and biochemical studies on muscle biopsies. Br J Ophthalmol 1987;71:531-536 31. Parke WD, Oley CA, Parks JK. A defect in rnitochondrial electron-transport activity (NADH-coenzyme Q oxidoreductase) in kber’s hereditary optic neuropathy. N Engl J Med 1989;320:1331-1333 32. Hatefi Y . The mitochondrial electron transport and oxidative phosphorylation system. Annu Rev Biochem 1985;54:10151069 33. DiMauro S, Bonilla E, Zeviani M, et al. Mitochondrial myopa[hies. Ann Neurol 1985;17:521-538 34. Lomax MI, Grossman LI. Tissue-specific genes for respiratory proteins. Trends Biochem Sci 1987;14:501-503 35. DiMauro S, Zeviani M, Servidei S, et al. Biochemical and molecular aspects of cyrochrome c oxidase deficiency. Adv Neurol 1988;48:93-105 36. Moraes CT, DiMauro S, Zeviani M, et al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and KearnsSayre syndrome. N Engl J Med 1989;320:1293-1299 37. King MP, Attardi G. Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation. Science 1989;246:500-503 38. Vilkki J. Mitochondrial inheritance in man: genetics of Leber hereditary optic neuroretinopathy. Thesis. Department of Biology, University of Turku, Finland, 1990 37. Roessman U, Schwartz JF. Familial striatal degeneration. Arch Neurol 1973;29:314-317 40. Seidenwurm D, Novotny E, Marshall W, Enzmann D. MR and CT in cytoplasmicdy inherited striatal degeneration. AJNR 1988;7:629-632
708 Annals of Neurology Vol 30 No 5
41. Koga Y, Nonaka I, Kobayashi M, et al. Findings in muscle in complex I (NADH coenzyme Q reductase) deficiency. Ann Neurol 1988;24:749-756 42. Berninger TA, Meyer L, Siess E, et al. Leber’s hereditary optic atrophy: further evidence for a defect of cyanide metabolism? Br J Ophthalmol 1989;73:314-316 43. Pallini R, Martelli P, Bardelli AM, et al. Normal rhodanese activity in leukocytes from Leber patients: enzyme characterization and activity levels. Neurology 1987;37:1878-1880 44.Ignarro LJ. Biosynthesis and metabolism of endotheliumderived nitric oxide. Annu Rev Pharmacol Toxicol 1990;30: 535-560 45. Vane JR, A-ird EE, Botting RM. Regulatory functions of the vascular endothelium. N Engl J Med 199032327-36 46. Wright CD, Mulsch A, Busse R, Osswald H. Generation of nitric oxide by human neutrophils. Biochim Biophys Acta 1989;160:813-819 47. Marletta MA, Yoon PS, Iyengar R, et al. Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 1988;27:8706-87 11 48. Granger DL, Lehninger AL. Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J Cell Biol 1982;95:527-535 49. Drapier JC, Hibbs JB. Murine cytotoxic activated macrophages inhibit aconicase in tumor cells. J Clin Invest 1986;78:790-797 50. Drapier JC, Hibbs JB. Differentiation of murine macrophages to express nonspecific cytotoxicity for tumor cells results in Larginine-dependent inhibition of mitochondrial iron-sulfur enzymes in the macrophage effector cells. J Immunol 1988;140: 2829-2838 51. Pellat C , Henry Y ,Drapier JC. IFN-,-activated macrophages: detection by electron paramagnetic resonance of complexes between L-arginine-derived nitric oxide and non-heme iron proteins. Biochem Biophys Res Commun 1990;166:119125 52. Leaf CD, Wishnok JS, Tannenbaum SR. L-Arginine is a precursor for nitrate biosynthesis in humans. Biochem Biophys Res Commun 1989;163:1032-1037 53. Mellion BT, Ignarro LJ, Ohlstein EH, et al. Evidence for the inhibitory role of guanosine 3’,5’-monophosphate in ADPinduced human platelet aggregation in the presence of nitric oxide and related vasodilators. Blood 1981;57:946-955
November 1991
We investigated a family with Leber’s hereditary optic neuropathy in which affected individuals were homoplasmic for the point mutation of the NADH-dehydrogenase 4 gene of mitochondrial DNA, described by Wallace and colleagues in 1988. The proband had bilateral optic atrophy, tremor, dystonia, and sharply defined lesions in the putamen on magnetic resonance images. Optic atrophy was found in another 3 of 13 investigated relatives on the maternal side. Additional neurological signs were found but only in patients with optic neuropathy. The morphological appearance and the respiratory chain function of muscle tissue were investigated in the proband, his mother, and 3 siblings. Polarographic