Journal of Neuro-Ophthalmology, December 2002, Volume 22, Issue 4

From Genotype to Phenotype in Leber Hereditary Optic Neuropathy

EDITORIAL From Genotype to Phenotype in Leber Hereditary Optic Neuropathy: Still More Questions than Answers Nancy J. Newman, MD In 1988, the first point mutation in the mitochondrial DNA ( mtDNA) to be associated with a human disease was reported in pedigrees with Leber hereditary optic neuropathy ( LHON) ( 1). An explosion of clinical, genetic, and biochemical studies followed ( 2- 5). The advent of molecular diagnosis for LHON allowed us to more easily make a definitive diag­nosis of LHON in patients without a family history of visual loss and expanded the clinical phenotype of the disorder to include some unusual features in individuals and in pedigrees. In the current edition of the Journal ofNeuro- Ophthalmology, the article by Howell et al. ( 6) highlights how observational clinical and genetic studies in LHON continue to help us sort through the various theories on pathogenesis and the determinants of expression of this disease. Three point mutations in the mtDNA, the primary LHON mutations, are believed to cause 90% to 95% of cases of LHON worldwide. They are located at mtDNA nucleotide positions 11778 ( 69% of cases), 3460 ( 13% of cases), and 14484 ( 14% of cases) ( 2- 5) ( Fig. 1). Screening for LHON in a patient with visual loss should begin with the three primary mutations. In those primary mutation- negative patients in whom suspicion remains high, testing for the other mtDNA mutations associated with LHON is probably warranted, es­pecially for those mutations deemed likely causal in a few previous pedigrees ( 2- 5,7) ( Fig. 1 ). Alternatively, because the majority of these other mtDNA mutations reside in genes encoding subunits of complex I, complete sequencing of complex I, perhaps beginning with the so- called " hot spot" ND6 gene ( 8), may also be considered. Finally, sequencing the entire mitochondrial genome is possible, although labor intensive. This should be per­formed only in those cases of high suspicion and interpreted by someone versed in the complexities of mitochondrial genetics ( 9). How do we get from a point mutation in the mtDNA of all the cells of an individual to a sudden, selective loss of optic nerve function primarily in males in their early adulthood? Some hints as to the mechanisms of the disease are provided by clinical and genetic studies performed over the past decade. Does the specific mtDNA mutation dictate particular clini­cal features? There are pedigrees with " LHON plus" in which certain mtDNA mutations seem to result in specific disease patterns of LHON- like visual loss combined with other neurologic syndromes, such as dystonia ( 4) ( Fig. 1). However, among the other LHON pedigrees, few significant clinical differences have been demonstrated to date among those patients with the three primary mutations, those with other mtDNA mutations, and those that are genetically unspecified. The only major exception is the far greater spontaneous recovery rate among patients with the 14484 mutation, as compared with those affected individuals with the 11778 mutation and even the 3460 mutation ( 3). Departments of Ophthalmology, Neurology, and Neurological Surgery, Emory University School of Medicine, Atlanta, Georgia. Address correspondence to Nancy J. Newman, MD, Neuro- Ophfhalmology Unit, Emory University School of Medicine, 1365- B Clifton Road, N. E., Atlanta, GA 30322, USA; E- mail: ophtnjn@ emory. edu Supported in part by an unrestricted departmental grant ( ophthalmology) from Research to Prevent Blindness, Inc. ( New York, New York) and by core grant P30- EY06360 ( Department of Ophthalmology) from the National Institutes of Health, Bethesda, Maryland. Dr. Newman is a Lew R. Wasserman merit award recipient. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 257 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 EDITORIAL 15812 15257 3308 3394 3398 3472 3505 , 3547 N. m 4136 4171* 4216 13730* 13708 9438 Fig. 1 . Mitochondrial genome showing the point mutations associated with Leber he­reditary optic neuropathy ( LHON). Over 90% of all cases of LHON are associated with the three primary mutations located inside the genome, and the other mutations are shown outside the genome. These other mutations vary markedly in their preva­lence, degree of evolutionary conservation of the encoded amino acids altered, and fre­quency among controls. Mutations marked * may be primary, but they each account for only one or a few pedigrees worldwide. Mu­tations marked ** are primary mutations as­sociated with LHON and dystonia. ( Adapted with permission [ 7], courtesy of Marie Lott). 