Am. J. Hum. Genet. 47:629-634, 1990
An Example of Leber Hereditary Optic Neuropathy Not Involving a Mutation in the Mitochondrial ND4 Gene Neil Howell and David McCullough Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston
Summary A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADHubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages-even those associated with a biochemical defect in mitochondrial respiratory chain Complex I-carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.
Introduction
Leber hereditary optic neuropathy (LHON) is characterized by a loss of central vision - usually permanent because of bilateral retinal degeneration (Nikoskelainen 1984). The most striking genetic feature of LHON is its strict maternal inheritance: no male within a LHON family has ever been shown to transmit the disease (van Senus 1963; Seedorf 1970; Nikoskelainen 1984; Nikoskelainen et al. 1987). The demonstration that the mitochondrial DNA (mtDNA) is maternally inherited in humans (Giles et al. 1980), as in other mammals, indicated that a mutation within the mitochondrial genome could be the proximate cause of LHON. Strong experimental support for this possibility was first obtained by Wallace et al. (1988; also see Singh et al. 1988), who showed that the mtDNA from nine of 11 LHON maternal lineages carried a G-to-A transition Received April 9, 1990; revision received June 13, 1990. Address for correspondence and reprints: Neil Howell, Ph.D., Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston, TX 77550. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4704-0007$02.00
at nucleotide position 11,778, a change causing loss of an SfaN1 restriction site. This mutation results in the replacement of a highly conserved arginine residue by histidine at position 340 of the ND4 protein. The ND4 gene product is one of the seven mitochondrially encoded subunits of complex I (NADH-ubiquinol oxidoreductase) of the electron-transport chain. More recent reports confirm the occurrence of this mutation in LHON families from different parts of the world. A Japanese LHON family has been shown to carry this ND4 mutation (Yoneda et al. 1989), and recent surveys of European LHON families have revealed that about half carry the sequence change at nucleotide position 11,778 (Holt et al. 1989; Vilkki et al. 1989). These studies were particularly important because they demonstrated that more than one specific mutation can cause LHON. However, since they were limited to screening for the SfaN1 restriction-site alteration, it could not be determined whether a mutation had occurred within the ND4 gene but at a different site, within one of the other mitochondrial complex I genes, or within one of the mitochondrial genes encoding a protein constituent of the other respiratory chain complexes. 629
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As a complement to these molecular genetic studies, Parker et al. (1989) have shown that members of the large Australian LHON pedigree studied by Wallace (1970) have statistically significant decreases, averaging about 50%, in the specific activity of mitochondrial complex I activity in platelets. There were also reductions in the specific activities of mitochondrial succinate-cytochrome c oxidoreductase (complexes II and III) and cytochrome oxidase (complex IV), but they were not statistically significant and may have reflected secondary effects of the complex I deficiency. As this is the only LHON pedigree for which a biochemical defect has been identified, it is important for the study of this mitochondrial disease to determine whether the members of the Australian pedigree carry a mutation at nucleotide 11,778 of the ND4 gene and, if not, whether a mutation elsewhere within the gene can be identified. Answering these questions is the subject of the present investigation. Material and Methods Preparation of DNA Samples Whole venous blood (approximately 50 ml/patient) was collected with informed consent by Dr. Davis Parker (Departments of Pediatrics and Neurology, Univer-
sity of Colorado Health Sciences Center). The crude white cell/platelet fractions were prepared, suspended in 50% glycerol, and then shipped on dry ice to this laboratory, where they were then stored at -800C. Whole cellular DNA was isolated by using the standard procedure involving lysis with SDS, digestion with proteinase K, multiple extractions with phenol followed by chloroform:isoamyl alcohol (24:1), and, finally, ethanol precipitation. This crude DNA was then resuspended in 1 ml of TE buffer (10 mM Tris-HCI, pH 8.0; 1.0 mM EDTA) and was stored at 40C. DNA samples were prepared from the following maternally related family members (Wallace 1970): IV2s, Vg, V17, and V18 (affected males); IV28 (affected female); and V1o, V16, VI1s, and V116 (unaffected females). The last two family members are clinically normal at present but just entering the age period when the optic atrophy is usually expressed. As an internal control, DNA was prepared from V4, the son of an affected male. External controls included DNA samples from clinically normal individuals and from patients with non-LHON neurological or mitochondrial diseases.
NDS
L
12.213
0.03 9
27
17
13
1 n
1s
5 24
M
Figure I Cloning and sequencing strategy for mitochondrial ND3 and ND4L/ND4 genes. The complex I genes are depicted by the large bars, while the tRNA genes are indicated by the black circles. The nucleotide coordinates of the genes are as follows: tRNAglu, 9,991-10,058; ND3 (cross-hatched bar), 10,059-10,404; tRNAarg, 10,405-10,469; ND4L (cross-hatched bar), 10,470-10,766; ND4 (solid bar), 10,760-12,317; and tRNAhis, 12,318-12,206. The arrows indicate the overlapping clones used for DNA sequence analysis, with the number of independent clones from the LHON subjects shown below or above the arrow. The arrows pointing to the right and left indicate whether the 5' or 3' end of the fragment, respectively, was inserted adjacent to the M13 origin. The double-headed arrows indicate fragments which were sufficiently small that the complete sequence of the entire fragment could be unambiguously determined; as a result, clones of the two insertion orientations have been pooled. Since the 389-bp ND4-3 fragment inserted into M13 in only one orientation, an "internal" sequencing primer was used to derive an unambiguous sequence of the entire mitochondrial insert.
