Mitochondria1 encephalomyopathy associated with a singlenucleotide pair deletion in the mitochondrial tRNALeu(UUR) gene J.M. Shoffner, MD; M.G. Bialer, MD; S.G. Pavlakis, MD; M. Lott, MS; A. Kaufman, PhD; J. Dixon, BS; S. Teichberg, MD; and D.C. Wallace, PhD Article abstract-The investigation of pathogenic mitochondrial DNA (mtDNA) mutations has revealed a complex relation between patient genotype and phenotype. For unknown reasons, some mtDNA mutations produce specific clinical manifestations such as chronic progressive external ophthalmoplegia; myoclonic epilepsy and ragged-red fiber disease (MERRF); and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). To enhance our understanding of the association between genotype and phenotype, we investigated a patient with mitochondrial encephalomyopathy and severe cerebral calcifications for a mtDNA mutation. There was a deletion of one of three T:A nucleotide pairs in the tRNALeu(UUR) gene of the mtDNA involving positions 3271 to 3273. Pedigree analysis suggested that this mutation may have occurred spontaneously in the proband. This analysis represents the smallest mtDNA deletion observed to date and is the first deletion identified within a mitochondrial tRNA. This observation emphasizes the importance of delineating the precise mutation responsible for an oxidative phosphorylation disease for patient diagnosis as well as for genetic counseling of maternal lineage relatives. NEUROLOGY 1995;45:286-292 Oxidative phosphorylation (OXPHOS) diseases are a heterogeneous group of disorders caused by mutations in the nuclear or the mitochondrial DNA (mtDNA). The mtDNA mutations can be divided into three classes: deletions, duplications, and point mutations.lX2 Many but not all of the pathogenic mtDNA mutations are heteroplasmic, meaning that they are present in tissues as mixtures of normal and mutant mtDNAs. Cell division results in the random distribution of the normal and abnormal mtDNAs to the daughter cells through a process called replicative segregation. The phenotype remains unaffected by the mutant mtDNAs until a critical number accumulate within a tissue and the threshold for phenotype expression is reached.l Once this threshold is exceeded, large changes in the phenotype can be observed with minor increases in the numbers of mutant mtDNAs. Organs with the highest adenosine triphosphate (ATP) requirements and the lowest regenerative capacities, such as brain, heart, and skeletal muscle, are sensitive to the effects of pathogenic mtDNA mutations. Although mtDNA deletions, duplications, and point mutations impair ATP production by OXPHOS, the clinical manifestations observed in severely affected individuals with high concentrations of a mtDNA mutation are surprisingly constant. For example, the c h ical manifestations observed in Kearns-Sayre and chronic progressive external ophthalmoplegia syndromes are generally associated with the presence of a mtDNA deletion or d~plication.~-~ Myoclonic epilepsy and ragged-red fiber disease6-*(MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes9-11( M E W ) are caused by point mutations in mitochondrial tRNAs, whereas Leber’s hereditary optic neuropathy can be caused by point commutations in OXPHOS subunits of complex 1,12-21 plex III,12J7and complex IV.22 Hence, recognition of the relation between phenotype and genotype is important for clinical diagnosis as well as for genetic counseling. In this report, we de- From the Departments of Genetics and Molecular Medicine (Drs. Shoffner, Kaufman, and Wallace, and M. Lott and J . Dixon) and Neurology (Drs. Shoffner and Wallace), Emory University School of Medicine, Atlanta, GA; and the Departments of Pediatrics (Dr. Bialer), Neurology (Dr. Pavlakis), and Laboratory Medicine (Dr. Teichberg), North Shore University Hospital-Come11University Medical College, Manhasset, NY. Conducted under the auspices of the Emory Neuromuscular Center and the Center for Molecular Medicine, Emory University School of Medicine. Supported by National Institutes of Health (NIH) grants NS21328, NS30164, HL45572, and GM46915 and a Muscular Dystrophy Foundation Clinical research grant awarded to D.C. Wallace; NIH grant NS0133601 awarded to J.M. Shoffner; an Emory Clinical Research Center grant (NIH grant MOlRR00039); and NIH grants IP50DK45215 and IP3OAG10139-01 supporting J.M. Shoffner and D.C. Wallace. Received March 22, 1994. Accepted in final form August 2, 1994. Address correspondence and reprint requests to Dr. John M. Shoffner, Department of Genetics and Molecular Medicine, Emory University School of Medicine, 1462 Clifton Road, Suite 276, Atlanta, GA 30322. 