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Variable Genotype of Leber's Hereditary Optic Neuropathy Patients
AMERICAN
OF
OPHTHALMOLOGY®
NUMBER 6
JUNE, 1990
JOURNAL
VOLUME 109
Variable Genotype of Leber's Hereditary Optic Neuropathy Patients Marie T. Lott, M.A., Alexander S. Voljavec, M.D., and Douglas C. Wallace, Ph.D. is a disease of young adults characterized by bilateral loss of central vision. In families with multiple affected individuals, all are related through the matemal lineage.P which suggests that Leber's hereditary optic neuropathy is caused by a mitochondrial DNA mutation.v' We confirmed this conclusion by identifying a single guanine to adenine transition mutation in the mitochondrial DNA (mtDNA) at nucleotide position 11778, which causes this disease. This mutation converts the 340th amino acid in the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 (ND4) gene from an arginine to a histidine. It also eliminates a recognition site for the restriction endonuclease SfaNI, thus providing a simple and rapid molecular diagnostic test for this disease."
Leber's hereditary optic neuropathy is caused by a single nucleotide change in the mitochondrial deoxyribonucleic acid (mtDNA). Each cell contains thousands of mitochondrial DNA molecules. We demonstrated that in certain isolated instances, the proband and close maternal lineage relatives can have mixtures of mutant and normal mitochondrial DNA molecules (heteroplasmy). The proportion of mutant mitochondrial DNA molecules was found to shift markedly across generations and within the tissues of an individual. One unaffected mother had 65% mutant mitochondrial DNA molecules whereas her affected son had essentially 100% mutant mitochondrial DNA molecules. Two affected individuals had predominantly mutant mitochondrial DNA in their blood, but significant normal mitochondrial DNA in their hair. The demonstration of heteroplasmy within maternal lineages and affected individuals means that the successful determination of the mitochondrial DNA genotype of a family or patient with Leber's hereditary optic neuropathy requires testing of more than one family member and more than one tissue from each individual.
LEBER'S HEREDITARY OPTIC NEUROPATHY
See also p. 726.
Analysis of the mitochondrial DNA molecules of Leber's hereditary optic neuropathy patients of different ethnic groups has indicated that this mutation has occurred many times independently.v" Because each human cell has thousands of mitochondrial DNA molecules.t the original mutant molecule must increase from one mutant among thousands of normal molecules to a substantial percentage of mutant molecules before a neuropathy would ensue. Thus, a patient who has visual loss must be preceded by ancestors with heteroplasmy (mixed mutant and normal molecules)." Because the normal molecules in the patients' cells would reduce the impact of the mutant genotype, detection and quantitation of heteroplasmy is of great importance in the diagnosis and counseling of these patients. JO We identified patients and their maternal relatives who were heteroplasmic for the Leber's
Accepted for publication March 23, 1990. From the Departments of Biochemistry (Ms. Lott and Dr. Wallace), Nephrology (Dr. Voljavec), Pediatrics (Dr. Wallace), and Neurology (Dr. Wallace), Emory University School of Medicine, Atlanta, Georgia. This study was supported by National Institutes of Health grant NS21328, a Muscular Dystrophy Clinical Research grant, and an Emory Neuromuscular Center grant (Dr. Wallace). Reprint requests to Douglas C. Wallace, M.D., Emory University School of Medicine, Department of Biochemistry, 3031 Rollins Research Ctr., Atlanta, GA 30329.
©AMERICAN JOURNAL OF OPHTHALMOLOGY
109:625-631,
JUNE,
1990
625
626
AMERICAN JOURNAL OF OPHTHALMOLOGY
hereditary optic neuropathy mutation. The ratio of mutant and normal mitochondrial DNA molecules can differ markedly both in the maternal lineages of pedigrees and between somatic tissues. The replicative segregation of heteroplasmic mitochondrial DNA molecules may play an important role in clinical variability of this unique form of optic neuropathy.
Material
and Methods
Whole blood, platelet or lymphocyte fractions, lymphoblastoid cell lines, and hairs (0.5 to 1 em of root end from four to five hairs) were studied in patients with Leber's hereditary optic neuropathy and their maternal relatives. Blood was collected in either sodium heparin or acid citrate dextrose after obtaining informed consent from the patients. Whole blood (200 ILl) was removed for immediate DNA extraction; the remainder of the sample was fractionated, and the lymphocytes were transformed with Epstein-Barr virus" to establish permanent cell lines. Blood samples were spotted onto filter paper and allowed to air dry. Hair samples included the root and shaft and were stored at room temperature. DNA was extracted from whole blood (200 ILl), small platelet or lymphocyte fractions (cells from 1 rnl), blood spots (1 em in diameter), and hair (0.5 to 1 em of the root end of four to five hairs). Lysis buffer [25 mM Tris(hydroxymethyl)-aminomethane (Tris), pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate, and 10 ILg proteinase K] was added to the samples to a final volume of 400 ILl before overnight incubation at 55 C. Samples were then heated to 95 C for ten minutes, cooled to room temperature, and incubated with 200 ILg of ribonuclease A for 15 minutes. Sodium dodecyl sulfate and proteins were precipitated with 2.55 M potassium acetate, and the supernatant was loaded onto an anion-exchange affinity column (Quiagen, Inc., Studio City, California). Contaminating proteins, heme, heparin, and RNA were eluted by following the manufacturer's recommendations, and the recovered DNA was used for amplification with the polymerase chain reaction." From most preparations, less than 1% of the sample was used as a template for a 100-ILI polymerase chain reaction. For suboptimal samples such as hair or dried blood spots, however, 20% to 50% of the preparation was used.
