Brain Pathology 2: 141-147 (1992)

Disorders Associated with Depletion of Mitochondria1 DNA

Enzo Ricci 1, Carlos T. Moraes 2, Serenella Servidei 1, Pietro Tonali 1, Eduardo Bonilla 3, and Salvatore DiMauro 3 UlLDM of Rome Research Center for Neuromuscular Diseases, Institute of Neurology, Catholic University, 00168 Rome, Italy 2 H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Department of Genetics and Development, Columbia-Presbyterian Medical Center, New York, NY 10032, U.S.A. 3 H. Houston Merritt Clinical Research Center for Muscular Dystrophy and Related Diseases, Department of Neurology, Columbia-Presbyterian Medical Center, New York, NY 10032, U.S.A.

Quantitative defects of mtDNA have been recently described in patients with fatal mitochondrial disease of early infancy or mitochondrial myopathy of childhood. There was variable tissue expression and depletion of up t o 98% of mtDNA in affected tissues. Pedigree analysis was compatible with mendelian inheritance, suggesting faulty communication between nuclear and mitochondrial genomes, but the primary molecular lesion is unknown. In muscle, morphological studies allowed to correlate mtDNA depletion, absence of mtDNA-encoded peptides, mitochondrial proliferation, and loss of cytochrome c oxidase (COX) activity in individual fibers. Introduction

The mitochondrial proteins are under a dual genetic control. Most of the approximately 100 subunits forming the five multi-enzymatic complexes of the respiratory chain are encoded by nuclear DNA (nDNA), while 13 of them, belonging t o complexes I, III, IV and V, are encoded by mitochondrial DNA (mtDNA). Therefore, mitochondrial diseases may be

due both t o defects of nDNA transmitted by mendelian inheritance and to defects of mtDNA, manifesting as sporadic cases or as maternally inherited traits (1). Although separate, the nuclear and mitochondrial genomes function coordinately and mtDNA replication, which is still poorly understood, is largely controlled by the nuclear genome (2,3). Depletion of mtDNA is the first hereditary mitochondrial disease characterized by a quantitative rather than a qualitative abnormality of mtDNA. It is also one of only two disorders apparently due t o faulty communication between the nuclear and the mitochondria1 genome, i.e., a nDNA defect transrnitted by mendelian inheritance but manifesting as mtDNA alteration (4). Two hereditary clinical phenotypes, a fatal mitochondrial disease of early infancy (5) and a mitochondrial myopathy of childhood (6) have been associated to severe or partial mtDNA depletion. The main clinical and laboratory features of all reported patients are summarized in Table 1. Although clinical and didactic considerations encourage us to preserve the distinction between a severe and a milder form of the disease, they are probably different clinical expressions of the same disease. As mentioned above, depletion of mtDNA probably is not the primary event, but rather the consequence of an unknown mutation in nuclear DNA. However, biochemical and pathological alterations appear to be the direct consequence of mtDNA depletion. Demonstration of mtDNA depletion is the diagnostic hallmark of the disease because ciinical, pathological and biochemical features are not different from those seen in other mitochondrial diseases (7). In addition to these hereditary forms of mtDNA depletion, an acquired reduction of muscle mtDNA content has been documented in zidovudine (AZT)-induced mitochondrial myopathy (8). Diagnostic Procedures

Corresponding author: Dr. E. Ricci, Institute of Neurology, Catholic University, 00168 Rome, Italy Tel. +39 (6) 301 54279; Fax +39 (6)305 1343

Diseases associated with mtDNA depletion have variable tissue expression (5) and the quantitative defects of mtDNA are observed in affected tissues only (5,6). Detection of mtDNA depletion may be easy when

E. Ricci et al: Disorders of rntDNA depletion

142

Table 1 Clinical and laboratory features of patients with rntDNA depletion

Early Onset

Late Onsei

Patients

1

2

3

4

5

6

7

8

9

10

Sex

F

F

F

F

M

M

M

M

F

M

Age a t onset

B

B

B

B

B

B

B

14 rno

12 rno.

12 rno.

Age at death

11 mo

+

+ +

Weakness Hypotonia

+ +

5 mo. 3 mo. 9 rno. 4 mo. 7 rno. 9 rno.