PCR Amplification and DNA Cloning/Sequencing
The 2.23-kb region of mtDNA spanning the ND3, ND4L, and ND4 genes was amplified as a series of overlapping fragments (fig. 1), by using the polymerase chain reaction (PCR) technique (Saiki et al. 1988) and the synthetic oligonucleotide primers listed in table 1. To facilitate M13 cloning, the terminal regions of all amplified fragments contain Sau3A restriction sites; these sites either occur within the mtDNA sequence or are engineered into the amplification primers. All nucleotide coordinates are taken from the standard Cambridge sequence of human mtDNA (Anderson et al. 1981). Since there is a naturally occurring Sau3A site at nucleotides 11,923-11,926, there is no sequence overlap between the ND4-4 and ND4-5 fragments (table 1). To ensure that there were no mutations occurring within the ND4 gene region complementary to the ND4-4 3' and ND4-5 5' primers, additional PCR amplifications were carried out using the ND4-4 5' and ND4-5 3' primers to generate the "double" fragment. This latter was then digested with Sau3A, and the resulting subfragments were cloned into M13 vectors for DNA sequencing. With this approach, there was no indication of a mutation either within the Sau3A site or in the bracketing regions. PCR amplification was carried out with Taq DNA polymerase (Cetus/Perkin-Elmer Corp.) by using the
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Table I Oligonucleotide Primers for PCR Amplification
Gene Fragment ND-3: 5' .......... 3' .......... ND4-1: 5' .......... 3' .......... ND4-2: 5' ......... 3' .......... ND4-3: 5' ......... 30 .......... ND4-4: 5' .......... 3' .......... ND4-5: 5' .......... 3' ..........
Primer Sequencea
Nucleotide Positions
CTC CAT CTA TTG ATC AGG GTC CAT TTG GTA AAT ATG ATC ATC
9,968-9,988 10,449-10,472
ACG AAT GAT CTC GAC TCA TTA AGG AAA AGG TTG GGG ATC AGC
10,425-10,445 10,911-10,931
TAT TAG GAT CAT CCC TCT ACT CAG GGG GTT TGG ATC AGA ATG
10,858-10,878
TAT TAG GAT CAT CCC TCT ACT ATA GAT CTT GGG CAG TGA GAG
10,858-10,878 11,298-11,318
GCA TAC TCT TCG ATC ACG CAC GTG ATA TTT GAT CAG GAG AAC
11,618-11,638 11,914-11,934
GTT CTC CTG ATC AAA TAT CAC AGC AGT TCT TGT GAT CTT TCT
11,914-11,934
11,659-11,679
12,208-12,228 a Sequences of both primers for each gene fragment are given in the 5'- to 3' direction; the designations 5' and 3' for the amplification primers are used to define from which end of the mtDNA fragment amplification is proceeding.
conditions recommended by the manufacturer. Total cellular DNA (100-700 ng) was added to each 100 il reaction volume, and the following temperatures and times were used: 940C and 90 s (denaturation), 720C and 150 s (primer annealing); and 550C and 80-150 s (template extension). Amplification was carried out for 25 cycles. For each mtDNA fragment amplified from a single subject, 8-10 independent reactions were pooled. This was done to ensure that any mutations occurring during the PCR amplification process itself would be sufficiently dilute to avoid being scored as authentic mtDNA mutations. After PCR amplification, the pooled reactions were extracted with phenol:chloroform:isoamyl alcohol and then with chloroform:isoamyl alcohol, and the DNA was precipitated with ethanol. After drying the DNA pellets under a vacuum, they were resuspended in a small volume of TE buffer. For the ND4-4 fragment, aliquots of DNA were digested with SfaN1, and the products were separated by electrophoresis in 4% agarose slab gels. For M13 cloning, aliquots of amplified DNA were digested with Sau3A, and the fragments were separated by electrophoresis through 3 % NuSieve:SeaPlaque (3:1 ratio)
agarose slab gels. The region of the gel containing the DNA fragment to be cloned was excised and used for in-gel ligation into BamHI-digested M13 mp18/19 (Crouse et al. 1983) and then for transformation into Escherichia coli DH5alpha F'. Recombinant plasmids were isolated and used for DNA sequencing with the dideoxy chain termination procedure (Sanger et al. 1977) by using 35S-ATP and gradient gels (Biggin et al. 1983). DNA sequences were stored and analyzed using the MUTANT program (Howell and Howell 1988). There are two reasons why these studies have relied on cloning and sequencing PCR-amplified mtDNA fragments rather than on direct nucleotide sequencing. First, it is our experience that the latter procedure frequently gives rise to ambiguous banding patterns in sequencing gels when it is applied to mtDNA. Second, when the direct sequencing method is used, heteroplasmy might be difficult to detect unambiguously, particularly if one genotype is relatively rare. This choice of experimental approaches has recently been vindicated by the finding that some LHON patients are heteroplasmic, carrying 5%-20% wild-type mitochondrial ND4 genes (Holt et al. 1989).
