Mitochondria1 DNA analysis in Leber’s hereditary optic neuropathy REFERENCES
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1. Nikoskelainen EK, Hoyt WF, Nummelin KU. Ophthalmoscopic findings in Leber’s hereditary optic neuropathy. Arch
Ophthalmol 1982; 100: 1597-1602. 2. Novotny EJ, Sing 0, Wallace DC et al. Leber’s disease and dystonia. A mitochondrial disease. Neurology 1986; 36: 1053-1060. 3. Nikoskelainen EK, Savontaus ML, Wanne OL, Katila MJ, Nummelin KU. Leber’s hereditary optic neuroretinopathy, a maternally inherited disease. Arch Ophthalmol 1987; 105 :665-671. 4. Newman NJ, Lott MT, Wallace DC. The clinical characteristics of pedigrees of Leber’s hereditary optic neuropathy with the 11778 mutation. Am J Ophthalmol 1991 ; 11 1 :750-762. 5 . Anderson S, Bankier AT, Barrel BG et al. Sequence and organization of the human mitochondrial genome. Nature 1981; 290:457-465. 6. Chomyn A, Mariottini P , Cleeter MWJ et al. Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase subunit. Nature 1985 ; 314: 592-597. 7. Chomyn A, Cleeter MWJ, Ragan CI, Riley M, Doolittle RF, Attardi G. URF6, last unidentified reading frame of human mt DNA, codes for an NADH dehydrogenase subunit. Science 1986; 234:614-618. 8. Giles RE, Blanc H , Cann HM, Wallace DC. Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 1980; 77:6715-6719. 9. Holt IJ, Harding AE, Morgan-Hughes JA. Deletion of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331 :717-719. 10. Lestienne P , Ponsot G. Kearns-Sayre syndrome with muscle mitochondrial DNA deletion. Cancet 1988; i : 885. 1 1 . Saifuddin Noer A, Marzuki S, Trounce I , Byrne E. Mitochondrial DNA deletions in encephalomyopathy. Lancet 1988; ii : 1253-1254. 12. Moraes CT, Di Mauro S, Zeviani M et al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N Engl J Med 1989; 320: 1293-1299. 13. Zeviani M, Servidei S, Gellera C , Bertini E, Di Mauro S, Di Donato S. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 1989; 339:309-311. 14. Poulton J, Deadman ME, Gardiner RM. Duplication of mitochondrial DNA in mitochondrial myopathy. Lancet 1989; i :236-239. 15. Holt IJ, Harding AE. Petty RKH, Morgan-Hughes JA. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990; 46:428-433. 16. Shoffner JM, Lott MT, Lezza AMS, Seibel P , Ballinger SW, Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNAlys mutation. Cell 1990; 61 :931-937. 17. Tanaka M, [no H, Ohno K et al. Mitochondrial mutation in fatal infantile cardiomyopathy. Lancet 1990; ii: 1452. 18. Tanaka M, Ino H, Ohno K et al. Mitochondrial DNA mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem Biophys Res Comm 1991 ; 174:861-868. 19. Zeviani M, Gellera C , Antozzi C et al. Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNALEu(uuR). Lancet 1991 ; 338: 143-147. 20. Wallace DC, Singh G , Loti MT et al. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 1988; 242: 1427-1430. 21. Hotta Y, Hayakawa M, Saito K , Kanai A, Nakajima A, Fujiki K. Diagnosis of Leber’s optic neuropathy by means of Polymerase Chain Reaction Amplification. Am J Ophthalmol 1989; 108:601-602. 22. Vilkki J, Savontaus ML, Nikoskelainen EK. Genetic heterogeneity in Leber’s hereditary optic neuroretinopathy revealed by mitochondrial DNA polymorphism. Am J Hum Genet 1989; 45 :206-211. 23. Yoneda M, Tsuji S, Yamauchi T et al. Mitochondrial DNA mutation in a family with Leber’s hereditary optic neuropathy. Lancet 1989; i : 1076-1077. 24. Stone EM, Coppinger JM, Kardon RH, Donelson J. Mae 111 positively detects the mitochondrial mutation associated with type I Leber’s hereditary optic neuropathy. Arch Ophthalmol 1990; 108: 1417-1420. 25. Jacobson DM, Stone EM. Difficulty in differentiating Leber’s from dominant optic neuropathy in patient with remote visual loss. J Clin Neuro-ophthalmol 1991 ; 11 : 152-157. 26. Holt IJ, Miller DH, Harding AE. Genetic heterogeneity and mitochondrial DNA heteroplasmy in Leber’s hereditary optic neuropathy. J Med Genet 1989; 26:739-743. 27. Bolhuis PA, Bleeker-Wagemakers EM, Ponne NJ et al. Rapid shift in genotype of human mitochondrial DNA in a
225
0 Aeolus Press Ophthalmic Paediatrics and Genetics 0167-6784/92/US$ 3.50
(Accepted 20 August 1992)
Mitochondrial DNA analysis in Leber's hereditary optic neuropathy
Ophthalmic Genet Downloaded from informahealthcare.com by Nyu Medical Center on 05/22/15 For personal use only.