286 NEUROLOGY 45 February 1996 scribe a novel mtDNA mutation that appeared to occur spontaneously within a family as a mitochondrial encephalomyopathy with unique manifestations, including severe intracerebral calcifications. The prominent localization of these calcifications within basal ganglia and cerebellar structures as well as the complex neurologic manifestations are consistent with the designation, "Fahr's syndrome." Although striopallidodentate calcifications can be produced by a variety of pathologic processes, this report indicates the importance of including mitochondria1 encephalomyopathies in the differential diagnosis of cerebral calcifications. Methods. Total genomic DNA was isolated from skeletal muscle as previously described.23The mtDNA was screened for mutations in the tRNALysgene at nucleotide pair (np) 8344 (M"K*MERRF8344Gl6 and in the tRNALeU(-) gene a t np 3243 (MTTLl*MELAS3243G) and 3271 (MTTL1*MELAS3271C).g-11 To screen for the MTTLl*MELAS3271C mutation, a 286 np fragment of mtDNA was amplified using a forward primer at np 30073032 and a reverse primer at np 3272-3292. The originally described T+C mutation at np 3271 was tested for by introducing a G+C mismatch into the reverse primer at np 3275, thus creating a Dde I site when the mutation was present. The reaction was performed at an annealing temperature of 53 "C, with standard reagents and amplification condit i o n ~ ?When ~ the 286 np fragment was digested with Dde I, the normal sequence generated three fragments: 126 np (np 3067-31921, 100 np (np 3193-32921, and 60 np (np 30073066). When the M'ITLl*MELAS3271Cmutation was present, a new Dde I site was created, and the 100 np fragment was cut into 79 np and 21 np fragments. Fragments were separated on an 8%polyacrylamide gel, stained with ethidium bromide, and visualized by ultraviolet fluorescence, and the proportions of mutant and normal mtDNAs were quantitated by laser densitometry of a photographic negative.23 Southern blot analysis was performed after Bum HI and EcoRV digestion of the mtDNA as previously de~cribed.2~ The mtDNA was sequenced directly from a mtDNA fragment amplified by the polymerase chain reaction using fluorescent terminator chemistry (Prism Ready Reaction Cycle SequencingKit, Applied Biosystems) and a model 3373A automated sequencer (Applied Biosystems). Reactions were run on 6% polyacrylamide gels from two manufacturers (National Diagnostics and Biorad). The tRNALeU(-) gene from this patient was sequenced from a 711 np mtDNA fragment that was amplified between np 3007 and np 3717 by using a forward primer at np 3007-3023 and a reverse primer at np 3701-3717 (annealing temperature, 51 "C). The heavy and light strands of the mtDNA were sequenced with a forward primer at np 3108-3127 and a reverse primer at np 33513370. In addition to mtDNA sequencing, the single nucleotide pair deletion was detected by amplifying a 129 np fragment, using a forward primer a t np 3242-3259 and a reverse primer at np 3351-3370. The amplification was performed at an annealing temperature of 51 "C as previously described23 except that the standard reaction mixture was supplemented with 15 pCi of [CX-~~PI~CTP. The amplified mtDNA fragment was purified using the Qiaex extraction kit (Qiagen, Chatsworth, CA) and digested with the restriction endonuclease, Mbo 11, which cuts the mtDNA at np 3286. Digestion of normal mtDNA produced 84 np (np 3287-3370) and 45 np (np 3242-3286) fragments, whereas digestion of mutant mtDNA produced 84 np and 44 np fragments when the np 3271 deletion was present. The restriction digest was purified using the Qiaex extraction kit (Qiagen),and 20 p1 of the [~x-~~PIdCTP-labeled mtDNA was electrophoresed through an 8% polyacrylamide gel with 8 M urea. The gel was dried (SGD4050 Savant gel drier) and the fragments visualized by autoradiography. Results. Case description. The proband's birth and development were