Journal of Neuro-Ophthalmology, September 1994, Volume 14, Issue 3

Cuban epidemic optic neuropathy.

OBJECTIVE: To search for mitochondrial DNA (mtDNA) mutations previously associated with Leber's hereditary optic neuropathy (LHON) in patients with an optic neuropathy that appeared in epidemic form in Cuba. METHODS: Twelve Cuban patients underwent a comprehensive neuro-ophthalmologic examination and were found to have a characteristic optic neuropathy, Cuban epidemic optic neuropathy (CEON). At the same time, one patient was diagnosed with typical LHON that occurred during the epidemic. Blood samples were taken from these patients as well as from 3 controls with normal neuro-ophthalmologic examinations. These samples were blindly analyzed for 9 LHON-associated mtDNA mutations by molecular genetic methods. RESULTS: CEON bore clinical and epidemiological similarity to LHON, however, family histories, systemic symptoms (especially weight loss and polyuria), and symptoms of peripheral neuropathy permitted a clinical distinction. None of the 12 patients with CEON or 3 controls had any of the LHON-associated mtDNA mutations. Only the patient with clinical LHON, who did not meet the case definition for CEON, harbored the 11778 mtDNA mutation. CONCLUSIONS: Known mtDNA mutations are not found frequently in CEON patients but they may contribute to some cases of Cuban optic neuropathy. CEON may represent an acquired variety of mitochondrial dysfunction induced by nutritional deficiencies, toxins, or both. Alternatively, CEON patients may also harbor as yet undiscovered mtDNA mutations that contribute to their genetic susceptibility.

journal of Ncuw- OphthalinohM 14(. V: 1M)~ B4, 1994 "'•' 1994 Raven Press, Ltd., New York Cuban Epidemic Optic Neuropathy Mitochondrial DNA Analysis Donald R. Johns, M. D., and Alfredo A. Sadun, M. D., Ph. D. Abstract: Objective: To search for mitochondrial DNA ( mtDNA) mutations previously associated with Leber's hereditary optic neuropathy ( LHON) in patients with an optic neu­ropathy that appeared in epidemic form in Cuba. Methods: Twelve Cuban patients underwent a com­prehensive neuro- ophthalmologic examination and were found to have a characteristic optic neuropathy, Cuban epidemic optic neuropathy ( CEON). At the same time, one patient was diagnosed with typical LHON that occurred during the epidemic. Blood samples were taken from these patients as well as from 3 controls with normal neuro- ophthalmologic examinations. These sam­ples were blindly analyzed for 9 LHON- associated mtDNA mutations by molecular genetic methods. Results: CEON bore clinical and epidemiological sim­ilarity to LHON, however, family histories, systemic symptoms ( especially weight loss and polyuria), and symptoms of peripheral neuropathy permitted a clinical distinction. None of the 12 patients with CEON or 3 controls had any of the LHON- associated mtDNA mu­tations. Only the patient with clinical LHON, who did not meet the case definition for CEON, harbored the 11778 mtDNA mutation. Conclusions: Known mtDNA mutations are not found frequently in CEON patients but they may con­tribute to some cases of Cuban optic neuropathy. CEON may represent an acquired variety of mitochondrial dys­function induced by nutritional deficiencies, toxins, or both. Alternatively, CEON patients may also harbor as yet undiscovered mtDNA mutations that contribute to their genetic susceptibility. Key Words: Optic neuropathy- Mitochondrial DNA- Leber's hereditary optic neuropathy- Toxic-nutritional amblyopia. From the Departments of Neurology and Ophthalmology, Harvard Medical School, Beth Israel Hospital, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts, and the Depart­ments of Ophthalmology and Neurosurgery, University of Southern California School of Medicine, Doheny Eye Institute, Los Angeles, California, U. S. A. This work was supported by grant NS 01359 from the Na­tional Institute of Neurological Disorders and Stroke, Bethesda, Maryland. Dr. Johns is the recipient of a Clinical Investigator Development Award from the National Institute of Neurologi­cal Disorders and Stroke and of Basil O'Connor Starter Scholar Research Award No. 5- FY93- 1049 from the March of Dimes Birth Defects Foundation, White Plain, New York. Address correspondence and reprint requests to Dr. Donald R. Johns, Harvard Medical School, Building Bl- 242, 220 Long-wood Avenue, Boston, MA 02115, U. S. A. In the spring of 1992 there was mounting evi­dence of an epidemic in Cuba of an optic neurop­athy characterized by bilateral subacute loss of vi­sion, dyschromatopsia, central or cecocentral sco­tomas, and, in a few cases, subjective neurologic complaints such as numbness and paresthesias. Initially, about 90% of the patients were heavy smokers and 90% were adult men ( 1- 3). However, over the next few months these risk factors became less prominent. As more of the patients mani­fested symptoms of peripheral neuropathy, the Cuban Ministry of