7444* 9101* Complex I gan& a [ NADH deh yd rogenase] | ComplM IV gonoB ' ( cytochrome c CUCidPK ) Complex [ II genBE ( ublqutnol: cytochrome c ox idored net as a} Complex V genes ( ATP synthase) Transfer RN A genes • ' ' Ribosomal RNAgenes Does heteroplasmy ( the coexistence of mutant and normal mtDNA) within individuals in a pedigree play a role in phenotypic expression of LHON? Although there are ex­ceptions, the mutant mtDNA content does seem to correlate positively with disease expression ( 10). However, once a person becomes symptomatic, there does not appear to be any clinical difference in expression. Furthermore, hetero­plasmy is found in only a minority of LHON pedigrees and, therefore, not in the majority of unaffected maternal rela­tives worldwide. Although it is theoretically possible that unrecognized heteroplasmy exists in the optic nerves of these individuals despite homoplasmy in their accessibly tested tissues, this remains highly unlikely. Although some investigators have claimed that hav­ing multiple LHON- associated mtDNA mutations may be necessary for visual loss, case reports of unaffected indi­viduals who even harbor two primary mutations make this claim improbable ( 3,6,11). Similarly, although the underly­ing mtDNA haplotype may influence the presence, pen­etrance, or expression of a mtDNA point mutation ( 12), this is unlikely to be a major factor. Nuclear- encoded factors modifying mtDNA expression, mtDNA products, or mito­chondrial metabolism may be necessary for phenotypic ex­pression of LHON ( 13). Although most studies have not been able to confirm X- linkage as an explanation for the male predominance of the disease, the X- linked hypothesis may still be viable. ( 4) Mitochondrial energy production decreases with age, and the timing of visual loss in patients at risk for LHON may reflect the threshold at which already reduced mito­chondrial function deteriorates to a critical level ( 14). Im­munologic factors have also been proposed, especially to explain the association of LHON with multiple sclerosis ( 15), but conclusive evidence is lacking. Finally, environ­mental factors, both internal and external, may play a role. Systemic illnesses, nutritional deficiencies, medications or toxins that stress or directly inhibit mitochondrial metabo­lism could conceivably initiate or increase phenotypic ex­pression of the disorder. However, widespread nutritional deficiency in Cuba did not appear to increase the expression Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 258 © 2002 Lippincott Williams & Wilkins EDITORIAL J Neuro- Ophthalmol, Vol. 22, No. 4, 2002 of LHON in one large 11778- positive pedigree ( 16). Simi­larly, although anecdotal reports suggest a possible role for tobacco and excessive alcohol use as precipitants of visual loss, other studies, including one large case- control study of sibships ( 17), failed to confirm this. Indeed, despite the extraordinary advances in our un­derstanding of mitochondrial genetics and disease over the past decade, the underlying pathophysiological mecha­nisms linking the specific gene defect with the clinical manifestations of LHON remain unknown ( 18- 20). Theo­ries must reconcile how multiple different mtDNA muta­tions located in different genes encoding different proteins result in an essentially identical clinical phenotype that is expressed only in the optic nerve and with sudden and bi­lateral involvement. The obvious place to begin in theorizing about the pathogenesis of LHON is at oxidative phosphorylation and the generation of ATP. The three primary LHON mutations are found in genes that code for protein subunits of complex I of the respiratory chain. However, even demonstration of a definite deficiency of complex I activity and/ or ATP syn­thesis as measured by enzyme assay has proved problem­atic ( 18), especially with the 11778 mutation, the most com­mon and severe mutation in terms of visual prognosis. Fur­thermore, although the central nervous system is very reliant on mitochondrial ATP, studies of the bioenergetics of vision do not indicate that ganglion cell function is mark­edly dependent upon mitochondrial energy production ( 18). Indeed, it is the photoreceptor layer of the retina, rather than the retinal ganglion