normal. At age 5 years, he was o b served to have hearing loss, which progressed to complete deafness by age eighteen. He was hyperactive and a discipline problem in school, but remained in normal classes until ninth grade and completed high school in special education classes. Five atonic seizures occurred during childhood that were not associated with fever. At age 20, an endocrine evaluation showed hypogonadism, hypothyroidism, and hypopituitarism. Calcifications on head CT were first noted at this time. By age 23, he had developed cataracts, glaucoma, and retinitis pigmentosa, which progressed rapidly to almost complete blindness. When he was 28 years old, he experienced a rapid worsening of his manifestations that was characterized by approximately 10 days of conhsion followed by generalized tonic-clonic seizures, obtundation, and renal failure. Head CT and MRI of the brain demonstrated extensive calcifications involving basal ganglia, thalamus, dentate nuclei, and centrum semiovale (figure 1). No stroke-like manifestations were observed. CSF analysis revealed a CSF protein of 134 mg/dl (normal, 15 to 45 mg/dl) in the first tube, which contained 3,663 RBC per p1 and 168 WBC per pl. The patient's growth was symmetrically retarded; he was 152.5 cm tall (<3rd percentile), weighed 85 pounds (<3rd percentile), andhad a head circumference of 53 cm (3rd percentile). His testes were small, measuring c1 ml with F'rader orchidometer. The renal disease was characterized by hyperkalemia, a nonselective proteinuria by urine electrophoresis, and echogenic kidneys on renal ultrasound. Urine organic acids were normal, and the serum lactate was at the upper limit of normal at 23.2 mg/dl (normal, 3 to 23). Urine amino acids revealed elevations in cysteine, ornithine, carnosine, and arginine. Plasma ammonia, plasma free and total carnitines, and numerous determinations of serum calcium were normal. A muscle biopsy showed a raggedred fiber myopathy with characteristic paracrystalline inclusions. After approximately 3 months of hospitalization, the patient died, with deteriorating renal function and probable sepsis. Autopsy was refixed. The patient's two older brothers, his mother, and his maternal lineage relatives were healthy and had no manifestations that would suggest the presence of an oxidative phosphorylation disease. Mutation analysis. Total genomic DNA isolated from the proband's skeletal muscle was tested for mtDNA deletions and duplications by Southern blot and screened for t h e MTTLl*MELAS3243G, MTTL 1"MELAS3271C, and MTTK*MERRF8344G mutations by directly analyzing the site with an appropriate restriction endonuclease. No mtDNA deletions or duplications were identified by Southern blot February 1996 NEUROLOGY 45 287 Figure 1. Brain CT showing marked bilateral calcification of the basal ganglia and thalamus (panels A and B), cerebellum (panel A), and white matter (panel C). analysis of mtDNA digested with BamHI or EcoRV. However, a homoplasmic EcoRV site loss was observed. Sequencing of this segment of mtDNA revealed a %A polymorphism in the cytochn>mec oxidase subunit I gene at np 6734 that produced a silent nucleotide change (ATIGlmethionine to AThJmethionine). Restriction endonuclease analysis for t h e MTTK*MERRF8344G and MTTLl"MELAS3243G mutations revealed a normal sequence at these sites. In tests for the M"K*MERRFS344G mutation, a homoplasmic Ban II site loss was encountered. Sequencing of this segment of mtDNA from other cases with this site loss revealed a silent &A polymorphism at np 8251 in the terminal region of the COII gene ("GIGlglycineto GGblglycine). Dde I restriction endonuclease analysis for the M"Ll*MELAS3271C mutation identified a heteroplasmic mutation at this site (figure2).Since the PCR primer was designed such that digestion by Dde I occurs only when a cytosine is present at np 3271, we Figure 2. Dde I identification of the single nucleotide pair deletion within the tRNAbuwuR)gene..Heteroplasmy for initially assumed that the previously described T+C this mutation is identifkd by the presence of both normal mutation had been detected. To investigate this asmtDNAs (126 np, 100 np, and 60 np fragments) and sumption, both the heavy and light strands of the mutant mtDNAs (79np and 20 np bands). The 20 np