Evidence for intramitochondrial complementation between deleted and normal mitochondrial DNA in some patients with mitochondrial myopathy

87

Journal of the Neurological Sciences. 107 (1992) 87-92 2" 1992 Elsevier Science Publishers B.V. All rights reserved (1022-51(1X/92/$05.00 JNS ('13676

Evidence for intramitochondrial complementation between deleted and normal mitochondrial D N A in some patients with mitochondrial myopathy S.R. Hammans,

M.G. Sweeney, I.J. Holt, J.M. Cooper, A. Toscano, J.B. Clark, J.A. M o r g a n - H u g h e s and A.E. Harding

Unit'ersiO' Department of Clinical Neurology, Institute of Neurolo,~9:, London (U. K. ) (Received 30 April, It)91) (Revised, received 19 August, 1991) (Accepted 28 August, 1991)

Key words: Mitochondrial DNA; Mitochondrial myopathy; Respiratory chain deficiency

Summary Twenty-three patients with mitochondrial myopathies and mitochondrial DNA deletions in muscle were studied by means of deletion mapping and sequencing, histochemistry and polarography. Histochemistry showed significantly less focal cytochrome oxidase deficiency relative to number of ragged red fibres when the deletion did not involve reading frames for cytochrome oxidase subunits. Polarography in such patients showed defects exclusively involving complex I, in contrast to the others with larger deletions who generally had more diffuse respiratory chain defects. Analysis of other published histochemical data showed similar findings to our own. It is concluded that translation of a proportion of deleted mitochondrial DNAs occurs in at least some patients with mitochondrial DNA deletions, implying that deleted and normal mitochondrial genomes share transfer RNAs within mitochondria in such cases.

Introduction Deletions of muscle mitochondrial D N A (mtDNA) have been observed in about 40% of adult patients with mitochondrial myopathies (Holt et al. 1988, 1989; Zeviani et al. 1988; Moraes et al. 1989). The presence of a mitochondrial D N A deletion is invariably accompanied by progressive external ophthalmoplegia (PEO) in patients with neurological disease. Some of these patients have one or more other elements of the Kearns-Sayre syndrome (KSS; P E O onset before 20 years of age, pigmentary retinopathy, ataxia, heart block, elevated CSF protein concentration; Berenberg et al. 1977). Attempts have been made to relate the size and site of m t D N A deletions to biochemical findings with conflicting results. Holt et al. (1988, 1989) found deletions which exclusively involved complex I ( N A D H dehydro-

Correspondence to: A.E. Harding, Institute of Neurology, Queen Square, London WCIN 3 BG, U.K. Tel: (44) 71 837 3611 (ext. 4255): Fax: (44) 71 278 5616.

genase; ND) subunit genes together with transfer R N A (tRNA) or ribosomal R N A (rRNA) genes in 4 cases. All of these patients had polarographic defects of the respiratory chain exclusively affecting complex I. Other patients with deletions involving subunits of more than one respiratory chain complex had more extensive loss of respiratory chain activity or normal polarography. No correlation between deletion site and biochemistry was found by others (Zeviani et al. 1988; Moraes et al. 1989; Gerbitz et al. 1990; Goto et al. 1990). Theoretically, a deletion involving one or more t R N A s would be expected to have a detrimental effect on translation of all mitochondrial encoded respiratory chain subunits. However, this effect may be lessened if complementation occurs between normal and deleted genomes which are physically close enough to share tRNAs. Nakase et al. (1990)studied transcription and translation of mitochondrial proteins in heteroplasmic clonal cultures of muscle from a patient with a deletion of m t D N A . The fusion m R N A arising from deleted mtDNAs appeared to be transcribed but no protein corresponding to the translation product was detected. It was suggested that failure of translation was caused by

88 failure of intramitochondrial complementation, possibly because of physical segregation of deleted and normal mtDNAs. The consequence of this hypothesis is that different mtDNA deletions are phenotypically equivalent. In this study we have examined in detail the relationship between the site of 23 mtDNA deletions and the biochemical and histochemical consequences. We discuss the implications of histochemical-genetic correlation in our own and other patients and conclude that there is evidence of translation from deleted mtDNA in at least some patients with mitochondrial myopathy.

