American Journal of Medical Genetics 41:301-305 (1991)
Mitochondria1 DNA Deletion in a Girl With Manifestations of Kearns-Sayre and Lowe Syndromes: An Example of Phenotypic Mimicry? Carlos T. Moraes, Massimo Zeviani, Eric A. Schon, Robert 0. Hickman, Brien W. Vlcek, and Salvatore DiMauro Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York (C.T.M., E.A.S.),Instituto Neurologic0 “C. Besta”, Milano, Italy (M.Z.), Division of Pediatric Nephrology, Children$ Hospital and Medical Center, Seattle, Washington (R.O.H.), Department of Neurology, Children$ Hospital and Medical Center, Seattle, Washington (B.W.V.), Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York (S.D.) Lowe oculocerebrorenal syndrome is an X-linked recessive disease whose locus has been assigned to Xp25. However, several reports of affected females without obvious chromosomal abnormalities suggest genetic heterogeneity of the Lowe phenotype. Although the biochemical defect in typical Lowe syndrome is not known,there is evidence s u g gesting that mitochondrial metabolism may be impaired. We have studied a girl who presented with an oculocerebrorenal syndrome, but later developed symptoms and signs of mitochondrial encephalomyopathy. Molecular genetic analysis of muscle mitochondrial DNA showed the presence of a population of partially deleted mtDNAs (heteroplasmy). The deletion was 7803 bp long and encompassed several genes encoding subunits of the respiratory chain enzymes. Our results suggest that mitochondrial DNA deletions may mimic several symptoms of the Lowe phenotype and reinforce the concept that a defect of mitochondrial metabolism could be involved in the pathogenesis of the X-linked disease.
KEY WORDS: Lowe syndrome, mtDNA deletions, mtDNA heteroplasmy, mitochondrial disease INTRODUCTION Lowe oculocerebrorenal syndrome (OCRL) is an X-linked recessive disorder characterized by bilateral Received for publication August 23, 1990; revision received March 16, 1991. Address reprint requests to Dr. Salvatore DiMauro, Department of Neurology, 4-420 College of Physicians and Surgeons, 630W 168th Street, New York, NY 10032.
0 1991 Wiley-Liss, Inc.
congenital cataracts, glaucoma, mental retardation, hypotonia, and defective renal tubular function [Lowe et al., 19521. Linkage analysis has assigned the OCRL locus to Xq24-q26 [Silver et al., 19871. The finding of a balanced de novo X/3 translocation in a girl with manifestations of Lowe syndrome has further localized the disorder to Xq25 [Hodgson et al., 19861. Tightly linked flanking polymorphic markers have also been described [Reilly et al., 19881. Though well established, the X-linked mode of inheritance is incompatible with a number of cases described in the literature. Matsuda et al. [19701 described a boy with typical Lowe syndrome whose father showed aminoaciduria after ornithine loading: the authors suggested an autosomal recessive variant. Svork et al. [1967], Denys and Corbeel [1964], and Scholten [19601 described affected females without detectable chromosome abnormalities, and Cyvin et al. 119731 reported a pair of male and female half-sibs allegedly of different fathers who were severely affected and died before the age of 6 months. These cases illustrate apparent heterogeneity of OCRL [McKusick, 1986; Curtis and Goel, 19821. Although the biochemical basis of OCRL is unknown, ultrastructural and biochemical studies have suggested involvement of mitochondrial metabolism. Ultrastructural abnormalities of mitochondria have been seen in renal [Schoen and Young, 1959; Ores, 1970;Sage1et al., 19701 and muscle [LM Turolla, personal communication] biopsies of patients with OCRL and biochemical studies of isolated muscle mitochondria were interpreted to suggest a defect in the respiratory chain [Gobernado et al., 19841. We have studied a girl who presented at age 3 years with manifestations of OCRL but who later developed neurosensory hearing loss, ataxia, and persistent lactic acidosis, which suggested the possibility of a mitochondrial encephalomyopathy and led to a muscle biopsy. Analysis of skeletal muscle mtDNA showed the presence of partially deleted mtDNAs coexisting with
302
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normal genomes (heteroplasmy). Our results support the idea that OCRL features may result from different genetic lesions, all of which cause mitochondrial dysfunction.
CLINICAL REPORT An 11-year-old girl apparently developed normally until age 3, when slowly progressive decline of cognitive function became evident. At age 5, Fanconi syndrome was diagnosed and impaired vision was attributed to bilateral cataracts. She also developed corneal crystals that were atypical, and cystinosis was excluded later by leukocyte and bone marrow studies. In the following years, she developed proximal limb weakness, bilateral ptosis and limitation of eye movements, nystagmus, truncal ataxia, and neurosensory hearing loss. Intermittently throughout the last 5 years she had episodesof encephalopathy with lethargy or stupor triggered by intercurrent infections. She also developed chronic renal insufficiency, and her vision deteriorated from 20/80 bilaterally to 20/400. Family history was noncontributory: both parents and an older sister were normal. Examination at age 11showed height 116.9 cm and weight 23.4 kg. She had severe bilateral ptosis and limited range of eye movements in all directions. F'undi could not be examined because of cataracts and marked cloudiness of both corneas. She had both gait and truncal ataxia. There was mild generalized weakness. Laboratory abnormalities included increased blood lactate (2.9-3.7 mM; normal, < 2.2 mM), increased cerebrospinal fluid lactate (5.6 mg/dl; normal < 2.0 mg/dl) and protein (230 mg/dl; normal < 45 mg/dl), glycosuria, phosphaturia, and generalized aminoaciduria (Fanconi syndrome). CT scan of the head showed mild brain atrophy and bilateral hypodense lesions in the putamen and in the regions around the third ventricle. At age 11she developed tachypnea, Kussmaul breathing, and had 3 cardiopulmonary arrests. Because of extreme cardiac arrhythmia, a temporary pacemaker was placed. After a transient improvement, she developed increasing respiratory distress, hypercarbia and acidosis. Despite intubation and i.v. sodium bicarbonate administration, the metabolic acidosis persisted, the renal insufficiency worsened, and she developed epidermal necrosis. She died of cardiopulmonary arrest at age 11.