PIERO BARBONI', VILMA MANTOVAN12,PASQUALE MONTAGNA3, MICHELA BRAGLIAN12, PIETRO CORTELL13, E L I 0 LUGARES13, PIERO PUDDU' and ROBERTO CARAMAZZA'*
Institute of Ophthalmology, Bologna University; 2Department of Pathology, Malpighi Hospital, Bologna; 31nstitute of Neurology, Bologna University; Italy ABSTRACT. A mitochondrial DNA (mtDNA) mutation at nucleotide 11778 has been reported as the genetic defect associated with Leber's hereditary optic neuropathy (LHON), but some pedigrees failed to reveal this mutation. The authors present the genetic analysis of a large Italian LHON family with three probands and 16 asymptomatic maternal relatives. The 11778 mtDNA mutation was present in this family and absent in 52 Italian healthy controls, confirming the association between this genetic defect and LHON. In addition, they found a variable proportion of mutated and wild-type mtDNA (heteroplasmy) in every maternally related family member, including the probands. Different patterns of heteroplasmy were present in the pedigree and lower levels of wild-type mtDNA seemed to correlate with the disease status. Moreover, the authors evaluated the mitotic segregation of mt genomes in blood, hair and urinary tract epithelia of the three patients and they found a similar level of heteroplasmy in these tissues. Key words: Leber's hereditary optic neuropathy ; mitochondrial DNA mutation
INTRODUCTION Leber's hereditary optic neuropathy (LHON) is a hereditary disease characterized by bilateral asynchronous optic nerve atrophy, cardiac dysrhythmias and occasionally dystonia in young adults with male predominance. Peripapillary microangiopathy is a typical feature of presymptomatically affected and asymptomatic family mernber~l-~. The inheritance of the disease does not follow Mendelian principles and the exclusive transmission through the maternal lineage has suggested a mitochondrial disorder. ~
*
Correspondence lo: Prof. Roberto Caramazza, Institute of Ophthalmology, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40100 Bologna, Italy.
Mitochondria are cytoplasmic organelles essential to cellular bioenergy containing several copies of their own DNA. Mitochondrial DNA (mtDNA) is a circular double-strand molecule of 16569 base pair (bp) coding for two rRNAs, 22 tRNAs and 13 of the 61 protein subunits of mitochondrial respiratory c o m p l e x e ~ ~ -The ~. mtDNA is transmitted through the maternal lineage and the mitochondria of the zygote are provided only by the ovum*. Recent clinical and genetic studies of some neuro-ophthalmic diseases have revealed various defects in mtDNA such as single and multiple deletions9-13, tandem d~plications'~and point A point mutation at nucleotide 11778 of
Ophthalmic Paediatrics and Genetics 1992. Voi. 13, No. 4, pp. 219-226 0Aeolus Press Buren (The Netherlands) 1992 ~
219
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P. Barboni et al. mtDNA as the genetic defect of LHON was first reported by Wallace et The mutation causes a transition from guanine to adenine converting a highly conserved arginine to histidine in NADH dehydrogenase subunit4. This replacement mutation was found in about 50% of LHON pedigrees and only the mutated mtDNA (homoplasmy) was detected in both patient and maternal relative^^'-^^. Holt et a1.26have found a variable proportion of mutated and wild-type mtDNA in some families. This condition is termed heteroplasmy and is a typical feature of mitochondria1 diseases. Until now the heteroplasmic condition in LHON has been described in only a small number of pedigree^^'-^^. We have reported the presence of the 11778 mtDNA mutation in three probands of an Italian LHON family; the mutated mt genomes appeared to be h o r n o p l a ~ m i c ~In~ .the present work we report the genetic analysis of the whole pedigree. We find the presence of heteroplasmy for the mutation in all maternally related family members including the probands, by using more sensitive methods to detect wild-type mtDNA. Moreover, we evaluate the segregation of the two mtDNA types in different tissues. SUBJECTS AND METHODS
The family. The pedigree included three male probands, 12 female and four male asymptomatic maternal relatives as well as two children
MT2 MT3
S-CTCACAGTCGCATCATAATC-3' 5-CTCACAGTCACATCATAATC-3
TABLE 1 . Oligonucleotide primers and probes
220