+ +

+ +

+ +

Nephropathy

+

Liver disease

Seizures

+ +

Abnormal EEG

+

Cardiopathy

Lactic acidosis

RRF

+

+

+

+ +

+

Family history % mtDNA (tissue)

% COX activity

f

8(M)

2(M)

2(M)

12(L)

14

2

2

7

+

+

+ +

+

+

+ +

+

+ +

3 year

+

+ + + + + +

1O(L) 3(M)

0

1

+ +

ND

+

+

+ +

17M

17M

34M

14-15(M)

5

8

42

19-5

+

Patients 1,2,8,9 and 10 correspond to patients 4,5,1,2 and 3, respectively, in Reference 5. Patients 3,4,6 and 7 correspond to patients 1,2,3 and 4, respectively, in Reference 6. The medical history of patient 5 was presented in poster form, by Mazziotta and Ricci et al. a t the 29th SSIEM Annual Symposium. held in London, U.K., 1991.

muscle tissue is involved, but may prove more difficult or even impossible during life in patients without muscle involvement (see Table 1, personal observation of patient 5). In addition to Southern blot analysis, techniques of in sitzi hybridization and immunohistochemistry, in muscle sections using anti-DNA antibodies (9), have been useful to document the reduction in mtDNA content (5,6). Biochemical assays can provide useful clues to mtDNA involvement, such as the demonstration of combined deficiencies of respiratory chain enzymes containing mtDNA-encoded proteins. However, respiratory chain defects are not specific of

mtDNA depletion and can also be due to qualitative alterations of mtDNA or to nDNA mutations (7). Morphological studies complement the results of biochemical and DNA analyses. A decrease in the activity of cytochrome c oxidase (COX) can be documented by histochemistry, while a selective lack of mtDNA-encoded polypeptides can be demonstrated by immunohistochemistry (10) (Fig. 1). Antibodies against generic DNA can also be used to demonstrate a reduction of mtDNA content (5,6,9). The histochemical reaction for succinate dehydrogenase (SDH), a mitochondrial enzyme encoded exclusively by nDNA, provides a good index of mitochondrial

E. Ricci et 81: Disorders of mtDNA depletion

143

C

f Figure 1 Absence of mitochondria1translation products in muscle of a patient with severe mtDNA depletion. a,d Muscle samples from patient 6 (lower panel) and from an age-matched control (upper panel) were stained for COX activity; b,e The samples were then imrnunoreacted for antibodies against COX subunits II; c,f lmmunoreacted for antibodies against COX subunit IV. The patient's muscle showed marked COX deficiency. The mtDNA-encoded COX II was markedly reduced, while the nDNA-encoded COX IV was normal or increased, especially in subsarcolemmal areas (arrow). Reproduced with permission from the American Journal of Human Genetics (1 991) (Reference 5).

proliferation (see Figures 2 and 3 ) . Electron microscopy provides only non-specific information about alterations in number, size and shape of mitochondria. However, when tissues other than muscle, such as liver, heart, or kidney, are biopsied in patients without muscle involvement, electron microscopy may provide the only evidence of mitochondrial etiology, because the limited amount of tissue available from biopsy does not allow complete biochemical or mtDNA analysis. Finally, in situ hybridization allows direct demonstration of mtDNA depletion (Figs. 2,3). Morphological examination of serial muscle sections permits to correlate mtDNA depletion with mitochondrial proliferation and with defects of mtDNAencoded peptides at the single cell level. This approach is especially useful in the less ,severe form of mtDNA depletion because it reveals the focal distribution of the lesions (see below).

Fatal Infantile Mitochondria1 Disease Associated with rntDNA Depletion

To date, seven infants have been reported (5,6) (see Table 1, patients 1-7) to have died from the fatal infantile mitochondrial disease associated with mtDNA depletion. In 1983, patients 3 and 4 had been described by Boustany et al. (ll),who found morphological changes of mitochondria and multiple defects of respiratory chain in muscle tissue of patient 3 and in liver tissue of patient 4. Recently, Moraes et al. documented mtDNA depletion in the same tissues from these patients ( 5 ) . Similar degrees of mtDNA depletion have been documented in five other patients (Table 1). ClinicaI manifestations include isolated myopathy (see Table 1, patients 1-3), myopathy associated with renal dysfunction (see Table 1, patients 6-7 diagnosed with DeToni-FanconiDebre syndrome), or neonatal liver failure (Table 1, patients 4-5). In addition, brain and heart tissues

