(NDl/L285P) has been found in one LHON family in which the optic neuropathy is accompanied by severe neurological abnormalities (HOWELL et al. 1991a).
Copyright 0 1993 by the Genetics Society of America
Letter to the Editor Leber’s Hereditary Optic Neuropathy:The Etiological Role of a Mutation in the Mitochondrial Cytochromeb Gene Leber’s hereditary optic neuropathy (LHON) is a mitochondrial genetic disease in which the defining clinical feature is a delayed onset loss of central vision and formation of centrocecal scotomata (LUNDSGARD 1944; VAN SENUS 1963;NIKOSKELAINEN et al. 1987). JOHNS and NEUFELD(1 99 1) and BROWN et al. (1 992a) have recently concluded that in a subset of LHON pedigrees, the primary etiological event is most likely to be a mutation at mtDNA nucleotide 15257 that results in the substitution of Asn for the Asp residue at position of 17 1 of the cytochrome b protein. For three reasons, these findings have great relevance for the etiology of LHON: First, previous studies have identified three primary LHON mutations and each has been within one of the seven mitochondrial genes encoding subunits of complex I of therespiratory chain (NADH-ubiquinone oxidoreductase). A mutation at nucleotide 11778 (ND4/R340H) occurs in approximately onehalf of all LHON pedigrees (WALLACEet al. 1988; POULTONet al. 1991) while a mutation at nucleotide 3460 (NDl/A52T) is found in about 15-25% of all LHON pedigrees (HOWELLet al. 1991b; HUOPONEN et al. 1991). A primary mutation at nucleotide 4160 (NDl/L285P) has been found in one LHON family inwhich the opticneuropathy is accompanied by severe neurological abnormalities(HOWELL et al. 1991a).Theseprimarymutations have never been detected in normalcontrols.BROWN et al. (1992a) concludedthat since LHON mutations affect two different respiratory complexes, LHON results from a nonspecific decrease in mitochondrial energy production. Second, JOHNS and BERMAN(1 991) identified nucleotide alterations within the mitochondrial complex I genes which they concluded were secondary LHON mutations. In contrast to primary LHON mutations, these secondary mutations were found in normal individuals but occurred at amuch higher frequency in LHON patients. Furthermore, comparedto those produced by primary LHON mutations, the resulting amino acid substitutions occurred at less highly conserved positions within the complex I subunits. JOHNS and NEUFELD(1991) and BROWNet al. (1992a) provide additional examples of secondary LHON mutations. The etiological or pathogenetic role(s) of these secondary mutations is not yet understood. Genetics 133: 133-136 (January, 1993)
Third, BROWNet al. (1992a) found that their four LHON families who carry the 15257 LHON mutation formed aphylogenetic cluster. This is the first description of such clustering among LHON families. In view of these important issues, it is worthwhile to consider further, and morecritically, the etiological role of the 15257 cytochrome b mutation in LHON. The main point of this communication is thatthe criteria that were used to deduce an etiological role for the 15257 mutation are not entirely robust and that unraveling the mitochondrial genetic etiology of LHON is unlikely to bestraightforward.However, we will present independent evidence which suggests that this mutation may have such a role. The same reservations that are raised here also apply tothe recent studies that have proposed etiological roles for additionalmitochondrialmutations(BROWN et al. 1992b,c). JOHNS and NEUFELD (1991) found the 15257 mutation in 8 of 80 LHON patients but in none of 240 normal and disease controls.BROWN et al. (1992a) detected the 15257 mutation in 4 of 23 non-11778 LHON patients but also in 1 of 362 normal individuals. This is the first instance inwhich aputative primary LHON mutation has been found in a normal control. Both groups cited the evolutionary conservation of the Asp’71residue in cytochrome b and deduced that mutational substitution with Asn would probably be deleterious to respiratory chain function (e.g., pp. 166 and 170 of BROWNet al. 1992a) althoughthis has not been established directly. The evolutionary conservation of the Asp171 residue,however, is not as stringent as that for the other amino acid residues that have been altered by previously identified primary LHON mutations. Although the residue equivalent to Asp’” is conserved in the cytochrome b proteins of plant and animal mitochondria, it is predicted to be Asn, Ser, Thr or Gly in those from more evolutionarily diverse species. In contrast, the Ala5*and LeuIs5 residues of the ND1 protein, which are altered by the 3460 and 4160 primary LHON mutations, respectively, show much greater sequence conservation (HOWELL et al. 199 la,b). Neither JOHNS and NEUFELD (199 1) nor BROWN et al. (1992a)foundLHON families thatcarriedthe 15257 mutation in association with another primary
I 34
N. Howell et al. TABLE 1
Loss of vision among males of Australian ND4/11778 LHON families LHON falnilya
T A S l (1600) Ql.I)2 (60) WAl (130) a
Secondary LHON mutations
4216
13708
15257
15812
-
-
-
-
+ +
+ +
+ -
+
-
Loss (%) of
visionb
15-20 33-43 47-58
The number in the parenthesis is the approximate number of
rnenlbers in the maternal lineage including both males and females.