June, 1990
Two different DNA fragments were prepared, which encompassed the nucleotide position 11778 Leber's marker: a 1435 base pair fragment (representing nucleotide positions 11141 to 12576) and a 212 base pair fragment (representing nucleotide positions 11673 to 11873). Forward (-) and reverse (-) oligonucleotide primers (Microchemical Facility of Molecular Biology, Emory University, Atlanta, Georgia) for the larger fragment were located at nucleotide positions 11141 to 11158(-) and nucleotide positions 12576 to 12557(-). Primers for the smaller fragment were located at nucleotide positions 11673 to 11691(-) and nucleotide positions 11873 to 11851(-). For suboptimal samples (hemolyzed blood samples, dried blood, or hair), the smaller fragment was more easily generated. Thermal profiles consisted of 35 cycles of denaturing for one minute at 94 C, annealing for 30 seconds at 53 C for the 1435 base pair primers, or 59 C for the 212 base pair primers, and extension for one minute at 72 C. Enriched mitochondrial DNA was obtained from Epstein-Barr virus transformed cell lines of a family with Leber's hereditary optic neuropathy (EUH297, EUH296, EUH321, EUH322, EUH323), a control Leber's patient (CDW0092/ GM10742), and a normal control subject (CDW0099). Approximately 3 x 10 7 cells were resuspended in 3 ml of isotonic buffer [2.5 mM EDTA, 20 mM N-[2-Hydroxyethyl] piperazineN' -[2-ethanesulfonic acid] (HEPES), 137 mM NaCl, 5 mM KCl, 0.7 mM dibasic sodium phosphate, 6 mM dextrose, pH 7.1], an equal volume of lysis buffer (0.01 M HE PES, 0.01 M EDTA, 1% sodium dodecyl sulfate, pH 7.0) was added and the mixture allowed to sit 20 minutes at room temperature to lyse the cells. Sodium chloride was added to a final concentration of 1 M, and the solution was chilled at 4 C overnight and clarified." The RNA was removed by addition of 200 ILg of ribonuclease A. Proteins were removed by further lysis with 0.5% sodium dodecyl sulfate folIowed by digestion at 55 C with 10 ILg/ml of proteinase K. Cellular debris and sodium dodecyl sulfate were precipitated by the addition of one half volume 2.55 M potassium acetate; the samples were brought to less than 0.85 M NaCl with 50 mM 3-(6-methoxyquinolino) propane sulfonate (MDPS), pH 7.0, and the DNA purified with an anionexchange affinity column. For genomic Southern blots, 4 ILg of enriched mitochondrial DNA from the lymphoblast celI lines was digested with 1 unit of the restriction endonuclease SfaN1 (New England Biolabs. Beverly, Massachusetts) at 37 C for eight to ten
Vol. 109, No.6
hours. The digested DNA was fractionated on 1.4% Tris-borate agarose gels and transferred onto nylon membrane (Magnagraph, MSI, Westboro, Massachusetts). To test for the Sfa N1 site in the 212 base pair DNA fragment, 200 to 300 ng of amplified ND4 DNA (10% to 20% of a polymerase chain reaction) was digested with 1 unit of the enzyme, and the fragments were separated on an agarose gel containing ethidium bromide and detected by ultraviolet fluorescence. To check for accidental cross-contamination of DNA, a sample of H 20 without DNA was carried through the entire extraction, amplification, and analysis process. Southern hybridization probes for the ND4 gene were prepared by random primer extension"-" of the 1435 base pair ND4 polymerase chain reaction fragment with digoxigenen-uridine-5'triphosphate, gel purified using Gene Clean II (Bio 101, Inc., La Jolla, California) and the hapten-labeled DNA detected with the Genius labeling and detection system (Boehringer Mannheim, Indianapolis, Indiana). The 1435 base pair fragment was directly sequenced using asymmetrically amplified mitochondrial DNA as templates.":" Agarose gels with ethidium bromide-stained DNA were photographed with Polaroid 55 PIN film. Quantitative densitometry was performed on the negatives with an UltroScan XL Laser Densitometer equipped with Gel-Scan XL software package, version 2.0 (Pharmacia LKB Biotechnology, Piscataway, New Jersey). Densi-
1317
bp-
79+638bp·
627
Variable Genotype and Leber's Optic Neuropathy
tometry was performed on positive film images of the hybridized filters.
Results To detect and quantitate mutant and wild type mitochondrial DNA molecules in family members with Leber's hereditary optic neuropathy, a 1435 base pair polymerase chain reaction fragment was prepared with the polymorphic SfaNI site in the center. SfaNI digestion of this fragment bearing the Leber's hereditary optic neuropathy mutation generates a 1317 base pair fragment (nucleotide positions 11141 to 12456) and a 119 base pair fragment (nucleotide positions 12456 to 12576). This latter fragment was allowed to migrate off the gel. Digestion of the fragment from wild type mitochondrial DNA molecules cuts the 1317 base pair fragment into 638 base pair (nucleotide positions 11141 to 11778) and 679 base pair (nucleotide positions 11778 to 12456) fragments. Because the 638 and 679 base pair fragments from wild type mitochondrial DNA initially superimpose on the gel, the DNA fluorescence in the 638 I 660 base pair and 1317 base pair bands are directly proportional to the number of wild type and mutant molecules. The blood cell mitochondrial DNA molecules of most isolated Leber's hereditary optic neuropathy cases gave a single band at nucleotide
·LHON
-Wl
Fig. 1 (Lott, Voljavec, and WaIlace). Heteroplasmy in patients with Leber's hereditary optic neuropathy and unaffected maternal lineage relatives. Mutant (Leber's hereditary optic neuropathy) and normal (wild type) mitochondrial DNA molecules detected by polymerase chain reaction amplification of a 1435 base pair ND4 fragment, digestion with SfaNI, and analysis by electrophoresis visualized by ethidium bromide staining. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp. base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained fuIl vision (unaffected carriers). [-] indicates lane with H20 sample only, a contamination control.
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AMERICAN JOURNAL OF OPHTHALMOLOGY
position 1317 and hence were essentially homoplasmic for the mutation (Fig. 1, lanes 1 and 12 from left). Similarly, random normal controls gave a band at about 638/660 base pair consistent with homoplasmic wild type (Fig. 1, lane 2). By contrast, occasional isolated cases gave both 1317 and 638/660 base pair bands indicating heteroplasmy (Fig. 1, lane 8). Densitometric analysis indicated that blood cells from this female patient with Leber's hereditary optic neuropathy contained 71 % mutant and 29% wild type mitochondrial DNA molecules. To determine if homoplasmic, isolated Leber's probands might have heteroplasmic relatives, we analyzed the blood cell mitochondrial DNA molecules of the three siblings and mother of one patient (Fig. 1, lanes 3 to 7). The mother (Fig. 1, lane 3) proved strongly heteroplasmic with 65% mutant and 35% wild type mitochondrial DNA molecules. One of the sisters (Fig. 1, lane 7) was comparably heteroplasmic (64% mutant, 36% wild type). The other sister (Fig. 1, lane 5) had about 7% normal mitochondrial DNA molecules and the other brother (Fig. 1, lane 6) had essentially pure mutant mitochondrial DNA molecules. Hence, in this family, three of the four children have segregated to essentially homoplasmic Leber's mutant mitochondrial DNA in one generation and one daughter retained the heteroplasmy of the mother. In a second family, the proband was observed to be mildly heteroplasmic with only about 25% wild type mitochondrial DNA molecules (Fig. 1, lane 11). Analysis of his unaffected mother disclosed 59 % mutant and 41 % wild type mitochondrial DNA molecules (Fig. 1, lane 10), whereas his grandmother had 42% mutant and 58% wild type mitochondrial DNA molecules (Fig. 1, lane 9). Hence, in this family, the Leber's hereditary optic neuropathy mutation progressively increased in frequency over three generations. Because the polymerase chain reaction amplifies even trace amounts of contaminating DNA to easily detectable levels, we confirmed the presence of heteroplasmy in genomic, nonamplified DNA by Southern blot analysis of lymphoblastoid cell lines from the two-generation heteroplasmic family (Fig. 1, lanes 3 to 7). Cellular DNA preparations enriched for mitochondrial DNA were digested with SfaNI, and the fragments were separated by agarose gel and detected by Southern blotting and hybridization with an ND4 probe (Fig. 2). The diagram
Q 1594
bp-
915
bp-
679
bp-
366
bp-
nCO
•••••
LHON
} WT
----1594 915
T 679
366
LHON
366
WT
.-"------'--.;;..;'--'
nt 1,nS Fig. 2 (Lott, Voljavec, and Wallace). Heteroplasmy detected by digestion of genomic DNA with SfaNI and detection of ND4 fragments by Southern blot hybridization. Upper panel, LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision (unaffected carriers). Lower panel, Restriction map of fragments detected by the ND4 hybridization probe. Vertical lines indicate SfaNI restriction sites; *, site of Leber's hereditary optic neuropathy mu tation; numbers in boxes, lengths of restriction fragments in nucleotide pairs.