MATERIALS AND METHODS Restriction enzymes were from Boehringer Mannheim and New England Biolabs. Klenow fragment ofE. coli DNA polymerase I was from Boehringer Mannheim. RNase A and chemicals were from Sigma. [a- 32PlDeoxycytidineand deoxyadenine triphosphate
(800 Ci/mmol)were from New England Nuclear. Singlestranded oligonucleotides primers were synthesized by Genetic Designs. The sequences of the primers were (mtDNA map positions according to Anderson et al., 1981) Primer If, 7,433 to 7,468; Ib, 15,600 to 15,774.
Biochemical Studies Mitochondrial enzyme activities were measured in muscle homogenates as described [DiMauro et al., 19871. Southern blot analysis. Total DNA was isolated from 50 mg of frozen muscle obtained by biopsy. DNA was purified by conventional methods as described previously [Zeviani et al., 19881. Purified mitochondrial DNA from normal liver was used as template for the hybridization probe in Southern analysis. The proportion of normal and mutant mtDNA genomes was estimated by densitometry of the X-ray film. The approximate site of the deleted region was determined by restriction mapping (using the enzymes PuuII, HindIII, PstI, XbaI, and EcoRI).The mapping procedure has been described elsewhere [Zeviani et al., 19881. PCR amplifications and sequencing. Polymerase chain reaction (PCR) amplifications were performed with the Ampli-Taq Kit from Perkin ElmerCetus. About 1 p,g of total DNA was used in the amplification reaction. All other conditions followed the recommended guidelines of the manufacturer. We have performed 35 cycles of amplification (1min 55"C,3 min 72"C, and 1min 94°C).Ten percent of the reaction mixture was analyzed by agarose gel electrophoresis. The fragment obtained was reamplified and purified from a low-melting agarose gel using the Gene-Clean kit (Bio 101).Double-strand direct sequencing was performed by the Sanger method [Sanger et al., 19771, modified by Winship [19891. About 1.5 pg of DNA template was used in the reaction. Sequenase kit (USB) was used in the sequencing reactions.
RESULTS Because this girl with initial diagnosis of OCRL had chronic lactic acidosis, a muscle biopsy was performed which showed ragged red fibers with the Gomori trichrome stain and abnormal mitochondria by electron microscopy (not shown). Biochemical analysis of mitochondrial enzymes showed markedly decreased activities of respiratory chain complex I (NADH-coenzymeQ oxireductase), I11 (coenzyme Q-cytochrome c oxireductase), and IV (cytochrome c oxidase), while the activities of complex I1 (succinate-coenzyme Q oxireductase), and citrate synthase, a matrix enzyme, were normal (Table I). Com-
TABLE I. Mitochondrial Enzymes Activity in Muscle Homogenates Enzymehespiratory chain complex" Patient (M.S.) Controls
I
I1
I + I11
I1 + I11
IV
cs
7.3 0.59 0.02 0.04 0.02 9.56 35.5 '-c 7.1 0.70 f0.23 1.02k0.38 0.70 ? 0.29 2.80 & 0.52 9.88 22.55 "Activities are Fmol substrate utilizedlrnidg fresh tissue. Controls expressed as mean 2 SD of n220. Enzymeskomplexes: I, NADH-coenzyme Q oxireductase; 11, succinate dehydrogenase; I + 111, NADH-cytochrome c oxireductase; I1 + 111, succinate-cytochrome c oxireductase; IV, cytochrome c oxidase; CS, citrate synthase.
mtDNA Deletion Causing Features of Lowe Syndrome plexes I, 111, and IV contain multiple subunits, some encoded by nuclear DNA others by mitochondrial DNA, but subunits of complex I1 and citrate synthase are encoded exclusively by nuclear DNA. Because the biochemical findings suggested that our patient might have a mtDNA mutation, we performed Southern analysis of total DNA purified from skeletal muscle. Using the restriction enzyme PuuII, which cuts only once in the mtDNA, the Southern blot showed the presence of two populations of mtDNA, one corresponding to full-length mtDNA (16.6 kb) and the other corresponding to a shorter molecule of approximately 8.8 kb (Fig. 1A). The location of the 7.8 kb deletion was determined by restriction mapping, and the information obtained was used to design a pair of polymerase chain reaction (PCR) primers that allowed us to amplify a DNA fragment spanning the deletion breakpoint [Schon, 19891. PCR primers If and Ib generated an amplified DNA fragment of approximately 360 bp that was used for double-strand direct sequencing. Southern analysis of mtDNA from muscle specimens of the patient’s mother and from a patient with typical X-linked OCRL showed no evidence of deletions (Fig. 1A). Availability of PCR primers specific to the patient’s breakpoint region allowed us to reexamine the muscle from the patient’s mother looking for small numbers of mutant mtDNAs. Even this more sensitive technique failed to show abnormal mitochondrial genomes. Normal controls, as well as the mother of the patient yielded nonspecific DNA fragments after the PCR reaction. The expected 360 bp DNA fragment spanning the deletion breakpoint was observed only after amplification of the patient’s DNA (Fig. 1B). The 360 bp DNA fragment spanning the deletion breakpoint was sequenced and the breakpoint determined by comparison with the published human mtDNA sequence [Anderson, 19811(Fig. 2A). The exact B
A 1
2
3
123M
4
303
A G
A T C
........_...