Health began describing two forms of the epidemic condition: primary optic ( neuropathy) and peripheral ( neuropathy). By December 1992, men constituted only 70% of the patients, and about 75% of the cases were of the " optic form" ( 1- 3). An analysis of risk factors from 240 patients presumed to have the epidemic disease showed that the most prominent risk fac­tors were cigarette smoking and rum drinking. No other environmental toxins were identified as risk factors for the epidemic ( 1). Deficiencies in folic acid, vitamin B,, vitamin B6, and vitamin B12 were also noted. However, blood samples from the non-involved spouses of these patients also had similar decreases in vitamin levels ( Personal communica­tion, Drs. J. Teja Perez, R. Rodriguez, R. Perez Cristia, L. Herrera, and G. Kouri at Biotechnical Center, Havana, Cuba on May 16, 1993). Virologic studies failed to support a viral etiology. By the time we visited Cuba for the second time ( September 1993), almost 50,000 cases of blindness from the epidemic had been reported ( 2). It was hypothesized that vitamin deficiencies ( probably folic acid and vitamin B12), acting in synergy with environmental toxins ( such as trace amounts of methanol found in some home- brewed rum), were playing an etiologic role. Impairment of mitochon­drial oxidative phosphorylation by these a n d other factors were postulated to produce an optic neu­ropathy resembling Leber's hereditary optic neu­ropathy ( LHON) that we term Cuban epidemic op­tic neuropathy ( CEON) ( 2). LHON and CEON 130 CUBAN EPIDEMIC OPTIC NEUROPATHY: mtDNA 131 share several clinical and epidemiologic features, including the painless loss of central vision with cecocentral scotomas and dyschromatopsia, selec­tive loss of the papillomacular bundle, a marked male predominance, and an association with alco­hol and tobacco use ( 1- 10). The majority of pa­tients with LHON harbor one of several mitochon­drial DNA ( mtDNA) mutations that have been pathogenetically associated with it ( 6- 9,11- 12). We therefore searched for LHON- associated mtDNA mutations in a representative group of well- characterized Cuban patients with optic neu­ropathy. SUBJECTS AND METHODS Twenty- one hospitalized patients were pre­sented ( to AAS) as typical cases of CEON. System­atic and comprehensive neuro- ophthalmologic ex­aminations, including psychophysical testing, were performed ( 2). All had assessments of visual acuity, pupils, and color vision. Neurologic exam­inations were performed and ocular motility mea­sured. Amsler grid and tangent field testing was performed. Funduscopy and fundus photographs were obtained with a customized portable Kawa camera. Of the 21 patients, 6 were found to have diseases other than CEON and blood samples were obtained from 12 of the 15 patients who met the case definition of CEON ( 2). While examining the hospitalized patients, a Cuban patient pre­sented to the outpatient department and was di­agnosed with probable LHON. Blood samples were obtained from these 13 pa­tients by fingerstick with blood drops being ab­sorbed in 8 to 10 zones onto filter paper. Once this had thoroughly dried, each filter paper was indi­vidually wrapped and isolated. Additionally, blood samples were similarly taken from three pa­tients without ophthalmologic or neurologic com­plaints and with normal neuro- ophthalmologic ex­aminations. All 16 sets of filter paper were coded, masked, and sent for mtDNA analysis. Blood was eluted from the dried blood spots and total DNA was extracted by standard proteinase K/ SDS methods. This DNA was then used as the template DNA for amplification of eight specific mtDNA regions by the polymerase chain reaction. The oligonucleotide primers and restriction endo-nuclease digestions were as described previously for mtDNA mutations at the following nucleotide positions: 3394, 3460, 11778, 13708, 14484, 15257, 15812 ( 13) and 9438 and 9804 ( 12). RESULTS Mitochondrial DNA Findings None of the 12 patients with CEON harbored an LHON- associated mtDNA mutation. The 11778 mutation, which is the most prevalent primary mtDNA mutation in LHON, was present in ho-moplasmic form in the patient with LHON clini­cally. None of the other 8 LHON- associated mtDNA mutations was found in any of the 12 CEON patients, the patient with LHON, or in the 3 Cuban controls. Clinical and Epidemiologic Findings Although the samples were sent blindly for mo­lecular genetic analysis, the patient with the 11778 mutation was identified prospectively on clinical grounds as being different from the other 12 Cu­ban optic neuropathy patients. We will first present the clinical and epidemiologic findings from the 12 CEON patients and then contrast them with those seen in the 11778 mutation- positive Cu­ban patient. Seven ( 58%) of the CEON patients consumed either homemade wine or homemade rum, or regularly ate cassava. At the time of diag­nosis, the 12 CEON patients had suffered an aver­age weight loss of 18 pounds. Most