at the bottom of Figure 2 illustrates the expected fragments: 1594 base pair for Leber's hereditary optic neuropathy mitochondrial DNA molecules, 915 base pair and 679 base pair for wild type mitochondrial DNA molecules with a common fragment of 366 base pair. All family member mitochondrial DNA preparations yielded predominantly the 1594 base pair Leber's hereditary optic neuropathy fragment, but the mother's mitochondrial DNA also produced a substantial proportion of the 915 base pair and 679 base pair wild type fragments, as did the youngest daughter's, but to a lesser extent. Hence, the heteroplasmy detected by SfaNI digestion of polymerase chain reaction amplified mitochondrial DNA is present in the original patient mitochondrial DNA. Differences in percentage between Figures 1 and 2 probably reflect replicative segregation during preparation of the lymphoblastoid cell lines. To confirm that the heteroplasmy detected by SfaNI digestion results from a mixture of mito-
Vol. 109, No.6
Variable Genotype and Leber's Optic Neuropathy
chondrial DNA molecules having an adenine or a guanine at nucleotide position 11778, we directly sequenced polymerase chain reaction amplified mitochondrial DNA from the mother (Fig. 1, lane 10) of the three-generation pedigree. The direct sequence disclosed both an adenine (Leber's hereditary optic neuropathy) and a guanine (wild type) at position 11778 (Fig. 3). Hence, the heteroplasmy detected by SfaNI digestion reflects heteroplasmy at the mitochondrial DNA nucleotide sequence level. Comparable results have been obtained for the mother (Fig. 1, lane 3) and daughter (Fig. 1, lane 7) of the two-generation pedigree. To determine if heteroplasmic mitochondrial DNA populations segregate during mitosis as well as meiosis, the proportion of Leber's hereditary optic neuropathy and wild type mitochondrial DNA molecules were compared in the blood (mesoderm) and hair (ectoderm) of several heteroplasmic individuals. One male proband had predominantly Leber's hereditary optic neuropathy mitochondrial DNA in his blood with a small amount of wild type mitochondrial DNA detectable only by the more sensitive Southern blot (Fig. 4, lane 4, lower panel). When hair follicles of this individual were examined, however, they were found to contain a high percentage of wild type mitochondrial DNA molecules with a smaller percentage of mutants (Fig. 4, lane 3, lower panel). For comparison, two female carriers homoplasmic for the Leber's hereditary optic neuropathy mutation are shown (Fig. 4, lanes 1 and 2). A second proband (Fig. 5, lanes 1 and 2) also had a predominance of mutant mitochondrial DNA in the blood and wild type in the hair. However, a third independent proband (Fig. 5, lanes 3 and 4) had predominantly mutant mitochondrial DNA molecules in both the blood and hair,
with wild type mitochondrial DNA being detectable only by the more sensitive Southern blotting. Differences between blood and hair were also observed in the three-generation pedigree of Figure 1 (Fig. 5, lanes 5 to 10). The grandmother and mother had comparable proportions of Leber's hereditary optic neuropathy and wild type mitochondrial DNA molecules in the two tissues (Fig. 5, lanes 5 and 8, 6 and 9). The proband (lanes 7 and 10), however, had substantially more wild type mitochondrial DNA in the hair than in the blood. Hence these results show that segregation of heteroplasmic mitochondrial DNA molecules occurs during the mitotic replication of development as well as during the meiotic replication of sexual reproduction.
Discussion
The discovery of heteroplasmy of the Leber's hereditary optic neuropathy mutation in our pedigrees confirms the recent observation of Holt, Miller, and Harding." Further, our demonstration of the accompanying rapid meiotic and mitotic segregation provides one explanation for the variability in expression of the mutation between members of the same family. Because the presence of wild type mitochondrial DNA molecules would reduce the extent of the respiratory complex I deficiency of Leber's hereditary optic neuropathy patients, individuals whose optic nerve cells were heteroplasmic would be less likely to lose their vision. Because meiotic segregation can result in major differences in the proportion of mutant mitochoI1drial DNA molecules between sib-
ACGT
A=
LHON
• •...........-
--
(
629
G=WT
Fig. 3 (Lott, Voljavec, and Wallace). Direct sequencing of polymerase chain reaction amplified mitochondrial DNA from a heteroplasmic individual. DNA was prepared from the blood of the individual in lane 10 of Figure 1. The A C G T (adenine, cytosine, guanine, and thymine) indicate the bases detected in each lane of the sequencing ladder read vertically from bottom (5') to top (3'). The heteroplasmic base is indicated. LHON indicates Leber's hereditary optic neuropathy; WT, wild type.
630
June, 1990
AMERICAN JOURNAL OF OPHTHALMOLOGY
HAIR
• •\ \
BLOOD
B
212 bp-
117 bp95 bp-
lHON 212 bp-
WT
212 bp-
lHON
117 bp95 bp-
WT
Fig. 4 (Lott, Voljavec, and Wallace). Heteroplasmy detected in the blood and hair from a patient. Upper panel, Heteroplasmy detected by polymerase chain reaction amplification of the 212 base pair ND4 fragment, digestion with SfaNI, and analysis by electrophoresis and ethidium bromide staining. Lower panel, Analysis of fragments from the upper panel by Southern blot and hybridization with the ND4 probe. Lanes 1 and 2 are blood cell DNA molecules from carriers of an independent lineage who have predominantly mutant mitochondrial DNA molecules. Lanes 3 and 4 are hair and blood from the proband. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision.
lings of the same family, some individuals will be genetically predisposed to visual loss and others will not. Further, because mitotic segregation can result in radical differences in the mitochondrial DNA genotype between tissues of the same individual, genotypic analysis of the mitochondrial DNA molecules of a single tissue such as blood is not necessarily indicative of the mitochondrial DNA genotype of the optic nerve and a reliable predictor of neuropathy. To increase the reliability of a genetic determination of Leber's hereditary optic neuropathy, individuals in the maternal lineage must be tested, and cells from several tissues (blood, hair, buccal mucosa, urinary tract epithelia) must be analyzed. The rapid meiotic segregation rate of the heteroplasmic family members with Leber's hereditary optic neuropathy is comparable to that
117 bp95 bp.
