Brain Pathology 2: 133-139 17 992)

Disorders Associated with Multiple Deletions of Mitochondria1 DNA

M a t t i Haltia 1, An u Suomalainen 2.4, Anna Majander 3 and Hannu Somer 2 Department of Pathology, 2 Department of Neurology, University of Helsinki,

SF-00290 Helsinki, Finland Department of Medical Chemistry, University of Helsinki, SF-00170 Helsinki, Finland 4 Department of Human Molecular Genetics, National Public Health institute, SF-00300 Helsinki, Finland

Multiple deletions o f mitochondria1 DNA (mtDNA) have recently been described in a number of patients with neurological disorders. M o s t cases have been clinically characterized b y autosomal dom inant inheritance, a d u l t onset, a n d a sl ow l y progressive course with external ophthalmoplegia and muscle weakness. Some patients have had evidence of central o r peripheral nervous system involvement or episodes of myoglobinuria. Muscle biopsy findings include ragged-red fibres (RRF), muscle fibres with absent COX-activity and abundant abnormal mitochondria with paracrystalline inclusions. Biochemically, a generalized reduction in t h e activities o f mtDNA-encoded enzymes i s observed in skeletal muscle. Southern bl otti ng o r PCR analysis reveal mu l ti p l e populations of deleted mtDNA. The deletions occur a t mul ti pl e sites between t h e replication initiation sites, involving a large portion of mtDNA, and most deletions seem t o b e flanked by direct sequence repeats, shown t o be "hot spots" in the case of single large deletions. Apparently, a defect in a nuclear gene results in m u l ti p l e deletions o f mtDNA. B oth clinical, genetic a n d molecular genetic observat ions indicate heterogeneity o f t h i s n e w disease category, apparently based o n a disturbance in t h e "cross-talk'' between t h e nuclear and the mitochondrial genomes. Corresponding author: Dr. M. Haltia, Department of Pathology, University of Helsinki, SF-00290 Helsinki, Finland Tel. +358 (0) 434 6337; Fax +358 (0)434 6700

The functional and structural integrity of mitochondria depends on the complementation of two separate genomes, nuclear and mitochondrial (1). While nuclear genes are transmitted as allelic Mendelian traits, mitochondrial DNA (mtDNA) is contributed mostly by the oocyte and is consequently inherited through the maternal lineage. Mutations of mtDNA have been associated with a number of human diseases ranging from mild myopathies to severe multisystem disorders (see Zeviani, this issue). However, matrilinear transmission has been unequivocally documented only in diseases associated with point mutations of mtDNA, such as Leber's hereditary optic atrophy ( 2 ) , myoclonus epilepsy with ragged-red fibres (3), mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS)-syndrome (4,5), as well as in a family with a multisystemic disorder (6). Large single deletions of mtDNA were first reported in mitochondrial myopathies (7), and apparently occur in most patients with Kearns-Sayre syndrome (8-lo), in about one-third of the cases with progressive external ophthalmoplegia (PEO) (lo), and in Pearson's syndrome (11-13). No correlation has been found between the location and size of the deletion and the clinical phenotype. Large single deletions are usually sporadic, and show variable tissue involvement (14). Although this suggests that the mutations are of somatic origin, it is still uncertain whether the primary molecular lesion is acquired or hereditary. Recently, Zeviani et al. have documented the occurrence of multiple deletions of mtDNA i n patients with adult onset familial PEO and mitochondrial myopathy showing autosomal dominant inheritance (15-17). Other reports have described multiple deletions of mtDNA associated with somewhat more varied clinical features (18-23).This review will summarize the clinical picture and the morphologic, biochemical and molecular genetic findings in this new group of disorders, apparently due to defects in a nuclear gene resulting in deletions of mtDNA. Clinical Findings Family histo-ry. The known families are of varying

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Table 1 Clinical and molecular genetic findings in patients with multiple deletions of mtDNA

Authors

Ophthalmic findings

Neurological findings

Other manifestations

Muscle findings, pathology

7issue with mtDNA deletion

Zeviani et al. 1989 and 1990, Servidei eta!. 1991

PEO Cataract

Sensorineural hypoacusia 1, Peripheral neuropathy

Dysphagia 1 Dysphonia 1

Weakness, RRF

Muscle + Leukocytes Fibroblasts -

Mizusawa et al. 1988 and Yuzaki et al., 1989

Ptosis Optic atrophy

Weaknessand atrophy, RRF

Muscle +

Otsuka et al. 1990

PEO

Weakness RRF

Muscle +

Cormier et al. 1991

Ptosis Ocular paralysis

Growth failure, Hypoparathyreoidism, Keto-acidotic comas

Weakness RRF

Muscle + Leukocytes + Fibroblasts (Leukocytes +)