measurements revealed complex I deficiency in the 5 investigated subjects. Morphological changes of mitochondria were found in 4 of these subjects. There was no decrease in complex I activity measured as NADH ferricyanide reductase or rotenone-sensitive NADH cytochrome c reductase activities. In other cases with complex I deficiency, good agreement between polarographic and spectrophotornetric measurements was found. This study showed that there is decreased activity of complex I of the respiratory chain in muscle and that cerebral striatal lesions occur in Leber’s hereditary optic neuropathy with the NADH-dehydrogenase 4 gene point mutation. Larsson NG, Andersen 0, Holme E, Oldfors A, Wahlstrom J. Leber’s hereditary optic neuropathy and complex I deficiency in muscle. Ann Neurol 1991;30:701-708
Leber’s hereditary optic neuropathy (LHON) (McKusick 308900) is a maternally inherited disease causing blindness predominantly in young men 11, 21. A point mutation (LHON mutation) that changes a highly conserved amino acid from arginine to histidine in subunit 4 of complex I (NADII ubiquinone oxidoreductase, EC 1.6.5.3)of the respiratory chain was found by Wallace and colleagues in patients with LHON { 3 } . The LHON mutation results in loss of a cleavage site for the restriction endonuclease SfaNI and is found in about half of the families with the disease 14-63. Some families with the LHON mutation are heteroplasmic; that is, mutated mitochondrial DNA (mtDNA) is found together with normal mtDNA in proportions that can vary both between individuals and between tissues of the same individual [5-8). In the early phase of the disease, tortuous and swollen retinal vessels are found. This peripapillary microangiopathy often precedes the visual loss {9, 101. Additional findings, that is, cardiac conduction defects [2, 11, 12) and neurological signs 113-161, have been described. The accumulated knowledge of LHON is based mostly on investigations of families where information concerning the LHON mutation is lacking. It is therefore important to do thorough investigations of
Patients A Swedish family with LHON was investigated (Fig 1). Fifteen members of the family (Cases III:9 and 15,IV:6 to 11 and 13, V:l to 5 and 13) were examined by an experienced neurologist (0.A,). For the other individuals, data were obtained from interviewswith relatives. Visual acuity was examined with a Hedin chart [17}. Color vision was investigated with Bostrom-Kugelberg pseudoisochromatic plates in persons below the age of 50 years [18) and with the Ishihara
From the Departments of +Clinical Chemistry, $Neurology, and Spathology, Gothenburg University, Sahlgren’s Hospital, and §Departrnent of Clinical Genetics, East Hospital, Gothenburg, Sweden.
Address correspondence to Dr N.-G. Iarsson, Department of Clinical Chemistry, Guthenburg University, Sahlgren’s Hospital, 5-413 45 Gothenburg, Sweden.
families that have the LHON mutation to delineate further the phenotype linked to this mutation. The LHON mutation affects a complex I gene, which implies deficient respiratory chain function. Most information on deficiencies in the respiratory chain function in mitochondrial disorders emanates from investigations of skeletal muscle, which is affected in the majority of patients. It is therefore of special interest to study muscle tissue from patients with LHON for comparison with findings in patients with other mitochondrial disorders. We report the clinical findings and the results of morphological and biochemical investigations of skeletal muscle in a family that is homoplasmic for the LHON mutation. Materials and Methods
Received Dec 17, 1990, and in revised form Mar 4 and Apr 29, 1991. Accepted for publication May 4, 1991.
Copyright 0 1991 by the American Neurological Association
701
I
Restriction Enzyme Analysis
m
Total DNA was isolated from blood, muscle, and cultivated fibroblasts and cleaved with the restriction endonuclease SfaNI (New England Biolabs, Beverly, MA) [31. The human mtDNA fragment from nucleotides 10254 to 11922 (fragment 13) 1251was used as aprobe. The methods for isolation of DNA, blotting, hybridization, and labeling of probes were as previously described {25].