144

appeared clinically affected in patients 1 and 6, who developed seizures and died of cardiac arrest. Unfortunately, an autopsy was not performed and thus, mtDNA depletion could only be documented in muscle tissue (5,6). The occurrence of isolated muscle or liver disease suggests that mtDNA depletion might also underlie isolated brain or heart diseases. As summarized in Table 1, the severe form of the disease is characterized by following: 1) onset at birth and death between 3 to 11months of age; 2 ) variable tissue expression, even in members of the same family; 3) depletion of up to 98% of mtDNA in affected tissues; 4) lactic acidosis and abundant ragged-red fibers (RRF) in patients with myopathy; 5 ) severe COX deficiency in affected tissues, roughly parallel to the degree of the mtDNA depletion. Patient 1 seemed to fall between the two groups of patients; neither severe or mild. The onset was at birth and death occurred at 11 months of age, as in the severe variant, but other features were more typical of the milder variant. These included delayed development of severe symptoms, muscle mtDNA content of 8%, residual COX activity of 14% and the presence of a few muscle fibers exhibiting normal COX activity by histochemistry. This case suggests that intermediate forms of the disease may occur and mtDNA depletion may cause a spectrum of clinical observations. Muscle biopsies from patients .with severe myopathy showed that COX activity was markedly decreased or absent in virtually all fibers (Fig. ld), while intrafusal fibers of muscle spindles and the walls of intramuscular arteries had normal COX activity (Fig. 2a). There were numerous RRF, and SDH stain was increased in all fibers reflecting excessive mitochondrial proliferation (Fig. 2c). Immunohistochemical studies showed an absence of mtDNA-encoded proteins (we tested subunit I1 of COX and subunit I of NADH-coenzyme Q oxidoreductase) ( 5 ) (Fig. le), while a nuclear DNA-encoded protein (subunit IV of COX) showed present in all fibers at normal or increased levels (5,lO) (Fig. If). Moreover, in using anti-DNA antibodies only the nuclei stained intensely and there was no cytoplasmic (i.e., mitochondrial) signal (5). Fina1ly;when using mtDNA probes, the signals for both mitochondrial DNA (Fig. 2e) and RNA (not shown) were much weaker than in control muscle. The very low quantity of mtDNA (as low as 2% of normal) in the face of abnormal mitochondrial proliferation suggests that, in these patients, many muscle mitochondria are completely devoid of DNA. Childhood Mitochondria1 Myopathy Associated with Partial mtDNA Depletion

In all three patients with this variant (see Table 1 patients 8-10), their symptoms appeared when the children started walking and the clinical picture was that of a proximal myopathy. There was rapid loss of

E.Ricci et al: Disorders of mtDNA depletion motor skills and the involvement of respiratory muscles in patient 8 (who died at the age of 3 years, from respiratory failure) and in patient 10 (who has been permanently ventilator-dependent since 18 months of age). Patient 9 (a sister of patient 8) had a milder course and at the age 19 months, she was diagnosed with a waddling gait and proximal limb weakness, but no respiratory insufficiency was noted. No lactic acidosis was found in patients 9 or 10, while patient 8 was not tested. The EMG result, in patient 8, showed neurogenic features with numerous fibrillation potentials and positive sharp waves at rest and high-amplitude, long-duration units on volitional activity. However, both motor and sensory nerve conduction velocities were normal and the postmortem histologic examination failed to show any abnormalities in the spinal cord. The EMG result was normal in patient 9 but showed myopathic features and abundant fibrillation potentials in patient 10 (6). The muscle biopsy showed severe but focal COX deficiency affecting between 60-80% of the fibers (Fig. 3a). There were many RRF and the SDH stain was increased in COX-negative fibers (Fig. 3c). Patient 3 underwent three muscle biopsies at the age of 12, 15 and 24 months. The mitochondrial alterations appeared only in the second biopsy and worsened dramatically in the third (6). In all three patients, immunohistochemistry showed that COX-negative fibers lacked mtDNA-encoded proteins, had normal amounts of the nDNA-encoded subunit IV of COX, and were depleted of mtDNA (as shown using anti-DNA antibodies) (6). Biochemical analysis showed decreased COX activity. Defects of complexes I and 111, relative to the controls, became evident only when the values were normalized to citrate synthase, a matrix enzyme used as a marker of mitochondrial volume. Southern blot analysis of mtDNA showed levels ranging between 14-34% of control. Two muscle biopsies from patient 10, obtained at the ages of 15 and 24 months, had essentially identical levels of mtDNA (14-15% of control) suggesting that the downhill clinical course and the progressive decrease in COX activity (from 19 to 5% of normal) were not associated with a progressive decrease of DNA (6). In situ hybridization showed a decreased mtDNA signal (6) (Fig. 3e), despite the presence of numerous ragged-red fibrers (RRF) (Fig. 3c). Pedigree Analysis

The four pedigrees of mtDNA depletion that included more than one case are shown in Figure 4. Patient 3 had a myopathy, yhile her second cousin (patient 4), related through the maternal grandfather, suffered from severe liver disease but did not have a myopathy ( 5 , l l ) . Patient 5 (personal observation) also had a n isolated liver disease, while two halfsiblings from a different mother died from an undi-