Patients and methods

The clinical and biochemical features of 19 of these patients have been described previously (Holt et al. 1989). Case 2 from the previous study has been excluded; his deletion is not comparable to the others as it includes not only ND subunit, tRNA and ribosomal RNA genes, but also the heavy strand promotor. It should thus lead to failure of transcription of 12 of the 13 mitochondrially encoded respiratory chain subunits. Four patients have been added; clinical data are shown in Table 3. Quantitative histochemical studies were performed in all 23 cases. The proportion of muscle fibres showing peripheral mitochondrial accumulations (ragged red fibres) was estimated using the succinic dehydrogenase stain. The method of Seligman et al. (1968) was used to demonstrate cytochrome oxidase activity in serial sections. In both cases 3 areas were chosen at random and 400-600 fibres scored, prior to calculation of percentage ragged red fibres ( % R R F ) and percentage of fibres devoid of cytochrome oxidase activity (%COX-ve). Biochemical studies were performed as described elsewhere as was extraction of DNA from blood and muscle (Holt et al. 1989). DNA was digested with a number of restriction endonucleases (from Bethesda Research Laboratories or Northumbria Biologicals Limited) prior to Southern blotting, hybridisation to HeLa cell mtDNA labelled with 32p by the random primer method and autoradiography (Holt et al. 1989). The proportions of normal and abnormal mtDNA

(%abnDNA) were quantified using an I,KB Ultroscan densitometcr, and modified to take thc smaller size of the deleted genomes into accounl. After restriction mapping, the regions adjacent to the deletion breakpoint were amplified by means of the polymcrase chain reaction (Saiki et al. 1988), using Taq polymerasc (Perkin Elmer Cetus) and a Hybaid Intelligent Heating Block. Details of the primers used are given in Table 1. DNA was amplified in 35 three-step cycles: denaturation (93°C, 61) s), annealing (5t)-55°C. 60 s) and extension (72°C, 120 s). A final extension step of 20 rain was allowed to fill in ragged ends. The amplified DNA was sequenced directly (Schon et al. 1989), with internal primers, a Sequenase kit (USB) and ce-~sS-ATP (Amersham), using the dideoxy method (Sanger c t a l . 1977). Labelled oligonucleotides were resolved on an 8% polyacrylamide gel by electrophoresis at 2500 V for 3h. Patients were divided into those with deletions involving COX subunits (group 1) and those with deletions not involving COX subunits (group 2). The Wilcoxon rank sum test was used to test for the significance of the difference between values of %COXv e / % R R F in the two groups. Correlation (between % C O X - v e / % R R F , % a b n D N A / % R R F and %abnD N A / % C O X - v e ) was analyzed by calculating Spearman's rank correlation coefficient.

Results

Thirteen patients appeared to have an identical deletion (the 'common deletion') on restriction enzyme analysis (Table 2, Fig. 1). Sequencing was performed in 3 of this group, showing an identical deletion of 4977bp with the breakpoint situated within a 13bp direct repeat at bp8470-8482 and bp13447-13459. A small deletion was observed in 5 patients (cases 16, 25, 85, 90, 110). Sequencing in all 5 showed an identical 2309bp deletion with a breakpoint within a 10bp direct repeat at bp12103-12112 and bp14412-14421; this 10bp sequence is part of an imperfect repeat of 13/14bp. One patient (95) had a large deletion of 6037bp between bp7439-13476 with a single base pair common to the flanking regions. Four further patients had different

TABLE 1 O L I G O N U C L E O T I D E PRIMERS U S E D IN A M P L I F I C A T I O N (a) AND S E Q U E N C I N G (s) ATPase8-ND5 deletion ND4-ND6 deletion

CO1-ND6 deletion

8171-8190 13525-13506 11835-11854 14490-14471 15002-14983 6949-6968 13523-13506

5 ' - T G C T C T G A A A T C T G T G G A G C - 3 ' a.s 5 ' - C G A T G A T G T G G T C T T T G G A G - 3 ' a,s 5 '-GACTTCTAGCAAGCCTCGCT-Y a,s 5'-GGAATGATGGTTGTCTTTGG-Y s 5 '-CATTGGCGTGAAGGTAGCGG-3' a 5 ' - C C G T A G G T G G C C T G A C T G G C - 3 ' a,s 5 '-ATGATGTGGTCWTTGG AG -3' a.s