-
03X’AClTCCCXXATCATAGC c02
-7,803
B
-
---W bp
............
-
’ I T A C I T C T m C T m
CYB
r.“”
c02
’
6
ATPase
Fig. 2. Location of the deletion breakpoint. (A) The 360 bp DNA fragment obtained by PCR (see Fig. 1)was sequenced, and the breakpoint determined by comparison with the published human mtDNA sequence [Anderson et al., 19811. Regions with significant homology, and possible role in the genesis of the deletion, are underlined. (B) Schematic representation of the patient’s mtDNA deletion. OH and OL, origins of heavy- and light-strand replication; 12 and 16 S, ribosomal RNA genes; ND1-6, genes corresponding to NADH-coenzyme Q oxidoreductase subunits 1-6; CO1-3, genes corresponding to cytochrome c oxidase subunits 1-3; CYT b, cytochrome b gene; ATPase 6 and 8, genes corresponding to ATP synthetase subunits 6 and 8; If and Ib PCR primers used to amplify the region spanning the breakpoint (see methods).
kb
-
-16.6
size of the deletion was 7803 bp, starting at position 7,636 a t the “left side” and ending at position 15,439 at c 360 bp the “right side”(Fig. 2B). Because no perfect direct repeats were observed at the breakpoint, this deletion falls into class 11, as defined by Mita et al. [19901. Short repeats as well as a long polypirimidine segment were Fig. 1. Detection of a heteroplasmic mtDNA deletion in a patient observed close to the breakpoint region (Fig. 2A), but with OCRL. (A) Southern analysis of total muscle DNA. About 5 pg of their contribution to the genesis of the deletion is untotal muscle DNA was digested with PuuII, electrophoresed through a certain. 0.8%agarose gel, transferred to a nitrocellulose membrane, and probed with 32P-labeledhuman mtDNA. 1, normal control; 2, patient’s mother; DISCUSSION 3, patient (M.S.); 4, typical OCRL (X-linked).A smaller mtDNA population (8.8 kb) is detected only in the patient’s muscle. (B)Analysls of We have found a partially deleted mtDNA population PCR products by agarose gel electrophoresis. Based on mapping information (see methods), oligonucleotideprimers were designed and used in skeletal muscle from a girl who presented a t age 3 to amplify selectively the region containing the deletion breakpoint. with manifestations of Lowe oculocerebrorenal syn1, normal control; 2, patient’s mother; 3, patient (M.S.); M, molecular drome. The deletion was 7803 bp in size and encomweight marker (pBR322 digested with BstNI). A specific 360 bp fragment is observed only in the patient sample. Control DNAs yield un- passed a number of tRNA genes and structural genes of respiratory chain complexes I, 111, IV, and V, but this specific DNA fragments. 8.8
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deletion did not differ significantly in size or location from those described previously in patients with mitochondrial encephalomyopathies [Moraes et al., 1989; Holt et al., 19891.The search for a mtDNA mutation was undertaken because the patient was a girl with normal karyotype, and because she had clinical, pathological, and laboratory abnormalities suggesting a mitochondrial disease. These included short stature, progressive external ophthalmoplegia (PEO), neurosensory hearing loss, ataxia, lactic acidosis, mitochondrial proliferation in skeletal muscle, and decreased activities of respiratory chain enzymes that contain mitochondrial-DNA encoded subunits. The association of OCRL features with a mtDNA mutation is puzzling. Nuclear DNA mutations can cause mtDNA deletions, as shown in a family with dominantly inherited progressive external ophthalmoplegia and multiple mtDNA deletions [Zeviani et al., 19891. However, a nuclear DNA mutation is unlikely in our patient because family history was negative and she showed a single deletion of mtDNA. On the other hand, there are many similarities between this patient and the many sporadic cases described with single mtDNA deletions [Moraes et al., 19891. These patients fall in two major clinical groups, which share the common abnormality of progressive external ophthalmoplegia (PEO). Some patients have a relatively benign condition characterized by a pure myopathy (ocular myopathy); others have a severe multisystem disorder usually conforming to the definition of Kearns-Sayre syndrome (KSS): onset before age 20, PEO, pigmentary degeneration of the retina, and a t least one of the following features: heart block, cerebellar syndrome, CSF protein above 100 mg/dl. Although our patient’s initial clinical presentation had several manifestations of OCRL (mental retardation, cataracts, renal tubular dysfunction), she also developed changes typical of KSS: ophthalmoparesis and ptosis, cerebellar ataxia, cardiac conduction defect, and CSF protein above 100 mg/dl. Only one of the cardinal abnormalities of KSS, pigmentary retinopathy, could not be established in this child because dense bilateral cataracts impeded adequate examination of the fundi. Like patients with KSS, she was a sporadic case and had ragged-red fibers in the muscle biopsy. Southern analysis of mtDNA isolated from postmortem tissues in patients with KSS has shown that mtDNA deletions are present in all tissues studied, although the percentage of mutant versus wild-type mitochondrial genomes varies widely from tissue to tissue [Shanske et al., 19901. It is generally assumed that in each tissue loss of function (and, therefore, clinical symptoms related to that tissue) will be manifested only when the number of mutant mtDNAs reaches a critical threshold [Shoffner and Wallace, 19901, which probably varies among tissues and may depend on their oxidative requirements. Thus, it is possible that the unusual association of OCRL and KSS manifestations in our patient was simply the result of an unusual distribution of mutant mtDNAs among tissues. Unfortunately, this hypothesis could not be tested directly because autopsy was not performed. This hypothesis does not explain why the