proband's mtDNA were sequenced between np 3144 band is not visualized in this &urn. Size marker = DNA and np 3707. Analysis of the sequence revealed that molecular weight marker V (Boehringer Manheim). one of the three T:A pairs involving np 3271 to np 3273 was deleted. This single nucleotide deletion gel, we avoided the effects of conformational changes moved the cytosine at np 3270 into a position that completed the recognition sequence for the Dde I site, in the mtDNA fragment on its migration. The restricthus accounting for detection of the mutant mtDNA by tion endonuclease digestion produced two small this restriction endonuclease analysis (figure3). Addi- mtDNA fragments, 84 n p + 45 n p from normal tional confirmation of the presence of this single numtDNA and 84 np + 44 np from mutant mtDNA. The cleotide pair deletion was obtained by incorporating single nucleotide pair deletion in the patient sample was easily detected by this approach (figure 4). [a-92PldCTPinto a 129 np fragment of the mtDNA and Densitometric determination of the percentages of digesting the fragment with Mbo I1 followed by electrophoresis through an 8%polyacrylamide gel containmutant and normal mtDNA detected by the Dde I ing 8 M urea. By using denaturing conditions in the analysis suggested that the mutant mtDNA was het888NEUFlOL.OGY46 February 1SS6 v v v Control 1 Proband Control 2 3266 3271 3275 Normalsequence T A A A A C T T T A C A G T C RimerMirmatchSequence T A A A A C T T T A G A G T C Primer Mismatch Sequenca +np 3271 T-t0-C mutation T A A A A c c T T A G A G T c Primer Mismatc&8 uence +np 3271 deledm I% I Recognition Sequence 3266 3270 3275 T A A A A C T T A G A G T C C T N A G Figure 3. Direct sequencing of the tRNALeu(uuR) gene from mtDNA isolated from the probandS skeletal muscle and from two controls, using fluorescent terminator chemistry, revealed a deletion of one of the T:Apairs involving np 3271 to 3273 (arrow). The relationships among the Dde I recognition site, the normal tRNALeUlUUR) gene sequence, the normal sequence + the primer mismatch (PM) that was incorporated during PCR, the PM +previously described T+C mutation at np 3271, and the single nucleofide pair deletion are diagrammed. eroplasmic and that it constituted approximately 65% of the total mtDNA in skeletal muscle (figure 2). However, both the Mbo I1 analysis and sequencing of this site indicated that the mutant mtDNA was actually present at much higher concentrations (figures 3 and 4).This difference may result from heteroduplex formation occurring during mtDNA amplification between mutant and normal mtDNAs, thus inhibiting cleavage of the mtDNA by Dde I. There was no other evidence of partial digestion at the other Dde I restriction sites, and the addition of excess Dde I did not result in additional cleavage at the 3271 site. When the evolutionary conservation of this site was evaluated by aligning the mtDNA sequences of varisequence variation at this site as well ous as in other nucleotides of the anticodon stem and loop was evident. In contrast to nuclear tRNA genes, which are highly conserved among various the mitochondrial tRNA sequences are estimated to have evolved approximately 100 times faster than the nuclear tRNA,38thus accounting for some of the difficulties encountered when using evolutionary conservation as a screening tool for evaluating candidate mutations in mitochondrial tRNA. To assess whether this single nucleotide deletion was maternally inherited, a mtDNA fragment was amplified from leukocyte total genomic DNA and platelet mtDNA from the proband's mother and tested for this mutation. Both the Dde I and Mbo I1 analyses revealed that the proband's mother had only normal mtDNA in these cells. The absence of clinical manifes- tations in other pedigree members, the absence of this mtDNA mutation in the mother's blood, and the clonal nature of the np 3271 deletion in the proband suggested that this mutation could have occurred as a sporadic event during embryogenesis. Since mtDNA mutations may segregate t o very low levels in hematopoietic cells and make mutation detection diffi~ u l t ,confirmation ~ ~ , ~ ~ of the inheritance pattern in this family would require analyzing DNA isolated h m the skeletal muscle of maternal lineage relatives. The