-
H
-_. -B
H
BLOOD
HAIR
.
lHON WT
Fig. 5 (Lott, Voljavec, and Wallace). Heteroplasmy detected in the blood and hair from two independent probands and the members of the three-generation family. Heteroplasmy assessed as in the upper panel of Figure 4. The three generation pedigree is the same as that in Figure 1. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision; B, blood; H, hair.
which has been described for mitochondrial DNA restriction fragment length polymerphisms along bovine maternal lineages.P'" This suggests that heteroplasmy may not be maintained for many generations. Additionally, large Leber's hereditary optic neuropathy pedigrees with many affected individuals would tend toward a homoplasmic mutant," thus accounting for the repeated transmission of the trait along the maternal lineage. By contrast, some isolated cases may represent the first clinical manifestation of a new mutation and, being closer to the origin of the Leber's hereditary optic neuropathy mitochondrial DNA mutation, more likely to be heteroplasmic. The rapid somatic segregation of the Leber's hereditary optic neuropathy mitochondrial DNA is surprising. Somatic cells have thousands of mitochondrial DNA molecules, and the equal division of the cell at cytokinesis should mean that each daughter cell would have a mitochondrial DNA genotype similar to that of the parental cell. Intracellular genetic drift of mitochondrial DNA proportion was thus expected to be slow and the mitochondrial DNA genotype between tissues similar. These data on heteroplasmic Leber's hereditary optic neuropathy patient blood and hair, however, do not support this view, because the mitochondrial DNA genotypes were found to differ drastically. This suggests that some additional factor may contribute to the extent of
Vol. 109, No.6
Variable Genotype and Leber's Optic Neuropathy
mitotic segregation in these patients. One possibility is that the cells of the tissues examined are mitotically active and therefore have had a greater opportunity to undergo mitotic segregation. Alternatively, the number of segregating mitochondrial DNA units is much smaller than the number of mitochondrial DNA molecules. Because segregating units might represent physically associated groups of mitochondrial DNA molecules (as in a mitochondrion) the smaller number of mitochondria to be distributed to daughter cells would increase the sampling error at cytokinesis and thus the effective rate of mitochondrial DNA segregation. It is clear that we must know more about the genetics of the mitochondrial DNA and Leber's hereditary optic neuropathy before we will be able to use molecular genotypes to make accurate predictions about clinical phenotypes.
References 1. Nikoskelainen, E. K.: New aspects of the genetic, etiologic, and clinical puzzle of Leber's disease. Neurology 34:1482, 1984. 2. Erickson, R. P.: Leber's optic atrophy, a possible example of maternal inheritance. Am. J. Hum. Genet. 24:348, 1972. 3. Giles, R. E., Blanc, H., Cann, H. M., and Wallace, D. c.: Maternal inheritance of human mitochondrial DNA. Proc. Natl. Acad. Sci. U.S.A. 77:6715, 1980. 4. Case, J. T., and Wallace, D. c.: Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts. Somatic Cell Genet. 7:103, 1981. 5. Wallace, D. c.. Singh, G., Lott, M. T., Hodge, J. A., Schurr, T. G., Lezza, A. M. S., Elsas, L. J., III, and Nikoskelainen, E.: Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242: 1427, 1988. 6. Singh, G., Lott, M. T., and Wallace, D. c.. A mitochondrial DNA mutation as a cause of Leber's hereditary optic neuropathy. N. Engl. J. Med. 320:1300,1989. 7. Yoneda, M., Tsuji, S., Yamauchi, T., Inuzuka, T., and Miyatake, T.: Mitochondrial DNA mutation in a family with Leber's hereditary optic neuropathy. Lancet 1:1076, 1989. 8. Shuster, R. C; Rubenstein, A. F., and Wallace, D. c.: Mitochondrial DNA in anucleate human blood
631
cells. Biochem. Biophys. Res. Commun. 155:1360, 1988. 9. Wallace, D. c.: Mitotic segregation of mitochondrial DNAs in human cell hybrids and expression of chloramphenicol resistance. Somatic Cell Mol. Genet. 12:41, 1986. 10. Newman, N. J., and Wallace, D. c. Mitochondria and Leber's hereditary optic neuropathy. Am. J. Ophthalmol. 109:xxx, 1990. 11. Wallace, D. c.. Yang, J., Ye, J., Lott. M. T., Oliver, N. A., and McCarthy, J.: Computer prediction of peptide maps. Assignment of polypeptides to human and mouse mitochondrial DNA genes by analysis of two-dimensional-proteolytic digest gels. Am. J. Hum. Genet. 38:461, 1986. 12. Saiki, R. K., Scharf, S., Faloona. T., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N.: Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350, 1985. 13. Feinberg, A. P., and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6, 1983. 14. Feinberg, A. P., and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137:266, 1984. 15. Shoffner, J. M., Lott, M. T., Voljavec, A. S., Soueidan, S. A., Costigan, D. A., and Wallace, D. c. Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion. A slip-replication model and metabolic therapy. Proc. Natl. Acad. Sci. U.S.A. 86:7952, 1989. 16. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A.: DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. U.S.A. 85:9436, 1988. 17. Holt, I. J., Miller, D. H., and Harding, A. E.: Genetic heterogeneity and mitochondrial DNA heteroplasmy in Leber's hereditary optic neuropathy. J. Med. Genet. 26:739, 1989. 18. Nauswirth, W. W., and Laipis, P. J.: Achievements and Perspectives of Mitochondrial Research, vol. II. Biogenesis. Amsterdam, Elsevier Science, 1985, pp. 49-59. 19. Laipis, P. J., Van de Walle, M. J., and Hauswirth, W. W.: Unequal partitioning of bovine mitochondrial genotypes among siblings. Proc. Natl. Acad. Sci. U.S.A. 85:8107, 1985. 20. Hauswirth, W. W., and Laipis, P. J.: Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc. Natl. Acad. Sci. U.S.A. 79:4686, 1982.
