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 in embryogenesis or have been inherited from phenotypically junctional regions associated with large deletions of mitochon- normal mothers. drial DNA found in four unrelated individuals with a pheno- To delineate further the mechanisms underlying the forma- type of chronic progressive external ophthalmoplegia. In each tion of the partial deletions of mtDNA, we determined the patient, the deletion breakpoint occurred within a directly nucleotide sequences of four additional junctional regions repeated sequence of 13-18 base pairs, present in different from unrelated patients with a phenotype of chronic progres- regions of the normal mitochondrial genome-separated by sive external ophthalmoplegia, disordered oxidative phos- 4.5-7.7 kilobases. In two patients, the deletions were identical. phorylation, and abnormalities ofmitochondrial morphology.¶ When all four repeated sequences are compared, a consensus In this report we show that each of thejunctions is associated sequence of 11 nucleotides emerges, similar to putative recom- with short (13-18 nts) directly repeated sequences. Within bination signals, suggesting the involvement of a recombina- these repeated regions, there is a consensus sequence of11 nts tional event. Partially deleted and normal mitochondrial DNAs that is similar to putative recombination signals, suggesting were found in all tissues examined, but in very different these deletions may arise as a consequence of recombination proportions, indicating that these mutations originated before events. In each of these patients, we could detect partially the primary cell layers diverged. deleted mtDNA in all examined tissues (albeit in very different proportions), suggesting that such mutations in symptomatic Large deletions in mitochondrial DNA (mtDNA) have been individuals arise before the primary cell layers diverge. found in the skeletal muscle of certain patients with mito- chondrial encephalomyopathies (1-9), a clinically and bio- MATERIALS AND METHODS chemically heterogeneous group of disorders, the common Patients Studied. Biopsy specimens ofskeletal muscle were features of which are dysfunction of oxidative phosphoryla- obtained from patients of the neurology service of the Johns tion and alterations of mitochondrial morphology (10). Al- Hopkins Medical Institutions (patients 3 and 4) or the Mas- though variable in location and size in different patients, sachusetts General Hospital (patient 2) with a phenotype of approximately a third of these deletions are identical by chronic progressive external ophthalmoplegia. Routine diag- Southern analysis (7). Recently, the nucleotide sequence of nostic studies included standard histological and histochem- the junctions surrounding this common deletion has been ical staining of frozen sections and polarographic analysis of determined in several unrelated patients, demonstrating that oxygen consumption by isolated mitochondria (patients 3 and these mutations are identical and that the junctions occur 4) (12). Frozen autopsy specimens were obtained from a within a directly repeated sequence of 13 nucleotides (nts), 13-year-old girl with chronic external ophthalmoplegia and suggesting slipped mispairing or recombination (7) as an cardiac dysfunction, studied clinically and biochemically at underlying mechanism. Independently, we have determined the Wayne State University School of Medicine (13). All the nucleotide sequence of the junctional region in another studies were approved by the Joint Committee on Clinical mitochondrial myopathy patient and found that the break- Investigation of the Johns Hopkins Medical Institutions and point did not occur in a repeated sequence (9). those of the referring institutions. In all cases thus far examined [except one (6)], Southern Preparation of DNA. DNA was extracted from mitochon- analysis has shown heteroplasmy (the presence ofnormal and drial and nuclear fractions of skeletal muscle tissue that were partially deleted mtDNAs) in skeletal muscle. The proportion prepared by differential centrifugation in preparation for of the partially deleted species was different in individual oxygen electrode polarography (patients 3 and 4) (12) and patients. In contrast, Southern analysis has failed to demon- from unfractionated homogenates of frozen muscle (patients strate the presence of mtDNA with a deletion in blood cells, 1 and 