II
V
Polymerase Chain Reaction and DNA Sequencing
VI
Fig 1. Pedigree of the family with Leber’s hereditary optic neuropathy. Key to the pedigree: healthy by history (0, healthy by examination blind by history @I, and optic neuropathy by examination ( .,a). The a m indicates the proband. Case V:3 had bilateral peripapillary microangiopatby.
(m, a),
o),
(a,
pseudoisochromatic plates for those above the age of 50 years, as well as with Sahlgren’s saturation test 117). Nystagmus was diagnosed only when phasic gaze-directed eye movements were recorded, and in no instance were pendular movements of iunaurotic eyes recorded as nystagmus. The vibration sensibility on fingers and toes was examined with a newly calibrated Bio-Thesiometer (Bio-Medical Company, Chagrin Falls, OH) at a vibration level appropriate for the age of the person 120). Muscle biopsy specimens were obtained from Cases IV:b and V:2 to 5. The control biopsy specimens were from 6 adults investigated for suspected neuromuscular disease without evidence of mitochondrial disease. M e have investigated another 151 patients for suspected mitochondrial disease. Fourteen had isolated complex I deficiency on polarographic measurements of the respiratory chain function in isolated muscle mitochondria. The biochemicd findings in these 14 patients were used for comparison with the patients with LHON. Three were adults (mean age, 37 years; range, 20 to 52 years) and 11 were children (mean age, 8 years; range, 1 to 15 years). All biopsy specimens were obtained after informed consent was obtained from the patients or the parents.
Muscle Biopsies Skeletal muscle specimens were obtained from the vastus lateralis muscle by open biopsy as described 1211. Local anesthesia of the skin was used in adults. In children general anesthesia was used.
A DNA fragment, 2 15 bp, was amplified by the polymerase chain reaction (PCR) [26], with the primers corresponding to nucleotides 11646 to 11665 on the heavy strand of mtDNA (NG12) and nucleotides 11841 to 11860 on the light strand of rntDNA (NG13). Total DNA (0.1 pg) was amplified in Tris-hydrochloric acid (HCI) buffer (10 mrnoVliter, p H 8.3) containing potassium chloride (KCI) (50 mmoliL), magnesium chloride (MgCl,) (1.5 mmol/L), gelatin (0.01 gm/L), 2-mercaptoethanol (50 mmol/L), deoxyribo-nucleotides (0.25 mrnol/L) (Pharmacia, Uppsala, Sweden), primers (50 pmol each), and TaqDNA-polymerase (5 units) (Perkin Elmer Cetus, Norwalk, CT), in a total volume of 50 p1. A DNA thermal cycler (Perkin Elmer Cetus) was used to perform 40 cycles of amplification with denaturation at 74°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 90 seconds. Samples of the amplification mixture (5 ~ 1 were ) digested with the restriction endonuclease SfaNI (1 unit) and separated in a gel with NuSieve GTG agarose (2.5%) (FMC BioProducts, Rockland, ME) and ultra pure agarose (0.7%). To generate single-stranded DNA for sequencing, the asymmetric primer method was used 1271. The singlestranded DNA was purified with microconcentrators, Centricon 30 (Amincon, Danvers, MA), according to the manufacturer. Sequencing was performed with the Sequenase version 2.0 kit (United States Biochemical, Cleveland, OH). Singlestranded DNA corresponding to the light strand of mtDNA was obtained with primer NG12 (4 pmol) and primer NG13 (50 pmol). An oligonucleotide corresponding to nucleotides 11691 to 11710 of the heavy strand of mtDNA (NG15) was used as the internal sequencing primer. Single-stranded DNA corresponding to the heavy strand of mtDNA was obtained with primer NG12 (50 pmol) and primer NG13 (4 pmol). An oligonucleotide corresponding to nucleotides 11821 to 11840 of the light strand of mtDNA (NG20) was used as the internal sequencing primer.
Morphological Examination of Muscle
Biochemist y
Enzyme histochemical and ultrastructural analysis of muscle was performed as described [21].
Polarographk measurtlments of the respiratory chain function were performed on isolated muscle mitochondria from fresh muscle tissue essentially as described by Scholte and colleagues [22, 231. The respiratory rate with ascorbate as substrate was measured in the presence of TMPD (N,N,N’,N’-tetramethyl-p-phenylenediamine) (500 pmol/L). A concentration of TMPD (600 FmoliL) was used by Scholte (personal commurilcation, 1788). Spectrophotometric measurements were done on freeze-thawed mitochondrial preparations as previously described 1241.