OCRL symptoms are not seen more frequently in patients with mtDNA mutations, but clinical variability is known to occur in these cases and is best exemplified by ocular myopathy and KSS. Even among patients with KSS, there is variability: besides the “obligatory”findings some patients have endocrine abnormalities, others have renal dysfunction, still others combine the features of KSS with those of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome), another mitochondrial encephalomyopathy. An extreme example of clinical variability caused by deletions in mtDNA is a rare condition known as Pearson pancytopenia. Deletions of mtDNA in patients with Pearson syndrome are similar to those of patients with OM or KSS, but the number of mutant mtDNAs is extremely high in blood cells and pancreas, thus probably causing these tissues to be primarily affected [Rotig et al., 19891. A partial defect of multiple respiratory chain complexes was also observed in patients with Kearns-Sayre syndrome and ocular myopathy [Moraes et al., 19891. Recent results from our laboratory suggest that the basis for this multienzyme defect is a failure of mitochondrial protein synthesis due to paucity of indispensable tRNAs [Mita et al., 1989; Nakase et al., 19901. Although the genetic cause of Lowe symptoms in our patient is different from that of typical X-linked OCRL, our findings are in agreement with biochemical data suggesting that mitochondrial metabolism may also be abnormal in X-linked OCRL. Gobernado et al. [19841 found that oxygen consumptionby muscle mitochondria from a patient with typical OCRL was markedly decreased with nicotinamide adenine dinucleotide or flavoprotein-linked substrates but returned to normal when ascorbate or N’N’N’N’-tetramethyl-p-phenylenediamine (TMPD) were used as substrates, suggesting a problem in the respiratory chain before complex IV. Taken together, our results and those of Gobernadoet al. [19841 suggest that mitochondrial dysfunction may play a role in the pathogenesis of OCRL. However, until the genetic or biochemical defect underlying typical OCRL is found, we cannot exclude the possibility that the mitochondrial dysfunction may be a secondary phenomenon.
ACKNOWLEDGMENTS Supported by Center Grant NS 11766 from the National Institute for Neurological Diseases and Stroke, by grants from the Muscular Dystrophy Association, the Aaron Diamond Foundation, and by a donation from Libero and Graziella Danesi (Milano, Italy). C.T.M. was supported by the Brazilian Research Council (CNPq). REFERENCES Anderson S, Bankier AT, Barrel BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH,Staden R, Young IG (1981):Sequence and organization of the human mitochondrial genome. Nature (London) 290: 457-465. Curtis JA, Goel KM (1982):Oculo-cerebro-renal syndrome (Lowe’s syndrome)-a report of three cases. The Practitioner 226:11591164.
mtDNA Deletion Causing Features of Lowe Syndrome Cyvin KB, Weidemann J, Bathen J (1973): Lowe’s syndrome. Acta Paediat Scand 62:309-312. Denys P, Corbeel L (1964): Acidosis renal, hypokaliemie, nanism et syndrome oculocer6bral. Ann Pediat 203:313-327. DiMauro S, Servidei S, Zeviani M, DiRocco M, De Vivo DC, DiDonato S, Uziel G, Berry K, Hoganson G, Johnsen SD, Johnson PC (1987): Cytochrome c oxidase deficiency in Leigh syndrome. Ann Neurol 22~498-506. Gobernado JM, Lousa M, Gimeno A, Gonsalvez M (1984): Mitochondrial defects in Lowe’s oculocerebrorenal syndrome. Arch Neurol 41:208-209. Hodgson SV, Heckmatt JZ, Hughes E, Crolla JA, Dubowitz V, Bobrow M (1986): A balanced do novo Wautosome translocation in a girl with manifestations of Lowe syndrome. Am J Med Genet 23:837--847. Holt IJ,Harding AE, Cooper JM, Schapira AHV, Toscano A, Clark JB, Morgan-Hughes JA (1989):Mitochondrial myopathies: Clinical and biochemical features of 30 patients with major deletions of muscle mitochondrial DNA. Ann Neurol 26599-708. Lowe CU, Terrey M, MacLachlan EA (1952): Organic-aciduria, decreased renal ammonia production, hydrophthalmos, and mental retardation. A clinical entity. Am J Dis Child 83: 164-184. Matsuda I, Sugai M, Kajii T (1970): Ornithine loading test in Lowe’s syndrome. Am J Dis Child 77: 127-129. McKusick VA (1986). Lowe oculocerebrorenal syndrome. In: “Mendelian Inheriatnce in Man,” 7th ed. Baltimore: The Johns Hopkins University Press, pp 1412-1413. Mita S, Rizzuto R, Moraes CT, Shanske S, Arnaudo E, Fabrizi G, Koga Y, DiMauro S and Schon EA (1990):Recombination via flanking direct repeats is a major cause of large-scale deletions of human mitochondrial DNA. Nucl Acids Res 18:561-567. Mita S, Schmidt B, Schon EA, DiMauro S, BonillaE (1989):Detection of “deleted” mitochondrial genomes in cytochrome-c oxidase-deficient muscle fibers of a patient with Kearns-Sayre syndrome. Proc Natl Acad Sci USA 86:9509-9513. Moraes CT, DiMauro S, Zeviani M, Lombes A, Shanske S, Miranda AF, Nakase H Bonilla, E., Werneck LC Servidei, S., Nonaka I, Koga Y, Spiro A, Brownell KW, Schmidt B, Schotland DL, Zupanc MD, De Vivo DC, Schon EA, Rowland LP (1989): Mitochondrial DNA deletions in progressive external ophthalmoplegia and KearnsSayre syndrome. N Engl J Med 320:1293-1299. Nakase H, Moraes CT, Rizzuto R, Lombes A, DiMauro S, Schon EA (1990):Transcription and translation of deleted mitochondrial genomes in Kearns-Sayre syndrome: implications for pathogenesis. Am J Hum Genet 46:418-427. Ores RO (1970): Renal changes in oculo-cerebro-renal syndrome of Lowe. Arch Path01 89:221-225.