89 TABLE 2 S E Q U E N C E S OF B R E A K P O 1 N T S A N D D E L E T E D R E A D I N G F R A M E S Patient numbers as in Holt et al. (1989). Direct repeats are underlined. CO, cytochrome oxidase (complex IV): ND, complex !; ATPase, complex V Patients

5'

3' .8470

1, 17, 18, 19,26, 27, 30, 44, 45, 48, 67, 68, 69

ATPase 8 Patient sequence ND5

ACCTCCCTCACCA AAGC

cr

CT ACCTCCCTCACCA TTGG

TA ACCTCCCTCACCA T T GG A13447

16, 25, 85, 90. 110

Glycine Histidine Serine 2 ( A G U / C ) Leucine 2 (CUN) Arginine

ND4, ND5, ND6

Histidine Serine 2 ( A G U / C ) Leucine 2 (CUN)

ATPase6, ATPase8, CO1, C O L CO3 ND4, ND4L, ND5,

Arginine, Glycine Serine 2 ( A G U / C ) Aspartic acid Lysine, Histidine Leucine 2 (CUN)

TC CCTCAACCCC TG GA CCTCAACCCC TG

A14421

v7439

COl Patient sequence ND5

CO3 ND3, ND4L, ND4, ND5 ATPase8, ATPase6

v12112

TC CCTCAACCCC GA

A14412

95

Deleted t R N A s

13459A

v12103

ND4 Patient sequence ND6

Deleted subunits

8482.

ATAAA A TCTAG

ATAAA A GCAGG GCATT A GCAGG

A13476

deletions which were defined by restriction mapping or amplification but not sequenced. The proportions of fibres with excess S D H or absent C O X activities are shown in Table 3, together with %COX-ve : % R R F ratios and proportions of abnormal mtDNA. Positive correlation was observed between %COX-ve and % R R F ; this was significantly different

from zero (P < 0.01, Fig. 2). Positive correlation was also observed between %abnDNA and %RRF (P < 0.05, Fig. 3). Positive correlation was present between %abnDNA and %COX-ve but this was not significantly different from zero. Group 2 patients (deletions with no COX subunits involved) had significantly lower % C O X - v e / % R R F ratios than group 1 patients (P <

Cases

38

-I II

Case

tl

83

I I Cases

16,25,85,90

& 110

I

Case

I

70

I-I

I -

Case

I-I

43

Cases

--I

I-I 95

I

Cases

1,17,18,19,26,27,30,44,45,48,67,68

I

0 H

N/C

OL

COII

A8

& 69

I COIII

ND3ND4L

ND6

I

I

l

I

I

I

I

I

0

2

4

6

8

10

12

14

N/C

I kb 16

Fig. 1. Linearised m a p of the mitochondrial genome showing the extent of the deletions in the 23 patients. Where the deletion breakpoint region has not been sequenced, the upper and lower limits are indicated by dotted lines, defined by restriction enzyme and PCR mapping.

90

o

40

RRF

a)

: C()X

w 0

•

i 0

-

%(

-

r~

or3 t3

30 c

°o

[]

Group 2

o

•

o

....

'

0

j D

ODO

2O El

o

o

o

• 40

00

•e

•

10 3C

0

,0

,

o

2fi

10

2'0

3'0

4'0

10

1

]

20

30

~ .....

q a,J

Percentage RRF

Percentage RRF

Fig. 3. Scattergram showing relationship between percentages of abnormal m t D N A and ragged red fibres. See text for definition of groups 1 and 2 and statistical analysis.

Fig. 2. Scattergram showing relationship between percentages of C O X negative and ragged red fibres. See text for definition of groups 1 and 2 and statistical analysis.

0.01). Results of polarographic studies are also shown in Table 3. The 4 patients studied from group 2 all had pure complex I defects.

combination and replication slippage have been suggested as mechanisms for these events (Shoffner et al. 1989; Mita et al. 1990). However, not all m t D N A deletions are associated with repeats (Mita et al, 1990), as observed in patient 95 who had a deletion not described to date. An association between lack of COX activity and a ragged red appearance in individual muscle fibres has

Discussion The breakpoint regions of the 4.97 and 2.30kb deletions, both flanked by direct repeats, have been reported previously (Mita et al. 1990). Intragenomic reTABLE 3