of a male patient. The diagnosis of LHON was based on the typical fundus change during the acute stage of the illness, with subsequent optic atrophy and on the maternal inheritance of the disease; other causes of optic atrophy were not evident. The controls. To test the absence of the 11778 mtDNA mutation in the Italian population, we included 52 unrelated healthy controls in the study. PCR amplification and SfaNI restriction analysis. Since the 11778 mtDNA mutation removes an SfaNI restriction site, we performed an endonuclease digestion with this enzyme, after polymerase chain reaction (PCR) amplification of rntDNA3,. Total DNA was extracted from leukocytes and epithelial cells of the urinary tract with phenol and chloroform-isoamylalcohol, precipitated with ethanol and solubilized in 10 mM Tris-HCVl mM EDTA pH 8.033. DNA preparation from hairs was performed by adding 0.15 mg proteinase K, 50 pl SDS 20070, 0.05 mg RNAse, 40 pM DTT for 70 h, before the conventional DNA extraction. The oligonucleotide primers MT1 and MT4 (Table 1) were synthesized in order to amplify a 415 bp region of mtDNA, including the SfaNI site. Twentyfive cycles of PCR amplification were completed with 1 pg of DNA in 100 pI volume reaction containing 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCI,, 100 pg/ml gelatin, 200 pM each dATP, dCTP, dGTP and dTTP, 1 pM of
1176411788 11769-11788
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Mitochondria1DNA analysis in Leber 's hereditary optic neuropathy each primer and 2.5 units Taq-polymerase (Perkin Elmer-Cetus, Norwalk, CO). Each cycle consisted of 94°C denaturation for 1.30 min, 55°C annealing for 2.30 rnin and 72°C extension for 4 min. The presence of the 11778 mtDNA mutation was detected by overnight digestion of the total amplified DNA (about 2 pg) with 10 units of SfaNI restriction enzyme (New England Biolabs, Beverly, MA, USA). The samples were then electrophoresed on 3% Nusieve plus 1% agarose gel 1 pg/ml ethidium bromide incl. and photographed under ultraviolet light. Southern blotting and hybridization. After staining, the gel was denatured in 0.4 N NaOH/ 0.6 M NaCl for 30 rnin at room temperature and neutralized in 1.5 M NaCVO.5 M Tris-HCl pH 7.5 for 30 rnin at room temperature. The DNA was then transferred overnight to a nylon membrane by capillary action in transfer buffer (3 M NaCV0.3 M sodium citrate, pH 7.0)34. The oligonucleotide probes MTI and MT4, 5'endlabeled with 32P-dATP,were used for hybridization. Filters were prehybridized for 1 h at 37°C in 5 x Denhardt's (0.1 Yo Ficoll/O. 1Yo polyvinylpyrrolidone/O. 1Vo bovine serum albumin),
5 x S S P E (750 mM NaCl/SO mM NaH,PO,/5 mM EDTA), 0.5% SDS and 200 mg/ml of denatured herring sperm, and then hybridized for 1 hr at 50°C in a solution consisting of the prehybridization solution plus the radiolabelled probes (2 x lo6 cpm/ml for each probe). After hybridization the filters were washed in 0.1 x SSPE/O. 1To SDS for 10 rnin at room temperature and for 10 rnin at 38°C. The filters were then exposed to Kodak XAR-5 at -80°C with intensifying screen for 2 hr and/or overnight. Dot blotting and hybridization. Approximately 100 ng (5 pl of the PCR reaction) of amplified mtDNA were denatured in 45 p1 of 0.5 N NaOH/1.5 M NaC1/25 mM EDTA for 5 rnin at room temperature and 3 rnin at 90"C, and then neutralized in 150 pl of 0.5 M Tris-HC1 pH 8.0/1.5 M NaCl. The samples were spotted onto duplicate nylon membrane by using a dotblot apparatus (BioDot, BioRad, Richmond, CA, USA) and DNAs were bound to the membranes by ultraviolet cross-linking. The first filter was hybridized with the radiolabeled oligonucleotide probe MT2, complementary to the
tV
M
C
41 5
243
166
1
Fig. I . LHON pedigree: filled symbols indicate affected individuals. The lower panel shows the related pattern obtained by SfaNI digestion and gel-electrophoresis of the total amplified mtDNA (about 2 p g ) , visualized by ethidium bromide staining. Fragment of 415 base pair indicates the mutated mtDNA. Fragments of 249and 166 base pair indicate the wild-type mtDNA. 25-50% wild-type mtDNA, M, size marker; C , healthy control; " < 5 % wild-type mtDNA, 5-25% wild-type mtDNA, 0000 100% wild-type mtDNA.
22 1
P. Barboni et al. wild-type mtDNA and the second filter with the probe MT3, complementary to the mutated mtDNA (Table 1). The conditions of prehybridization, hybridization and washing were the same as used for Southern blotting, but the stringent wash was carried out at 40°C.