The fa~nilieshas designated according to whether they are located principally i n Tasmania, Queensland, or Western Australia. A range is given to account for uncertainties in dates of birth and death for some family members. The figures given are the percentages of affected males relative to the total number of males aged 25 years or older.
LHON mutation. However, we haveidentified t w o LHON Families who carryboththeprimaryND4 mutation at nucleotide 1 1 778 and the 15257 cytochrome b mutation (N. HOWELL, manuscriptin preparation). To determine if the 15257 mutationis markedly changing the penetrance of the optic neuropathy when it is associated with the 11778 primary LHON mutation, we have compared the frequencies of affected males from three Australian LHON families (Table 1). These preliminary results suggest that penetrance may beincreased by the4216 plus 13708 secondary mutations but that there is no discernible effect of thecytochrome b mutationsuponpenetrance. One possibility is that the low penetrance of the optic neuropathy in the Tasl LHON family (Table 1) is due to heteroplasmy of the 11778 mutation. HOWever, we have sequenced 173 independent M 13 clones from seven Family members (both affected and unaffected) and all 173 clones carry the 11778 mutation (N. HOWELL and S. GIBSON,unpublished data). O n theotherhand,there may besomeheteroplasmic individuals in the LHON family from Western Aust ralia (SUDOYO et al. 1992) although the penetranceis highest in this family (Table 1). Since it is not feasible to routinely analyze the affected tissue (there is also a question as to which tissue in the eye is the primary site of the lesion), caution is required when extrapolating from one tissue to another. Nonetheless, there is no evidencethatthedifferences in penetrance among these three LHON families can be explained b y heteroplasrny (for an example of a LHON family in which heteroplasmycanexplain the low penetrance, see HOWELL, MCCULLOUGH and BODIS-WOLLNER 1992). Anotherobservation is thatthe15257mutation showsa greater association with secondary LHON mutations compared to previously identified primary LHON mutations. Seven of the eight (88%) 15257 1,HON patients of JOHNS and NEUFELD (1 99 l), and all four in the cluster of Brown et al. (199Ba)also
carried the secondary N D 5 LHON mutation at nucleotide13708. T h e correspondingfrequenciesfor the13708secondarymutation in theirLHON patients with theprimarymutationsatnucleotides 1 1778and3460were17%and 33%, respectively (JOHNS and NEUFELD 1991). BROWNet al. (1992a) found that their four 15257 LHON Families formedaphylogeneticcluster (see their Figure 4). There is insufficient information to determine how many of the 15257 LHONpatients of JOHNS and NEUFELD (1 99 1) are members of thesame cluster. We have recently found two Australian LHON families that are also members of this phylogenetic cluster and have a mild form of the disease (MACKEYand HOWELL 1992; HOWELL, manuscript in preparation). Neither of these LHON families carries the cytochrome b mutations at nucleotides 15257 or 158 12. The important point about their phylogenetic cluster is that the 15257 mutation most probably did not occur independently in the four LHON families of BROWNet al. Instead, as shown by these investigators, this mutation appears to have arisen once in a common ancestor. Thus, theiruse of a chi-squaretest to show that the 15257 mutation occurs more often in non-11778 LHON families (their Table2) does not appear appropriate. We have recently obtained results which may bear upon the etiological role of the 15257 mutation in LHON. A young Australian woman lost vision with the ophthalmologicalcharacteristicsof LHON although there was no previous family history. During a preliminary screening of mtDNA restrictionsites, it