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1
4
3
5
6
M
Figure 2 SfaN1 digests of PCR-amplified fragment of mitochondrial ND4 gene. Lane 1, Undigested ND4 fragment. DNA in lanes 2-6 was digested with SfaN1 and was isolated from the following individuals: lane 2, non-LHON control; lane 3, LHON family member V,7 (affected male); lane 4, LHON family member V16 (unaffected female); lane 5, internal non-LHON control V4 (son of affected male); lane 6, LHON family member IV28 (affected female). The outside lane marked with an "M" contains HaeIII-digested phiX174 DNA size markers.
Results To determine whether the maternally related members of the Australian LHON lineage carry the mitochondrial ND4 mutation first described by Wallace et al. (1988), a 300-bp mtDNA fragment spanning nucleotide 11,778 was PCR amplified with DNA isolated from
nine family members both clinically afflicted and asymptomatic and was analyzed for retention or loss of the diagnostic SfaN1 site. If the SfaN1 site is intact, then two fragments of about 150 and 140 bp will be produced. Representative results are shown in figure 2: it was found that all individuals from this lineage re-
-
tain an intact restriction site; that is, they do not carry
the ND4 mutation detected in several other LHON families. Careful inspection of the results shown in figure 2 reveals that there are faint DNA bands of higher molecular weight, in addition to the two SfaN1 fragments. One of these has the same mobility as the uncut ND4 fragment (see arrows in fig. 2) and probably represents
the result of an incomplete digestion, since the same minor fragment also appears among the digestion products from non-LHON controls. To directly establish that the members of this LHON lineage did not carry the ND4 mutation at nucleotide 11,778, amplified fragments were subjected to DNA sequencing. At the present time, the sequence has been determined for 55 independently cloned fragments from seven of the family members: no clone had a mutation at nucleotide 11,778. As an extension to this experiment, the minor gel bands containing amplified DNA not cut by SfaN1 (fig. 2) were excised and used for cloning and nucleotide sequencing. Twelve independent clones were analyzed, and all contained an intact SfaN1 restriction site. These results confirm, therefore, that these LHON patients do not carry a minor subpopulation of mtDNA molecules in their white cell/platelet fraction which contains the mutation at nucleotide 11,778. The failure to find in the members of this family an association between LHON and a mutation at nucleotide 11,778 raises the possibility that there is a causative mutation in the mitochondrial ND4 gene -but at a different site. This question has been answered by sequencing a 2.23-kb mtDNA region which includes the entire ND4 gene as well as the overlapping ND4L, upstream ND3, and contiguous tRNA genes. For each mtDNA subfragment of this region, a number of independent clones were sequenced (fig. 1), so that mutations would be detected even if they occurred among a relatively small proportion of mtDNA molecules. Also, the results for each mtDNA subregion represent pooled results for at least three maternally related LHON family members. More than 58,000 bp of mtDNA sequence have been determined thus far, and in the ND3, ND4L, or ND4 genes there is no evidence for a mutation which could explain either the reduced flux through mitochondrial complex I or the LHON symptoms in this family. Four homoplasmic sequence changes have been found in the ND4 gene of members of this LHON family, but all are transitions which constitute silent polymorphisms of amino acid residues ALA191, ASN192, LEU236, and GLY320. Two of these polymorphisms (ASN192 and GLY320) have also been found in the non-LHON controls. No changes from the prototype sequence were found in the ND3, ND4L, or tRNA genes. Single base changes were detected in 17 of the clones analyzed. These sequence alterations most probably represent errors occurring during the PCR amplification, as each was detected in only a single clone and the overall fre-
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Leber Hereditary Optic Neuropathy quency (about 1/4,000 bp) is the same as that for the ND3/ND4L/ND4 genes of non-LHON controls and for other mitochondrial genes from both mouse and human cells (N. Howell, unpublished data). Finally, experiments have shown that there are no internal deletions within this mtDNA region from the LHON family members (data not shown). Discussion