drank alcohol heavily, smoked tobacco heavily, or did both. A number of systemic symptoms were frequently noted, including polyuria ( 75%), fatigue ( 67%), and hearing loss ( 17%). None of the CEON pa­tients had any signs or symptoms of congestive heart failure. Most patients had visual acuities in the range of 20/ 400 in both eyes. All had severe dyschromatop­sia ( average number of AOHRR color plates cor­rectly identified slightly less than 2 of 8), and many had sluggish pupillary reflexes. Ocular motility demonstrated cogwheeling smooth pursuits. Am­sler grid or tangent field testing demonstrated very dense central or cecocentral scotomas. On fundus examination mild temporal pallor of the optic discs and a wedge of severe nerve fiber layer loss in the papillomacular bundle were noted. Many patients also demonstrated opacification and marked swelling of nerve fibers immediately above and below the papillomacular area. On neu­rologic examination, decreased sensation to pin prick and vibration was noted in 3 ( 25%) CEON patients. Subjective dysesthesias and paresthesias were also described by 5 ( 42%) patients. None of the 12 CEON patients had a positive family history of visual loss and none had parents, siblings, or children affected with the epidemic / Neuro- Ophthalmol, Vol. 14, No. 3. 1994 132 D. R. JOHNS AND A. A. SADUN disease, except for one man whose sister was sim­ilarly affected. Although the brother and sister lived apart, they consumed about 2 to 3 bottles per week of home brewed rum from the same source that was found to contain about 1% methanol ( 2). The CEON patients showed dramatic visual re­sponse to treatment with B complex vitamins and folic acid with the average visual acuity improving to about 20/ 50 and color plate identification im­proving to an average of 7 of 8 ( 2). One patient had a clinical presentation that was distinctive from the remainder of those in the ep­idemic. A 23- year- old man had painless visual loss in one eye about 3 months earlier and then in the second eye 1 week before presentation. There was no history of weight loss, fatigue, polyuria, or any neurologic symptoms typical for the epidemic. He did not smoke and drank rum ( not home- brewed) only occasionally. He did not regularly eat cassava. His neuro- ophthalmologic examination showed bi­lateral decreased visual acuities ( 20/ 200 to 20/ 400) and severe dyschromatopsia ( 0 of 9 AOHRR color plates OU) in association with central scoto­mas and defects of the papillomacular bundle. However, he did not have cogwheeling smooth pursuits nor any of the neurologic signs associated with the epidemic. He described a maternal uncle who lost vision in both eyes decades earlier for no apparent reason, but he denied any relatives who were affected during the epidemic. He was af­fected in the second eye only 1 week prior to ex­amination, and therefore visual recovery could not be assessed. DISCUSSION Our clinical and molecular genetic findings in this group of Cuban optic neuropathy patients in­dicate that the epidemic form of optic neuropathy in Cuba ( CEON) is similar to, yet remains distin­guishable from typical LHON. Subacute painless, central visual loss, in association with selective and severe loss of the papillomacular bundle, is the central feature of both CEON and LHON. The ad­jacent nerve fiber layer often shows subtle inflam­matory changes in LHON and more obvious marked swelling in CEON. Both show a marked male predominance and are associated with expo­sure to tobacco and alcohol. However, CEON may be distinguished from LHON by the occurrence of prodromal symptoms ( e. g., weight loss, fatigue, polyuria) and the fre­quency of neurologic accompaniments ( e. g., deaf­ness, peripheral neuropathy). CEON is also re­markable for the high incidence of associated risk factors: all 12 CEON patients had suffered severe dietary restrictions as well as exposure to alcohol, tobacco, or cassava. Cogwheeling smooth pursuit movements were also unique to CEON patients. The marked propensity for prompt visual recovery in CEON patients after vitamin therapy and the successful prevention of CEON with prophylactic vitamins is also distinctive and supports a nutri­tional etiology for the epidemic. Visual recovery does occur in patients with LHON, but only a mi­nority of patients recover and they do so only after several months of stable visual loss ( 7- 9,14- 15). A positive family history of visual loss in a maternal relative was only obtained in our case of LHON. The single LHON patient also differed from the CEON group in not having suffered from polyuria or marked weight loss. LHON is further character­ized by the high incidence of an associated mtDNA mutation [ 84% of our LHON families harbor a known primary LHON- associated mtDNA muta­tion ( 12)] and by the infrequent occurrence of dis­ease outside of the optic nerve. Unusual cases of LHON do exhibit peripheral ( 8) or central nervous system involvement ( 16) and