OF
OPHTHALMOLOGY®
NUMBER 6
JUNE, 1990
JOURNAL
VOLUME 109
Variable Genotype of Leber's Hereditary Optic Neuropathy Patients Marie T. Lott, M.A., Alexander S. Voljavec, M.D., and Douglas C. Wallace, Ph.D. is a disease of young adults characterized by bilateral loss of central vision. In families with multiple affected individuals, all are related through the matemal lineage.P which suggests that Leber's hereditary optic neuropathy is caused by a mitochondrial DNA mutation.v' We confirmed this conclusion by identifying a single guanine to adenine transition mutation in the mitochondrial DNA (mtDNA) at nucleotide position 11778, which causes this disease. This mutation converts the 340th amino acid in the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4 (ND4) gene from an arginine to a histidine. It also eliminates a recognition site for the restriction endonuclease SfaNI, thus providing a simple and rapid molecular diagnostic test for this disease."
Leber's hereditary optic neuropathy is caused by a single nucleotide change in the mitochondrial deoxyribonucleic acid (mtDNA). Each cell contains thousands of mitochondrial DNA molecules. We demonstrated that in certain isolated instances, the proband and close maternal lineage relatives can have mixtures of mutant and normal mitochondrial DNA molecules (heteroplasmy). The proportion of mutant mitochondrial DNA molecules was found to shift markedly across generations and within the tissues of an individual. One unaffected mother had 65% mutant mitochondrial DNA molecules whereas her affected son had essentially 100% mutant mitochondrial DNA molecules. Two affected individuals had predominantly mutant mitochondrial DNA in their blood, but significant normal mitochondrial DNA in their hair. The demonstration of heteroplasmy within maternal lineages and affected individuals means that the successful determination of the mitochondrial DNA genotype of a family or patient with Leber's hereditary optic neuropathy requires testing of more than one family member and more than one tissue from each individual.
LEBER'S HEREDITARY OPTIC NEUROPATHY
See also p. 726.
Analysis of the mitochondrial DNA molecules of Leber's hereditary optic neuropathy patients of different ethnic groups has indicated that this mutation has occurred many times independently.v" Because each human cell has thousands of mitochondrial DNA molecules.t the original mutant molecule must increase from one mutant among thousands of normal molecules to a substantial percentage of mutant molecules before a neuropathy would ensue. Thus, a patient who has visual loss must be preceded by ancestors with heteroplasmy (mixed mutant and normal molecules)." Because the normal molecules in the patients' cells would reduce the impact of the mutant genotype, detection and quantitation of heteroplasmy is of great importance in the diagnosis and counseling of these patients. JO We identified patients and their maternal relatives who were heteroplasmic for the Leber's
Accepted for publication March 23, 1990. From the Departments of Biochemistry (Ms. Lott and Dr. Wallace), Nephrology (Dr. Voljavec), Pediatrics (Dr. Wallace), and Neurology (Dr. Wallace), Emory University School of Medicine, Atlanta, Georgia. This study was supported by National Institutes of Health grant NS21328, a Muscular Dystrophy Clinical Research grant, and an Emory Neuromuscular Center grant (Dr. Wallace). Reprint requests to Douglas C. Wallace, M.D., Emory University School of Medicine, Department of Biochemistry, 3031 Rollins Research Ctr., Atlanta, GA 30329.
©AMERICAN JOURNAL OF OPHTHALMOLOGY
109:625-631,
JUNE,
1990
625
626
AMERICAN JOURNAL OF OPHTHALMOLOGY
hereditary optic neuropathy mutation. The ratio of mutant and normal mitochondrial DNA molecules can differ markedly both in the maternal lineages of pedigrees and between somatic tissues. The replicative segregation of heteroplasmic mitochondrial DNA molecules may play an important role in clinical variability of this unique form of optic neuropathy.
Material
and Methods
Whole blood, platelet or lymphocyte fractions, lymphoblastoid cell lines, and hairs (0.5 to 1 em of root end from four to five hairs) were studied in patients with Leber's hereditary optic neuropathy and their maternal relatives. Blood was collected in either sodium heparin or acid citrate dextrose after obtaining informed consent from the patients. Whole blood (200 ILl) was removed for immediate DNA extraction; the remainder of the sample was fractionated, and the lymphocytes were transformed with Epstein-Barr virus" to establish permanent cell lines. Blood samples were spotted onto filter paper and allowed to air dry. Hair samples included the root and shaft and were stored at room temperature. DNA was extracted from whole blood (200 ILl), small platelet or lymphocyte fractions (cells from 1 rnl), blood spots (1 em in diameter), and hair (0.5 to 1 em of the root end of four to five hairs). Lysis buffer [25 mM Tris(hydroxymethyl)-aminomethane (Tris), pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulfate, and 10 ILg proteinase K] was added to the samples to a final volume of 400 ILl before overnight incubation at 55 C. Samples were then heated to 95 C for ten minutes, cooled to room temperature, and incubated with 200 ILg of ribonuclease A for 15 minutes. Sodium dodecyl sulfate and proteins were precipitated with 2.55 M potassium acetate, and the supernatant was loaded onto an anion-exchange affinity column (Quiagen, Inc., Studio City, California). Contaminating proteins, heme, heparin, and RNA were eluted by following the manufacturer's recommendations, and the recovered DNA was used for amplification with the polymerase chain reaction." From most preparations, less than 1% of the sample was used as a template for a 100-ILI polymerase chain reaction. For suboptimal samples such as hair or dried blood spots, however, 20% to 50% of the preparation was used.