The results presented here provide the first positive experimental evidence that LHON is not exclusively due to mutations within the mitochondrial ND4 gene. Furthermore, the data indicate that the causative mutation in this LHON family does not lie either within the .ND3 or ND4L genes or within the tRNAglu, tRNAarg, or tRNAhis genes. If the proximate cause of LHON is a mutation within one of the mitochondrial genes encoding subunits of complex I-and this is strongly indicated by the biochemical studies by Parker et al. (1989)-then sequencing analyses of the other four mitochondrial complex I genes in members of this family should identify the mutation. The finding that LHON is not associated with a mutation in a single mitochondrial gene may help to resolve one of the more puzzling clinical features of the disease. In addition to its strict maternal inheritance, LHON is defined by a specific set of ophthalmological abnormalities, although among individuals these vary markedly as regards severity, time of onset, and reversibility (no comprehensive and current literature review of LHON is available, but see Nikoskelainen 1984; Nikoskelainen et al. 1987). Superimposed, however, upon the characteristic optic atrophy is a striking heterogeneity in the presentation of additional systemic deficits. For example, it has been observed that some LHON families have a high incidence of cardiovascular abnormalities (Rose et al. 1970; Nikoskelainen et al. 1987). In contrast, within the Australian LHON family, there are late-onset neurological abnormalities including tremors, ataxia, dysarthria, skeletal deformities, posterior column signs, and spasticity (Wallace 1970). In addition, in this family there have been at least nine instances of a severe infantile encephalopathy, which was fatal in three cases. Similar neurological deficits have been authenticated in several other wellstudied European LHON families (Bruyn and Went 1964; Went 1964; Adams et al. 1966). There is also evidence that the ophthalmological changes themselves are influenced by the site of the mitochondrial mutation. Holt et al. (1989) noted that affected males from
LHON families lacking the ND4 mutation often show significant recovery from their visual defects; in contrast, those families with the ND4 mutation had a poor record of recovery or improvement. When the clinical differences among LHON families are considered in conjunction with the available molecular genetic studies, a case can be made that LHON is a closely related group of diseases or syndrome in which the common features are a maternally inherited visual affliction and a biochemical defectstructural or catalytic- in mitochondrial complex I. Further molecular analyses of different LHON families will reveal how many of the seven mitochondrial complex I genes are involved in the etiology of LHON and whether there is a correlation between the gene which is mutated and the expression of neurological or cardiovascular abnormalities.
Acknowledgments This research was supported by National Institutes of Health grants RO1 GM33683 and by the John Sealy Memorial Endowment Fund. Technical assistance was provided by Iwona Kubacka and Mian Xu. We gratefully acknowledge the contributions to this project that were made by Dr. Davis Parker and Jan Parks of the University of Colorado Health Sciences Center and by Dr. Christine Oley of Mater Mother's Hospital, Brisbane. Appreciation is also extended to Drs. Davis Parker and Frank Frerman (University of Colorado) and to Drs. Douglas Turnbull and Lawrence Bindoff (University of Newcastle-upon-Tyne) for their comments and suggestions on earlier drafts of the manuscript.
References Adams JH, Blackwood W, Wilson J (1966) Further clinical and pathological observations on Leber's optic atrophy. Brain 89:15-26 Anderson S, Bankier AT, Barrell BG, de Brujin MHL, Coulson AR, Drouin J, Eperon IC, et al (1981) Sequence and organization of the human mitochondrial genome. Nature 290:457-465 Biggin MD, Gibson JJ, Hong GF (1983) Buffer gradient gels and S35 label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci USA 80:3963-3965 Bruyn GW, Went LN (1964) A sex-linked heredo-degenerative neurological disorder, associated with Leber's optic atrophy. I. Clinical studies. J Neurol Sci 1:59-80 Crouse GF, Frischauf A, Lehrach H (1983) An integrated and simplified approach to cloning into plasmids and singlestranded phages. Methods Enzymol 101:78-89 Giles RE, Blanc H, Cann HM, Wallace DC (1980) Maternal inheritance of human mitochondrial DNA. Proc Natl Acad Sci USA 77:6715-6719
634 Holt IJ, Miller DH, Harding AE (1989) Genetic heterogeneity and mitochondrial DNA heteroplasmy in Leber's hereditary optic neuropathy. J Med Genet 26:739-743 Howell N, Howell B (1988) A string-searching program for mutational analysis. J Mol Biol 203:618 Nikoskelainen E (1984) New aspects of the genetic, etiologic, and clinical puzzle of Leber's disease. Neurology 34: 1482-1484 Nikoskelainen E, Savontaus M-L, Wanne OP, Katila MJ, Nummelin KU (1987) Leber's hereditary optic neuroretinopathy, a maternally inherited disease: a genealogic study in four pedigrees. Arch Ophthalmol 105:665-671 Parker WD, Oley CA, Parks JK (1989) A defect in mitochondrial electron transport activity (NADH-coenzyme Q oxidoreductase) in Leber's hereditary optic neuropathy. N Engl J Med 320:1331-1333 Rose FC, Bowden AN, Bowden PM (1970) The heart in Leber's optic atrophy. Br J Ophthalmol 54:388-393 Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491 Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467