E. Ricci et al: Disorders of mtDNA depletion

145

a

b

C

d

e

f Figure 2 Morphological evidence of mtDNA depletion in a patient with early-onset myopathy (patient 6 in Table 1). a,b Frozen sections of muscle from the patient (left panel) and from an age-matched control (right panel) were stained for COX; c,d The muscle tissue was then stained for SDH activity: e,f The sections were hybridized with a 35 S- labeled human mtDNA probe; In the patient, only intrafusal fibers of muscle spindles showed normal COX activity (a), while the mtDNA signal was very low (el. despite abnormal mitochondrial proliferation indicated by increased SDH stain (c).

agnosed muscle disorder at three months of age. These two pedigrees show variable intrafamilial tissue expression, but exclude maternal inheritance of mtDNA depletion. The pedigree of patient 5 is compatible with autosomal dominant inheritance with incomplete penetrance. mtDNA Depletion i s a Specific Disorder of Unknown Etiology

Two lines of evidence suggest that mtDNA depletion is not a secondary phenomenon in mitochondrial

diseases: 1) The levels of mtDNA in patients with other mitochondrial disorders associated with COX deficiency (fatal infantile myopathy, benign infantile myopathy, Kearns-Sayre syndrome and Leigh disease) were either normal or higher than normal (6); 2) RRF were extensively analyzed by in sihi hybridization in more than 30 patients with mitochondrial diseases. Both in cases with unknown etiology and in cases with well-defined genetic defects, such as large-scale deletions, multiple deletions, and MERRF or MELAS point-mutations, mutant mtDNA and mtRNA levels

146

E. Ricci et al: Disorders of mtDNA depletion

a

b

C

d

e

f Figure 3 Morphological evidence of partial mtDNA depletion in a patient with childhood myopathy (patient 9. in Table 1). Frozen serial sections of muscle from the patient (left panel) and from a 3 year old boy with mitochondrial myopathy of unknown etiology (right panel) were stained as in Figure 2. In both patients, COX and SDH stains showed similar results: a,b COX activity was present in a small proportion of fibers; c.d SDH stain was markedly increased and there were numerous RRF. e f Opposite results were observed by in situ hybridization: The mtDNA signal was increased in the disease control (f). The mtDNA signal was diffusely reduced in the patient with mtDNA depletion (el.

were dramatically increased in all RRF which reflects mitochondrial proliferation (Ricci and Moraes, unpublished observations) (Figs. 3d,3f). Conversely, in patient 9, who had the highest residual levels of mtDNA (34% of normal) and of COX activity (42% of normal), no increase in mtDNA and mtRNA content was observed in RRF (Figs. 3c,3e). These results show that proliferation of mitochondria virtually devoid of mtDNA represents the pathological hallmark of mtDNA depletion, regardless of the residual amount of mtDNA.

The etiology of mtDNA depletion is unknown. Pedigree analysis suggests that disorders associated with mtDNA depletion are transmitted by mendelian inheritance. This could be due to the mutations in nuclear genes that control mtDNA levels in early development (5,6). Depletion of mtDNA in AZT-Induced Wlyopathy

A less severe, secondary form of mtDNA depIetion (8) has been documented in muscle tissue from patients with AIDS and mitochondrial myopathy after pro-

147

E. Ricci et al: Disorders of mtDNA depletion

Dr. Moraes was supported by the Brazilian Research Council (CNPq).

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References

v

H 5

I

1. Shoffner JM, Wallace DC (1 990) Oxidative phosphorylation diseases: Disorders of t w o genomes. In: Advances in Human Genetics, Harrys H, Hirschhorn K (eds.), Vol. 19, pp. 267-330, Plenum Press: New York 2. Attardi G, Schatz G (1988) Biogenesis of mitochondria. Ann Rev Cell Biol 4: 289-333

3. Chang DD, Clayton DA (1989) Mouse RNAase MRP RNA is encoded by a nuclear gene and contains a decamer sequence complementary to a conserved region of mitochondrial RNA substrate. Cell 56: 131-139

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4. DiMauro S, Moraes CT, Schon EA (1991) Mitochondrial encephalomyopathies: Problems of classification. In: Progress in Neuropathology, Takeshi S. DiMauro S (eds.), Vol. 7, pp. 113-127, Raven Press: New York 6

8

9

Figure 4 Pedigrees of patients with mtDNA depletion. The patients are numbered as in Table 1.