Alcohol intolerance

Weakness, Episodes of rnyoglobinuria RRF

Muscle +

Ohno et al.. 1991

*

*

Ciafaloni et al.. 1991

PEO 2

Own patients

PEO

1 =

1

*

= Present in a sporadic case

= Not specified ( ) = Clinically unaffected family members

*

*

Mental retardation, Ataxia

*

Peripheral neuropathy 3

*

Present in some of the affected family members

2 = Found in some families 3

Peripheral neuropathy

Affective disorder Multiple lipomas 3

*

Muscle

Weakness and atrophy, RRF

+

Muscle +

M. Haltia et al: Multiple deletions of mtDNA

ethnic background, at least three being of Italian heritage (16), three of Japanese descent (18,19,21,23), one of French (ZO), and one of Finnish extraction (Suomalainen, Majander, Somer and Haltia, in press). In the three Italian pedigrees, comprising altogether 18 affected individuals in three or four generations, t h e trait was considered autosomal dominant, because transmission was through both the paternal and maternal lineages and both sexes were affected in subsequent generations (16). Maternal inheritance was ruled out by the recurrent transmission of the clinical and the molecular traits to patrilinear descendants (15). Similar pattern of inheritance prevailed in the Finnish family (Fig. 1) and possibly also in the French family (ZO), as well as in the families reported by Ciafaloni et al. (22) from the United States. In contrast, within one of the Japanese pedigrees (18,19) the unaffected parents of the two affected brothers were first cousins, suggesting autosomal recessive inheritance.

Symptoms and signs. Both the age of onset and the clinical manifestations have been highly variable (Table 1). Several patients had a childhood onset (18-20) while the members of the large Italian and Finnish families had their first clinical manifestations at adult age. Common clinical features are bilateral blepharoptosis and/or PEO without pigmentary changes of the retina, as well as generalized muscle weakness. In the largest Italian family the onset was in the third decade with bilateral ptosis, premature fatiguability and occasional transient diplopia as the presenting signs (17). The patients of a11 three Italian families gradually developed PEO, mild limb muscle weakness and wasting, respiratory weakness, dysphagia, exercise intolerance and sensorineural hypoacusia. Bilateral cataracts were present in the older patients. Symptoms steadily progressed with the increasing age of the patients. Tremor, ataxia, and chronic sensorimotor peripheral neuropathy occurred in all eight patients of one of the Italian pedigrees (16). Two Japanese brothers (18) suffered from optic atrophy and blepharoptosis since childhood, and later developed weakness and wasting of limb muscles, as well as peripheral neuropathy. The only clinically affected member of the French pedigree (20) first presented at five years of age with repetitive attacks of ataxia, drowsiness and ketoacidotic coma. He then developed bilateral cerebellar ataxia, ptosis, deafness, growth failure and mental retardation. Hypoglycemia and hypoparathyroidism were also noted. While the subsequent course of the childhood onset cases is not known, many of the Italian adult patients have died prematurely in their 40's or 50's. They apparently all died during febrile illnesses presumably due to the combined effect of metabolic crisis and respiratory failure (17). The two Japanese brothers (23) manifested with repeated episodes of

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Figure 1 A pedigree of familial PEO suggesting autosornal dominant pattern of inheritance. Black characters, PEOpatients; Open characters, healthy individuals; A slash over a character indicates the deceased individuals.

myoglobinuria and muscle wasting were without other neurological signs or symptoms. One sporadic case, a 74-year-old man, had peripheral neuropathy and multiple symmetrical lipomas without PEO or other neurological manifestations (22).

Neurophysiological studies. All of the Italian patients had myopathic changes at their electromyographic examination. In affected members of the Italian family with peripheral neuropathy, the motor and sensory nerve conductions- were profoundly reduced in amplitude, while their conduction velocities were normal, indicating predominantly axonal involvement (16). The Japanese brothers, with disease onset in childhood (18,19), showed signs of axonal neuropathy. Laboratory findings.The serum creatine kinase (CK) values were often slightly elevated. Peripheral blood lactate was usually slightly to moderately elevated

Figure 2 Subtotal loss of muscle fibres (arrows) and fibrosis in an extraocular muscle of a patient with autosornal dominant PEO. Note the remaining prominent intramuscular nerves. Cryostat section, Haernatoxylin and Eosin. x 100

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M. Haltia et al: Multiple deletions of mtDNA

Figure 3 Subsarcolernmal region of a muscle fibre packed with abnormal mitochondria from an extraocular muscle of a patient with autosornal dominant PEO. Several mitochondria contain concentric membranous whorls. Electron micrograph. x 15 000

at rest and rose to abnormal values after exercise (16-18). Elevated lactate/pyruvate and elevated ketone body molar ratios in plasma were recorded in the French boy (20). Morphological Findings