Results Clinical Findings Bilateral severe optic atrophy was found in 4 subjects. These all had gaze-evoked nystagmus and 3 had decreased vibratioa sensibility in the feet. The 3 males suffered severe visual loss at the age of 18 (Case IV:7), 26 (Case V:l), and 36 years (Case V:2). The female (Case II1:9) became blind at the age of 72 years. Bilat-
702 Annals of Neurology
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N o 5 November 1971
Fig 3. Restriction fragment analyJis of mtDNA in djfferent tissues showing homoplasmyfor the Leber’s hweditaty optic neuropathy malation. The polymerase chain reaction was used to amplifv a 215-bp mtDNA fragment sarrounding the point matation. The amplifiedfragment was cieaved with the restriction endonuclease SfaNI and separated in NuSiPve agarose with ethidiumbromide. The analysis of D N A from blood, muscle, and fibroblasts (FIBRO)from Cases IV:8 and V:2 to j (lanes 1 t o 5, respectivelyi and D N A from blood from a control (lane C) are shown. A size marker is shown in lane M. Fig 2. M R I of the brain of the proband (Case V:2). T l weighted seqaences show symmetrical, sharply defined low-signal areas in the putamen. The lesions are indicated with arrouir.
eral peripapillary microangiopathy was found in 1 female, aged 39 years (Case V:3). She had no additional neurological signs. There were no symptoms or signs of abnormal ophthalmological or neurological function in the other examined subjects. The proband, Case V:2 (see Fig l), was a farm worker with a history of alcohol abuse and smoking. He was healthy until the age of 36 years when he experienced progressive bilateral loss of vision. He was virtually blind within 6 months. From the age of 37 years, he noted a disturbance of equilibrium and a tremor in the left hand. At the age of 38 years, vision was hand movements. Bilateral severe optic atrophy without specific diagnostic clues was found. H e presented a cerebellar-extrapyramidal tremor of his left arm and moderate left-side rigidity. He had bilateral ankle clonus and extensor plantar response. H e had bilateral gaze-evoked nystagmus. Magnetic resonance imaging (MRI) of the brain showed bilateral sharply defined lesions in the putamen (Fig 2). After diagnostic workup and advice, he totally abstained from alcohol. At an investigation at the age of 39 years, the left-side tremor and the rigidity were reduced, but ophthalmological findings were unchanged. MRIs of the brain of his mother (Case IV:8) and a brother (Case V:4) appeared normal. Electrocardiograms in Cases IV:8 and V:2 to 5 were essentially normal. Preexcitation was not found.
mtDNA Analysis To detect the LHON mutation, D N A from blood was digested with the endonuclease SfuNI. The restriction enzyme site around nucleotide 11778 was lost in the proband and all maternally related relatives (Cases IIk9 and 15; IV:6 to 8, 11, and 13; V:l to 5 and 13). Cases IV:9 and 10 were paternally related to the proband and did not have the point mutation. No heteroplasmy was found after prolonged exposure of the blots. These results were confirmed by PCR amplification of blood DNA and SfaNI digestion of the 215-bp fragment surrounding the point mutation. Muscle D N A from the proband (Case V:2) and muscle and fibroblast DNA from the mother of the proband (Case IV:8) and his 3 siblings (Cases V:3-5) were also examined. All examined tissues were homoplasmic for the point mutation. N o normal mtDNA could be detected after SfaNI digestion of total D N A or the 215-bp PCR fragment (Fig 3 ) obtained from these tissues. By using PCR and direct sequencing of both mtDNA strands of the region surrounding the point mutation, the previously described guanine to adenine conversion at nucleotide 11778 {3] was identified in the proband. Biochemistry
A muscle biopsy specimen was taken from the proband, his 3 siblings, and his mother. Polarographic measurements of the respiratory chain function in muscle mitochondria showed reduced oxidation rates for complex I-linked substrates (pyruvate plus malate and glutamate plus malate) whereas normal oxidation rates were found for substrates dependent on complex 11, Iarsson et al: LHON and Complex 1 Deficiency