The heteroplasmic condition was revealed by the presence of all three fragments. An initial SfaNI digestion of 10 pl (about 200 ng) of amplified mtDNA and the following photograph of gel electrophoresis revealed heteroplasmy in only four maternal relatives (1-1, 1-2, 1-3, 11-1). The relative amount of wild-type mtDNA could be estimated at about 2550%. The three probands and the other maternal family members seemed to be homoplasmic for the mutation. When the SfaNI digestion was performed on the total amplified mtDNA (about 2 pg), the gel photograph revealed the presence of mild heteroplasmy also in six other maternal relatives (11-2, 111-1, 111-2, 1-4, 11-6, 111-9) (5-25070 wildtype mtDNA) (Fig. 1). In order to improve the sensitivity of the analysis and to detect even trace amounts of wild-type mtDNA we performed a Southern transfer and hybridization with radiolabeled oligo-probes. We also performed a dot-blot analysis and hybridization with wild-type and mutation specific oligo-
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RESULTS The presence of the 11778 mtDNA mutation removes an SfaNI restriction site. The endonuclease SfaNI was not able to digest the 415 bp amplified mtDNA when the mutation was present; but the SfaNI digestion resulted in two fragments of 249 and 166 bp when the mutation was absent. The three LHON patients and all maternally related family members possessed the 11778 mtDNA mutation. The individuals from the paternal lineage, including the offspring of a male patient, retained the SfaNI site (Fig. 1). All the 52 unrelated healthy controls showed the SfaNI site.
2
C
1-3 11-3 111-3 11-4 111-4 111-5 111-6 IV-1 11-5 111-10 C
1-2
C
C
1-3 11-3 111-3 11-4 111-4 111-5 111-6 IV-1 TI 5 111-10 C
I2
C
C
C
11-5 11-5 I114 111-4 111-3 111-3 U
H
U
H
U
H
11-5 11-5 111-4 111-4 I11 3 I11 3 U
H
U
ti
U
C
C
C
C
H
Fig. 2. Dot-blot analysis of amplified mtDNA of some LHON family rneinbers (see Fig. 1) and controls ( C ) . Filter A was hybridized with wild-type mtDNA probe MT2. Filter B was hybridized with the mutation specific mtDNA probe MT3. U, urinary tract epithelia; H, hair.
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Mitochondria1 DNA analysis in Leber's hereditary optic neuropathy probes. Using these methods we found heteroplasrny for the 11778 mtDNA mutation in every maternal member of the LHON family, including the three probands ( < 5 % wild-type mtDNA) (Fig. 2). Analysis of hair and epithelial cells of the urinary tract obtained from the three patients was performed to evaluate if there was a different segregation of the two mtDNA types in these tissues. A similar level of heteroplasmy was detected in both tissues of probands with the hybridization methods, although the photograph of the gel electrophoresis did not disclose wild-type mtDNA (Figs. 2 and 3). DISCUSSION The point mutation in nucleotide 11778 of the mtDNA NADH dehydrogenase subunit 4 gene has been reported as the most frequent genetic defect associated with LHON20-25.31. Nevertheless, some LHON pedigrees lack this mutation, suggesting a genetic heterogeneity in the dis111-3 U
ease20-22.Other point mutations of mtDNA were recently proposed as alternative genetic defects associated with the d i ~ e a s e ~ ~ - ~ ~ . The LHON family investigated in this study showed the 11778 mutation. In addition, the absence of this mutation in 52 Italian healthy controls studied, makes it unlikely that the mutation is an ethnic-specific polymorphism. Our results confirm the association between this genetic defect and LHON in Italian patients. The heteroplasmic condition, an otherwise typical feature of mitochondria1 diseases, seems to be unusual in LHON27-30. The mtDNA analysis of this Italian pedigree showed the presence of heteroplasmy in all maternally related family members, including the three probands. At first, the SfaNI digestion of amplified mtDNA revealed heteroplasmy only in three siblings and a nephew of the probands' maternal grandmother. These individuals belonged to a pedigree branch without optic atrophy. When the SfaNI digestion was performed on a larger amount of amplified DNA
11- 5 H
u
H
1-2
1-3
0
6
bp 415249.-
166-
41%
249-
3 Fig. 3. Analysis of urinary tract epithelia and hair mtDNA. The upper panel shows the SfaNI digestion of amplified mtDNA of two probands (wild-type mtDNA < 5 % ) ; two maternal relatives were included as heteroplasmic controls (wild-type mtDNA 25-50'70). The lower panel shows the subsequent Southern blot and hybridization with MTI/MT4 probes. U , urinary tract epithelia; H, hair; B, blood; bp, base pair.