these manifestations differ among the various LHON- associated mtDNA mutations. The presentation of a typical LHON patient in the midst of an epidemic of optic neuropathy illus­trates the diagnostic difficulties that arise in the evaluation of patients during such an epidemic. Is the occurrence of symptomatic LHON during the epidemic simply a coincidence or is there a plau­sible mechanism that provides a pathogenetic ex­planation? LHON patients may be more likely to be ascertained as part of the Cuban epidemic both because of the heightened interest in and screen­ing for optic neuropathy, and because of epige-netic factors that might render these genetically at- risk patients symptomatic. The timing of the symptomatic occurrence of LHON may have been influenced by the environmental factors triggering the epidemic. A brief review of our current conceptualization of the pathogenesis of visual loss in LHON may clarify the potential relationship of CEON and LHON. Visual loss in LHON appears to occur as a consequence of a complex interplay between ge­netic factors ( primary and secondary mtDNA mu­tations, possibly nuclear DNA mutations) and epi-genetic risk factors ( 6- 11,17- 19). Potential risk fac­tors in LHON that have been identified to date include alcohol and tobacco abuse, vitamin and nutritional deficiency, diabetes mellitus, occupa­tional exposure, and head trauma ( 6- 10,18- 19). In this schema, mtDNA mutations render an individ- / Neuw- Ophlhalmol, Vol. 14, No. .3, 1994 CUBAN EPIDEMIC OPTIC NEUROPATHY: mtDNA 133 ual genetically susceptible to visual loss but they are not the sole determinants. The interaction of genetic and epigenetic factors is demonstrated by the analysis for LHON-associated mtDNA mutations in 12 patients diag­nosed with tobacco- alcohol amblyopia in another study ( 10). Two ( 17%) of these patients harbored a known primary LHON- associated mtDNA muta­tion ( 10). Further evidence for the importance of epigenetic factors is evident in the existence of identical twins who are discordant for visual loss in LHON ( 19). The patients harbored the 11778 mtDNA mutation in homoplasmic form and were proven by zygosity analysis to have the same nu­clear DNA and thus were shown to harbor the identical genetic constitutions. Despite this genetic identity, one brother became permanently blind, while his identical twin brother remained visually normal 6I/ 2 years later ( 19). While it seems unlikely that such a large number of Cuban patients would suddenly manifest a ge­netic disease, it is possible that a mtDNA mutation might be present in a portion of the Cuban popu­lation. In fact, the Cuban epidemic was character­ized by its nonuniform occurrence and it never affected more than 0.5% of the entire Cuban pop­ulation ( 1). One explanation of the selective vul­nerability of only a minority of the Cuban popula­tion is that these individuals harbor an mtDNA mutation that would not be manifest except under the additional burden of further insults to their mitochondria. In a study of mtDNA mutations in a different population of Cuban optic and peripheral neuropathy patients, 2 ( 22%) of 9 patients har­bored cytochrome c oxidase mtDNA mutations ( 20). Both of these patients had prominent periph­eral nerve involvement. Environmental factors, such as nutritional defi­ciencies or extrinsic toxins, might cause decom­pensation of an intrinsic genetic disorder. Potential toxins that have been implicated in CEON include tobacco, ethanol, methanol ( as a contaminant in home- brewed spirits), and cassava ( which con­tains cyanide). Each of these substances may im­pair mitochondrial oxidative phosphorylation and thus both genetic and epigenetic factors may inter­act to impair mitochondrial production of adeno­sine triphosphate ( ATP). Axonal transport is ex­tremely ATP- dependent ( 21- 22). Interference with the production of ATP could impair axonal trans­port in the optic and peripheral nerves and cause optic and peripheral neuropathy. Only a limited number of Cuban disease and control patients were available for clinical and mo­lecular genetic study and the epidemiologic limita­tions of any conclusions drawn from our data must be explicitly recognized, particularly with respect to an epidemic of these proportions. Our sample of patients with CEON demonstrated a clinical pic­ture quite similar to that of LHON, which may reflect the common underlying feature of mito­chondrial dysfunction. In this regard, the frequent occurrence of hearing loss in CEON is reminiscent of that seen in a number of different molecularly verified mitochondrial disorders ( 23). However, the data in the present study demonstrate that most CEON patients do not harbor a known LHON- associated mtDNA mutation. In CEON it is possible that the cumulative effect of multiple del­eterious factors results in an acquired mitochon­drial deficiency that phenotypically resembles LHON. 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