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Reilly DS, Lewis RA, Ledbetter DH, Nussbaum RL (1988): Tightly linked flanking markers for the Lowe oculocerebrorenal syndrome, with application to carrier assessment. Am J Hum Genet 42: 748-755. Rotig A, Colonna M, Bonnefont JP, Blanche S, Fisher A, Saudubray JM, Munnich A (1989): Mitochondrial DNA deletion in Pearson’s marrow/pancreas syndrome. Lancet 1:902-903. Sage1I, Ores RO, Yuceoglu AM (1970):Renal function and morphology in a girl with oculocerebrorenal syndrome. J Pediatr 77:124-128. Sanger F, Nicklen S, Coulson AR (1977):DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci U.S.A. 74:5463-5467. Schoen EJ, Young G (1959):“Lowe’ssyndrome”: Abnormalities in renal tubular function in combination with other congenital defects. Am J Med 27:781-792. Scholten HG (1960): Een meisje met het syndroom van Lowe. Maandschr Kindergeneesk 28:251-255. Schon EA, Rizzuto R, Moraes CT, Nakase H, Zeviani M, DiMauro S (1989):A direct repeat is a hotspot for large-scale deletions of human mitochondrial DNA. Science 244346-349. Shanske S, Moraes CT, Lombes A, Miranda AF, Bonilla E, Lewis P, Whelan MA, DiMauro S (1990): Widespread tissue distribution of mitochondrial DNA deletions in Kearns-Sayre syndrome. Neurology 40:24-28. Shoffner JM, Wallace DC. (1990). Oxidative phosphorylation diseases: disorders of two genomes. In Harris H, and Hirschhorn K (eds): “Advances in Human Genetics.” New York: Plenum Press, pp 267-330. Silver DN, Lewis RA, Nussbaum RL (1987): Mapping the Lowe oculocerebrorenal syndrome to Xq24-q26 by use of restriction fragment length polymorphisms. J Clin Invest 79:282-285. Svork J , Masopust J, Komarkova A, Macek M, Hyanek J (1967):Oculocerebrorenal syndrome in a female child. Am J Dis Child 114:186-190. Winship PR (1989):An improved method for directly sequencing PCR amplified material using dimethyl sulphoxide. Nucleic Acids Res 17:1266. Zeviani M, Moraes CT, DiMauro S, Nakase H, Bonilla E, Schon EA, Rowland LP (1988): Deletions of mitochondrial DNA in KearnsSayre syndrome. Neurology 38:1339-1346. Zeviani M, Servidei S, Gellera C, Bertini E, DiMauro S, DiDonato S (1989):An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature (London) 339:309-3 11.
Mitochondria1 DNA Deletion in a Girl With Manifestations of Kearns-Sayre and Lowe Syndromes: An Example of Phenotypic Mimicry? Carlos T. Moraes, Massimo Zeviani, Eric A. Schon, Robert 0. Hickman, Brien W. Vlcek, and Salvatore DiMauro Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, New York (C.T.M., E.A.S.),Instituto Neurologic0 “C. Besta”, Milano, Italy (M.Z.), Division of Pediatric Nephrology, Children$ Hospital and Medical Center, Seattle, Washington (R.O.H.), Department of Neurology, Children$ Hospital and Medical Center, Seattle, Washington (B.W.V.), Department of Neurology, College of Physicians and Surgeons, Columbia University, New York, New York (S.D.) Lowe oculocerebrorenal syndrome is an X-linked recessive disease whose locus has been assigned to Xp25. However, several reports of affected females without obvious chromosomal abnormalities suggest genetic heterogeneity of the Lowe phenotype. Although the biochemical defect in typical Lowe syndrome is not known,there is evidence s u g gesting that mitochondrial metabolism may be impaired. We have studied a girl who presented with an oculocerebrorenal syndrome, but later developed symptoms and signs of mitochondrial encephalomyopathy. Molecular genetic analysis of muscle mitochondrial DNA showed the presence of a population of partially deleted mtDNAs (heteroplasmy). The deletion was 7803 bp long and encompassed several genes encoding subunits of the respiratory chain enzymes. Our results suggest that mitochondrial DNA deletions may mimic several symptoms of the Lowe phenotype and reinforce the concept that a defect of mitochondrial metabolism could be involved in the pathogenesis of the X-linked disease.
KEY WORDS: Lowe syndrome, mtDNA deletions, mtDNA heteroplasmy, mitochondrial disease INTRODUCTION Lowe oculocerebrorenal syndrome (OCRL) is an X-linked recessive disorder characterized by bilateral Received for publication August 23, 1990; revision received March 16, 1991. Address reprint requests to Dr. Salvatore DiMauro, Department of Neurology, 4-420 College of Physicians and Surgeons, 630W 168th Street, New York, NY 10032.