2), peripheral blood (patients 3 and 4), and urinary suggesting that the deletions may have arisen as somatic sediment (patient 4). Total DNA was extracted by standard mutations, after the muscle lineage diverged. However, we proteinase K procedures (14). Samples containing 5 pg of were able to detect a minor proportion of partially deleted DNA were digested with restriction endonucleases (Be- mtDNA in blood and urinary epithelial cells in two patients thesda Research Laboratories and New England Biolabs) with mitochondrial encephalomyopathy by using a polymer- according to the manufacturers' instructions, subjected to ase chain reaction (8, 9). In the one patient from whom we had electrophoresis on 1.1% (wt/vol) agarose gels, and trans- obtained multiple tissue specimens, large proportions of the ferred to nitrocellulose. They were then hybridized with a partially deleted mtDNA were present in brain, heart, liver, cloned probe prepared from HeLa mtDNA [complementary kidney, and muscle (11). These observations suggest that the to nt 16,453-3245 of the Cambridge mtDNA sequence (15)] pathogenic mutations in these patients must have arisen early Abbreviation: nt, nucleotide. tTo whom reprint requests should be addressed. The publication costs of this article were defrayed in part by page charge IThe sequences reported in this paper have been deposited in the payment. This article must therefore be hereby marked "advertisement" GenBank data base (accession nos. M27283, patients 1 and 2; in accordance with 18 U.S.C. §1734 solely to indicate this fact. M27284, patient 3; and M27285, patient 4). 8059 Downloaded by guest on September 25, 2021 8060 Medical Sciences: Johns et al. Proc. Natl. Acad. Sci. USA 86 (1989) that had been radioactively labeled by the random-primer method using 32P-labeled deoxynucleotides (16) and visual- ized by autoradiography. Proportions of deleted and unde- leted mtDNA species were estimated by laser densitometry (LKB Bromma) of the exposed x-ray film. Polymerase Chain Reaction. The polymerase chain reac- _~~~~~~~~~~/Vf I00:tsus tions were performed on 100 ng of template DNA, using primer pairs (Table 1) that closely bracketed the mtDNA z _: 4142 12,427 _ deletions, but whose recognition sites on normal mtDNA were spaced too widely to permit amplification of the unde- leted 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 microconcen- trator (Centricon-30, Amicon) and sequenced directly with 32P-end-labeled oligonucleotide primers, internal to those used for amplification (Table 1), by the dideoxy chain- 4tCOI AB termination method (17). RESULTS Clinical Studies. All four patients reported in this study FIG. 1. Partial deletions of human mtDNA in four patients. The deletions observed in our patients with mitochondrial encephalomy- were women a with phenotype ofptosis, chronic progressive opathy are indicated by the arcs. They are 4.98 kb (patients 1 and 2), external ophthalmoplegia, and elevations oflactic acid in the 4.51 kb (patient 3), and 7.67 kb (patient 4) in length. The right blood. Patients 1 and 2 demonstrated additional signs of breakpoints are clustered within 550 nts in the NADH 5 gene. The pigmentary retinopathy, cerebellar ataxia, cardiac dysfunc- genes are abbreviated as follows: 12S and 16S, rRNAs; NADH 1, 2, tion, and sensorineural deafness: clinical evidence of multi- 3, 4L, 4, 5, and 6, subunits of NADH-coenzyme Q reductase system disease of the "Kearns-Sayre" type. The family (respiratory complex I); CO I, II, and III, subunits of cytochrome was in all three oxidase (respiratory complex IV); A8 and A6, subunits of ATP history negative instances, including college- synthetase (respiratory complex V); and Cyt b, cytochrome b aged offspring of patient 3. (respiratory complex III). The origins ofreplication for the heavy and Histologic analysis of frozen muscle sections demon- light chain are indicated by OH and OL, respectively. The 22 transfer strated an excessive accumulation of mitochondria in some RNAs are represented by the small unfilled spaces. The numerals type I muscle fibers, seen as typical "ragged-red fibers" on refer to the nucleotide position according to the Cambridge sequence the Gomori trichrome stain and increased subsarcolemmal (15). deposits of reaction products on standard oxidative stains (18). Polarographic analysis of isolated skeletal muscle mi- hybridization with cloned probes specific to other regions of tochondria