proband's mother declined a muscle biopsy, thus preventing resolution of this issue. Discussion. Mitochondrial encephalomyopathies can be caused by a variety of pathogenic mtDNA mutations.lr2In this report, a novel mtDNA mutation is presented in which a single nucleotide pair in the tRNAhU(-) gene involving positions 3271 to 3273 of the mtDNA is deleted. We believe this mutation is the cause of OF patient's mitochondrial encephalomyopathy for the following reasons. First, a heteroplasmic T+C transition mutation at np 3271 was present in four individuals with MELAS,S,ll thus s i g n i ~ n gthe importance of this nucleotide in OXPHOS disease pathogenesis. This mutation was not present in 50 normal controls representing different ethnic groups, making it unlikely that this site is polymorphic.ll Second, the deletion of this nucleotide is predicted to prevent pairing of the terminal nucleotide pair of the anticodon stem in the tRNALeu(UUR). The conformational may be adversely afstructure of the tRNAhu(UUR) February 1995 NEUROLOGY 45 289 Other manifestations in MELAS patients may include a ragged-red fiber myopathy, lactic acidemia, hearing loss, short stature, dementia, psychiatric disorders such as schizophrenia, pigmentary retinal degeneration, cardiomyopathy, cardiac conduction abnormali. ~ -al~l ~ ~ ~ ~ ~ ~ ties, and basal ganglia c a l ~ i f i ~ a t i ~ n sNot patients with the MTTLl"MELAS3243G mutation have MELAS. Individuals can also present with Kearns-Sayre or chronic progressive external ophthalmoplegia s y n d r o m e ~ , ~ ~ mitochondrial *~0~4~ encephalomyopathies with or without a ragged-red fiber myopathy,40,43 mitochondrial myopathy,",& or diabetes To date, the only phenotype mellitus and deafhes~.~O associated with the MITLl*MELAs3271C mutation is M E W . However, as more individuals with this mutation are identified, the range of phenotypes is likely to grow. In this regard, the single nucleotide deletion involving np 3271 to 3273 did not produce the stroke-like episodes observed in the previously reported patients with the T+C mutation at np 3271.9J1However, our patient did have features that are encountered in mitochondrial encephalomyopathies such as hearing loss, retinitis pigmentosa, short stature, renal Mure,generalized tonic-clonic seizures, and a mitochondrial myopathy. Several unusual clinical manifestations were identified in this patient and included cataracts, glaucoma, hypogonadism, and severe intracranial calcifications. Although intracranial calcifications o h n occur in patients with MELAS,11,43v49*51-N the cerebral calcifications observed in this patient (figure 1) were much more extensive than those found in previously described cases and were not associated with abnormal serum calcium levels. Investigation of other individuFigure 4. Detection of the single nucleotide deletion on an als with the 3271 deletion will be important in assess8% polyacrylamide, 8 M urea gel of a mtDNA fragment ing whether these manifwtations are unique to this amplified from leukocyte DNA and digested with Mbo II. patient or are characteristic of this mutation. Digestion of the normal mtDNA gives 84 np and 45 np fragments. When the single nucleotide deletion is present, In thiscase,there were severe intracranial calcifica84 np and 44 np fragments are observed. These tions and progressive encephalopathy consistent with fragments were first denatured and then electrophoresed a designation of Fahr's syndrome (McKusick no. under denaturing conditions as single-stranded 213600). Fahr's syndrome is a nonspecific clinical clasmolecules. sification that designates a disorder with nonarteriosclerotic bilateral calcification of the cortex, pallidum, striatum, centrum semiovale, and dentate nufeded, resulting in abnormal mitochondrial protein ~ l e u sAssociated . ~ ~ ~ ~clinical manifestations are disynthesis. Third, our patient had a mitochondrial enverse and can include seizures, dementia, psychiatric cephalopathy and a ragged-red fiber myopathy. Point disorders, parkinsonism, retinitis pigmentosa, glaumutations in the tRNAhU(-) gene at np 3302,4l np coma, and endocrine abnormalities such as growth 3303,42np 3243,9JO~~~ np 3250,44and np 32606 of the hormone deficiency, hyperparathyroidism, hymtDNA are associated with