Howell and McCullough Seedorf T (1970) The inheritance of Leber's disease: a genealogical follow-up study. Acta Ophthalmol 63:135-145 Singh G, Lott MA, Wallace DC (1989) A mitochondrial DNA mutation as a cause of Leber's hereditary optic neuropathy. N Engl J Med 320:1300-1305 van Senus AHC (1963) Leber's disease in the Netherlands. Doc Ophthalmol 17:1-162 Vilkki J, Savontaus M-L, Nikoskelainen EK (1989) Genetic heterogeneity in Leber hereditary optic neuroretinopathy revealed in mitochondrial DNA polymorphism. Am J Hum Genet 45:206-211 Wallace DC (1970) A new manifestation of Leber's disease and a new explanation for the agency responsible for its unusual pattern of inheritance. Brain 93:121-132 Wallace DC, Singh G, Lott MT, Hodge JA, Schurr TG, Lezza AMS, Elsas LJ, et al (1988) Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 242:1427-1430 Went LN (1964) A sex-linked heredo-degenerative neurological disorder associated with Leber's optic atrophy: genetical aspects. Acta Genet (Basel) 14:220-239 Yoneda M, Tsuji S, Yamaguchi T, Inuzuka T, Miyatake T, Horai S, Ozawa T (1989) Mitochondrial DNA mutation in family with Leber's hereditary optic neuropathy. Lancet 1:1076-1077
An Example of Leber Hereditary Optic Neuropathy Not Involving a Mutation in the Mitochondrial ND4 Gene Neil Howell and David McCullough Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston
Summary A large Australian family afflicted with Leber's Hereditary Optic Neuropathy (LHON) is analyzed at the nucleotide sequence level in this report. Biochemical assays of platelet mitochondria isolated from members of this family have demonstrated a significant decrease in the specific activity of Complex I (NADHubiquinol oxidoreductase) of the electron transport chain. It is shown here, however, that neither this biochemical lesion nor the optic neuropathy are due to the mutation at nucleotide position 11,778 of the mitochondrial ND4 gene first identified by Wallace et al. in several LHON pedigrees. Furthermore, extensive DNA sequencing studies reveal no candidate mutations within the mitochondrial ND3 gene, the ND4L/ND4 genes, or the contiguous tRNA genes. These studies provide the first direct evidence that not all LHON lineages-even those associated with a biochemical defect in mitochondrial respiratory chain Complex I-carry a mutation in the ND4 gene. Members of the Australian LHON family exhibit neurological abnormalities in addition to the well-characterized ophthalmological changes. It is hypothesized that LHON may be a syndrome or set of related diseases in which the clinical abnormalities are a function, at least in part, of the mitochondrial Complex I gene in which the proximate mutation occurs.
Introduction
Leber hereditary optic neuropathy (LHON) is characterized by a loss of central vision - usually permanent because of bilateral retinal degeneration (Nikoskelainen 1984). The most striking genetic feature of LHON is its strict maternal inheritance: no male within a LHON family has ever been shown to transmit the disease (van Senus 1963; Seedorf 1970; Nikoskelainen 1984; Nikoskelainen et al. 1987). The demonstration that the mitochondrial DNA (mtDNA) is maternally inherited in humans (Giles et al. 1980), as in other mammals, indicated that a mutation within the mitochondrial genome could be the proximate cause of LHON. Strong experimental support for this possibility was first obtained by Wallace et al. (1988; also see Singh et al. 1988), who showed that the mtDNA from nine of 11 LHON maternal lineages carried a G-to-A transition Received April 9, 1990; revision received June 13, 1990. Address for correspondence and reprints: Neil Howell, Ph.D., Biology Division, Department of Radiation Therapy, University of Texas Medical Branch, Galveston, TX 77550. © 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4704-0007$02.00
at nucleotide position 11,778, a change causing loss of an SfaN1 restriction site. This mutation results in the replacement of a highly conserved arginine residue by histidine at position 340 of the ND4 protein. The ND4 gene product is one of the seven mitochondrially encoded subunits of complex I (NADH-ubiquinol oxidoreductase) of the electron-transport chain. More recent reports confirm the occurrence of this mutation in LHON families from different parts of the world. A Japanese LHON family has been shown to carry this ND4 mutation (Yoneda et al. 1989), and recent surveys of European LHON families have revealed that about half carry the sequence change at nucleotide position 11,778 (Holt et al. 1989; Vilkki et al. 1989). These studies were particularly important because they demonstrated that more than one specific mutation can cause LHON. However, since they were limited to screening for the SfaN1 restriction-site alteration, it could not be determined whether a mutation had occurred within the ND4 gene but at a different site, within one of the other mitochondrial complex I genes, or within one of the mitochondrial genes encoding a protein constituent of the other respiratory chain complexes. 629