was found that she carried the 15257 mutation in the mitochondrial cytochrome b gene. Subsequently, the seven mitochondrial complex I and the cytochromeb genes were sequenced in their entirety using the polymerase chain reaction-based approach of this laboratory (HOWELL et al. 1991 a).Relative to the standard Cambridge sequence of human mtDNA (ANDERSON et al. 198 l ) , 12 sequence changes were found (Table 2). In addition to the 15257 mutation, 10 nucleotide changes were silent polymorphisms while there was one conservative substitution polymorphism in a region of the cytochromeb protein that is not conserved during evolution(HOWELL1989).Wehavenot included in Table 2 the seven sites within the complex I genes, and the onewithin the cytochrome6 gene, at which the Cambridge sequence appears to be in error or the common polymorphisms at nucleotides 4769 and4985(HOWELL et al. 1992).Itshould also be noted this patient is not a memberof the phylogenetic cluster of BROWNet al. (compare the polymorphisms in Table 2 with those in their Table 1). This indicates an independent origin of the 15257 mutation in this patient(HOWELL,manuscript in preparation).We I1ave also confirmed that this patient does not carry the putative L,HON mutation in themitochondrial
Letter to the Editor
TABLE 2 Nucleotide changesin a singleton case of LHON
Sequence
Amino acid changeb
A+G C-T A+G A+G A+G G+A G+A C+Y T-C T+C G+A C+T
K58K I981 M27M L236L T180T G320G L12L E169E F18L L149L D171N L377L
changea Nucleotide Gene
NDI N D2 ND4L N D4
12372 ND5 14167 N D6 CYT b
3480 4763 10550 11299 1 1467 11719
14798 15191 15257 15877
T h e nucleotide change is that of the L-strand sequence. T h e first letter is the amino acidin the proteins of the standard o r wildtype; the number is the residue number within the protein: the last letter is the amino acid in the LHON patient. a
135
One working hypothesis for the etiology of LHON is as follows. A primary LHON mutation establishes a risk fordeveloping the opticneuropathythat is sufficient to lead to development of the optic neuropathy at a substantial frequency (to simplify the discussion, homoplasmy of mitochondrial mutations is assumed). However, this risk, as expressed by the penetrance, is also a function of other factors, both genetic and environmental (e.g., VILKKIet al. 199 1 ; BROWN et al. 1992b). Thus, the penetrance may be increased when secondary LHON mutations are present within themitochondrialgenome(Table 1). Ina complementary fashion, other mitochondrial nucleotide changes may decrease the risk, or amelioratethe pathological changes(HOWELL et al. 1991a). This hypothesis provides a possible explanation for theloss of vision in the patient described here. Under typical circumstances, the 15257 mutation alone is not sufficient to lead to the optic neuropathy. In this patient, however, nuclear genetic or environmental factors may have played an unusually substantial etiological role. Our studies have provided additional evidence for LHON families that lack a primary mutation but carrysecondarymutations (MACKEYandHOWELL 1992).Furthermore,theoccurrence of the15257 mutation in a normal control (BROWNet al. 1992a) is also consistent with this model since one can envision that in this individual, other etiologically important factors have been insufficient to “trigger” the development of the optic neuropathy. Thus far, LHON is the only mitochondrial genetic disease in which such a complex, multistep etiology has been suggested. In view of the accumulating evidence that defects in mitochondrial energy metabolismmay have a role in several major neurological disordersincluding Parkinson’s, Huntington’sand Alzheimer’s disease (WALLACE1992), LHON may provide a useful model system for the experimental and theoretical analysis of these diseases.