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P. Barboni et al. we were able to disclose heteroplasmy in six further family members. Only the three probands and some of their close relatives seemed to be homoplasmic (Fig. 1). A very small amount of wild-type mtDNA was revealed also in the latter individuals by using the more sensitive Southern and dot-blot hybridization methods (Fig. 2 ) . This suggests that the sensitivity of the method employed is crucial for the detection of heteroplasmy in those LHON families in which few residual wild-type mt genomes are present. In addition, in our pedigree the disease status seems to correlate with lower levels of wild-type mtDNA. For example, the probands’ grandmother (I-4) was less heteroplasmic than her siblings (1-1, 1-2, 1-3) (Fig. 1); the three patients (111-3, 111-4, 11-5) showed the lowest level of wild-type mtDNA, detectable only by hybridization methods (Fig. 2). A similar low level of heteroplasmy (
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1. Nikoskelainen EK, Hoyt WF, Nummelin KU. Ophthalmoscopic findings in Leber’s hereditary optic neuropathy. Arch
Ophthalmol 1982; 100: 1597-1602. 2. Novotny EJ, Sing 0, Wallace DC et al. Leber’s disease and dystonia. A mitochondrial disease. Neurology 1986; 36: 1053-1060. 3. Nikoskelainen EK, Savontaus ML, Wanne OL, Katila MJ, Nummelin KU. Leber’s hereditary optic neuroretinopathy, a maternally inherited disease. Arch Ophthalmol 1987; 105 :665-671. 4. Newman NJ, Lott MT, Wallace DC. The clinical characteristics of pedigrees of Leber’s hereditary optic neuropathy with the 11778 mutation. Am J Ophthalmol 1991 ; 11 1 :750-762. 5 . Anderson S, Bankier AT, Barrel BG et al. Sequence and organization of the human mitochondrial genome. Nature 1981; 290:457-465. 6. Chomyn A, Mariottini P , Cleeter MWJ et al. Six unidentified reading frames of human mitochondrial DNA encode components of the respiratory-chain NADH dehydrogenase subunit. Nature 1985 ; 314: 592-597. 7. Chomyn A, Cleeter MWJ, Ragan CI, Riley M, Doolittle RF, Attardi G. URF6, last unidentified reading frame of human mt DNA, codes for an NADH dehydrogenase subunit. Science 1986; 234:614-618. 8. Giles RE, Blanc H , Cann HM, Wallace DC. Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 1980; 77:6715-6719. 9. Holt IJ, Harding AE, Morgan-Hughes JA. Deletion of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331 :717-719. 10. Lestienne P , Ponsot G. Kearns-Sayre syndrome with muscle mitochondrial DNA deletion. Cancet 1988; i : 885. 1 1 . Saifuddin Noer A, Marzuki S, Trounce I , Byrne E. Mitochondrial DNA deletions in encephalomyopathy. Lancet 1988; ii : 1253-1254. 12. Moraes CT, Di Mauro S, Zeviani M et al. Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N Engl J Med 1989; 320: 1293-1299. 13. Zeviani M, Servidei S, Gellera C , Bertini E, Di Mauro S, Di Donato S. An autosomal dominant disorder with multiple deletions of mitochondrial DNA starting at the D-loop region. Nature 1989; 339:309-311. 14. Poulton J, Deadman ME, Gardiner RM. Duplication of mitochondrial DNA in mitochondrial myopathy. Lancet 1989; i :236-239. 15. Holt IJ, Harding AE. Petty RKH, Morgan-Hughes JA. A new mitochondrial disease associated with mitochondrial DNA heteroplasmy. Am J Hum Genet 1990; 46:428-433. 16. Shoffner JM, Lott MT, Lezza AMS, Seibel P , Ballinger SW, Wallace DC. Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNAlys mutation. Cell 1990; 61 :931-937. 17. Tanaka M, [no H, Ohno K et al. Mitochondrial mutation in fatal infantile cardiomyopathy. Lancet 1990; ii: 1452. 18. Tanaka M, Ino H, Ohno K et al. Mitochondrial DNA mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS). Biochem Biophys Res Comm 1991 ; 174:861-868. 19. Zeviani M, Gellera C , Antozzi C et al. Maternally inherited myopathy and cardiomyopathy: association with mutation in mitochondrial DNA tRNALEu(uuR). Lancet 1991 ; 338: 143-147. 20. Wallace DC, Singh G , Loti MT et al. Mitochondrial DNA mutation associated with Leber’s hereditary optic neuropathy. Science 1988; 242: 1427-1430. 21. Hotta Y, Hayakawa M, Saito K , Kanai A, Nakajima A, Fujiki K. Diagnosis of Leber’s optic neuropathy by means of Polymerase Chain Reaction Amplification. Am J Ophthalmol 1989; 108:601-602. 22. Vilkki J, Savontaus ML, Nikoskelainen EK. Genetic heterogeneity in Leber’s hereditary optic neuroretinopathy revealed by mitochondrial DNA polymorphism. Am J Hum Genet 1989; 45 :206-211. 23. Yoneda M, Tsuji S, Yamauchi T et al. Mitochondrial DNA mutation in a family with Leber’s hereditary optic neuropathy. Lancet 1989; i : 1076-1077. 24. Stone EM, Coppinger JM, Kardon RH, Donelson J. Mae 111 positively detects the mitochondrial mutation associated with type I Leber’s hereditary optic neuropathy. Arch Ophthalmol 1990; 108: 1417-1420. 25. Jacobson DM, Stone EM. Difficulty in differentiating Leber’s from dominant optic neuropathy in patient with remote visual loss. J Clin Neuro-ophthalmol 1991 ; 11 : 152-157. 26. Holt IJ, Miller DH, Harding AE. Genetic heterogeneity and mitochondrial DNA heteroplasmy in Leber’s hereditary optic neuropathy. J Med Genet 1989; 26:739-743. 27. Bolhuis PA, Bleeker-Wagemakers EM, Ponne NJ et al. Rapid shift in genotype of human mitochondrial DNA in a
225
0 Aeolus Press Ophthalmic Paediatrics and Genetics 0167-6784/92/US$ 3.50
(Accepted 20 August 1992)
Mitochondrial DNA analysis in Leber's hereditary optic neuropathy
Ophthalmic Genet Downloaded from informahealthcare.com by Nyu Medical Center on 05/22/15 For personal use only.