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As a complement to these molecular genetic studies, Parker et al. (1989) have shown that members of the large Australian LHON pedigree studied by Wallace (1970) have statistically significant decreases, averaging about 50%, in the specific activity of mitochondrial complex I activity in platelets. There were also reductions in the specific activities of mitochondrial succinate-cytochrome c oxidoreductase (complexes II and III) and cytochrome oxidase (complex IV), but they were not statistically significant and may have reflected secondary effects of the complex I deficiency. As this is the only LHON pedigree for which a biochemical defect has been identified, it is important for the study of this mitochondrial disease to determine whether the members of the Australian pedigree carry a mutation at nucleotide 11,778 of the ND4 gene and, if not, whether a mutation elsewhere within the gene can be identified. Answering these questions is the subject of the present investigation. Material and Methods Preparation of DNA Samples Whole venous blood (approximately 50 ml/patient) was collected with informed consent by Dr. Davis Parker (Departments of Pediatrics and Neurology, Univer-
sity of Colorado Health Sciences Center). The crude white cell/platelet fractions were prepared, suspended in 50% glycerol, and then shipped on dry ice to this laboratory, where they were then stored at -800C. Whole cellular DNA was isolated by using the standard procedure involving lysis with SDS, digestion with proteinase K, multiple extractions with phenol followed by chloroform:isoamyl alcohol (24:1), and, finally, ethanol precipitation. This crude DNA was then resuspended in 1 ml of TE buffer (10 mM Tris-HCI, pH 8.0; 1.0 mM EDTA) and was stored at 40C. DNA samples were prepared from the following maternally related family members (Wallace 1970): IV2s, Vg, V17, and V18 (affected males); IV28 (affected female); and V1o, V16, VI1s, and V116 (unaffected females). The last two family members are clinically normal at present but just entering the age period when the optic atrophy is usually expressed. As an internal control, DNA was prepared from V4, the son of an affected male. External controls included DNA samples from clinically normal individuals and from patients with non-LHON neurological or mitochondrial diseases.
NDS
L
12.213
0.03 9
27
17
13
1 n
1s
5 24
M
Figure I Cloning and sequencing strategy for mitochondrial ND3 and ND4L/ND4 genes. The complex I genes are depicted by the large bars, while the tRNA genes are indicated by the black circles. The nucleotide coordinates of the genes are as follows: tRNAglu, 9,991-10,058; ND3 (cross-hatched bar), 10,059-10,404; tRNAarg, 10,405-10,469; ND4L (cross-hatched bar), 10,470-10,766; ND4 (solid bar), 10,760-12,317; and tRNAhis, 12,318-12,206. The arrows indicate the overlapping clones used for DNA sequence analysis, with the number of independent clones from the LHON subjects shown below or above the arrow. The arrows pointing to the right and left indicate whether the 5' or 3' end of the fragment, respectively, was inserted adjacent to the M13 origin. The double-headed arrows indicate fragments which were sufficiently small that the complete sequence of the entire fragment could be unambiguously determined; as a result, clones of the two insertion orientations have been pooled. Since the 389-bp ND4-3 fragment inserted into M13 in only one orientation, an "internal" sequencing primer was used to derive an unambiguous sequence of the entire mitochondrial insert.
PCR Amplification and DNA Cloning/Sequencing
The 2.23-kb region of mtDNA spanning the ND3, ND4L, and ND4 genes was amplified as a series of overlapping fragments (fig. 1), by using the polymerase chain reaction (PCR) technique (Saiki et al. 1988) and the synthetic oligonucleotide primers listed in table 1. To facilitate M13 cloning, the terminal regions of all amplified fragments contain Sau3A restriction sites; these sites either occur within the mtDNA sequence or are engineered into the amplification primers. All nucleotide coordinates are taken from the standard Cambridge sequence of human mtDNA (Anderson et al. 1981). Since there is a naturally occurring Sau3A site at nucleotides 11,923-11,926, there is no sequence overlap between the ND4-4 and ND4-5 fragments (table 1). To ensure that there were no mutations occurring within the ND4 gene region complementary to the ND4-4 3' and ND4-5 5' primers, additional PCR amplifications were carried out using the ND4-4 5' and ND4-5 3' primers to generate the "double" fragment. This latter was then digested with Sau3A, and the resulting subfragments were cloned into M13 vectors for DNA sequencing. With this approach, there was no indication of a mutation either within the Sau3A site or in the bracketing regions. PCR amplification was carried out with Taq DNA polymerase (Cetus/Perkin-Elmer Corp.) by using the
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Table I Oligonucleotide Primers for PCR Amplification
Gene Fragment ND-3: 5' .......... 3' .......... ND4-1: 5' .......... 3' .......... ND4-2: 5' ......... 3' .......... ND4-3: 5' ......... 30 .......... ND4-4: 5' .......... 3' .......... ND4-5: 5' .......... 3' ..........