COX I gene at nucleotide 7444 (BROWN et al. 1992c; data not shown). Thus far, this is the only patient in whichlossof visioncan be associated with the15257mutation alone, in the absence of any other known primary or secondary LHON mutation in the mitochondrial genome. Two caveats, however, must be noted. In the first place, in the absence of a positive family history of maternal transmission, a diagnosis of LHON is not robust. I t is statistically highly unlikely, on the other hand, thatthe15257mutation has occurredindependently of the optic neuropathy.Secondly, it cannot yet be ruled out that there aresecondary (or primary) LHON mutations-not yet identified in any other study-within regions of the mitochondrial genome of this patientthat were not included without our sequencing analyses (which cumulatively span about 50% of the entire genome). Taking all the results discussed here into consideration, an etiological role for the 15257 mutation in This research was supported by research grant PO1 HD08315 from the National Institutes o f Health (N.H.) LHON is likely, particularly as it appearsto have arisen independently on two occasions. However, relNEIL HOWELL,’ IWONA KUBACKAand STEVEN ative to the more common primary LHON mutations HALVORSON at nucleotides 3460 and 11778, it behaves more simDepartment of Radiation Therapy ilarly to a secondary LHON mutation. What do these Department of Human Biological Chemistry & results, and those of JOHNS and NEUFELD(1991) and Genetics BROWN et al. (1992a), suggest about the mitochondrial The University of Texas Medical Branch genetic etiology of LHON? Before attempting to proGalveston, Texas 77550 vide atentative answer tothatquestion, it merits reemphasizing the incomplete penetrance of LHON. DAVIDMACKEY T h e incidence of affected individuals within a LHON The Murdoch Institute f:,mily is much less than 100%; classically 40-50% of The Royal Children’s Hospital the males and about 10% of the females [LUNDSGARD Melbourne, Victoria 3052 1944; VAN SENUS(1963); Table 13. Thus, while the Australia mitochondrial genotype establishes the risk of developingtheopticneuropathy, it is not sufficient to ensure its occurrence. ’ To whom correspondence should be addressed.
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L. SAVONTAUS, 1991 A new mtDNA mutation associated with Leber hereditary optic neuroretinopathy. Am. J. Hum. Genet. ANDERSON, S., A. T . BANKIER, B. G . BARRELL, M. H. L. DEBRUIJN, 48: 1147-1 153. A. R. COULSON, J. DROUIN,I. C. EPERON, D. P. NIERLICH,B. JOHNS, D. R., andJ.BERMAN, 1991 Alternative,simultaneous A. ROE,F. SANGER, P. H . SCHRIER, A. J. H. SMITH, R. STADEN complex 1 mitochondrial DNA mutations in Leber’s hereditary and I. G . YOUNG, 1981 Sequenceandorganizationofthe opticneuropathy.Biochem. Biophys. Res. Commun. 174: 1324-1330. human mitochondrial genome. Nature 290: 457-465. JOHNS,D. R., and M. J. NEUFELD,1991 Cytochrome b mutations BROWN,M. D., A. S. VOLJAVEC,M. T. LOTT, A. TORRONI,C.-C. in Leber hereditary optic neuropathy. Biochem. Biophys. Res. YANG and D. C. WALLACE,1992a Mitochondrial DNA comCommun. 