PIERO BARBONI', VILMA MANTOVAN12,PASQUALE MONTAGNA3, MICHELA BRAGLIAN12, PIETRO CORTELL13, E L I 0 LUGARES13, PIERO PUDDU' and ROBERTO CARAMAZZA'*
Institute of Ophthalmology, Bologna University; 2Department of Pathology, Malpighi Hospital, Bologna; 31nstitute of Neurology, Bologna University; Italy ABSTRACT. A mitochondrial DNA (mtDNA) mutation at nucleotide 11778 has been reported as the genetic defect associated with Leber's hereditary optic neuropathy (LHON), but some pedigrees failed to reveal this mutation. The authors present the genetic analysis of a large Italian LHON family with three probands and 16 asymptomatic maternal relatives. The 11778 mtDNA mutation was present in this family and absent in 52 Italian healthy controls, confirming the association between this genetic defect and LHON. In addition, they found a variable proportion of mutated and wild-type mtDNA (heteroplasmy) in every maternally related family member, including the probands. Different patterns of heteroplasmy were present in the pedigree and lower levels of wild-type mtDNA seemed to correlate with the disease status. Moreover, the authors evaluated the mitotic segregation of mt genomes in blood, hair and urinary tract epithelia of the three patients and they found a similar level of heteroplasmy in these tissues. Key words: Leber's hereditary optic neuropathy ; mitochondrial DNA mutation
INTRODUCTION Leber's hereditary optic neuropathy (LHON) is a hereditary disease characterized by bilateral asynchronous optic nerve atrophy, cardiac dysrhythmias and occasionally dystonia in young adults with male predominance. Peripapillary microangiopathy is a typical feature of presymptomatically affected and asymptomatic family mernber~l-~. The inheritance of the disease does not follow Mendelian principles and the exclusive transmission through the maternal lineage has suggested a mitochondrial disorder. ~
*
Correspondence lo: Prof. Roberto Caramazza, Institute of Ophthalmology, Policlinico S. Orsola-Malpighi, via Massarenti 9, 40100 Bologna, Italy.
Mitochondria are cytoplasmic organelles essential to cellular bioenergy containing several copies of their own DNA. Mitochondrial DNA (mtDNA) is a circular double-strand molecule of 16569 base pair (bp) coding for two rRNAs, 22 tRNAs and 13 of the 61 protein subunits of mitochondrial respiratory c o m p l e x e ~ ~ -The ~. mtDNA is transmitted through the maternal lineage and the mitochondria of the zygote are provided only by the ovum*. Recent clinical and genetic studies of some neuro-ophthalmic diseases have revealed various defects in mtDNA such as single and multiple deletions9-13, tandem d~plications'~and point A point mutation at nucleotide 11778 of
Ophthalmic Paediatrics and Genetics 1992. Voi. 13, No. 4, pp. 219-226 0Aeolus Press Buren (The Netherlands) 1992 ~
219
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P. Barboni et al. mtDNA as the genetic defect of LHON was first reported by Wallace et The mutation causes a transition from guanine to adenine converting a highly conserved arginine to histidine in NADH dehydrogenase subunit4. This replacement mutation was found in about 50% of LHON pedigrees and only the mutated mtDNA (homoplasmy) was detected in both patient and maternal relative^^'-^^. Holt et a1.26have found a variable proportion of mutated and wild-type mtDNA in some families. This condition is termed heteroplasmy and is a typical feature of mitochondria1 diseases. Until now the heteroplasmic condition in LHON has been described in only a small number of pedigree^^'-^^. We have reported the presence of the 11778 mtDNA mutation in three probands of an Italian LHON family; the mutated mt genomes appeared to be h o r n o p l a ~ m i c ~In~ .the present work we report the genetic analysis of the whole pedigree. We find the presence of heteroplasmy for the mutation in all maternally related family members including the probands, by using more sensitive methods to detect wild-type mtDNA. Moreover, we evaluate the segregation of the two mtDNA types in different tissues. SUBJECTS AND METHODS
The family. The pedigree included three male probands, 12 female and four male asymptomatic maternal relatives as well as two children
MT2 MT3
S-CTCACAGTCGCATCATAATC-3' 5-CTCACAGTCACATCATAATC-3
TABLE 1 . Oligonucleotide primers and probes
220