longed therapy with AZT (12). The causal role of AZT in this acquired form of mtDNA depletion is suggested by three lines of evidence: 1) Southern blot analysis documented severe depletion (up to 78%) of muscle mtDNA in nine AZT-treated patients, but not in two untreated AIDS patients. The content of mtDNA increased markedly in one patient after discontinuation of AZT treatment (8); 2 ) Exposure of human muscle cultures to AZT induced alterations of mitochondria, which resolved partially after AZT withdrawal (13). Rats treated with AZT for three months developed a mitochondrial myopathy with RRF, lactic acidosis and impaired oxidation-phosphorylation coupling of isolated muscle mitochondria (13); 3) In rats, AZT treatment also caused ultrastructural changes of cardiac mitochondria and depressed mRNA expression in a dose- and time-related fashion, while nuclear mFNAs were unaffected (14). Thus, at least in experimental animals, AZT is toxic for both striated and cardiac muscles, and should be considered a potential cause of cardiopathy in humans as well. The mechanism of AZT toxicity has been related to the fact that the drug is readily incorporated into mtDNA by polymerase gamma, and thus, in turn, causes inhibition of mtDNA replication (8,14,15).

Acknowledgements This work was supported by the following granting organizations: a grant from Telethon Italia 1990, grant NS11766 from the National Institute of Health, a grant from the Muscular Dystrophy Association and a donation from Libero and Graziella Danesi.

5. Moraes CT, Shanske S, Tritschler HJ, Aprille JR, Andreetta F, Bonilla E, Schon EA, DiMauro S (1991) mtDNA depletion with variable tissue expression: A novel genetic abnormality in rnitochondrial diseases. Am J Hum Genet 48: 492-501 6. Tritschler HJ, Andreetta F, Moraes CT, Bonilla E. Arnaudo E, Danon MJ, Glass S, Zelaya BM, Vamos E, Telerman-Toppet N, Shanske S, Kadenback B, DiMauro S, Schon EA (1992) Mitochondrial rnyopathy of childhood associated with depletion of mitochondrial DNA. Neurology 42: 209-21 7

7. Moraes CT, Schon EA, DiMauro S (1992) Mitochondrial diseases: Toward a rational classification. In: Current Neurology, Appel SH (ed.). Vol. 11, pp. 83-119, Mosby Year Book: St. Louis 8. Arnaudo E, Dalakas M. Shanske S.Moraes CT, DiMauro S, Schon EA (1 991) Depletion of muscle rnitochondrial DNA in AIDS patients with zidovudine-induced myopathy. Lancet 337: 508-510 9. Andreetta F, Tritschler HJ, Schon EA, DiMauro S, Bonilla E (1991) Localization of mitochondrial DNA in normal and pathological muscle using immunological probes - a new approach to the study of mitochondrial myopathies. J Neurol Sci 105: 88-92 10. Ricci E, Andreetta F, Moraes CT, Minetti C, Schon EA, DiMauro S, Bonilla E (1991) Immunodeficiency of mtDNAencoded proteins in muscle from patients with deletionmutation-depletion of rntDNA. Neurology 41 : 208 11. Boustany RN, Aprille JR. Halperin J, Levy H, DeLong GR (1983) Mitochondrial cytochrorne deficiency presenting as a myopathy with hypotonia. external ophthalmoplegia, and lactic acidosis in an infant and as fatal hepatopathy in a second cousin. Ann Neurol 14: 462-470 12. Dalakas M, llla I, Pezeshkpour GH, Laukaitas JP, Cohen B, Griffin JL (1990) Mitochondrial myopathy caused by longterm zidovudine therapy. N EnglJ M e d 322: 1098-1105 13. Dalakas M, Larnperth L, Dagani F (1991) Abnormal muscle mitochondria induced by AZT in vitro and in an animal model: Morphological, enzymatic, and oxygen-consumption studies. Neurology 41 : 375 14. Lewis W, Papoian T, Gonzalez B, Louie H, Kelly DP, Payne RM, Grody W (1991) Mitochondrial ultrastructural and molecular changes induced by zidovudine in rat hearts. Lab lnvest 65: 228-236 15. Sirnpson MV, Chin CD, Keilbaugh SA. Lin T-S. Prusoff WH (1989) Studies of the inhibition of mitochondrial DNA replication by 3'-azido-3'-deoxythymidine and other dideoxynucleoside analogs which inhibit HIV-1 replication. Biochem Pharmacol 38: 1033-1036

Disorders associated with depletion of mitochondrial DNA.

Quantitative defects of mtDNA have been recently described in patients with fatal mitochondrial disease of early infancy or mitochondrial myopathy of ...
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