June, 1990
Two different DNA fragments were prepared, which encompassed the nucleotide position 11778 Leber's marker: a 1435 base pair fragment (representing nucleotide positions 11141 to 12576) and a 212 base pair fragment (representing nucleotide positions 11673 to 11873). Forward (-) and reverse (-) oligonucleotide primers (Microchemical Facility of Molecular Biology, Emory University, Atlanta, Georgia) for the larger fragment were located at nucleotide positions 11141 to 11158(-) and nucleotide positions 12576 to 12557(-). Primers for the smaller fragment were located at nucleotide positions 11673 to 11691(-) and nucleotide positions 11873 to 11851(-). For suboptimal samples (hemolyzed blood samples, dried blood, or hair), the smaller fragment was more easily generated. Thermal profiles consisted of 35 cycles of denaturing for one minute at 94 C, annealing for 30 seconds at 53 C for the 1435 base pair primers, or 59 C for the 212 base pair primers, and extension for one minute at 72 C. Enriched mitochondrial DNA was obtained from Epstein-Barr virus transformed cell lines of a family with Leber's hereditary optic neuropathy (EUH297, EUH296, EUH321, EUH322, EUH323), a control Leber's patient (CDW0092/ GM10742), and a normal control subject (CDW0099). Approximately 3 x 10 7 cells were resuspended in 3 ml of isotonic buffer [2.5 mM EDTA, 20 mM N-[2-Hydroxyethyl] piperazineN' -[2-ethanesulfonic acid] (HEPES), 137 mM NaCl, 5 mM KCl, 0.7 mM dibasic sodium phosphate, 6 mM dextrose, pH 7.1], an equal volume of lysis buffer (0.01 M HE PES, 0.01 M EDTA, 1% sodium dodecyl sulfate, pH 7.0) was added and the mixture allowed to sit 20 minutes at room temperature to lyse the cells. Sodium chloride was added to a final concentration of 1 M, and the solution was chilled at 4 C overnight and clarified." The RNA was removed by addition of 200 ILg of ribonuclease A. Proteins were removed by further lysis with 0.5% sodium dodecyl sulfate folIowed by digestion at 55 C with 10 ILg/ml of proteinase K. Cellular debris and sodium dodecyl sulfate were precipitated by the addition of one half volume 2.55 M potassium acetate; the samples were brought to less than 0.85 M NaCl with 50 mM 3-(6-methoxyquinolino) propane sulfonate (MDPS), pH 7.0, and the DNA purified with an anionexchange affinity column. For genomic Southern blots, 4 ILg of enriched mitochondrial DNA from the lymphoblast celI lines was digested with 1 unit of the restriction endonuclease SfaN1 (New England Biolabs. Beverly, Massachusetts) at 37 C for eight to ten
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hours. The digested DNA was fractionated on 1.4% Tris-borate agarose gels and transferred onto nylon membrane (Magnagraph, MSI, Westboro, Massachusetts). To test for the Sfa N1 site in the 212 base pair DNA fragment, 200 to 300 ng of amplified ND4 DNA (10% to 20% of a polymerase chain reaction) was digested with 1 unit of the enzyme, and the fragments were separated on an agarose gel containing ethidium bromide and detected by ultraviolet fluorescence. To check for accidental cross-contamination of DNA, a sample of H 20 without DNA was carried through the entire extraction, amplification, and analysis process. Southern hybridization probes for the ND4 gene were prepared by random primer extension"-" of the 1435 base pair ND4 polymerase chain reaction fragment with digoxigenen-uridine-5'triphosphate, gel purified using Gene Clean II (Bio 101, Inc., La Jolla, California) and the hapten-labeled DNA detected with the Genius labeling and detection system (Boehringer Mannheim, Indianapolis, Indiana). The 1435 base pair fragment was directly sequenced using asymmetrically amplified mitochondrial DNA as templates.":" Agarose gels with ethidium bromide-stained DNA were photographed with Polaroid 55 PIN film. Quantitative densitometry was performed on the negatives with an UltroScan XL Laser Densitometer equipped with Gel-Scan XL software package, version 2.0 (Pharmacia LKB Biotechnology, Piscataway, New Jersey). Densi-
1317
bp-
79+638bp·
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Variable Genotype and Leber's Optic Neuropathy
tometry was performed on positive film images of the hybridized filters.
Results To detect and quantitate mutant and wild type mitochondrial DNA molecules in family members with Leber's hereditary optic neuropathy, a 1435 base pair polymerase chain reaction fragment was prepared with the polymorphic SfaNI site in the center. SfaNI digestion of this fragment bearing the Leber's hereditary optic neuropathy mutation generates a 1317 base pair fragment (nucleotide positions 11141 to 12456) and a 119 base pair fragment (nucleotide positions 12456 to 12576). This latter fragment was allowed to migrate off the gel. Digestion of the fragment from wild type mitochondrial DNA molecules cuts the 1317 base pair fragment into 638 base pair (nucleotide positions 11141 to 11778) and 679 base pair (nucleotide positions 11778 to 12456) fragments. Because the 638 and 679 base pair fragments from wild type mitochondrial DNA initially superimpose on the gel, the DNA fluorescence in the 638 I 660 base pair and 1317 base pair bands are directly proportional to the number of wild type and mutant molecules. The blood cell mitochondrial DNA molecules of most isolated Leber's hereditary optic neuropathy cases gave a single band at nucleotide
·LHON
-Wl
Fig. 1 (Lott, Voljavec, and WaIlace). Heteroplasmy in patients with Leber's hereditary optic neuropathy and unaffected maternal lineage relatives. Mutant (Leber's hereditary optic neuropathy) and normal (wild type) mitochondrial DNA molecules detected by polymerase chain reaction amplification of a 1435 base pair ND4 fragment, digestion with SfaNI, and analysis by electrophoresis visualized by ethidium bromide staining. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp. base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained fuIl vision (unaffected carriers). [-] indicates lane with H20 sample only, a contamination control.
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position 1317 and hence were essentially homoplasmic for the mutation (Fig. 1, lanes 1 and 12 from left). Similarly, random normal controls gave a band at about 638/660 base pair consistent with homoplasmic wild type (Fig. 1, lane 2). By contrast, occasional isolated cases gave both 1317 and 638/660 base pair bands indicating heteroplasmy (Fig. 1, lane 8). Densitometric analysis indicated that blood cells from this female patient with Leber's hereditary optic neuropathy contained 71 % mutant and 29% wild type mitochondrial DNA molecules. To determine if homoplasmic, isolated Leber's probands might have heteroplasmic relatives, we analyzed the blood cell mitochondrial DNA molecules of the three siblings and mother of one patient (Fig. 1, lanes 3 to 7). The mother (Fig. 1, lane 3) proved strongly heteroplasmic with 65% mutant and 35% wild type mitochondrial DNA molecules. One of the sisters (Fig. 1, lane 7) was comparably heteroplasmic (64% mutant, 36% wild type). The other sister (Fig. 1, lane 5) had about 7% normal mitochondrial DNA molecules and the other brother (Fig. 1, lane 6) had essentially pure mutant mitochondrial DNA molecules. Hence, in this family, three of the four children have segregated to essentially homoplasmic Leber's mutant mitochondrial DNA in one generation and one daughter retained the heteroplasmy of the mother. In a second family, the proband was observed to be mildly heteroplasmic with only about 25% wild type mitochondrial DNA molecules (Fig. 1, lane 11). Analysis of his unaffected mother disclosed 59 % mutant and 41 % wild type mitochondrial DNA molecules (Fig. 1, lane 10), whereas his grandmother had 42% mutant and 58% wild type mitochondrial DNA molecules (Fig. 1, lane 9). Hence, in this family, the Leber's hereditary optic neuropathy mutation progressively increased in frequency over three generations. Because the polymerase chain reaction amplifies even trace amounts of contaminating DNA to easily detectable levels, we confirmed the presence of heteroplasmy in genomic, nonamplified DNA by Southern blot analysis of lymphoblastoid cell lines from the two-generation heteroplasmic family (Fig. 1, lanes 3 to 7). Cellular DNA preparations enriched for mitochondrial DNA were digested with SfaNI, and the fragments were separated by agarose gel and detected by Southern blotting and hybridization with an ND4 probe (Fig. 2). The diagram
Q 1594
bp-
915
bp-
679
bp-
366
bp-
nCO
•••••
LHON
} WT
----1594 915
T 679
366
LHON
366
WT
.-"------'--.;;..;'--'
nt 1,nS Fig. 2 (Lott, Voljavec, and Wallace). Heteroplasmy detected by digestion of genomic DNA with SfaNI and detection of ND4 fragments by Southern blot hybridization. Upper panel, LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision (unaffected carriers). Lower panel, Restriction map of fragments detected by the ND4 hybridization probe. Vertical lines indicate SfaNI restriction sites; *, site of Leber's hereditary optic neuropathy mu tation; numbers in boxes, lengths of restriction fragments in nucleotide pairs.