0 1991 Wiley-Liss, Inc.
congenital cataracts, glaucoma, mental retardation, hypotonia, and defective renal tubular function [Lowe et al., 19521. Linkage analysis has assigned the OCRL locus to Xq24-q26 [Silver et al., 19871. The finding of a balanced de novo X/3 translocation in a girl with manifestations of Lowe syndrome has further localized the disorder to Xq25 [Hodgson et al., 19861. Tightly linked flanking polymorphic markers have also been described [Reilly et al., 19881. Though well established, the X-linked mode of inheritance is incompatible with a number of cases described in the literature. Matsuda et al. [19701 described a boy with typical Lowe syndrome whose father showed aminoaciduria after ornithine loading: the authors suggested an autosomal recessive variant. Svork et al. [1967], Denys and Corbeel [1964], and Scholten [19601 described affected females without detectable chromosome abnormalities, and Cyvin et al. 119731 reported a pair of male and female half-sibs allegedly of different fathers who were severely affected and died before the age of 6 months. These cases illustrate apparent heterogeneity of OCRL [McKusick, 1986; Curtis and Goel, 19821. Although the biochemical basis of OCRL is unknown, ultrastructural and biochemical studies have suggested involvement of mitochondrial metabolism. Ultrastructural abnormalities of mitochondria have been seen in renal [Schoen and Young, 1959; Ores, 1970;Sage1et al., 19701 and muscle [LM Turolla, personal communication] biopsies of patients with OCRL and biochemical studies of isolated muscle mitochondria were interpreted to suggest a defect in the respiratory chain [Gobernado et al., 19841. We have studied a girl who presented at age 3 years with manifestations of OCRL but who later developed neurosensory hearing loss, ataxia, and persistent lactic acidosis, which suggested the possibility of a mitochondrial encephalomyopathy and led to a muscle biopsy. Analysis of skeletal muscle mtDNA showed the presence of partially deleted mtDNAs coexisting with
302
Moraes et al.
normal genomes (heteroplasmy). Our results support the idea that OCRL features may result from different genetic lesions, all of which cause mitochondrial dysfunction.
CLINICAL REPORT An 11-year-old girl apparently developed normally until age 3, when slowly progressive decline of cognitive function became evident. At age 5, Fanconi syndrome was diagnosed and impaired vision was attributed to bilateral cataracts. She also developed corneal crystals that were atypical, and cystinosis was excluded later by leukocyte and bone marrow studies. In the following years, she developed proximal limb weakness, bilateral ptosis and limitation of eye movements, nystagmus, truncal ataxia, and neurosensory hearing loss. Intermittently throughout the last 5 years she had episodesof encephalopathy with lethargy or stupor triggered by intercurrent infections. She also developed chronic renal insufficiency, and her vision deteriorated from 20/80 bilaterally to 20/400. Family history was noncontributory: both parents and an older sister were normal. Examination at age 11showed height 116.9 cm and weight 23.4 kg. She had severe bilateral ptosis and limited range of eye movements in all directions. F'undi could not be examined because of cataracts and marked cloudiness of both corneas. She had both gait and truncal ataxia. There was mild generalized weakness. Laboratory abnormalities included increased blood lactate (2.9-3.7 mM; normal, < 2.2 mM), increased cerebrospinal fluid lactate (5.6 mg/dl; normal < 2.0 mg/dl) and protein (230 mg/dl; normal < 45 mg/dl), glycosuria, phosphaturia, and generalized aminoaciduria (Fanconi syndrome). CT scan of the head showed mild brain atrophy and bilateral hypodense lesions in the putamen and in the regions around the third ventricle. At age 11she developed tachypnea, Kussmaul breathing, and had 3 cardiopulmonary arrests. Because of extreme cardiac arrhythmia, a temporary pacemaker was placed. After a transient improvement, she developed increasing respiratory distress, hypercarbia and acidosis. Despite intubation and i.v. sodium bicarbonate administration, the metabolic acidosis persisted, the renal insufficiency worsened, and she developed epidermal necrosis. She died of cardiopulmonary arrest at age 11.
MATERIALS AND METHODS Restriction enzymes were from Boehringer Mannheim and New England Biolabs. Klenow fragment ofE. coli DNA polymerase I was from Boehringer Mannheim. RNase A and chemicals were from Sigma. [a- 32PlDeoxycytidineand deoxyadenine triphosphate
(800 Ci/mmol)were from New England Nuclear. Singlestranded oligonucleotides primers were synthesized by Genetic Designs. The sequences of the primers were (mtDNA map positions according to Anderson et al., 1981) Primer If, 7,433 to 7,468; Ib, 15,600 to 15,774.
Biochemical Studies Mitochondrial enzyme activities were measured in muscle homogenates as described [DiMauro et al., 19871. Southern blot analysis. Total DNA was isolated from 50 mg of frozen muscle obtained by biopsy. DNA was purified by conventional methods as described previously [Zeviani et al., 19881. Purified mitochondrial DNA from normal liver was used as template for the hybridization probe in Southern analysis. The proportion of normal and mutant mtDNA genomes was estimated by densitometry of the X-ray film. The approximate site of the deleted region was determined by restriction mapping (using the enzymes PuuII, HindIII, PstI, XbaI, and EcoRI).The mapping procedure has been described elsewhere [Zeviani et al., 19881. PCR amplifications and sequencing. Polymerase chain reaction (PCR) amplifications were performed with the Ampli-Taq Kit from Perkin ElmerCetus. About 1 p,g of total DNA was used in the amplification reaction. All other conditions followed the recommended guidelines of the manufacturer. We have performed 35 cycles of amplification (1min 55"C,3 min 72"C, and 1min 94°C).Ten percent of the reaction mixture was analyzed by agarose gel electrophoresis. The fragment obtained was reamplified and purified from a low-melting agarose gel using the Gene-Clean kit (Bio 101).Double-strand direct sequencing was performed by the Sanger method [Sanger et al., 19771, modified by Winship [19891. About 1.5 pg of DNA template was used in the reaction. Sequenase kit (USB) was used in the sequencing reactions.