Primer Sequencea
Nucleotide Positions
CTC CAT CTA TTG ATC AGG GTC CAT TTG GTA AAT ATG ATC ATC
9,968-9,988 10,449-10,472
ACG AAT GAT CTC GAC TCA TTA AGG AAA AGG TTG GGG ATC AGC
10,425-10,445 10,911-10,931
TAT TAG GAT CAT CCC TCT ACT CAG GGG GTT TGG ATC AGA ATG
10,858-10,878
TAT TAG GAT CAT CCC TCT ACT ATA GAT CTT GGG CAG TGA GAG
10,858-10,878 11,298-11,318
GCA TAC TCT TCG ATC ACG CAC GTG ATA TTT GAT CAG GAG AAC
11,618-11,638 11,914-11,934
GTT CTC CTG ATC AAA TAT CAC AGC AGT TCT TGT GAT CTT TCT
11,914-11,934
11,659-11,679
12,208-12,228 a Sequences of both primers for each gene fragment are given in the 5'- to 3' direction; the designations 5' and 3' for the amplification primers are used to define from which end of the mtDNA fragment amplification is proceeding.
conditions recommended by the manufacturer. Total cellular DNA (100-700 ng) was added to each 100 il reaction volume, and the following temperatures and times were used: 940C and 90 s (denaturation), 720C and 150 s (primer annealing); and 550C and 80-150 s (template extension). Amplification was carried out for 25 cycles. For each mtDNA fragment amplified from a single subject, 8-10 independent reactions were pooled. This was done to ensure that any mutations occurring during the PCR amplification process itself would be sufficiently dilute to avoid being scored as authentic mtDNA mutations. After PCR amplification, the pooled reactions were extracted with phenol:chloroform:isoamyl alcohol and then with chloroform:isoamyl alcohol, and the DNA was precipitated with ethanol. After drying the DNA pellets under a vacuum, they were resuspended in a small volume of TE buffer. For the ND4-4 fragment, aliquots of DNA were digested with SfaN1, and the products were separated by electrophoresis in 4% agarose slab gels. For M13 cloning, aliquots of amplified DNA were digested with Sau3A, and the fragments were separated by electrophoresis through 3 % NuSieve:SeaPlaque (3:1 ratio)
agarose slab gels. The region of the gel containing the DNA fragment to be cloned was excised and used for in-gel ligation into BamHI-digested M13 mp18/19 (Crouse et al. 1983) and then for transformation into Escherichia coli DH5alpha F'. Recombinant plasmids were isolated and used for DNA sequencing with the dideoxy chain termination procedure (Sanger et al. 1977) by using 35S-ATP and gradient gels (Biggin et al. 1983). DNA sequences were stored and analyzed using the MUTANT program (Howell and Howell 1988). There are two reasons why these studies have relied on cloning and sequencing PCR-amplified mtDNA fragments rather than on direct nucleotide sequencing. First, it is our experience that the latter procedure frequently gives rise to ambiguous banding patterns in sequencing gels when it is applied to mtDNA. Second, when the direct sequencing method is used, heteroplasmy might be difficult to detect unambiguously, particularly if one genotype is relatively rare. This choice of experimental approaches has recently been vindicated by the finding that some LHON patients are heteroplasmic, carrying 5%-20% wild-type mitochondrial ND4 genes (Holt et al. 1989).
Howell and McCullough
632 2
1
4
3
5
6
M
Figure 2 SfaN1 digests of PCR-amplified fragment of mitochondrial ND4 gene. Lane 1, Undigested ND4 fragment. DNA in lanes 2-6 was digested with SfaN1 and was isolated from the following individuals: lane 2, non-LHON control; lane 3, LHON family member V,7 (affected male); lane 4, LHON family member V16 (unaffected female); lane 5, internal non-LHON control V4 (son of affected male); lane 6, LHON family member IV28 (affected female). The outside lane marked with an "M" contains HaeIII-digested phiX174 DNA size markers.