ragged-red fiber mypoparathyroidism, hypothyroidism, and a generalized opathies. Fourth, the single nucleotide pair deletion dysfunction of hypothalamic pituitary axis.55-58 The observed in our patient was heteroplasmic, a common cerebral calcifications observed in Fahr's syndrome are feature of pathogenic mtDNA rnutati0ns.l Hence, the deletion of one of the three T:A nucleotide pairs involv- localized primarily to blood vessels. Although the pathophysiology of the process is unknown, this cliniing np 3271 to np 3273 is likely to be the cause for this cal-pathologic association is relevant because of the patient's oxidative phosphorylation disease. prominent mitochondrial abnormalities in the blood Extensive phenotype information is available for invessels of patients with M E W and the important dividuals harboring the MTTLl"MELAS3243G and role of mitochondria in intracellular calcium homeM"Ll*MELAS3271C mutations. Individuals who inostasis. Autosomal recessive inheritance is invoked to herit hgh concentrations of these mutations often deexplain most familial cases,59but in one reported famMELAS is first suspected velop MELAS.9-11~4°~43~*6-48 ily with three generations of affected individuals, no when patients have focal or generalized seizures, recurrent migraine headaches, vomiting, and str0ke.4~ male-to-male transmission was evident,BOthus sug890 NEUROLOGY 45 February 1995 and practice [editorial]. Am J Hum Genet 1992;51:1179-1186. gesting the possibility of a maternally transmitted 3. Holt IJ, Harding AE, Morgan HJA. Deletions of muscle mimtDNA mutation. Hence, patients who are classified tochondrial DNA i n p a t i e n t s w i t h mitochondrial myas having Fahr's syndrome should be examined for evopathies. Nature 1988331:717-719. idence of an OXPHOS disease. 4. Moraes CT, DiMauro S, Zeviani M, e t al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and Based on the Mbo I1 restriction endonuclease analyKearns-Sayre syndrome. N Engl J Med 1989;320:1293-1299. sis, the proband from this pedigree had high levels 5. Poulton J, Deadman ME, Gardiner R.M. Duplications of mi(>95%) of the mutant mtDNA in skeletal muscle. The tochondrial DNA i n mitochondrial myopathy. Lancet quantities of pathogenic mtDNA mutations vary dra1989;1:236-240. 6. Shoffner JM, Lott MT, Lezza AM, Seibel P, Ballinger SW, matically, even among individuals with similar phenoWallace DC. Myoclonic epilepsy and ragged-red fiber disease types and between tissues of a single individual. For (MERRF) i s a s s o c i a t e d w i t h a m i t o c h o n d r i a l DNA example, concentrations of the M'ITLl"MELAS3243G tRNA(Lys) mutation. Cell 1990;61:931-937. mutation can vary between approximately 25% and 7. Silvestri G, Moraes CT, Shanske S, Oh SJ, DiMauro S. A 95% in skeletal muscle from individuals with new mutation in the tRNA-Lys gene associated with my* clonic epilepsy and ragged-red fibers (MERRF). Am J Hum MELAS.10v43r46,48,61-64 Autopsy tissues f h m patients with Genet 1992;51:1213-1217. MELAS have shown similar degrees of variation be8. Zeviani ML, M u n t o n i F, S a v a r e s e N, e t a l . A tween tissues, with concentrations of the mutant rangMERRF/MELAS overlap syndrome associated with a new 79 ing from approximately 32 to 81%in one point mutation of mitochondrial DNA tRNA-Lys gene. Eur J Hum Genet 1993;1:80-87. to 88%in a second and 30 to 80%in a third 9. Tokunaga M, Mita S, S a k u t a R, Nonaka I, Araki S. Inpatient.62 Although oligosymptomatic and asympcreased mitochondrial DNA in blood vessels and ragged-red tomatic individuals generally have lower concentrafibers in mitochondrial myopathy, encephalopathy, lactic tions of the MTTLl"MELAS3243G m ~ t a t i o n , ~ ~ , ~acidosis, ~ , ~ ~and stroke-like episodes (MELAS). Ann Neurol variation in the mutant concentration among an indi1993;33:275-280. 10. Goto Y, N o n a k a I, H o r a i S. A m u t a t i o n i n t h e vidual's organs limits the value of quantitating the tRNA(Leu)(UUR) gene associated with the MELAS subpercentage of mutant mtDNA in a single tissue in degroup of mitochondrial encephalomyopathies. N a t u r e termining the prognosis for these individuals. 