CLINICAL, H I S T O C H E M I C A L , G E N E T I C A N D B I O C H E M I C A L D A T A ON 23 P A T I E N T S W I T H m t D N A D E L E T I O N S Patient numbers up to 85 as in Holt et al. (1989). O, ophthalmoplegia; R, retinopathy; M, myopathy; A, ataxia; H, heart block; D, deafness; S, stroke-like episodes; N.D., not done; N, normal. * Predominantly complex 1 defect Patient number

Clinical features

Percent abnormal mtDNA

Percent RRF

Percent COXnegative fibres

N u m b e r of deleted tRNAs

C O X subunits deleted

%COX-ve/%RRF

Polarography: site of defect

1 17 18 19 26 27 30 44 45 48 67 68 69 83 70 43 95 38 16 25 85 90 110

OM ORMDAH ORMDAHS ORMAH ORM ORM ORM ORM OM OM ORMDAH ORM ORMAH ORM OM OM ORMDAH OM ORMDA ORMDH OM ORMDAH ORMD

50 65 45 50 60 78 70 60 60 45 55 75 65 65 30 50 50 55 80 65 50 80 85

9 25 4 7 17 32 12 14 13 4 5 14 4 13 5 8 3 23 6 18 17 24 19

20 39 11 3 26 47 16 16 33 11 6 33 5 23 12 24 4 15 l) 13 12 11 6

5 5 5 5 5 5 5 5 5 5 5 5 5 6 6-7 8 8 4 3 3 3 3 3

3 3 3 3 3 3 3 3 3 3 3 3 3 2, 3 2, 3 1, 2, 3 1, 2, 3 0 0 0 (1 0 0

2.22 1.56 2.75 0.43 1.53 1.47 1.33 1.14 2.54 2.75 1.20 2.36 t .25 1.77 2.40 3.00 1.33 0.65 0.00 0.72 (1.71 0.46 0.32

l-III 1-1V N.D. !--III l-IV 1-tli * N N 1- IV I- tli N N N I-IV * N I-Ill N N ;D. I 1 I I N.D.

91 been widely reported in mitochondrial myopathies (Shoubridge et al. 1990). Many of the fibres counted in this study had both these features, and the correlation between % C O X - v e fibres and % R R F is not surprising. In situ hybridisation studies suggest that R N A transcribed from deleted m t D N A largely exists in R R F (Shoubridge et al. 1990). A correlation between % a b n D N A and % R R F could therefore be expected. Although there was a positive correlation between % a b n D N A and % C O X - v e fibres, this did not reach significance, largely as a result of group 2 patients having fewer C O X - r e fibres. It has long been recognised that a characteristic feature of mitochondrial myopathy is the focal nature of the biochemical defect as demonstrated histochemically. This is not always associated with complex IV deficiency oll enzyme assay or polarography (Holt et al. 1989). The present study was in part suggested by the observations that some R R F do exhibit C O X activity histochemically, and that one patient with 6% R R F had no COX-vc fibres (patient 16; Holt et al. 1989); as in other patients, some fibres were COX-deficient. Quantitatively the relationship between R R F and C O X negativity can be examined by the ratio of COX-ve fibres to RRF. This ratio was significantly different between group 1 and group 2, suggesting that the site of the deletion may have specific biochemical consequences. Polarographic studies showed pure complex 1 defects in 4 out of 20 patients (patients 16, 25, 85, 90) and a further two had predominant complex I defects (27, 83). On genetic analysis, all 4 of the patients with pure complex I defects were shown to have deletions not affecting C O X subunits. In contrast, all of our patients with deletions involving more than one respiratory chain complex had more diffuse respiratory chain defects or normal polarography. The latter can be explained by the relative insensitivity of assaying biochemical function in pooled mitochondria; the patients with normal polarography tended to have fewer ragged red and C O X - r e fibres (Table 3). Enzyme assays and cytochrome concentrations showed no clear pattern in relation to deletion site (Holt et al. 1989). Measuring oxygen uptake in intact mitochondria is likely to be a more reliable method of assessing oxidative function in vivo than other methods. There are two main differences between the two groups, which may explain the differences in biochemical and histochemical data. G r o u p 2 deletions involve complex 1 subunits but not C O X subunits. Also, the 2.3kb deletion is small and only involves 3 tRNAs. Both of these factors may be important. It has been suggested that imbalance between t R N A s may play a role in causing defects of translation in patients with m t D N A deletions (Shoubridge et al. 1990). This effect may be expected to be more pronounced when more t R N A s are involved in the deletion. In our patients the

