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We acknowledge many valuable and pleasurable discussions with Drs. Haig Kazazian, Stylianos Antonarakis, Victor. McKusick, Pamela Talalay, and Richard T.
Proc. Natl. Acad. Sci. USA Vol. 86, pp. 8059-8062, October 1989 Medical Sciences

Directly repeated sequences associated with pathogenic mitochondrial DNA deletions (chronic progressive external ophthalmoplegia/mutation/recombination/polymerase chain reaction/heteroplasmy)

DONALD R. JOHNS*t, S. LANE RUTLEDGEt, 0. COLIN STINE§, AND OREST HURKO*§ Departments of *Neurology, tPediatrics, and

WMedicine, The Johns Hopkins University School of Medicine, 600 North Wolfe Street, Baltimore, MD 21205

Communicated by John W. Littlefield, July 10, 1989

ABSTRACT We determined the nucleotide sequences of junctional regions associated with large deletions of mitochondrial DNA found in four unrelated individuals with a phenotype of chronic progressive external ophthalmoplegia. In each patient, the deletion breakpoint occurred within a directly repeated sequence of 13-18 base pairs, present in different regions of the normal mitochondrial genome-separated by 4.5-7.7 kilobases. In two patients, the deletions were identical. When all four repeated sequences are compared, a consensus sequence of 11 nucleotides emerges, similar to putative recombination signals, suggesting the involvement of a recombinational event. Partially deleted and normal mitochondrial DNAs were found in all tissues examined, but in very different proportions, indicating that these mutations originated before the primary cell layers diverged.

in embryogenesis or have been inherited from phenotypically normal mothers. To delineate further the mechanisms underlying the formation of the partial deletions of mtDNA, we determined the nucleotide sequences of four additional junctional regions from unrelated patients with a phenotype of chronic progressive external ophthalmoplegia, disordered oxidative phosphorylation, and abnormalities of mitochondrial morphology.¶ In this report we show that each of the junctions is associated with short (13-18 nts) directly repeated sequences. Within these repeated regions, there is a consensus sequence of 11 nts that is similar to putative recombination signals, suggesting these deletions may arise as a consequence of recombination events. In each of these patients, we could detect partially deleted mtDNA in all examined tissues (albeit in very different proportions), suggesting that such mutations in symptomatic individuals arise before the primary cell layers diverge.

Large deletions in mitochondrial DNA (mtDNA) have been found in the skeletal muscle of certain patients with mitochondrial encephalomyopathies (1-9), a clinically and biochemically heterogeneous group of disorders, the common features of which are dysfunction of oxidative phosphorylation and alterations of mitochondrial morphology (10). Although variable in location and size in different patients, approximately a third of these deletions are identical by Southern analysis (7). Recently, the nucleotide sequence of the junctions surrounding this common deletion has been determined in several unrelated patients, demonstrating that these mutations are identical and that the junctions occur within a directly repeated sequence of 13 nucleotides (nts), suggesting slipped mispairing or recombination (7) as an underlying mechanism. Independently, we have determined the nucleotide sequence of the junctional region in another mitochondrial myopathy patient and found that the breakpoint did not occur in a repeated sequence (9). In all cases thus far examined [except one (6)], Southern analysis has shown heteroplasmy (the presence of normal and partially deleted mtDNAs) in skeletal muscle. The proportion of the partially deleted species was different in individual patients. In contrast, Southern analysis has failed to demonstrate the presence of mtDNA with a deletion in blood cells, suggesting that the deletions may have arisen as somatic mutations, after the muscle lineage diverged. However, we were able to detect a minor proportion of partially deleted mtDNA in blood and urinary epithelial cells in two patients with mitochondrial encephalomyopathy by using a polymerase chain reaction (8, 9). In the one patient from whom we had obtained multiple tissue specimens, large proportions of the partially deleted mtDNA were present in brain, heart, liver, kidney, and muscle (11). These observations suggest that the pathogenic mutations in these patients must have arisen early

