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Kearns-Sayre syndrome: Different amounts of deleted mitochondrial DNA are present in several autoptic tissues
Journal of the Neurological Sciences, 1990, 96:207-210
207
Elsevier JNS 03316
Kearns-Sayre syndrome" different amounts of deleted mitochondrial D N A are present in several autoptic tissues C. Ponzetto 1, N. Bresolin 2, A. Bordoni 2, M. Moggio z, G. Meola z, L. Bet 2, A. Prelle 2 a n d G. Scarlato z IDipartimento di Scienze Biomediche e Oncologia Umana, Universit?t di Torino, Torino (Italy), and Zlstituto di Clinica Neurologica, Centro Dino Ferrari, Universit?; di Milano, Milano (Italy)
(Received 15 September, 1989) (Revised, received 20 December 1989) (Accepted 20 December, 1989)
SUMMARY A population of deleted mitochondrial D N A (mtDNA) was found in different amounts in autoptic muscle, heart, cortex, cerebellum, liver and kidney of a patient who died of Kearn-Sayre Syndrome (KSS). The widespread occurrence of the deletion correlates with the multisystem nature of KSS and supports the hypothesis that this is a genetic disease due to an alteration of mtDNA presumably arising in the oocyte or early embryo.
Key words: mtDNA deletions; Kearns-Sayre syndrome; Mitochondrial myopathies
INTRODUCTION Kearns-Sayre syndrome (KSS) is a multisystem disease characterized by the presence of ragged-red fibers (RRF) in muscle, but also affecting other tissues such as retina, brain, cerebellum, liver and heart (Bresolin et al. 1987; Rowland et al. 1988). mtDNA molecules beating large deletions have been found in muscles of the majority of patients with KSS so far examined (Holt et al. 1988a; Zeviani et al. 1988; Moares Correspondence to: Dr. C. Ponzetto, Dipartimento di Scienze Biomediehe e Oncologia Umana, Universit~ di Torino, Corso Massimo D'Azeglio 52, 1-10126Torino, Italy.
0022-510X/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
208 et al. 1989). Similar deletions are also found in muscle mtDNA of a high percentage of patients affected by various mitochondrial myopathies with progressive external ophtalmoplegia (CPEO) and RRF (Moares et al. 1989). An initial report suggested that the presence of deleted mtDNA was probably confined to muscle in these patients, since no deletions were found in leukocytes (Holt et al. 1988b). A recent paper shows that a mtDNA deletion identical to that found in muscle is also present in blood cells from one patient with KSS, in cultured fibroblasts from three KSS patients, and in brain and liver from a fourth KSS patient (Moares et al. 1989). In this study we examined a number of autoptic tissues of a patient who died of KSS. A population of deleted mtDNA is present in all the tissues, albeit in different abundance. This finding strengthens the hypothesis of a causative role of the mtDNA deletion in the pathogenesis of KSS and confnans that the disease is of genetic origin.
MATERIALS AND METHODS mtDNA analysis was performed on frozen specimens of pectoral and psoas muscle, heart, liver, cortex, cerebellum and kidney which had been obtained two hours post mortem from a patient who died of KSS. Muscle biopsies of the same patient had been the subject of an earlier investigation showing an increased number of RRF and a progressive loss in muscle cytochrome c oxidase activity, concomitant with the worsening of the clinical signs of the disease. Examination of post-mortem tissues had shown spongiosis in the frontal cortex, diffuse loss of Purkinje cells in the cerebellum, liver steatosis, and heart fibrosis with mitochondrial abnormalities (Bresolin et al. 1987). Total DNA was isolated from approximately 30 mg frozen tissues and digested with the enzymes PvulI and PstI. Restriction digests were electrophoresed on an 0.8 ~/o agarose gel, transferred to nitrocellulose and hybridized with probes encompassing the entire human mitochondrial genome (kindly provided by Dr. G. Attardi, California Institute of Technology, Pasadena). Techniques of DNA extraction, restriction enzyme digestion, agarose gel electrophoresis and Southern blotting are described in Maniatis et al. (1982). PvulI cuts once in the mitochondrial genome producing a linearized DNA molecule of approx. 16.5 kb. PstI cuts twice producing two fragments, one of about 14.4 and the other of about 2.1 kb. Finding an additional band in the PvulI and PstI digests indicates the presence of a deleted mtDNA population.