RESULTS Because this girl with initial diagnosis of OCRL had chronic lactic acidosis, a muscle biopsy was performed which showed ragged red fibers with the Gomori trichrome stain and abnormal mitochondria by electron microscopy (not shown). Biochemical analysis of mitochondrial enzymes showed markedly decreased activities of respiratory chain complex I (NADH-coenzymeQ oxireductase), I11 (coenzyme Q-cytochrome c oxireductase), and IV (cytochrome c oxidase), while the activities of complex I1 (succinate-coenzyme Q oxireductase), and citrate synthase, a matrix enzyme, were normal (Table I). Com-
TABLE I. Mitochondrial Enzymes Activity in Muscle Homogenates Enzymehespiratory chain complex" Patient (M.S.) Controls
I
I1
I + I11
I1 + I11
IV
cs
7.3 0.59 0.02 0.04 0.02 9.56 35.5 '-c 7.1 0.70 f0.23 1.02k0.38 0.70 ? 0.29 2.80 & 0.52 9.88 22.55 "Activities are Fmol substrate utilizedlrnidg fresh tissue. Controls expressed as mean 2 SD of n220. Enzymeskomplexes: I, NADH-coenzyme Q oxireductase; 11, succinate dehydrogenase; I + 111, NADH-cytochrome c oxireductase; I1 + 111, succinate-cytochrome c oxireductase; IV, cytochrome c oxidase; CS, citrate synthase.
mtDNA Deletion Causing Features of Lowe Syndrome plexes I, 111, and IV contain multiple subunits, some encoded by nuclear DNA others by mitochondrial DNA, but subunits of complex I1 and citrate synthase are encoded exclusively by nuclear DNA. Because the biochemical findings suggested that our patient might have a mtDNA mutation, we performed Southern analysis of total DNA purified from skeletal muscle. Using the restriction enzyme PuuII, which cuts only once in the mtDNA, the Southern blot showed the presence of two populations of mtDNA, one corresponding to full-length mtDNA (16.6 kb) and the other corresponding to a shorter molecule of approximately 8.8 kb (Fig. 1A). The location of the 7.8 kb deletion was determined by restriction mapping, and the information obtained was used to design a pair of polymerase chain reaction (PCR) primers that allowed us to amplify a DNA fragment spanning the deletion breakpoint [Schon, 19891. PCR primers If and Ib generated an amplified DNA fragment of approximately 360 bp that was used for double-strand direct sequencing. Southern analysis of mtDNA from muscle specimens of the patient’s mother and from a patient with typical X-linked OCRL showed no evidence of deletions (Fig. 1A). Availability of PCR primers specific to the patient’s breakpoint region allowed us to reexamine the muscle from the patient’s mother looking for small numbers of mutant mtDNAs. Even this more sensitive technique failed to show abnormal mitochondrial genomes. Normal controls, as well as the mother of the patient yielded nonspecific DNA fragments after the PCR reaction. The expected 360 bp DNA fragment spanning the deletion breakpoint was observed only after amplification of the patient’s DNA (Fig. 1B). The 360 bp DNA fragment spanning the deletion breakpoint was sequenced and the breakpoint determined by comparison with the published human mtDNA sequence [Anderson, 19811(Fig. 2A). The exact B
A 1
2
3
123M
4
303
A G
A T C
........_...
-
03X’AClTCCCXXATCATAGC c02
-7,803
B
-
---W bp
............
-
’ I T A C I T C T m C T m
CYB
r.“”
c02
’
6
ATPase
Fig. 2. Location of the deletion breakpoint. (A) The 360 bp DNA fragment obtained by PCR (see Fig. 1)was sequenced, and the breakpoint determined by comparison with the published human mtDNA sequence [Anderson et al., 19811. Regions with significant homology, and possible role in the genesis of the deletion, are underlined. (B) Schematic representation of the patient’s mtDNA deletion. OH and OL, origins of heavy- and light-strand replication; 12 and 16 S, ribosomal RNA genes; ND1-6, genes corresponding to NADH-coenzyme Q oxidoreductase subunits 1-6; CO1-3, genes corresponding to cytochrome c oxidase subunits 1-3; CYT b, cytochrome b gene; ATPase 6 and 8, genes corresponding to ATP synthetase subunits 6 and 8; If and Ib PCR primers used to amplify the region spanning the breakpoint (see methods).
kb
-
-16.6
size of the deletion was 7803 bp, starting at position 7,636 a t the “left side” and ending at position 15,439 at c 360 bp the “right side”(Fig. 2B). Because no perfect direct repeats were observed at the breakpoint, this deletion falls into class 11, as defined by Mita et al. [19901. Short repeats as well as a long polypirimidine segment were Fig. 1. Detection of a heteroplasmic mtDNA deletion in a patient observed close to the breakpoint region (Fig. 2A), but with OCRL. (A) Southern analysis of total muscle DNA. About 5 pg of their contribution to the genesis of the deletion is untotal muscle DNA was digested with PuuII, electrophoresed through a certain. 0.8%agarose gel, transferred to a nitrocellulose membrane, and probed with 32P-labeledhuman mtDNA. 1, normal control; 2, patient’s mother; DISCUSSION 3, patient (M.S.); 4, typical OCRL (X-linked).A smaller mtDNA population (8.8 kb) is detected only in the patient’s muscle. (B)Analysls of We have found a partially deleted mtDNA population PCR products by agarose gel electrophoresis. Based on mapping information (see methods), oligonucleotideprimers were designed and used in skeletal muscle from a girl who presented a t age 3 to amplify selectively the region containing the deletion breakpoint. with manifestations of Lowe oculocerebrorenal syn1, normal control; 2, patient’s mother; 3, patient (M.S.); M, molecular drome. The deletion was 7803 bp in size and encomweight marker (pBR322 digested with BstNI). A specific 360 bp fragment is observed only in the patient sample. Control DNAs yield un- passed a number of tRNA genes and structural genes of respiratory chain complexes I, 111, IV, and V, but this specific DNA fragments. 8.8
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Moraes et al.