181: 1358-1364. plex I and 111 mutationsassociatedwithLeber’shereditary LUNDSGARD, R., 1944 Leber’sdisease: a genealogic, genetic and optic neuropathy. Genetics 1 3 0 163-173. clinical study of 101 cases of retrobulbar optic neuritis in 20 BROWN,M. D., A. S. VOLJAVEC, M. T. LOTT, I. MACDONALD and Danish families. Acta Ophthalmol. 21(Suppl. 3): 3-306. D. C. WALLACE,1992b Leber’s hereditary optic neuropathy: 1992 A variant of Leber hereditary a model for mitochondrial neurodegenerative diseases. FASEB MACKEY, D., and N. HOWELL, optic neuropathy characterized by recovery of vision and an J. 6 2791-2799. unusual mitochondrial genetic etiology. Am. J. Hum. Genet. BROWN,M. D., C.-C. YANG, I. TROUNCE, A. TORRONI, M. T . L o n 51: (in press). and D. C. WALLACE,1992c AmitochondrialDNAvariant, E. K., M.-L. SAVONTAUS, 0. P. WANNE,M. J. identified in Leber hereditary optic neuropathy patients, which NIKOSKELAINEN, KATILAand K . U. NUMMELIN, 1987 Leber’s hereditary optic extendstheaminoacidsequenceofcytochrome c oxidase neuropathy, a maternally inherited disease: a genealogic study subunit I . Am. J. Hum. Genet. 51: 378-385. in four pedigrees. Arch. Ophthalmol. 105: 665-671. HOWELL, N.,1989 Evolutionary conservation of protein regions POULTON, J., M. E. DEADMAN, J. BRONTE-STEWART, W. S. FOULDS in the protonmotive cytochrome b and their possible roles in and R. M. GARDINER, 1991 Analysis of mitochondrial DNA redox catalysis.]. Mol. Evol. 29: 157-169. in Leber’shereditaryopticneuropathy.J.Med.Genet. 28: HOWELL, D. N., MCCULLOUGH and 1. BODIS-WOLLNER, 765-770. 1992 Molecular genetic analysis of a sporadic case of Leber SUDOYO,H., S. MARZUKI,F. MASTAGLIAandW.CARROLL, hereditary optic neuropathy. Am. J. Hum. Genet. 50: 4431992 Molecular genetics of Leber’s hereditary optic neuropa446. thy: study of a six-generation family from Western Australia. HOWELL,N., 1. KUBACKA,M. XUand D. A.MCCULLOUGH, J. Neurol. Sci. 108: 7-17. 199 la Leber hereditary optic neuropathy: involvement of the VAN SENUS,A. H. C., 1963 Leber’sdisease in theNetherlands. mitochondrial NDI gene and evidence for an intragenic supDoc. Ophthalmol. 17: 1-162. pressor mutation. Am. J. Hum. Genet. 48: 935-942. VILKKI, J., J. OTT, M.-L. SAVONTAUS, P. AULAand E. K. NIKOSKEHOWELL, N.,L. A. BINDOFF,D. A. MCCULLOUGH, 1. KUBACKA, J. LAINEN,1991 Optic atrophy in LeberhereditaryopticneuPOULTON,D. MACKEY,L. TAYLOR and D. M. TURNBULL, roretinopathy is probably determined by an X-chromosomal 1991 b Leber hereditary optic neuropathy: identification of gene closely linked to DXS7. Am. J. Hum. Genet. 48: 484the same mitochondrial NDI mutationin six pedigrees. Am. J. 491. WALLACE,D. C., 1992 Mitochondrialgenetics:aparadigmfor Hum. Genet. 49: 939-950. aging and degenerativediseases? Science 256: 628-632. HOWELL, N.,D. A. MCCULLOUGH, I. KUBACKA, S. HALVORSON and WALLACE,D. C., G . SINGH, M. T . LOTT, J. A. HODGE,T. G . D. MACKEY, 1992 T h e sequence of human mtDNA: the quesSCHURR, A.M. S. LEZZA,L. J. ELSASI1 and E. K. NIKOSKELAItion of errors versus polymorphisms. Am. J. Hum. Genet. 5 0 NEN, 1988 Mitochondrial DNA mutation associated with Le1333-1337. ber’s hereditary optic neuropathy. Science242: 1426-1430. HUOPONEN, K., J. VILKKI,P. AULA,E. K. NIKOSKELAINEN and M.-
LITERATURE CITED