RESULTS Southern analysis of the mtDNA of the various tissues is shown in Fig. 1. All tissues examined contained two populations of mtDNA (normal and bearing a deletion of about 4.5 kb). The two mtDNA populations were present in different relative amounts in the various tissues and organs. Densitometric measurement showed that the deleted mtDNA species was 57 ~o of the total in pectoral muscle, and 72 ~o of the total in psoas. In cerebral cortex the deleted mtDNA population was 4 0 ~ of the total, in
209 C 1
M1 2
3
M2 4
5
co
H 7
L 8
9
15 10
11
K 12
13
14
-- 12.0
--
2.1
Fig. 1. A normal mtDNA population (16.5 kb) and a deleted mtDNA population (12.0 kb) is present in everyautoptic tissue. Odd lanes: PvulI digests;even lanes: PstIdigests. C: control muscle;M 1: K SS pectoral muscle; M2: KSS psoas muscle; H: KSS heart; L: KSS liver; B: KSS cerebral cortex; K: KSS kidney. Cerebellum is not shown. liver and cerebellum (not shown) 30~o, while in heart was 1 7 ~ . In kidney the deleted m t D N A was barely detectable (5 ~ ) . The approximate location of the deletion was mapped through analysis o f m t D N A digests obtained using accessory enzymes (EcoRI, HindlI and XbaI, not shown) according to Zeviani et al. (1988). The deletion lies between the XbaI site located at nucleotide 8287 (retained) and the EcoRI site located at nucleotide 126418 (deleted), and includes the gene for subunit III of Cox. This deletion seems to map to the same m t D N A region found to be missing in 11 out of 29 patients with mitochondrial myopathies who had deleted m t D N A (Moares et al. 1989).
DISCUSSION The study of this patient confwrns the observation published by others (Holt et al. 1988b; Zeviani et al. 1988; Moares et al. 1989) that KSS is frequently associated to the presence of deleted m t D N A in muscle. Muscle biopsies that we have obtained from three additional KS S patients do show comparable amounts o f m t D N A bearing similar
210 albeit not identical deletions (to be published elsewhere). Furthermore, the availability of autoptic specimens o f this K S S patient has made it possible to confirm that the deleted m t D N A is widespread to all the tissues which have been examined, extending previous observations limited to cultured fibroblasts, blood cells, liver and brain (Moares et al. 1989). The presence o f the same deletion in various tissues is most easily explained postulating a mutational event occurring in the oocyte or in the early embryo. The different amounts of deleted m t D N A in the various tissues could be due either to unequal distribution of the altered m t D N A during embryogenesis or could result from negative selection against the altered m t D N A during cell proliferation. The degree of morphological abnormalities present in the various tissues correlates well with the relative amount o f deleted m t D N A . In the patient's kidney, which appeared to be normal at light and electron microscopy, only a very low amount of deleted m t D N A is detectable. The threshold value above which the m t D N A defect translates itself into clinical manifestation is probably dictated by how much every tissue or organ relies on oxidative metabolism.
REFERENCES Bresolin, N., M. Moggio, L. Bet et al. (1987) Progressive cytochrome c oxidase deficiency in a case of Kearns-Sayre syndrome: morphological, immunological, and biochemical studies in muscle biopsies and autopsy tissues. Annals of Neurology, 21: 564-572. Holt, I, J., J. M. Cooper, J. A. Morgan-Hughes and A. E. Harding (1988) Deletions of muscle mitochondrial DNA. Lancet, 1: 1462. Holt, I.J., A.E. Harding, and J.A. Morgan-Hughes (1988) Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature, 331: 717-719. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning. A laboratory manual. Cold Spring Harbor Laboratory, NY. Moares, C.T., S. Di Mauro, M. Zeviani et al. (1989) Mitochondrial DNA deletions in progressive external ophtalmoplegia and Kearns-Sayre Syndrome, N. Engl. J. Med., 320: 1293-1299. Rowland, L.P., I. Hausmanowa-Petrusewicz, B. Barduska et al. (1988) Kearns-Sayre syndrome in twins. Lethal dominant or acquired disease? Neurology, 38: 1399-1402. Zeviani, M., C.T. Moares, S. Di Mauro et al. (1988) Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology, 38: 1339-1346.