703
*
Table I . PolavographicMeasurements of the Respiratovy Chain Function in Isolated Muscle Mitochondria f+om Fi%deMembws ofthe Affected Family Age at Biopsy
C0.X IV:8 v:2 V.3 V:4 V.5 Controls (n = 6 ) Mean t SD hnge
Isolated Miruchondna
Pyruvare
+ Malate
Glut-a~e * Malare
Succinate
+
Ascorbace
Rorcnonc
+ TMPD
(mgpmtrmlgm
!yr)
nu.ir/ri
64 39 39 38 29
3.29 1.65 3.70 3.40
35 t 17 21-61
2 76 t 1.89 1.26-6.28
3.69
RCI
P:O
v
RCI
P:O
v
3.2 35 37 5.4 30
61 4 73.0 58.7 45.6 77.7
4.5 27 1.8
3.2 39
1.8
69.9
5.6 4.8 4.3 1.8 5.3
2.4
5.3 5.0
117 139 105 121 134
107 ? 9 95.4-118
6.6 f 2.3 3.2-9.2
3.7 0.49 3.3-4.7
114 t 14 95.4-128
3.6 ? 0.93 2.6-5 2
4 5 f 0.81 3 7-5.7
122 t 18 98.1-151
55.4 72.5 55.9 36.7
*
59
RCI
P:O
v
RC.1
3.7
23 I 322 249 246 285
16
1.5
15 1.5 1.6 1.5
1.7
2.9 3.4
2.2 2.3 2.6 2.4 2.4
1.8 2 1.2 2.0-5.0
2.4 t 0.30 2.1-2.9
290 44 214-339
15
2.5
26
P.0
1.8 19 1.7
*
0.17 1.2-1.7
1 8 t 0 16 1.5-2.0
v = respiratory rate in the presence of ADP, expressed in natoms O/mg proteinimin; RCI = respiratory control index, the stimulation of respiratory rate by ADP; P : O = moles ADP phosphory1ated:atoms 0 consumed.
111, and IV activities in all investigated subjects (Table 1). There was no correlation between the degree of complex I deficiency in muscle and the occurrence of blindness or other neurological symptoms. The blind proband (Case V:2) had the highest complex I activity, whereas his clinically unaffected brother (Case V:4) had the lowest activity. Among other patients investigated for mitochondrial disease, we found 14 with deficient complex I activity in muscle in the polarographic assay. In this group, there is a good correlation between the respiratory rates in the presence of the complex I-linked substrates pyruvate plus malate (r = 0.86) (Fig 4) or glutamate plus malate (r = 0.89) and spectrophotometric measurements of complex I activity (NADH ferricyanide reductase). Such correlation was not found in the investigated members of the LHON family (see Fig 4). The enzyme activities of complex I (NADH ferricyanide reductase and rotenone-sensitive N A D H cytochrome c reductase), complex I1 and I11 (succinate cytochrome c reductase), and complex IV (cytochrome c oxidase), as well as the activity of the citric acid cycle enzyme, citrate synthase, were normal (Table 2). The activities of fumarase, glutamate dehydrogenase, and malate dehydrogenase in muscle were also normal. The resting plasma lactate concentration (reference interval, 0.8 to 1.8 mmol/L) was slightly elevated to 1.9 mmol/L in Case V:2 and normal, 1.5, 1.O, 1.8, and 1.4 mmol/L in Cases IV:8 and V:3 to 5. Lactate in cerebrospinal Auid was measured in the proband (V:2), and the concentration was slightly elevated to 1.9 mmol/L (reference interval, 0.8 to 1.8 mmol/L).
Morphological Examination of Muscle All but 1 patient (IV:8) showed mitochondrial abnormalities. In 4, large subsarcolemmal accumulations of mitochondria were observed in the muscle fibers (Figs 5a and 5b). The subsarcolemmal mitochondria were frequently enlarged. In 1 patient paracrystalline inclusions were found in occasional muscle fibers (Fig 5c). In 2 of the patients several mitochondria with large
I
-1
0
20 40 60 Ox i da t i o n r a t e ip y r u v a t e/ma l a t el nmol O/min mg p r o t e i n
80
Fig 4. Corre(ation between polarographic and spectrophotometric measurements of complex I activity in isolated muscle mitocbondriu. The open circles s h w the activities in 14 patients with isolated complex I deficiency on pohrogruphic analysis. The regression line has been calcakated with the leust square method from the results in this group (r = 0.86). Thejlled circles s h w the activities in the jive investigated members of the family with Leber's hereditavy optic newrapatby (Cases IV:8 and V:2 to 51.
electron-dense inclusions were present (Fig 5d). The amount of lipid appeared to be slightly increased in scattered muscle fibers in all subjects. The intermyofibrillar network was slightly irregular in 2 subjects.