differing number of deleted t R N A s does not explain all the findings for several reasons. First, of the group 2 patients, patient 38 had 4 t R N A s deleted and a low C O X - v e / R R F ratio. Second, 4 out of 4 group 2 patients studied biochemically had isolated deficiency of complex I on biochemical studies, suggesting a qualitative as well as a quantitative difference. Furthermore, patients with smaller deletions are as severely affected clinically in terms of muscle weakness as those with larger ones, and this presumably reflects biochemical deficiency to some extent. The most attractive explanation for the predominant complex I lesion and less marked focal C O X deficiency in group 2 patients is the occurrence of translation of the reading frames for all m t D N A encoded COX subunits of at least some of the deleted genomes, but not the deleted subunits of complex I. Support for our observations has been sought from other workers' results. Only Goto et al. (1990) have provided sufficient biochemical and histochemical data (on 21 patients) to allow a similar analysis. Their respiratory chain enzyme assays did not show a correlation with deletion site; no polarographic studies were performed. Unfortunately, in estimating numbers of R R F they used either the modified Gomori trichrome or succinic dehydrogenase technique without specifying which in individual patients. The numbers of R R F were less than in our study, probably because the trichrome technique is less sensitive. Group 1 and group 2 may be defined by the same criteria as our own. The mean C O X - v e / R R F ratio is less in group 2; this reaches significance ( % C O X - v e / % R R F in group 1 is equal or less than group 2 is rejected, P < 0.05). Our data suggest that some deleted m t D N A s in group 2 patients are translated; there is no evidence that this is the case in group 1, but it cannot be excluded. The work of Nakase et al. (1990) shows the apparent absence of a fusion protein in a patient with a large (group 1) m t D N A deletion. No excess of undeleted subunits was found. These findings might be expected if the abnormal fusion protein and unassembled subunits were rapidly degraded. Alternatively the large deletion studied by Nakase and colleagues (9 reading frames and 7 tRNAs), may impair intergenomic complementation, so that m t D N A genomes with larger deletions show lower or absent levels of translated products. Longitudinal sections of R R F show one or more segments of mitochondrial proliferation separated by apparently normal segments. In the centre of the ragged red segments, where the ratio of normal to abnormal IntDNA is lowest (Shoubridge et al. 1990), complementation is less likely to be possible and most deleted genomes fail to be translated. Complementation is more likely to take place in the ' p e n u m b r a ' of ragged red segments. Group 2 patients may have COX activity partially extending into the ragged red seg-

92

ments, thus causing a decrease in the C O X - v e / R R F ratio when the fibres are viewed transversely. In the pathogenesis of mitochondrial myopathy, deleted mtDNAs are likely to be causal. Point mutations in tRNAs have been shown to be associated wit', myoclonic epilepsy and ragged red fibres (Shoffner et al. 199(/)mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (Goto et al. 1990), and a maternally inherited myopathy with cardiac involvement (Zeviani et al. 1991). The clinical phenotype of large deletions of m t D N A is generally different. The mechanism of their effects may well be heterogeneous, and cannot be assumed to be the same as point mutations. It is likely that loss of tRNAs is important but it is our contention that the loss of reading frames for respiratory chain subunits also plays a role in pathogenesis of these disorders. Acknowledgments We wish to thank Ma0orie Ellison and Camilla Kurucz for technical assistance, and the Brain Research Trust, the Musct, lar Dystrophy Group of Great Britain and Northern Ireland, and the Medical Research Council for financial support.

References Berenberg, R.A., Pellock, J.M., DiMauro, S., Schotland D.L., Bonilla, E., Eastwood, A., Hays, A., Vicale, C.T., Behrens, M., Chutorian, A. and Rowland, L.P. (1977) Lumping or splitting? "Ophthalmoplegia-plus" or Kearns-Sayre syndrome? Ann. Neurol., 1: 3754. Gerbitz, K-D., Obermaier-Kusser, B., Zierz, S., Pongratz, D., Miiller-H6cker, J. and Lestienne, P. (1990) Mitochondrial myopathies: divergencies of genetic deletions, biochemical defects and the clinical syndromes. J. Neurol., 237: 5-10. Goto, Y., Koga, Y., Horai, S, and Nonaka, I. (1990) Chronic progressive external ophthalmoplegia: a correlative study of mitochondrial DNA deletions and their phenotypic expression in muscle biopsies. J. Neurol. Sci., 100: 63-69. Goto, Y., Nonaka, I. and Horai, S. (1990) A mutation in the tRNALeu(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature, 348: 651-653. Holt, I.J., Harding, A.E. and Morgan-Hughes, J.A. (1988) Deletions of mitochondrial DNA in patients with mitochondrial myopathies. Nature, 331: 717-719. Holt, I.J., ttarding, A.E., Cooper, J.M. Schapira, A M . V , Toscano, A., Clark, J.B. and Morgan-Hughes, J.A. (19891 Mitochondrial myopathies: clinical and biochemical features in 30 cases with major deletions of muscle mitochondrial DNA. Ann. Neural., 26: 699-708.