Biopsies of limb muscles show evidence of typical mitochondria1 myopathy, usually of mild to moderate degree, even in presymptomatic patients. The number of ragged-red fibres (RRF) vary from a few to about one third of all fibres. By the use of electron microscopy, the affected muscle fibres show large accumulations of mitochondria under the sarcolemma but also between the myofibrils. The mitochondria are often enlarged and contain paracrystalline inclusions and abundant, abnormally oriented cristae which often form concentric whorls (17,18). In the Italian cases, the histo-enzymatic reactions for cytochrome oxidase (COX) and succinate dehydrogenase (SDH) showed that either one or both enzyme activities were lacking in a number of fibres. RRF were frequently COX-negative and reacted strongly for SDH, although some of them were COX-positive. In longitudinal sections, adjacent segments of the same fibre could be positive only for COX, only for SDH, or for both, suggesting the presence of hetero-

plasmic populations of mitochondria in different parts of the same fibre (17). Specimens of the extra-ocular muscles of a Finnish patient showed advanced loss of muscle fibres and fibrosis (Fig. 2 ) . The majority of the remaining fibres were RRF and showed an abnormal distribution of oxidative enzyme activity. Electron micrographs revealed that such fibres were packed with highly abnormal mitochondria, often characterized by excessive whorled cristae and electron-dense inclusions (Fig. 3). Biochemical Findings

Biochemical analysis of both crude muscle extracts and/or isolated mitochondria shows a general respiratory chain defect, affecting enzymes encoded by mtDNA. In crude muscle extracts a partial defect of COX was shown in clinically affected members of all three Italian pedigrees (16), ranging from about 50% to 70% of normal among members of the largest family (17). In these patients, the other mitochondria1 enzymes showed less impressive changes, but the activities of the succinate cytochrome c reductase (complexes I1 and 111), NADH cytochrome c reductase (complexes I and 111), and NADH dehydrogenase (complex I) were also consistently decreased. The

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activity of the nuclear encoded enzyme citrate synthase was unaffected. In the mitochondria1 extracts of one patient a marked decrease of the immunologically reactive COX enzyme protein was demonstrated by immunotitration (17). In the isolated muscle mitochondria of two Japanese patients the rotenone-sensitive NADH cytochrome c reductase was most severely affected (with residual activity of 10-20%), followed by COX and antimycin A-sensitive succinate cytochrome c reductase. The activity of succinate dehydrogenase encoded by nuclear genes was not affected in the mitochondria of these Japanese patients (18), while reduced activity was reported in the crude muscle extracts of some of the Italian cases (17). Markedly reduced NADH-CoQreductase (complex I) activity in muscle mitochondria and decreased oxygen consumption by intact lymphocytes were reported in the French boy (20). Mitochondria1 DNA Analysis

Southern blot analysis of total muscle DNA digested with the restriction endonuclease Pvu I1 (which cleaves the mtDNA at a single position linearizing the molecule) shows the presence of multiple bands after hybridization with human mtDNA probes (Fig. 4) (15,16,19). In addition to the band produced by the normal 16.6 kb mtDNA, numerous additional bands are seen corresponding to smaller, faster moving molecules of partially deleted mtDNA (Fig. 4). The size of the deletions varies from about 2 kb to 10 kb. No deletions were found in mtDNA extracted from fibroblasts or lymphocytes of five Italian patients (17). Members of the French family showed a somewhat deviating picture. The muscle mtDNA of the affected proband showed only one extra 10 kb band, while two abnormal populations of 8 kb and 10 kb were present in his leukocyte mtDNA (20). No rearrangement of mtDNA was found in his cultured fibroblasts. Surprisingly, his clinically unaffected mother and aunt also had abnormal mtDNA fragments in their lymphocytes, but their muscle mtDNA was not studied. The deletions were located between the two replication initiation sites (Fig. 5) in all of the families. The deletions were shown sometimes to start from the displacement loop region (15), but breakpoints were also located in coding regions (16,17,19). Sequencing of several deleted mtDNA populations showed that the deletions were often flanked by short sequence repeats (15,16). Pathogenetic Mechanisms

The main unifymg features of most patients discussed are the PEO with autosomal inheritance and the presence of multiple populations of partially deleted mtDNA in muscle. Although autosomal dominant transmission was documented in the majority of families (15-17), autosomal recessive inheritance may

Figure 4 Southern blot hybridization analysis of biopsy specimens from deltoid (lane 2) and vastus lateralis (lane 3) muscles of a patient with autosomal dominant PEO. Compared to the control (lane 1) several hybridizing signals are seen: 16.6 kb signal represents the normal-sized rntDNA and the numerous smaller bands represent the deleted rntDNA populations. Total human mtDNA was used as a hybridizing probe. The molecule sizes (kb) were determined according to lambda-bacteriophage DNA digested with Hind Ill as a size-standard.