MATERIALS AND METHODS Patients Studied. Biopsy specimens of skeletal muscle were obtained from patients of the neurology service of the Johns Hopkins Medical Institutions (patients 3 and 4) or the Massachusetts General Hospital (patient 2) with a phenotype of chronic progressive external ophthalmoplegia. Routine diagnostic studies included standard histological and histochemical staining of frozen sections and polarographic analysis of oxygen consumption by isolated mitochondria (patients 3 and 4) (12). Frozen autopsy specimens were obtained from a 13-year-old girl with chronic external ophthalmoplegia and cardiac dysfunction, studied clinically and biochemically at the Wayne State University School of Medicine (13). All studies were approved by the Joint Committee on Clinical Investigation of the Johns Hopkins Medical Institutions and those of the referring institutions. Preparation of DNA. DNA was extracted from mitochondrial and nuclear fractions of skeletal muscle tissue that were prepared by differential centrifugation in preparation for oxygen electrode polarography (patients 3 and 4) (12) and from unfractionated homogenates of frozen muscle (patients 1 and 2), peripheral blood (patients 3 and 4), and urinary sediment (patient 4). Total DNA was extracted by standard proteinase K procedures (14). Samples containing 5 pg of DNA were digested with restriction endonucleases (Bethesda Research Laboratories and New England Biolabs) according to the manufacturers' instructions, subjected to electrophoresis on 1.1% (wt/vol) agarose gels, and transferred to nitrocellulose. They were then hybridized with a cloned probe prepared from HeLa mtDNA [complementary to nt 16,453-3245 of the Cambridge mtDNA sequence (15)] Abbreviation: nt, nucleotide. tTo whom reprint requests should be addressed. IThe sequences reported in this paper have been deposited in the GenBank data base (accession nos. M27283, patients 1 and 2; M27284, patient 3; and M27285, patient 4).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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that had been radioactively labeled by the random-primer method using 32P-labeled deoxynucleotides (16) and visualized by autoradiography. Proportions of deleted and undeleted mtDNA species were estimated by laser densitometry (LKB Bromma) of the exposed x-ray film. Polymerase Chain Reaction. The polymerase chain reactions were performed on 100 ng of template DNA, using primer pairs (Table 1) that closely bracketed the mtDNA deletions, but whose recognition sites on normal mtDNA were spaced too widely to permit amplification of the undeleted species (9). Oligonucleotide primers were synthesized by Operon Technologies (San Pablo, CA). DNA Sequencing. The products of the polymerase chain reactions were collected in an ultrafiltration microconcentrator (Centricon-30, Amicon) and sequenced directly with 32P-end-labeled oligonucleotide primers, internal to those used for amplification (Table 1), by the dideoxy chaintermination method (17). RESULTS Clinical Studies. All four patients reported in this study were women with a phenotype of ptosis, chronic progressive external ophthalmoplegia, and elevations of lactic acid in the blood. Patients 1 and 2 demonstrated additional signs of pigmentary retinopathy, cerebellar ataxia, cardiac dysfunction, and sensorineural deafness: clinical evidence of multisystem disease of the "Kearns-Sayre" type. The family history was negative in all instances, including three collegeaged offspring of patient 3. Histologic analysis of frozen muscle sections demonstrated an excessive accumulation of mitochondria in some type I muscle fibers, seen as typical "ragged-red fibers" on the Gomori trichrome stain and increased subsarcolemmal deposits of reaction products on standard oxidative stains (18). Polarographic analysis of isolated skeletal muscle mitochondria (patients 1, 3, and 4) demonstrated decreased oxygen consumption with succinate, NADH-linked substrates, as well as with N,N,N',N'-tetramethyl-p-phenylenediamine and ascorbic acid-a pattern consistent with dysfunction of respiratory complex IV. Southern Analysis. In all four patients, Southern analysis of total muscle DNA, after digestion with BamHI [which cleaves only at nt 14,258 in the Cambridge human mtDNA sequence (15)] and hybridization with cloned DNA complementary to a region of human mtDNA that spans the origin of heavy chain replication, demonstrated two populations of mtDNA. In addition to a normal 16.6-kilobase (kb) species, patients 1 and 2 demonstrated an 11.6-kb species; patient 3, a 12.1-kb species; and patient 4, an 8.9-kb species. The partially deleted species comprised 83%, 86%, 55%, and 25% of all mtDNA molecules in the muscle specimen of each patient, respectively. In contrast to skeletal muscle, Southern analysis of total DNA extracted from blood (patients 3 and 4) demonstrated only one species of mtDNA of normal electrophoretic mobility after digestion with BamHI. The approximate extent of each deletion (Fig. 1) was estimated by digestion with multiple restriction enzymes and

(1989)

Vf I00:tsus _~~~~~~~~~~/

z

_:

12,427

4142

4tCOI

_

AB

FIG. 1. Partial deletions of human mtDNA in four patients. The deletions observed in our patients with mitochondrial encephalomyopathy are indicated by the arcs. They are 4.98 kb (patients 1 and 2), 4.51 kb (patient 3), and 7.67 kb (patient 4) in length. The right breakpoints are clustered within 550 nts in the NADH 5 gene. The genes are abbreviated as follows: 12S and 16S, rRNAs; NADH 1, 2, 3, 4L, 4, 5, and 6, subunits of NADH-coenzyme Q reductase (respiratory complex I); CO I, II, and III, subunits of cytochrome oxidase (respiratory complex IV); A8 and A6, subunits of ATP synthetase (respiratory complex V); and Cyt b, cytochrome b (respiratory complex III). The origins of replication for the heavy and light chain are indicated by OH and OL, respectively. The 22 transfer RNAs are represented by the small unfilled spaces. The numerals refer to the nucleotide position according to the Cambridge sequence (15).

hybridization with cloned probes specific to other regions of the mitochondrial genome (9). Each deletion removed genes encoding several tRNAs and components of respiratory complexes I and IV; the deletions of patients 1, 2, and 4 also removed components of respiratory complex V (Fig. 1). Polymerase Chain Reaction. We had demonstrated previously that the use of appropriate primer pairs in a polymerase chain reaction leads to preferential amplification of partially deleted mtDNA even when the normal mtDNA is present in 1000-fold excess (9). Amplification by the polymerase chain reaction with total DNA from skeletal muscle of patients 1 and 2 using DRJ-3 and -16 (Table 1) produced a 1.4-kb fragment; of patient 3 using primers DRJ-12 and -8, a 0.8-kb fragment; and of patient 4, primers DRJ-12 and -10, a 1.3-kb fragment. Identical reaction products were obtained by using total DNA in blood (patients 3 and 4) or urinary epithelial cells (patient 4), indicating the presence of a minor population of partially deleted mtDNAs. Sequence Analysis. The amplified DNA obtained was sequenced directly, using 32P-end-labeled oligonucleotide primers internal to those used for amplification (Table 1). Each deletion breakpoint occurs in a directly repeated sequence that is present in two widely separated regions of the normal mitochondrial genome (Fig. 2). The direct repeats associated

Table 1. Oligonucleotide primers used for amplification and sequencing of deletion junctions Amplification primer L strand Patients H strand Sequencing primer* 1 and 2 DRJ-3 (7407-7425) DRJ-16 (13,789-13,809) DRJ-18 (8417-8434) 3 DRJ-8 (9151-9169) DRJ-12 (14,452-14,470) DRJ-19 (9282-9299) 4 DRJ-10 (5533-5551) DRJ-12 (14,452-14,470) DRJ-17 (6291-6308) All nucleotide designations (in parentheses) refer to the Cambridge sequence (15). *Sequencing primers all complementary to the L strand.

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Proc. Natl. Acad. Sci. USA 86 (1989)

al.

8470:

AC:'CTCCCTCACCAi

1&2

&2Y

96 ~AC:,CTCCCTCACCA 9361 - > * * ~~~~ V 13447: 0. A -CTAACCAACACACT

L

3 i) 3** RCTA CC TMCCAACAMCT 6325 V

1J,,-CCTCCGT AGACCTAACCA COCTCC -T:AGACCTAACCT

~~~~~~~~~~~13868

38

A 13989

consensus

CTA CCTMCCA'

FIG. 2. mtDNA sequences emcompassing four independent deletion junctions. The directly repeated sequences are numbered as in Fig. 1 and are aligned in pairs above the consensus sequence. The direct repeats share 13 consecutive, 16/18 (10 consecutive), and 16/18 (11 consecutive) nts, respectively. The nucleotides in the consensus are found in 4, 4, 4, 5, 6, 6, 4, 6, 6, 6, and 5 of the sequences, respectively. The mismatched nucleotides are marked with an asterisk. The arrows indicate the actual sequence of each patient's mtDNA.

with the deletions found in patients 1 and 2 are identical at 13 of 13 base pairs (bp); those in patient 3, 16 or 18 bp; those in patient 4, 16 of 18 bp (Fig. 2). In each case, the two strands maintain their integrity throughout the junctional region without any nucleotide insertions. The breakpoint cannot be determined precisely because junction occurs within a nucleotide sequence that is identical on both strands. When the direct repeats are compared, a consensus sequence of 11 bp can be identified (Fig. 2). Search for mtDNA Insertions. We considered that these deletions might have arisen from unequal intermolecular recombination events and predicted that the reciprocal products would be mtDNA species with inverted insertions of the same size as the corresponding deletion. No mtDNA species of a size appropriate to such inserted species was found in Southern blots of skeletal muscle in any of the four patients. In patient 1, the search was extended further through the use of the polymerase chain reaction with primers designed to overlap the junction of the predicted insertion species: DRJ20, complementary to nt 8539-8520 of the H strand of the Cambridge sequence (15), and DRJ-21, complementary to nt 13,360-13,379 of the L strand. No reaction product was detected when 100 ng of total DNA from skeletal muscle was used as a template.

DISCUSSION Our results demonstrate that directly repeated sequences present in widely separated regions of mtDNA are commonly involved in deletions associated with genetic disease. Such repeated sequences occur frequently in the mitochondrial genome: a computerized search (MICROGENIE, Beckman) of the published human mtDNA sequence (15) reveals 198 duplicated sequences of at least 10 consecutive matching bases, which lie within a segment bracketed by the origins of replication for the light and heavy strands. (Repeated sequences that straddle either origin would be predicted to give rise to partially deleted species that would be incapable of replication. Hence, they were excluded from consideration.) The inappropriate junctions in patients 1 and 2 occur within one of two such duplications that contain 13 consecutive identical nucleotides. This deletion junction has been found independently in several other patients (7). Deletions in other systems appear to be promoted by the presence of flanking short, directly repeated sequences,

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including those in lacI-lacZ fusion strands of Escherichia coli (19), the human /3-globin gene (20), and some mtDNA deletions in yeast (21). In addition, directly repeated Alu sequences have been found at deletion junctions of pathogenic mutations in several disease states, including familial hypercholesterolemia (22) and Tay-Sachs disease (23). The presence of direct repeats may contribute to the formation of deletions by "slipped mispairing" during DNA replication, as has been proposed to account for the most frequently observed mtDNA deletion found in association with certain mitochondrial encephalomyopathies (7). Alternatively, such direct repeats could contribute to independent recombination events mediated by enzymes that recognize specific short sequences (19, 20). In other systems, the frequency with which deletions occur appears to correlate with the length of repeated sequences, the distance between them, and the nature of neighboring sequences. Furthermore, the precise nucleotide sequence of the direct repeat itself has been shown to have a strong effect on the rate of formation of deletions

(19).

In our patients, the regions in which the deletion breakpoints occur are similar over a stretch of 11 nts, the consensus sequence of which is CTACCTAACCA (Fig. 2). This sequence is similar to the core repeats in the hypervariable minisatellite regions described by Jeffreys et al. (24), which have been proposed to be recombination signals in human nuclear DNA (Fig. 3). Additionally, it is similar to portions of the chi sequence, a putative recombination signal in E. coli

(25) (Fig. 3).

We suggest that this consensus sequence may mediate recombination in the human mitochondrial genome. However, the sequence data do not distinguish between intra- and intermolecular recombination. An intramolecular looping mechanism has been suggested to explain the duplication and inversion of portions of the mitochondrial genome in lizards (26). Alternatively, the deletions observed in our patients may have arisen from intermolecular recombination. The presence of 2-10 mtDNA molecules per human mitochondrion (27) eliminates any obvious physical barrier to intermolecular recombination. Such intermolecular recombination between two misaligned molecules would produce a deleted molecule and a molecule with a corresponding inverted insertion. Our initial attempts to identify the corresponding insertions were unsuccessful. However, the putative larger, inserted species would be expected to suffer a replicative disadvantage, and thus its absence is not unexpected. There is ample genetic (28) and physical (29) evidence for intermolecular recombination of mtDNA in yeast, where it may contribute to the formation of certain rho- deletions (30). Intermolecular recombination is generally assumed not

TGG G**C AGGAAG lambda 33.11 * junction consensus 1 ** 2* TGGGC AGGTGG lambda 33.15 chi

GCTGGTGG 2 ,1

2

FIG. 3. Comparison of the sequences associated with partial deletions of mtDNA to the heavy (opposite) strand of the consensus sequence associated with deletion junctions in mtDNA. The mtDNA deletion sequence shares 8 of 11 nts with the core region from two minisatellite clones of Jeffreys et al. (24). A 33.11 and 33.15. The mismatched nucleotides are marked with an asterisk. In addition, the first four (1) and last five (2) bases of the consensus are similar to the chi sequence (25), a known recombination signal in E. coli.

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to occur in animal mtDNA (31), but two lines of evidence suggest that it may indeed occur. (i) In human population studies, two polymorphic restriction sites have been found in all combinations in individuals who are known to differ only at those two sites (32). This observation could be explained by the highly unlikely occurrence of two independent muta-

tions in a single site (with a probability of