of a male patient. The diagnosis of LHON was based on the typical fundus change during the acute stage of the illness, with subsequent optic atrophy and on the maternal inheritance of the disease; other causes of optic atrophy were not evident. The controls. To test the absence of the 11778 mtDNA mutation in the Italian population, we included 52 unrelated healthy controls in the study. PCR amplification and SfaNI restriction analysis. Since the 11778 mtDNA mutation removes an SfaNI restriction site, we performed an endonuclease digestion with this enzyme, after polymerase chain reaction (PCR) amplification of rntDNA3,. Total DNA was extracted from leukocytes and epithelial cells of the urinary tract with phenol and chloroform-isoamylalcohol, precipitated with ethanol and solubilized in 10 mM Tris-HCVl mM EDTA pH 8.033. DNA preparation from hairs was performed by adding 0.15 mg proteinase K, 50 pl SDS 20070, 0.05 mg RNAse, 40 pM DTT for 70 h, before the conventional DNA extraction. The oligonucleotide primers MT1 and MT4 (Table 1) were synthesized in order to amplify a 415 bp region of mtDNA, including the SfaNI site. Twentyfive cycles of PCR amplification were completed with 1 pg of DNA in 100 pI volume reaction containing 50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgCI,, 100 pg/ml gelatin, 200 pM each dATP, dCTP, dGTP and dTTP, 1 pM of
1176411788 11769-11788
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Mitochondria1DNA analysis in Leber 's hereditary optic neuropathy each primer and 2.5 units Taq-polymerase (Perkin Elmer-Cetus, Norwalk, CO). Each cycle consisted of 94°C denaturation for 1.30 min, 55°C annealing for 2.30 rnin and 72°C extension for 4 min. The presence of the 11778 mtDNA mutation was detected by overnight digestion of the total amplified DNA (about 2 pg) with 10 units of SfaNI restriction enzyme (New England Biolabs, Beverly, MA, USA). The samples were then electrophoresed on 3% Nusieve plus 1% agarose gel 1 pg/ml ethidium bromide incl. and photographed under ultraviolet light. Southern blotting and hybridization. After staining, the gel was denatured in 0.4 N NaOH/ 0.6 M NaCl for 30 rnin at room temperature and neutralized in 1.5 M NaCVO.5 M Tris-HCl pH 7.5 for 30 rnin at room temperature. The DNA was then transferred overnight to a nylon membrane by capillary action in transfer buffer (3 M NaCV0.3 M sodium citrate, pH 7.0)34. The oligonucleotide probes MTI and MT4, 5'endlabeled with 32P-dATP,were used for hybridization. Filters were prehybridized for 1 h at 37°C in 5 x Denhardt's (0.1 Yo Ficoll/O. 1Yo polyvinylpyrrolidone/O. 1Vo bovine serum albumin),
5 x S S P E (750 mM NaCl/SO mM NaH,PO,/5 mM EDTA), 0.5% SDS and 200 mg/ml of denatured herring sperm, and then hybridized for 1 hr at 50°C in a solution consisting of the prehybridization solution plus the radiolabelled probes (2 x lo6 cpm/ml for each probe). After hybridization the filters were washed in 0.1 x SSPE/O. 1To SDS for 10 rnin at room temperature and for 10 rnin at 38°C. The filters were then exposed to Kodak XAR-5 at -80°C with intensifying screen for 2 hr and/or overnight. Dot blotting and hybridization. Approximately 100 ng (5 pl of the PCR reaction) of amplified mtDNA were denatured in 45 p1 of 0.5 N NaOH/1.5 M NaC1/25 mM EDTA for 5 rnin at room temperature and 3 rnin at 90"C, and then neutralized in 150 pl of 0.5 M Tris-HC1 pH 8.0/1.5 M NaCl. The samples were spotted onto duplicate nylon membrane by using a dotblot apparatus (BioDot, BioRad, Richmond, CA, USA) and DNAs were bound to the membranes by ultraviolet cross-linking. The first filter was hybridized with the radiolabeled oligonucleotide probe MT2, complementary to the
tV
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41 5
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166
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Fig. I . LHON pedigree: filled symbols indicate affected individuals. The lower panel shows the related pattern obtained by SfaNI digestion and gel-electrophoresis of the total amplified mtDNA (about 2 p g ) , visualized by ethidium bromide staining. Fragment of 415 base pair indicates the mutated mtDNA. Fragments of 249and 166 base pair indicate the wild-type mtDNA. 25-50% wild-type mtDNA, M, size marker; C , healthy control; " < 5 % wild-type mtDNA, 5-25% wild-type mtDNA, 0000 100% wild-type mtDNA.
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P. Barboni et al. wild-type mtDNA and the second filter with the probe MT3, complementary to the mutated mtDNA (Table 1). The conditions of prehybridization, hybridization and washing were the same as used for Southern blotting, but the stringent wash was carried out at 40°C.