Discussion We found that in a family with homoplasmy for the LHON mutation there is a functional deficiency of complex I in intact muscle mitochondria. This deficiency was not only found in patients with optic neuropathy, but also in healthy individuals with the mutation. The deficiency was moderate and accompanied by morphological changes of mitochondria in 4 of the 5 investigated subjects. Similar morphological changes of muscle mitochondria in LH ON have been reported by others [28-30). Parker and associates reported de-
704 Annals of Neurology Vol 30 No 5 November 1991
Table 2. Spectrophotometric Measurements o f Respivatoty Chain Enzyme Actizlities in Isolated Muscle Mitochondria fmm Five Members of the A#ected Fami4 Rotenone-
Sensitive NADH
NADH
Ferricyanide Reductase
Cytochrome c
Succinate Cytochrome c Reductase
Cytochrome c Oxidase
Citrate Synthase (pnollmin mg
ikimg protein)
protein)
245 444 262 468 485
9.34 11.6 13.1 17.7 13.7
2.12 2.50 2.45 2.28
263 f 41 207-309
10.0 +- 1.37 7.70-11.8
2.03 2 0.44 1.26-2.54
(pmoNmin mg protein}
Reductase (nmoltmin tng protein)
(nmolimin mg protein)
6.08 5.02 7.51 7.41 6.74
24 3 150 248 25 1 289
6.39 ? 1.87 3.08-8.50
281 ? 149 139-523
Case 1V:B v:2 v:3 v:4 V:5
Controls (n Mean
Range
?
=
SD
6)
creased complex I activity in thrombocytes measured as the rotenone-sensitive N A D H ubiquinone reductase activity [3 I}. Uemura and colleagues reported normal complex I activity in muscle, measured as the rotenone-sensitive N A D H cytochrome c reductase activity [30], which was also found in our patients using the same method. This is at variance with our experience from other mitochondrial disorders, where good agreement is found between polarographic measurements of complex I activity in intact mitochondria and the spectrophotometric assays. Complex I consists of at least 25 different polypeptides and very little is known about the function of the different subunits and how they interact with each other and with other parts of the oxidative phosphorylation system 1321. Measurements of the respiratory rate in intact mitochondria are more likely to reflect the in vivo activity of complex I than are the different spectrophotometric assays. There is no coupling to ADP phosphorylation in these assays. The different electron acceptors used interact with the respiratory chain in different ways and may be more or less sensitive to defects in different parts of the complex. Another possibility is that the only effect of the mutation in complex I is an extraordinary sensitivity to endogenous or exogenous inhibitors resulting in a depressed in vivo activity. If there is a reversible inhibition of complex I, the preparation and assay procedures would be crucial for the detection of a depressed activity. Highly energydependent tissues (i.e., the central nervous system, the heart, and skeletal muscle) are preferentially affected in mitochondrial disorders [33]. These tissues, however, are not affected in a uniform pattern. In some disorders, for example, cytochrome c oxidase deficiency, the variable clinical pictures may be due to tissue-specific isoenzymes [34, 351. In the disorders due to mutations of mtDNA, heteroplasmy
1.95
occurs with a different percentage of mutated D NA in different organs [S, 25, 361. This may determine whether symptoms from a certain organ occur or not. From work with transmitochondrial cell lines, it has been shown that mitochondria with the same genotype have different rates of respiration in different nuclear backgrounds [37). This suggests that nuclear genes influence to what extent a certain mtDNA mutation affects the respiratory rate in an organ. The occurrence of optic neuropathy in six Finnish families with the LHON mutation is possibly linked to a locus on the X chromosome {38]. hnkage to the X chromosome would explain a higher vulnerability in men. In our family, which is homoplasmic for the W O N mutation, the optic nerve is obviously the most susceptible tissue. There were additional neurological signs, but these were only found together with optic neuropathy. In several large pedigrees with LHON, no associated neurological symptoms have been described [1, 21. Discrete signs of central nervous system involvement may not have been diagnosed since no neurological examination