1990;348:651-653. Based on the current analysis, the inheritance of 11. Goto Y-I, Nonaka I, Horai S. A new mutation in the tRNALeu(UUR) gene associated with mitochondrial myopathy, this single nucleotide pair deletion is difficult to deterlactic acidosis, and stroke-like episodes. Biochim Biophys mine definitively and possibilities include a sporadic Acta 1991;1097:238-240. mtDNA deletion that occurred during embryogenesis 12. Brown MD, Voljavec AS, Lott MT, Torroni A, Yang C-C, or a maternally inherited mutation. The absence of the Wallace DC. Mitochondrial DNA complex I and 111 mutamutation in leukocyte and platelet mtDNA from the tions associated with Leber's hereditary optic neuropathy. Genomics 1992;130:163-173. proband's mother and the absence of symptoms of an 13. Howell N, Bindoff L, McCullough DA, e t al. Leber herediOXPHOS disease in maternal-lineage family members tary optic neuropathy: identification of the same mitochonfavors the hypothesis that the single nucleotide deledrial ND1 mutation i n six pedigrees. Am J Hum Genet tion occurred as a sporadic event in the proband. How1991;49:939-950. 14. Huoponen K, Vilkki J, Aula P, Nikoskelainen EK, Savonever, mtDNA deletions and point mutations can be diftaus M-L. A new mtDNA mutation associated with Leber ficult to detect in leukocytes and platelets from pae d i t a r y optic neuropathy. Am J H u m Genet 1991; tients and maternal-lineage r e l a t i v e ~ . ~ ~Tissues ~ ~ ~ * @ , ~ h48: ~e r1147-1153. with high rates of cell division, like blood, may select 15. Wallace DC, Singh G, Lott MT, e t al. Mitochondrial DNA against cells that harbor the mutant, thus favoring the mutation associated with Leber's hereditary optic neuropathy. Science 1988;242:1427-1430. replication of cells with normal mtDNAs. Hence, ex16. Johns DR, Berman J. Alternative, simultaneous complex I miclusion of maternal transmission of this mutation tochondrial DNA mutations in Leber's hereditary optic neuwould require analysis of a tissue from the proband's ropathy. Biochem Biophys Res Commun 1991;174:1324-1330. mother, such as skeletal muscle, which has a limited 17. Johns DR, Neufeld MJ. Cytochrome b mutations in Leber hereditary optic neuropathy. Biochem Biophys Res Commun capacity for cell division. 1991;181:1358-1364. This report broadens the range of the OXPHOS 18. Brown MD, Voljavec AS, Lott MT, MacDonald I, Wallace diseases produced by mutations in the mitochondrial DC. Leber's hereditary optic neuropathy: a model for mitotRNALeu(UUR) gene by demonstrating that patients c h o n d r i a l n e u r o d e g e n e r a t i v e d i s e a s e s . FASEB J may harbor pathogenic mtDNA mutations in this 1992;6:2791-2799. 19. Johns DR, Neufeld MJ, Park RD. An ND-6 mitochondrial gene even though clinical analysis of maternal-linDNA mutation associated with Leber hereditary optic neueage family members is normal. In addition, mtDNA ropathy. Biochem Biophys Res Commun 1992;187:1551-1557. deletions involving one or possibly even a few nu20. Mackey D, Howell N. A variant of Leber hereditary optic cleotide pairs should be considered as possible neuropathy characterized by recovery of vision and by a n unusual mitochondrial genetic etiology. Am J Hum Genet causes for an OXPHOS disease and investigated at 1992;51:1218-1228. centers experienced in mtDNA analysis, particularly 21. Johns DR, Hehrer KL, Miller NR, Smith KH. 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Mitochondria1 encephalomyopathy associated with a single nucleotide pair deletion in the mitochondrial tRNA Leu(UUR) gene J. M. Shoffner, M. G. Bialer, S. G. Pavlakis, et al. Neurology 1995;45;286-292 DOI 10.1212/WNL.45.2.286 This information is current as of February 1, 1995 Updated Information & Services including high resolution figures, can be found at: http://www.neurology.org/content/45/2/286.full.html Citations This article has been cited by 7 HighWire-hosted articles: http://www.neurology.org/content/45/2/286.full.html##otherarticles Permissions & Licensing Information about reproducing this article in parts (figures,tables) or in its entirety can be found online at: http://www.neurology.org/misc/about.xhtml#permissions Reprints Information about ordering reprints can be found online: http://www.neurology.org/misc/addir.xhtml#reprintsus Neurology ® is the official journal of the American Academy of Neurology. 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