deletion did not differ significantly in size or location from those described previously in patients with mitochondrial encephalomyopathies [Moraes et al., 1989; Holt et al., 19891.The search for a mtDNA mutation was undertaken because the patient was a girl with normal karyotype, and because she had clinical, pathological, and laboratory abnormalities suggesting a mitochondrial disease. These included short stature, progressive external ophthalmoplegia (PEO), neurosensory hearing loss, ataxia, lactic acidosis, mitochondrial proliferation in skeletal muscle, and decreased activities of respiratory chain enzymes that contain mitochondrial-DNA encoded subunits. The association of OCRL features with a mtDNA mutation is puzzling. Nuclear DNA mutations can cause mtDNA deletions, as shown in a family with dominantly inherited progressive external ophthalmoplegia and multiple mtDNA deletions [Zeviani et al., 19891. However, a nuclear DNA mutation is unlikely in our patient because family history was negative and she showed a single deletion of mtDNA. On the other hand, there are many similarities between this patient and the many sporadic cases described with single mtDNA deletions [Moraes et al., 19891. These patients fall in two major clinical groups, which share the common abnormality of progressive external ophthalmoplegia (PEO). Some patients have a relatively benign condition characterized by a pure myopathy (ocular myopathy); others have a severe multisystem disorder usually conforming to the definition of Kearns-Sayre syndrome (KSS): onset before age 20, PEO, pigmentary degeneration of the retina, and a t least one of the following features: heart block, cerebellar syndrome, CSF protein above 100 mg/dl. Although our patient’s initial clinical presentation had several manifestations of OCRL (mental retardation, cataracts, renal tubular dysfunction), she also developed changes typical of KSS: ophthalmoparesis and ptosis, cerebellar ataxia, cardiac conduction defect, and CSF protein above 100 mg/dl. Only one of the cardinal abnormalities of KSS, pigmentary retinopathy, could not be established in this child because dense bilateral cataracts impeded adequate examination of the fundi. Like patients with KSS, she was a sporadic case and had ragged-red fibers in the muscle biopsy. Southern analysis of mtDNA isolated from postmortem tissues in patients with KSS has shown that mtDNA deletions are present in all tissues studied, although the percentage of mutant versus wild-type mitochondrial genomes varies widely from tissue to tissue [Shanske et al., 19901. It is generally assumed that in each tissue loss of function (and, therefore, clinical symptoms related to that tissue) will be manifested only when the number of mutant mtDNAs reaches a critical threshold [Shoffner and Wallace, 19901, which probably varies among tissues and may depend on their oxidative requirements. Thus, it is possible that the unusual association of OCRL and KSS manifestations in our patient was simply the result of an unusual distribution of mutant mtDNAs among tissues. Unfortunately, this hypothesis could not be tested directly because autopsy was not performed. This hypothesis does not explain why the
OCRL symptoms are not seen more frequently in patients with mtDNA mutations, but clinical variability is known to occur in these cases and is best exemplified by ocular myopathy and KSS. Even among patients with KSS, there is variability: besides the “obligatory”findings some patients have endocrine abnormalities, others have renal dysfunction, still others combine the features of KSS with those of mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome), another mitochondrial encephalomyopathy. An extreme example of clinical variability caused by deletions in mtDNA is a rare condition known as Pearson pancytopenia. Deletions of mtDNA in patients with Pearson syndrome are similar to those of patients with OM or KSS, but the number of mutant mtDNAs is extremely high in blood cells and pancreas, thus probably causing these tissues to be primarily affected [Rotig et al., 19891. A partial defect of multiple respiratory chain complexes was also observed in patients with Kearns-Sayre syndrome and ocular myopathy [Moraes et al., 19891. Recent results from our laboratory suggest that the basis for this multienzyme defect is a failure of mitochondrial protein synthesis due to paucity of indispensable tRNAs [Mita et al., 1989; Nakase et al., 19901. Although the genetic cause of Lowe symptoms in our patient is different from that of typical X-linked OCRL, our findings are in agreement with biochemical data suggesting that mitochondrial metabolism may also be abnormal in X-linked OCRL. Gobernado et al. [19841 found that oxygen consumptionby muscle mitochondria from a patient with typical OCRL was markedly decreased with nicotinamide adenine dinucleotide or flavoprotein-linked substrates but returned to normal when ascorbate or N’N’N’N’-tetramethyl-p-phenylenediamine (TMPD) were used as substrates, suggesting a problem in the respiratory chain before complex IV. Taken together, our results and those of Gobernadoet al. [19841 suggest that mitochondrial dysfunction may play a role in the pathogenesis of OCRL. However, until the genetic or biochemical defect underlying typical OCRL is found, we cannot exclude the possibility that the mitochondrial dysfunction may be a secondary phenomenon.
ACKNOWLEDGMENTS Supported by Center Grant NS 11766 from the National Institute for Neurological Diseases and Stroke, by grants from the Muscular Dystrophy Association, the Aaron Diamond Foundation, and by a donation from Libero and Graziella Danesi (Milano, Italy). C.T.M. was supported by the Brazilian Research Council (CNPq). REFERENCES Anderson S, Bankier AT, Barrel BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Schreier PH, Smith AJH,Staden R, Young IG (1981):Sequence and organization of the human mitochondrial genome. Nature (London) 290: 457-465. Curtis JA, Goel KM (1982):Oculo-cerebro-renal syndrome (Lowe’s syndrome)-a report of three cases. The Practitioner 226:11591164.
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