was performed. Minor neurological signs, including reflex abnormalities, have been described in some LHON families [ 151. In other famtlies major neurological signs occurred in childhood. Both an acute encephalopathy with respiratory difficulties [ 131 and an extrapyramidal-pyramidal syndrome [16}, similar to familial striatal degeneration { 391, have been described. In the family of Novotny and coworkers 1161 the adult manifestation of W O N and the childhood manifestation of striatal degeneration occurred both isolated and in combination. This family had a maternal pattern of inheritance, but the LHON mutation was not found IS]. The most consistent computed tomography (CT) findings in this family were symmetrical low-attenuation areas in the putamen [40). These findings are similar to the MRI findings in our most Larsson et al: LHON and Complex I Deficiency
705
Fig 5 . Morphological changes in muscle. (a) rncubation for succinate dehydrogenase shows subsarcolemmal accumulation of stained material in the majority of the muscle fibers (Case V:4). (b) Large subsarcolemmal accumulations of mitochondria (Case V:3). (c) Paracrystalline inclusions in mitochondria (Case V:Sj. (d) Large electron-dense inclusion in a mitochondrion (Case V:2).
706 Annals of Neurology Vol 30 No 5
November 1991
affected individual (Case V:2). The history of this patient agrees with the notion that the clinical manifestation of LHON not only is governed by genetic factors but also is influenced by exogenous factors. The acute or subacute occurrence of blindness indicates that it is precipitated by a sudden event that compromises the metabolism in the affected tissue. Signs of sudden metabolic incompensation are also seen in other mitochondrial disorders, most marked in the stroke-like episodes of MELAS (myopathy, encephalopathy, lactic acidosis, and stroke-like episodes), where a deficiency of complex I is often found {411. Exogenous factors, for example, cyanide in cigarette smoke, have been suggested to be of impotrance for the development of LHON 129, 42, 431. Cyanide, however, is most effective as a cytochrome c oxidase inhibitor. A better candidate would be a complex I inhibitor, for example, nitric oxide. Nitric oxide, which has been identified as the active principle of endothelid-derived relaxation factor (EDRF) 144, 451 is released not only from endothelial cells but also by other cells (e.g., neutrophils and activated macrophages) 146, 47). Activated macrophages were shown to inhibit the first complexes of the respiratory chain in target cells by Granger and Lehninger 1481. In these cells nitric oxide was convincingly shown to be the effector molecule, whch binds to iron-sulphur ciusters and inhibits complex I and I1 activity of the respiratory chain and the citric acid cycle enzyme aconitase 148-5 I}. Heavy exercise increases the urinary excretion of nittic oxide-derived nitrate, and in infection the excretion is increased several hundred-fold 1521. The effects of nitric oxide could also be mimicked by cigarette smoke, which contains 500 to 1,000ppm of nitric okide in the filtered gaseous phase 1537. It is possible that increased nitric oxide exposure or production may be the trigger that precipitates an acute metabolic incompensation and subsequent tissue damage in susceptible individuals.
5.
6.
7.
8.
9.
10.
11.
12. 13.
14.
15. 16.
7. 8.
9. 20. 21.
This study was supported by grants from the Swedish Medical Research Council (project numbers 585 and 7122), the Kronprinsessan Margaretas Arbetsnhnd for synskadade, First of May Flower Annual Campaign, The Goteborg Medical Society, Linnea and Josef Carlsson foundation, SvenJerring foundation, and Wilhelm and Martina Lundgren foundation.
22.
23.
References 1. Seedorff T. The inheritance of Leber’s disease. A genealogical follow-up study. Acta Ophthalmol 1985;63:13 5-145 2. Nikoskelainen EK, Savontaus ML, Wanne OP, et al. kber’s herehtary optic neuroretinopathy, a maternally inherited disease. Arch Ophthalmol 1987;105:665-671 3. Wallace DC, Singh G , Lott MT, et al. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 1988;242:1427-1430 4. Vilkki J, Savontaus ML, Nikoskelainen EK. Genetic heterogeneity in Leber hereditary optic neuroretinopathy revealed by
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