at the bottom of Figure 2 illustrates the expected fragments: 1594 base pair for Leber's hereditary optic neuropathy mitochondrial DNA molecules, 915 base pair and 679 base pair for wild type mitochondrial DNA molecules with a common fragment of 366 base pair. All family member mitochondrial DNA preparations yielded predominantly the 1594 base pair Leber's hereditary optic neuropathy fragment, but the mother's mitochondrial DNA also produced a substantial proportion of the 915 base pair and 679 base pair wild type fragments, as did the youngest daughter's, but to a lesser extent. Hence, the heteroplasmy detected by SfaNI digestion of polymerase chain reaction amplified mitochondrial DNA is present in the original patient mitochondrial DNA. Differences in percentage between Figures 1 and 2 probably reflect replicative segregation during preparation of the lymphoblastoid cell lines. To confirm that the heteroplasmy detected by SfaNI digestion results from a mixture of mito-
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Variable Genotype and Leber's Optic Neuropathy
chondrial DNA molecules having an adenine or a guanine at nucleotide position 11778, we directly sequenced polymerase chain reaction amplified mitochondrial DNA from the mother (Fig. 1, lane 10) of the three-generation pedigree. The direct sequence disclosed both an adenine (Leber's hereditary optic neuropathy) and a guanine (wild type) at position 11778 (Fig. 3). Hence, the heteroplasmy detected by SfaNI digestion reflects heteroplasmy at the mitochondrial DNA nucleotide sequence level. Comparable results have been obtained for the mother (Fig. 1, lane 3) and daughter (Fig. 1, lane 7) of the two-generation pedigree. To determine if heteroplasmic mitochondrial DNA populations segregate during mitosis as well as meiosis, the proportion of Leber's hereditary optic neuropathy and wild type mitochondrial DNA molecules were compared in the blood (mesoderm) and hair (ectoderm) of several heteroplasmic individuals. One male proband had predominantly Leber's hereditary optic neuropathy mitochondrial DNA in his blood with a small amount of wild type mitochondrial DNA detectable only by the more sensitive Southern blot (Fig. 4, lane 4, lower panel). When hair follicles of this individual were examined, however, they were found to contain a high percentage of wild type mitochondrial DNA molecules with a smaller percentage of mutants (Fig. 4, lane 3, lower panel). For comparison, two female carriers homoplasmic for the Leber's hereditary optic neuropathy mutation are shown (Fig. 4, lanes 1 and 2). A second proband (Fig. 5, lanes 1 and 2) also had a predominance of mutant mitochondrial DNA in the blood and wild type in the hair. However, a third independent proband (Fig. 5, lanes 3 and 4) had predominantly mutant mitochondrial DNA molecules in both the blood and hair,
with wild type mitochondrial DNA being detectable only by the more sensitive Southern blotting. Differences between blood and hair were also observed in the three-generation pedigree of Figure 1 (Fig. 5, lanes 5 to 10). The grandmother and mother had comparable proportions of Leber's hereditary optic neuropathy and wild type mitochondrial DNA molecules in the two tissues (Fig. 5, lanes 5 and 8, 6 and 9). The proband (lanes 7 and 10), however, had substantially more wild type mitochondrial DNA in the hair than in the blood. Hence these results show that segregation of heteroplasmic mitochondrial DNA molecules occurs during the mitotic replication of development as well as during the meiotic replication of sexual reproduction.
Discussion
The discovery of heteroplasmy of the Leber's hereditary optic neuropathy mutation in our pedigrees confirms the recent observation of Holt, Miller, and Harding." Further, our demonstration of the accompanying rapid meiotic and mitotic segregation provides one explanation for the variability in expression of the mutation between members of the same family. Because the presence of wild type mitochondrial DNA molecules would reduce the extent of the respiratory complex I deficiency of Leber's hereditary optic neuropathy patients, individuals whose optic nerve cells were heteroplasmic would be less likely to lose their vision. Because meiotic segregation can result in major differences in the proportion of mutant mitochoI1drial DNA molecules between sib-
ACGT
A=
LHON
• •...........-
--
(
629
G=WT
Fig. 3 (Lott, Voljavec, and Wallace). Direct sequencing of polymerase chain reaction amplified mitochondrial DNA from a heteroplasmic individual. DNA was prepared from the blood of the individual in lane 10 of Figure 1. The A C G T (adenine, cytosine, guanine, and thymine) indicate the bases detected in each lane of the sequencing ladder read vertically from bottom (5') to top (3'). The heteroplasmic base is indicated. LHON indicates Leber's hereditary optic neuropathy; WT, wild type.
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HAIR
• •\ \
BLOOD
B
212 bp-
117 bp95 bp-
lHON 212 bp-
WT
212 bp-
lHON
117 bp95 bp-
WT
Fig. 4 (Lott, Voljavec, and Wallace). Heteroplasmy detected in the blood and hair from a patient. Upper panel, Heteroplasmy detected by polymerase chain reaction amplification of the 212 base pair ND4 fragment, digestion with SfaNI, and analysis by electrophoresis and ethidium bromide staining. Lower panel, Analysis of fragments from the upper panel by Southern blot and hybridization with the ND4 probe. Lanes 1 and 2 are blood cell DNA molecules from carriers of an independent lineage who have predominantly mutant mitochondrial DNA molecules. Lanes 3 and 4 are hair and blood from the proband. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision.
lings of the same family, some individuals will be genetically predisposed to visual loss and others will not. Further, because mitotic segregation can result in radical differences in the mitochondrial DNA genotype between tissues of the same individual, genotypic analysis of the mitochondrial DNA molecules of a single tissue such as blood is not necessarily indicative of the mitochondrial DNA genotype of the optic nerve and a reliable predictor of neuropathy. To increase the reliability of a genetic determination of Leber's hereditary optic neuropathy, individuals in the maternal lineage must be tested, and cells from several tissues (blood, hair, buccal mucosa, urinary tract epithelia) must be analyzed. The rapid meiotic segregation rate of the heteroplasmic family members with Leber's hereditary optic neuropathy is comparable to that
117 bp95 bp.