have occurred in some cases (18),suggesting genetic heterogeneity. Heterogeneity is also suggested by the molecular genetic finding of only a few deleted populations of mtDNA in the Southern blot analysis of the French family (ZO), while the other families have shown over ten deleted fragments. The Mendelian inheritance pattern indicates that a mutation in a nuclear gene may result in the multiple deletions of mtDNA. The deletions vary in number, size and location. Although the 5' breakpoints of the deletions were originally mapped to the end of the displacement loop region (H-strand replication sense) (15), it was later found that all the deletions do not originate from this area (16,17,19). In fact, deletions have been found to occur at multiple sites between the replication initiation sites, involving a large portion of mtDNA. Most deletions seem to be flanked by direct sequence repeats (16,17). Such repeats have been shown to be deletion "hot spots" i n the case of single large deletions (24-26) and a slip-replication hypothesis between these repeats has been proposed (16,27), although the possibility of intragenomic recombina-

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Figure 5 Mitochondrial DNA. The deletions occur between the origins of replication of the heavy and light chains (arrows). Abbreviations: OH, heavy chain origin of replication; OL, light chain origin of replication; CO 1-3, genes encoding subunits of cytochrome c oxidase; ND 1-5, genes encoding subunits of NADH-dehydrogenase; ATPase, genes encoding subunits of ATPase; CYTB, cytochrome b; Small grey boxes, tRNA-genes.

tion cannot be formally ruled out. The hypothetical nuclear mutation could reside in a gene encoding for a protein involved in the replication of mtDNA. Candidates include mtDNA-binding proteins believed to prevent branch migration during the synthesis of the nascent H-strand (2829). A defect in such a protein could make mtDNA susceptible to slippage and mispairing of single strands during replication, resulting in deletions. Another, still more hypothetical theory is that a deleterious external factor hits the "hot spots" of mtDNA causing deletions. The possible role of free radicals has recently been considered in the context of mtDNA deletions in aging (30). The deletions involve regions coding for a number of enzyme proteins such as five subunits of complex I (NADH dehydrogenase), the first three subunits of complex IV (COX), subunits 6 and 8 of complex V (ATPase), cytochrome b, as well as for several tRNAs. However, deficient function of enzymes requiring mtDNA-encoded subunits has been observed irrespective of the exact location or extent of the deletions. The most consistent finding has been a partial defect of COX activity and a reduction of immunologically reactive COX protein, suggesting a partial defect of enzyme assembly, probably due to the lack of mtDNA-encoded COX subunits (17). The single mtDNA deletions involve a variety of tissues in the multisystemic Kearns-Sayre syndrome

(10,14). Also, in the patients with pure ocular myopathy small amounts of deleted mtDNA were found in lymphocytes and fibroblasts by PCR amplification (31,32). However, even after PCR amplification, Servidei et al. (17) did not observe deletions in lymphocytes or fibroblasts of their Italian patients with multiple deletions and suggested a selective tissuespecific distribution of the deleted mtDNAs. In contrast, deletions were found in the lymphocytes of the French family (20). In the three Italian families, all the clinically affected and none of the unaffected tested individuals had multiple deletions, and the mtDNA heteroplasmy was considered to be the cause of the clinical phenotype (16). Morphologic and biochemical abnormalities were more evident in the oldest and clinically most affected patients. Furthermore, the clinical phenotype was roughly proportional to the amount of deletions, as well as to the number of RRF and the COX deficiency (17). It was suggested that the deletions increase with time, perhaps due to the combined effects of a permanent nuclear lesion and a possible replicative advantage of deleted mitochondrial genomes (17,33). A very different situation was observed in the French family where the clinical heterogeneity among carriers of the mtDNA deletions not only raises the hypothesis of variable explessivity of the trait in different tissues but also addresses the question of whether the deletions of the mtDNA actually cause the clinical expression of the disease (20). Further studies of autopsied tissues and of clinically unaffected family members are needed to establish these questions. The methods of both direct and reverse genetics may be applied to approach the ultimate goal, the identification of the putative nuclear gene(s) responsible for the multiple mtDNA deletions. References 1. Wallace AC (1987) Maternal genes: Mitochondrial diseases. In: Medical and Experimental Mammalian Genetics: A Perspective, McKusick VA, Roderick TH, Mori J. Paul NW (eds.), Vol. 23, Birth defects: Original article series, pp. 137190, Alan R. Liss: New York 2. Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AMS, Elsas II LJ, Nikoskelainen EK (1988) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242: 1427-1430

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