Mira, S.. Rizzuto. R.., Moraes, ('.1., bhanskc S, +\t~latldo, t ' Fabrizi, G.M., Koga, Y,, DiMauro, S. and 5,choll I'Z,\. (l',~q!l) Recombination via flanking direct repeat> ~, a major cau~,c ol large-scale deletions of human mitochondrial I)NA Nucleic +\cid', Res., 18: 561-567. Moraes, C.T.. DiMauro, S., Zeviani, M., Ix)robes, A.. Shanske, S.. Miranda, A.F., Nakase, H., Bonilla, E., Werneck, 1.('., Sem'idci, S.. Nonaka, I., Koga, Y., Spiro, A.J., Brownell, A.KW., Schmidt, B., Schotland, D.L., Zupanc, M., De Vivo, I).C., Sol/on, E.A, aad Rowland, l,.P. (1989) Mitochondrial DNA deletions m progressive external ophthahnoplegia and Kearns-Sayre s}ndrome. N. Engl. J. Med., 320: 1293-1299. Nakasc, tt., Moraes. C.T., Rizzuto, R., Lombes, A., l)lMauro, S. and Schon E.A. (199(I) Transcription and translation of deleted mitochondrial genomes in Kearns-Sayre syndrome: implications [or pathogenesis. Am. J. Hum. Genet., 46:418 427. Saiki. R.K., Gelfand, D.H., Stoffel. S.. Scharf. S.,I:. tliguchi. R., Horn, G.T., Mullis, K.B. and Erlich, It.A. (19881 Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239: 487-491. Sanger, F.. Nickten, S., and Coulson, A.R. (19771 DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A., 74: 5463-5467. Schon, E.A., Rizzulo, R., Moraes. C.T., Nakase. It., Zeviani, M. and DiMauro, S. (1989) A direct repeat is a hotspot for large-scale deletion of human mitochondrial DNA. Science, 244: 346-349: Seligman, A.M., Karaw)wsky, M.J., Wasserkrug, tt.L. and Hawker, J.S. (1968) Non-droplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmophilic reagent, diaminobenzidine. J. Cell. Biol., 38: 1-14. Shoffner, J.M., l,ott, M.T,, Voljavec, A.. Soucidan, S., Cosligan, D.A, and Wallace, D.C. (19891 Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion: a slip replication model and metabolic therapy. Proc. Natl. Acad. Sci. U.S.A., 86: 7952-795~). Shoffner, J.M.. Lott, M.T., Lczza, A.M.S., Seibel, P., Ballinger, S.W. and Wallace, D.C. (lq90) Myoclonic epilepsy and ragged red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA ly~ nm|ation. Cell, 6l: 931-937. Shoubridge, E.A., Karpafi, G. and tlastings, K, (199(11 Deletion mutants arc functionally dominant over wild-type mitochondrial genomes m skeletal muscle fiber segments in mitocbondrial disease. Cell, 62: 43~49. Zeviani, M., Moracs, C.T., DiMauro, S., Nakase. 1t., Bonilta, E., Schon, E.A. anti Rowland, L.P. (19881 Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology, 38: 1339-1346. Zeviani, M., Gellera, C., Pannacci, M., Uziel, G.. Prelte, A., Servidei, S. and DiDonato, S. (1990) Tissue distribution and transmission of mitochondrial I)NA deletions in milochondrial myopathies. Ann. Neurol., 28: 94.-97. Zeviani, M., Gellera, C., Antozzi, C., Rimoldi, M., Morandi, L., Villani, F., Tiranti, V. and DiDonato, S. (1991) Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNA t ~'lc~tJm. Lancet, 338 143-147.