cell layer, that is richest in enzymes for oxidative metabolism ( 18). These observations have led some to speculate that the pathogenesis in LHON is not via direct effects on oxidative phosphorylation but rather via more indirect mechanisms. Complex I is the site of NADH reoxidation, and it may be that maintenance of the proper NAD/ NADH redox balance is particularly nec­essary for normal maintenance and function of the ganglion cells or their axons, especially for axonal transport ( 18,21). Transporter defects involving channels and pumps particu­larly reliant on mitochondrial ATP production could have specific tissue effects ( 20). Another avenue of investigation involves free radical damage ( 19,22- 24). Inhibition of the respiratory chain causes increased generation of free radi­cals. The respiratory chain may, in turn, be inhibited by oxi­dative damage. This self- amplifying cycle of oxidative damage and respiratory chain dysfunction could cause di­rect or indirect damage to vulnerable tissues such as the ganglion cell or its axon. The different mtDNA mutations may cause different amounts of free radical production at the level of complex I, and this increased production of re­active oxygen species may occur particularly in a neuronal cell environment ( 24,25). What are the subsequent mechanisms by which respi­ratory chain dysfunction or free radical production result in irreversible damage to the ganglion cells and their axons? Mitochondria play a major role in the induction of apoptosis ( 26). The collapse of the mitochondrial transmembrane po­tential and opening of the permeability transition pore leads to the release of proapoptotic proteins, including cyto­chrome c, and resultant cell death ( 19,27). Many physi­ologic and pathologic stimuli can directly trigger opening of the mitochondrial transition pore. Multiple consequences of mitochondrial dysfunction- collapse of the transmem­brane potential, uncoupling of the respiratory chain, hyper-production of superoxide anions, disruption of mitochon­drial biogenesis, outflow of matrix calcium and glutathione, and release of soluble intermembrane proteins- probably in­directly activate apoptosis or necrosis ( 27). Alternatively, excitotoxic death can be initiated directly by reduced mito­chondrial energy production, which leads to activation of the NMDA- type glutamate receptors ( 19). Free radicals are key components in some excitotoxicity pathways and may also trigger apoptotic cell death directly. The next step in theorizing how any of these proposed pathophysiologic processes result in selective damage to the optic nerve is fraught with even more uncertainty. Vari­ous mechanisms have been proposed, but supportive evi­dence is scarce. Based on the abnormal appearance of the papillary and peripapillary vasculature in many LHON pa­tients, a primary vascular process with subsequent ischemia has been proposed ( 28). However, vascular changes may be secondary, especially given the intact vascular endothelium as demonstrated by the lack of fluorescein leakage and the absence of fundus abnormalities in many patients. A further hypothesis is that the acute angle turn made by optic nerve axons in the prelaminar region creates a " chokepoint" region of impaired or labile axoplasmic trans­port ( 18). Small- caliber, constantly firing P- cells may be particularly vulnerable ( 29). Histochemical studies of the optic nerve in animals have shown a high degree of mito­chondrial respiratory activity within this unmyelinated, pre­laminar portion of the optic nerve, suggesting a particularly high requirement for mitochondrial function in this region ( 30- 32). Of great interest in this regard is the recent identi­fication of the causative gene product in autosomal domi­nant optic atrophy ( another inherited disorder that selec­tively involves the optic nerve) as a nuclear- encoded pro­tein destined for the mitochondria and important in the formation and maintenance of the mitochondrial network ( 33). Similarly, a recent genetically induced mouse model of complex I deficiency shows the histopathologic features of optic nerve degeneration and demyelination ( 34). These studies support the theory of a selective vulnerability of the retinal ganglion cell and its axon to defects in mitochon­drial function. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 259 JNeuro- Ophthalmol, Vol. 22, No. 4, 2002 EDITORIAL The two independent pedigrees described in the ar­ticle by Howell et al ( 6) both harbor two primary LHON mutations, the 14484 homoplasmic and the 11778 hetero-plasmic. This genetic " double whammy" has been reported in only two previous families ( 3,11). Yet even harboring two primary mutations, these pedigrees exhibit no excep­tional presentation of visual loss, including penetrance, the role of heteroplasmy, timing, severity, and even eventual recovery. Although one branch of the Baltimore pedigree has an extremely high incidence of a fatal infantile enceph­alopathy, the absence of this manifestation in other branches of the family and in the other pedigrees previously reported makes it unlikely to be a direct manifestation of the combination of the two primary LHON mutations. This branch of the Baltimore family is reminiscent of the Australian pedigree with maternally related LHON- like visual loss, movement disorders, and episodic infantile en­cephalopathy who harbor both the 14484 mutation and an ND1 mutation at mtDNA position 4160, the latter presum­ably responsible for the additional neurologic manifesta­tions ( 35). However, sequencing of the entire mtDNA of the Baltimore family, including those neurologically affected individuals, failed to reveal any variance in mtDNA to ex­plain these additional manifestations. Although the influ­ence of a nuclear DNA factor has been proposed, that is a somewhat unsatisfactory explanation for this highly pen­etrant phenotype in a single generation. How can we reconcile the observations of Howell et al. ( 6) with the various proposed theories of LHON patho­genesis? Clearly, having a primary mtDNA mutation asso­ciated with LHON, or even two, is not sufficient for the expression of the disease. Even if the two mutations com­bine to cause a more severe biochemical defect in mito­chondrial respiratory chain activity ( 11), this is not trans­lated into increased clinical severity, therefore suggesting a more indirect pathogenesis of optic nerve dysfunction. The mtDNA mutation, or mutations, may set the stage for visual loss, but unknown internal or external precipitants deter­mine its occurrence. Further elucidation of these triggers of the pathologic cascade in susceptible individuals will re­quire more genetic, biochemical, physiological, and patho­logic studies. Development of mouse models for LHON and other mitochondrial diseases should help focus our ef­forts toward directed therapies for both prevention and re­covery ( 34,36). [ See also Biousse et al. ( 37) in this issue.] REFERENCES 1. Wallace DC, Singh G, Lott MT, et al. Mitochondrial DNA mutation associated with Leber's hereditary optic neuropathy. Science 1988; 242: 1427- 30. 2. Mackey DA, Oostra RJ, Rosenberg T, et al. Primary pathogenic mtDNA mutations in multigeneration pedigrees with Leber heredi­tary optic neuropathy. Am J Hum Genet 1996; 59: 481- 5. 3. Riordan- Eva P, Sanders MD, Go van GG, et al. The clinical features of Leber's hereditary optic neuropathy defined by the presence of a pathogenic mitochondrial DNA mutation. Brain 1995; 118: 319- 37. 4. Man PY, Turnbull DM, Chinnery PF. Leber hereditary optic neu­ropathy. J Med Genet 2002; 39: 162- 9. 5. Huopenen K. Leber hereditary optic neuropathy: clinical and mo­lecular genetic findings. Neurogenetics 2001; 3: 119- 25. 6. Howell N, Miller NR, Mackey DA, et al. Lightning strikes twice: Leber hereditary optic neuropathy families with two pathogenic mtDNA mutations. J Neuro- Ophthalmol 2002; 22: 262- 269. 7. MITOMAP: a human mitochondrial genome database. Atlanta, GA: Center for Mol Med, Emory University. Available at: http:// www. mitomap. org. 8. Chinnery PF, Brown DT, Andrews RM, et al. The mitochondrial ND6 gene is a hot spot for mutations that cause Leber's hereditary optic neuropathy Brain 2001; 124: 209- 18. 9. Howell N, Bogolin C, Jamieson R, et al. mtDNA mutations that cause optic neuropathy: how do we know? Am J Hum Genet 1998; 62: 196- 202. 10. Chinnery PF, Andrews RM, Turnbull DM, et al. Leber hereditary optic neuropathy: does heteroplasmy influence the inheritance and expression of the G11778A mitochondrial DNA mutation? Am J Med Genet 2001; 98: 235- 43. 11. Brown MD, Allen JC, Van Stavern GP, et al. Clinical, genetic, and biochemical characterization of a Leber hereditary optic neuropathy family containing both the 11778and 14484 primary mutations. Am J Med Genet 2001; 104: 331- 8. 12. Brown MD, Starikovskaya E, Derbeneva O, et al. The role of mtDNA background in disease expression: a new primary mutation associated with Western Eurasian haplogroup J. Hum Genet 2002; 110: 130- 8. 