-
H
-_. -B
H
BLOOD
HAIR
.
lHON WT
Fig. 5 (Lott, Voljavec, and Wallace). Heteroplasmy detected in the blood and hair from two independent probands and the members of the three-generation family. Heteroplasmy assessed as in the upper panel of Figure 4. The three generation pedigree is the same as that in Figure 1. LHON indicates Leber's hereditary optic neuropathy; WT, wild type; bp, base pairs; solid symbols, blind subjects; stippled symbols, maternal lineage relatives who retained full vision; B, blood; H, hair.
which has been described for mitochondrial DNA restriction fragment length polymerphisms along bovine maternal lineages.P'" This suggests that heteroplasmy may not be maintained for many generations. Additionally, large Leber's hereditary optic neuropathy pedigrees with many affected individuals would tend toward a homoplasmic mutant," thus accounting for the repeated transmission of the trait along the maternal lineage. By contrast, some isolated cases may represent the first clinical manifestation of a new mutation and, being closer to the origin of the Leber's hereditary optic neuropathy mitochondrial DNA mutation, more likely to be heteroplasmic. The rapid somatic segregation of the Leber's hereditary optic neuropathy mitochondrial DNA is surprising. Somatic cells have thousands of mitochondrial DNA molecules, and the equal division of the cell at cytokinesis should mean that each daughter cell would have a mitochondrial DNA genotype similar to that of the parental cell. Intracellular genetic drift of mitochondrial DNA proportion was thus expected to be slow and the mitochondrial DNA genotype between tissues similar. These data on heteroplasmic Leber's hereditary optic neuropathy patient blood and hair, however, do not support this view, because the mitochondrial DNA genotypes were found to differ drastically. This suggests that some additional factor may contribute to the extent of
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Variable Genotype and Leber's Optic Neuropathy
mitotic segregation in these patients. One possibility is that the cells of the tissues examined are mitotically active and therefore have had a greater opportunity to undergo mitotic segregation. Alternatively, the number of segregating mitochondrial DNA units is much smaller than the number of mitochondrial DNA molecules. Because segregating units might represent physically associated groups of mitochondrial DNA molecules (as in a mitochondrion) the smaller number of mitochondria to be distributed to daughter cells would increase the sampling error at cytokinesis and thus the effective rate of mitochondrial DNA segregation. It is clear that we must know more about the genetics of the mitochondrial DNA and Leber's hereditary optic neuropathy before we will be able to use molecular genotypes to make accurate predictions about clinical phenotypes.
References 1. Nikoskelainen, E. K.: New aspects of the genetic, etiologic, and clinical puzzle of Leber's disease. Neurology 34:1482, 1984. 2. Erickson, R. P.: Leber's optic atrophy, a possible example of maternal inheritance. Am. J. Hum. Genet. 24:348, 1972. 3. Giles, R. E., Blanc, H., Cann, H. M., and Wallace, D. c.: Maternal inheritance of human mitochondrial DNA. Proc. Natl. Acad. Sci. U.S.A. 77:6715, 1980. 4. Case, J. T., and Wallace, D. c.: Maternal inheritance of mitochondrial DNA polymorphisms in cultured human fibroblasts. Somatic Cell Genet. 7:103, 1981. 5. Wallace, D. c.. Singh, G., Lott, M. T., Hodge, J. A., Schurr, T. G., Lezza, A. M. S., Elsas, L. J., III, and Nikoskelainen, E.: Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242: 1427, 1988. 6. Singh, G., Lott, M. T., and Wallace, D. c.. A mitochondrial DNA mutation as a cause of Leber's hereditary optic neuropathy. N. Engl. J. Med. 320:1300,1989. 7. Yoneda, M., Tsuji, S., Yamauchi, T., Inuzuka, T., and Miyatake, T.: Mitochondrial DNA mutation in a family with Leber's hereditary optic neuropathy. Lancet 1:1076, 1989. 8. Shuster, R. C; Rubenstein, A. F., and Wallace, D. c.: Mitochondrial DNA in anucleate human blood
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cells. Biochem. Biophys. Res. Commun. 155:1360, 1988. 9. Wallace, D. c.: Mitotic segregation of mitochondrial DNAs in human cell hybrids and expression of chloramphenicol resistance. Somatic Cell Mol. Genet. 12:41, 1986. 10. Newman, N. J., and Wallace, D. c. Mitochondria and Leber's hereditary optic neuropathy. Am. J. Ophthalmol. 109:xxx, 1990. 11. Wallace, D. c.. Yang, J., Ye, J., Lott. M. T., Oliver, N. A., and McCarthy, J.: Computer prediction of peptide maps. Assignment of polypeptides to human and mouse mitochondrial DNA genes by analysis of two-dimensional-proteolytic digest gels. Am. J. Hum. Genet. 38:461, 1986. 12. Saiki, R. K., Scharf, S., Faloona. T., Mullis, K. B., Horn, G. T., Erlich, H. A., and Arnheim, N.: Enzymatic amplification of 13-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350, 1985. 13. Feinberg, A. P., and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6, 1983. 14. Feinberg, A. P., and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Addendum. Anal. Biochem. 137:266, 1984. 15. Shoffner, J. M., Lott, M. T., Voljavec, A. S., Soueidan, S. A., Costigan, D. A., and Wallace, D. c. Spontaneous Kearns-Sayre/chronic external ophthalmoplegia plus syndrome associated with a mitochondrial DNA deletion. A slip-replication model and metabolic therapy. Proc. Natl. Acad. Sci. U.S.A. 86:7952, 1989. 16. Innis, M. A., Myambo, K. B., Gelfand, D. H., and Brow, M. A.: DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. U.S.A. 85:9436, 1988. 17. Holt, I. J., Miller, D. H., and Harding, A. E.: Genetic heterogeneity and mitochondrial DNA heteroplasmy in Leber's hereditary optic neuropathy. J. Med. Genet. 26:739, 1989. 18. Nauswirth, W. W., and Laipis, P. J.: Achievements and Perspectives of Mitochondrial Research, vol. II. Biogenesis. Amsterdam, Elsevier Science, 1985, pp. 49-59. 19. Laipis, P. J., Van de Walle, M. J., and Hauswirth, W. W.: Unequal partitioning of bovine mitochondrial genotypes among siblings. Proc. Natl. Acad. Sci. U.S.A. 85:8107, 1985. 20. Hauswirth, W. W., and Laipis, P. J.: Mitochondrial DNA polymorphism in a maternal lineage of Holstein cows. Proc. Natl. Acad. Sci. U.S.A. 79:4686, 1982.