207
Elsevier JNS 03316
Kearns-Sayre syndrome" different amounts of deleted mitochondrial D N A are present in several autoptic tissues C. Ponzetto 1, N. Bresolin 2, A. Bordoni 2, M. Moggio z, G. Meola z, L. Bet 2, A. Prelle 2 a n d G. Scarlato z IDipartimento di Scienze Biomediche e Oncologia Umana, Universit?t di Torino, Torino (Italy), and Zlstituto di Clinica Neurologica, Centro Dino Ferrari, Universit?; di Milano, Milano (Italy)
(Received 15 September, 1989) (Revised, received 20 December 1989) (Accepted 20 December, 1989)
SUMMARY A population of deleted mitochondrial D N A (mtDNA) was found in different amounts in autoptic muscle, heart, cortex, cerebellum, liver and kidney of a patient who died of Kearn-Sayre Syndrome (KSS). The widespread occurrence of the deletion correlates with the multisystem nature of KSS and supports the hypothesis that this is a genetic disease due to an alteration of mtDNA presumably arising in the oocyte or early embryo.
Key words: mtDNA deletions; Kearns-Sayre syndrome; Mitochondrial myopathies
INTRODUCTION Kearns-Sayre syndrome (KSS) is a multisystem disease characterized by the presence of ragged-red fibers (RRF) in muscle, but also affecting other tissues such as retina, brain, cerebellum, liver and heart (Bresolin et al. 1987; Rowland et al. 1988). mtDNA molecules beating large deletions have been found in muscles of the majority of patients with KSS so far examined (Holt et al. 1988a; Zeviani et al. 1988; Moares Correspondence to: Dr. C. Ponzetto, Dipartimento di Scienze Biomediehe e Oncologia Umana, Universit~ di Torino, Corso Massimo D'Azeglio 52, 1-10126Torino, Italy.
0022-510X/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)
208 et al. 1989). Similar deletions are also found in muscle mtDNA of a high percentage of patients affected by various mitochondrial myopathies with progressive external ophtalmoplegia (CPEO) and RRF (Moares et al. 1989). An initial report suggested that the presence of deleted mtDNA was probably confined to muscle in these patients, since no deletions were found in leukocytes (Holt et al. 1988b). A recent paper shows that a mtDNA deletion identical to that found in muscle is also present in blood cells from one patient with KSS, in cultured fibroblasts from three KSS patients, and in brain and liver from a fourth KSS patient (Moares et al. 1989). In this study we examined a number of autoptic tissues of a patient who died of KSS. A population of deleted mtDNA is present in all the tissues, albeit in different abundance. This finding strengthens the hypothesis of a causative role of the mtDNA deletion in the pathogenesis of KSS and confnans that the disease is of genetic origin.
MATERIALS AND METHODS mtDNA analysis was performed on frozen specimens of pectoral and psoas muscle, heart, liver, cortex, cerebellum and kidney which had been obtained two hours post mortem from a patient who died of KSS. Muscle biopsies of the same patient had been the subject of an earlier investigation showing an increased number of RRF and a progressive loss in muscle cytochrome c oxidase activity, concomitant with the worsening of the clinical signs of the disease. Examination of post-mortem tissues had shown spongiosis in the frontal cortex, diffuse loss of Purkinje cells in the cerebellum, liver steatosis, and heart fibrosis with mitochondrial abnormalities (Bresolin et al. 1987). Total DNA was isolated from approximately 30 mg frozen tissues and digested with the enzymes PvulI and PstI. Restriction digests were electrophoresed on an 0.8 ~/o agarose gel, transferred to nitrocellulose and hybridized with probes encompassing the entire human mitochondrial genome (kindly provided by Dr. G. Attardi, California Institute of Technology, Pasadena). Techniques of DNA extraction, restriction enzyme digestion, agarose gel electrophoresis and Southern blotting are described in Maniatis et al. (1982). PvulI cuts once in the mitochondrial genome producing a linearized DNA molecule of approx. 16.5 kb. PstI cuts twice producing two fragments, one of about 14.4 and the other of about 2.1 kb. Finding an additional band in the PvulI and PstI digests indicates the presence of a deleted mtDNA population.