13. Cock HR, Tabrizi SJ, Cooper JM, et al. The influence of nuclear background on the biochemical expression of 3460 Leber's heredi­tary optic neuropathy. Ann Neurol 1998; 44: 187- 93. 14. Trounce I, Byrne E, Marzuki S. Decline in skeletal muscle mito­chondrial respiratory chain function: possible factor in ageing. Lan-cet 1989; 1: 637- 9. 15. Bhatti MT, Newman NJ. A multiple sclerosis- like illness in a man harboring the mtDNA 14484 mutation. J Neuro- Ophthalmol 1999; 19: 28- 33. 16. Newman NJ, Torroni A, Brown MD, et al. Epidemic neuropathy in Cuba not associated with mitochondrial DNA mutations found in Leber's hereditary optic neuropathy patients. Am J Ophthalmol 1994; 118: 158- 68. 17. Kerrison JB, Miller NR, Hsu FC, et al. A case- control study of to­bacco and alcohol consumption in Leber hereditary optic neuropa­thy. Am J Ophthalmol 2000; 130: 803- 12. 18. Howell N. Leber hereditary optic neuropathy: respiratory chain dys­function and degeneration of the optic nerve. Vis Res 1998; 38: 1495- 504. 19. Schapira AH. Mitochondrial disorders: an overview. J Bioenerget-icsBiomemb 1997; 29: 105- 7. 20. Schon EA, Bonilla E, DiMauro S. Mitochondrial DNA mutations and pathogenesis. JBioenergetics Biomemb 1997; 29: 131- 49. 21. Levin LA, Schalow K. Differential susceptibility of retinal ganglion cells to redox modulation: a possible link to mitochondrial optic neuropathy [ abstract]. Invest Ophthalmol Vis Sci 1998; 39: S808. 22. Klivenyi P, Karg E, Rozsa C, et al. a- Tocopherol/ lipid ratio in blood is decreased in patients with Leber's hereditary optic neuropathy and asymptomatic carriers of the 11778 mtDNA mutation. J Neurol Neurosurg Psychiatry 2001; 70: 359- 62. 23. Kirkinezos IG, Moraes CT. Reactive oxygen species and mitochon­drial diseases. Seminars in Cell & DevBiol 2001; 12: 449- 57. 24. Wong A, Cavelier L, Collins- Schramm HE, et al. Differentiation-specific effects of LHON mutations introduced into neuronal NT2 cells. Hum Mol Genet 2002; 11: 431- 8. 25. Lodi R, Carelli V, Cortelli P, et al. Phosphorous MR spectroscopy shows a tissue specific in vivo distribution of biochemical expres­sion of the G3460A mutation in Leber's hereditary optic neuropa­thy. J Neurol Neurosurg Psychiatry 2002; 72: 805- 7. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 260 © 2002 Lippincott Williams & Wilkins EDITORIAL J Neuro- Ophthalmol, Vol. 22, No. 4, 2002 26. Danielson SR, Wong A, Carelli V, et al. Cells bearing mutations causing Leber's hereditary optic neuropathy are sensitized to Fas-induced apoptosis. J Biol Chem 2002; 277: 5810- 5. 27. Zamzami N, Hirsch T, Dallaporta B, et al. Mitochondrial implica­tion in accidental and programmed cell death: apoptosis and necro­sis. J Bioenergetics Biomemb 1997; 29: 185- 93. 28. Nikoskelainen EK, Huoponen K, Juvonen V, et al. Ophthalmologic findings in Leber hereditary optic neuropathy, with special refer­ence to mtDNA mutations. Ophthalmology 1996; 103: 504- 14. 29. Sadun AA, Win PH, Ross- Cisneros FN, et al. Leber's hereditary optic neuropathy differentially affects smaller axons in the optic nerve. Tr Am Ophth Soc 2000; 98: 223- 35. 30. Lessell S, Horovitz B. Histochemical study of enzymes of optic nerve of monkey and rat. Am J Ophthalmol 1972; 74: 118- 26. 31. Andrews RM, Griffiths PG, Johnson MA, et al. Histochemical lo­calisation of mitochondrial enzyme activity in human optic nerve and retina. Br J Ophthalmol 1999; 83: 231- 5. 32. Bristow EA, Griffiths PG, Andrews RM, et al. The distribution of mitochondrial activity in relation to optic nerve structure. Arch Ophthalmol 2002; 120: 791- 6. 33. Delettre C, Lenaers G, Pelloquin L, et al. OPA1 ( Kjer type) domi­nant optic atrophy: a novel mitochondrial disease. Mol GenetMetab 2002; 75: 97- 107. 34. Qi X, Lewin A, Guy J. Ribozymes against mitochondrial genes in­duce optic nerve degeneration in the mouse: an animal model for Leber hereditary optic neuropathy [ abstract]. Neurology 2002; 58( suppl 3): A507. 35. Howell N, Kubacka I, Xu M, et al. Leber hereditary optic neuropa­thy: involvement of the mitochondrial ND1 gene and evidence for an intragenic suppressor mutation. Am J Hum Genet 1991 ; 48: 935^ 12. 36. Wallace DC. Mitochondrial diseases in man and mouse. Science 1999; 283: 1482- 8. 37. Biousse VB, Pardue MT, Wallace DC, et al. The eyes of mito-mouse: mouse models of mitochondrial disease. J Neuro- Ophthalmol 2002; 22: 279- 85. Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 261