Results To determine whether the maternally related members of the Australian LHON lineage carry the mitochondrial ND4 mutation first described by Wallace et al. (1988), a 300-bp mtDNA fragment spanning nucleotide 11,778 was PCR amplified with DNA isolated from
nine family members both clinically afflicted and asymptomatic and was analyzed for retention or loss of the diagnostic SfaN1 site. If the SfaN1 site is intact, then two fragments of about 150 and 140 bp will be produced. Representative results are shown in figure 2: it was found that all individuals from this lineage re-
-
tain an intact restriction site; that is, they do not carry
the ND4 mutation detected in several other LHON families. Careful inspection of the results shown in figure 2 reveals that there are faint DNA bands of higher molecular weight, in addition to the two SfaN1 fragments. One of these has the same mobility as the uncut ND4 fragment (see arrows in fig. 2) and probably represents
the result of an incomplete digestion, since the same minor fragment also appears among the digestion products from non-LHON controls. To directly establish that the members of this LHON lineage did not carry the ND4 mutation at nucleotide 11,778, amplified fragments were subjected to DNA sequencing. At the present time, the sequence has been determined for 55 independently cloned fragments from seven of the family members: no clone had a mutation at nucleotide 11,778. As an extension to this experiment, the minor gel bands containing amplified DNA not cut by SfaN1 (fig. 2) were excised and used for cloning and nucleotide sequencing. Twelve independent clones were analyzed, and all contained an intact SfaN1 restriction site. These results confirm, therefore, that these LHON patients do not carry a minor subpopulation of mtDNA molecules in their white cell/platelet fraction which contains the mutation at nucleotide 11,778. The failure to find in the members of this family an association between LHON and a mutation at nucleotide 11,778 raises the possibility that there is a causative mutation in the mitochondrial ND4 gene -but at a different site. This question has been answered by sequencing a 2.23-kb mtDNA region which includes the entire ND4 gene as well as the overlapping ND4L, upstream ND3, and contiguous tRNA genes. For each mtDNA subfragment of this region, a number of independent clones were sequenced (fig. 1), so that mutations would be detected even if they occurred among a relatively small proportion of mtDNA molecules. Also, the results for each mtDNA subregion represent pooled results for at least three maternally related LHON family members. More than 58,000 bp of mtDNA sequence have been determined thus far, and in the ND3, ND4L, or ND4 genes there is no evidence for a mutation which could explain either the reduced flux through mitochondrial complex I or the LHON symptoms in this family. Four homoplasmic sequence changes have been found in the ND4 gene of members of this LHON family, but all are transitions which constitute silent polymorphisms of amino acid residues ALA191, ASN192, LEU236, and GLY320. Two of these polymorphisms (ASN192 and GLY320) have also been found in the non-LHON controls. No changes from the prototype sequence were found in the ND3, ND4L, or tRNA genes. Single base changes were detected in 17 of the clones analyzed. These sequence alterations most probably represent errors occurring during the PCR amplification, as each was detected in only a single clone and the overall fre-
633
Leber Hereditary Optic Neuropathy quency (about 1/4,000 bp) is the same as that for the ND3/ND4L/ND4 genes of non-LHON controls and for other mitochondrial genes from both mouse and human cells (N. Howell, unpublished data). Finally, experiments have shown that there are no internal deletions within this mtDNA region from the LHON family members (data not shown). Discussion
The results presented here provide the first positive experimental evidence that LHON is not exclusively due to mutations within the mitochondrial ND4 gene. Furthermore, the data indicate that the causative mutation in this LHON family does not lie either within the .ND3 or ND4L genes or within the tRNAglu, tRNAarg, or tRNAhis genes. If the proximate cause of LHON is a mutation within one of the mitochondrial genes encoding subunits of complex I-and this is strongly indicated by the biochemical studies by Parker et al. (1989)-then sequencing analyses of the other four mitochondrial complex I genes in members of this family should identify the mutation. The finding that LHON is not associated with a mutation in a single mitochondrial gene may help to resolve one of the more puzzling clinical features of the disease. In addition to its strict maternal inheritance, LHON is defined by a specific set of ophthalmological abnormalities, although among individuals these vary markedly as regards severity, time of onset, and reversibility (no comprehensive and current literature review of LHON is available, but see Nikoskelainen 1984; Nikoskelainen et al. 1987). Superimposed, however, upon the characteristic optic atrophy is a striking heterogeneity in the presentation of additional systemic deficits. For example, it has been observed that some LHON families have a high incidence of cardiovascular abnormalities (Rose et al. 1970; Nikoskelainen et al. 1987). In contrast, within the Australian LHON family, there are late-onset neurological abnormalities including tremors, ataxia, dysarthria, skeletal deformities, posterior column signs, and spasticity (Wallace 1970). In addition, in this family there have been at least nine instances of a severe infantile encephalopathy, which was fatal in three cases. Similar neurological deficits have been authenticated in several other wellstudied European LHON families (Bruyn and Went 1964; Went 1964; Adams et al. 1966). There is also evidence that the ophthalmological changes themselves are influenced by the site of the mitochondrial mutation. Holt et al. (1989) noted that affected males from
LHON families lacking the ND4 mutation often show significant recovery from their visual defects; in contrast, those families with the ND4 mutation had a poor record of recovery or improvement. When the clinical differences among LHON families are considered in conjunction with the available molecular genetic studies, a case can be made that LHON is a closely related group of diseases or syndrome in which the common features are a maternally inherited visual affliction and a biochemical defectstructural or catalytic- in mitochondrial complex I. Further molecular analyses of different LHON families will reveal how many of the seven mitochondrial complex I genes are involved in the etiology of LHON and whether there is a correlation between the gene which is mutated and the expression of neurological or cardiovascular abnormalities.
Acknowledgments This research was supported by National Institutes of Health grants RO1 GM33683 and by the John Sealy Memorial Endowment Fund. Technical assistance was provided by Iwona Kubacka and Mian Xu. We gratefully acknowledge the contributions to this project that were made by Dr. Davis Parker and Jan Parks of the University of Colorado Health Sciences Center and by Dr. Christine Oley of Mater Mother's Hospital, Brisbane. Appreciation is also extended to Drs. Davis Parker and Frank Frerman (University of Colorado) and to Drs. Douglas Turnbull and Lawrence Bindoff (University of Newcastle-upon-Tyne) for their comments and suggestions on earlier drafts of the manuscript.
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