RESULTS Southern analysis of the mtDNA of the various tissues is shown in Fig. 1. All tissues examined contained two populations of mtDNA (normal and bearing a deletion of about 4.5 kb). The two mtDNA populations were present in different relative amounts in the various tissues and organs. Densitometric measurement showed that the deleted mtDNA species was 57 ~o of the total in pectoral muscle, and 72 ~o of the total in psoas. In cerebral cortex the deleted mtDNA population was 4 0 ~ of the total, in
209 C 1
M1 2
3
M2 4
5
co
H 7
L 8
9
15 10
11
K 12
13
14
-- 12.0
--
2.1
Fig. 1. A normal mtDNA population (16.5 kb) and a deleted mtDNA population (12.0 kb) is present in everyautoptic tissue. Odd lanes: PvulI digests;even lanes: PstIdigests. C: control muscle;M 1: K SS pectoral muscle; M2: KSS psoas muscle; H: KSS heart; L: KSS liver; B: KSS cerebral cortex; K: KSS kidney. Cerebellum is not shown. liver and cerebellum (not shown) 30~o, while in heart was 1 7 ~ . In kidney the deleted m t D N A was barely detectable (5 ~ ) . The approximate location of the deletion was mapped through analysis o f m t D N A digests obtained using accessory enzymes (EcoRI, HindlI and XbaI, not shown) according to Zeviani et al. (1988). The deletion lies between the XbaI site located at nucleotide 8287 (retained) and the EcoRI site located at nucleotide 126418 (deleted), and includes the gene for subunit III of Cox. This deletion seems to map to the same m t D N A region found to be missing in 11 out of 29 patients with mitochondrial myopathies who had deleted m t D N A (Moares et al. 1989).
DISCUSSION The study of this patient confwrns the observation published by others (Holt et al. 1988b; Zeviani et al. 1988; Moares et al. 1989) that KSS is frequently associated to the presence of deleted m t D N A in muscle. Muscle biopsies that we have obtained from three additional KS S patients do show comparable amounts o f m t D N A bearing similar
210 albeit not identical deletions (to be published elsewhere). Furthermore, the availability of autoptic specimens o f this K S S patient has made it possible to confirm that the deleted m t D N A is widespread to all the tissues which have been examined, extending previous observations limited to cultured fibroblasts, blood cells, liver and brain (Moares et al. 1989). The presence o f the same deletion in various tissues is most easily explained postulating a mutational event occurring in the oocyte or in the early embryo. The different amounts of deleted m t D N A in the various tissues could be due either to unequal distribution of the altered m t D N A during embryogenesis or could result from negative selection against the altered m t D N A during cell proliferation. The degree of morphological abnormalities present in the various tissues correlates well with the relative amount o f deleted m t D N A . In the patient's kidney, which appeared to be normal at light and electron microscopy, only a very low amount of deleted m t D N A is detectable. The threshold value above which the m t D N A defect translates itself into clinical manifestation is probably dictated by how much every tissue or organ relies on oxidative metabolism.
REFERENCES Bresolin, N., M. Moggio, L. Bet et al. (1987) Progressive cytochrome c oxidase deficiency in a case of Kearns-Sayre syndrome: morphological, immunological, and biochemical studies in muscle biopsies and autopsy tissues. Annals of Neurology, 21: 564-572. Holt, I, J., J. M. Cooper, J. A. Morgan-Hughes and A. E. Harding (1988) Deletions of muscle mitochondrial DNA. Lancet, 1: 1462. Holt, I.J., A.E. Harding, and J.A. Morgan-Hughes (1988) Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature, 331: 717-719. Maniatis, T., E.F. Fritsch and J. Sambrook (1982) Molecular Cloning. A laboratory manual. Cold Spring Harbor Laboratory, NY. Moares, C.T., S. Di Mauro, M. Zeviani et al. (1989) Mitochondrial DNA deletions in progressive external ophtalmoplegia and Kearns-Sayre Syndrome, N. Engl. J. Med., 320: 1293-1299. Rowland, L.P., I. Hausmanowa-Petrusewicz, B. Barduska et al. (1988) Kearns-Sayre syndrome in twins. Lethal dominant or acquired disease? Neurology, 38: 1399-1402. Zeviani, M., C.T. Moares, S. Di Mauro et al. (1988) Deletions of mitochondrial DNA in Kearns-Sayre syndrome. Neurology, 38: 1339-1346.