The heteroplasmic condition was revealed by the presence of all three fragments. An initial SfaNI digestion of 10 pl (about 200 ng) of amplified mtDNA and the following photograph of gel electrophoresis revealed heteroplasmy in only four maternal relatives (1-1, 1-2, 1-3, 11-1). The relative amount of wild-type mtDNA could be estimated at about 2550%. The three probands and the other maternal family members seemed to be homoplasmic for the mutation. When the SfaNI digestion was performed on the total amplified mtDNA (about 2 pg), the gel photograph revealed the presence of mild heteroplasmy also in six other maternal relatives (11-2, 111-1, 111-2, 1-4, 11-6, 111-9) (5-25070 wildtype mtDNA) (Fig. 1). In order to improve the sensitivity of the analysis and to detect even trace amounts of wild-type mtDNA we performed a Southern transfer and hybridization with radiolabeled oligo-probes. We also performed a dot-blot analysis and hybridization with wild-type and mutation specific oligo-
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RESULTS The presence of the 11778 mtDNA mutation removes an SfaNI restriction site. The endonuclease SfaNI was not able to digest the 415 bp amplified mtDNA when the mutation was present; but the SfaNI digestion resulted in two fragments of 249 and 166 bp when the mutation was absent. The three LHON patients and all maternally related family members possessed the 11778 mtDNA mutation. The individuals from the paternal lineage, including the offspring of a male patient, retained the SfaNI site (Fig. 1). All the 52 unrelated healthy controls showed the SfaNI site.
2
C
1-3 11-3 111-3 11-4 111-4 111-5 111-6 IV-1 11-5 111-10 C
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C
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1-3 11-3 111-3 11-4 111-4 111-5 111-6 IV-1 TI 5 111-10 C
I2
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11-5 11-5 I114 111-4 111-3 111-3 U
H
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11-5 11-5 111-4 111-4 I11 3 I11 3 U
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ti
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Fig. 2. Dot-blot analysis of amplified mtDNA of some LHON family rneinbers (see Fig. 1) and controls ( C ) . Filter A was hybridized with wild-type mtDNA probe MT2. Filter B was hybridized with the mutation specific mtDNA probe MT3. U, urinary tract epithelia; H, hair.
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Mitochondria1 DNA analysis in Leber's hereditary optic neuropathy probes. Using these methods we found heteroplasrny for the 11778 mtDNA mutation in every maternal member of the LHON family, including the three probands ( < 5 % wild-type mtDNA) (Fig. 2). Analysis of hair and epithelial cells of the urinary tract obtained from the three patients was performed to evaluate if there was a different segregation of the two mtDNA types in these tissues. A similar level of heteroplasmy was detected in both tissues of probands with the hybridization methods, although the photograph of the gel electrophoresis did not disclose wild-type mtDNA (Figs. 2 and 3). DISCUSSION The point mutation in nucleotide 11778 of the mtDNA NADH dehydrogenase subunit 4 gene has been reported as the most frequent genetic defect associated with LHON20-25.31. Nevertheless, some LHON pedigrees lack this mutation, suggesting a genetic heterogeneity in the dis111-3 U
ease20-22.Other point mutations of mtDNA were recently proposed as alternative genetic defects associated with the d i ~ e a s e ~ ~ - ~ ~ . The LHON family investigated in this study showed the 11778 mutation. In addition, the absence of this mutation in 52 Italian healthy controls studied, makes it unlikely that the mutation is an ethnic-specific polymorphism. Our results confirm the association between this genetic defect and LHON in Italian patients. The heteroplasmic condition, an otherwise typical feature of mitochondria1 diseases, seems to be unusual in LHON27-30. The mtDNA analysis of this Italian pedigree showed the presence of heteroplasmy in all maternally related family members, including the three probands. At first, the SfaNI digestion of amplified mtDNA revealed heteroplasmy only in three siblings and a nephew of the probands' maternal grandmother. These individuals belonged to a pedigree branch without optic atrophy. When the SfaNI digestion was performed on a larger amount of amplified DNA
11- 5 H
u
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bp 415249.-
166-
41%
249-
3 Fig. 3. Analysis of urinary tract epithelia and hair mtDNA. The upper panel shows the SfaNI digestion of amplified mtDNA of two probands (wild-type mtDNA < 5 % ) ; two maternal relatives were included as heteroplasmic controls (wild-type mtDNA 25-50'70). The lower panel shows the subsequent Southern blot and hybridization with MTI/MT4 probes. U , urinary tract epithelia; H, hair; B, blood; bp, base pair.
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P. Barboni et al. we were able to disclose heteroplasmy in six further family members. Only the three probands and some of their close relatives seemed to be homoplasmic (Fig. 1). A very small amount of wild-type mtDNA was revealed also in the latter individuals by using the more sensitive Southern and dot-blot hybridization methods (Fig. 2 ) . This suggests that the sensitivity of the method employed is crucial for the detection of heteroplasmy in those LHON families in which few residual wild-type mt genomes are present. In addition, in our pedigree the disease status seems to correlate with lower levels of wild-type mtDNA. For example, the probands’ grandmother (I-4) was less heteroplasmic than her siblings (1-1, 1-2, 1-3) (Fig. 1); the three patients (111-3, 111-4, 11-5) showed the lowest level of wild-type mtDNA, detectable only by hybridization methods (Fig. 2). A similar low level of heteroplasmy (
