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Show Journal of Neuro- Ophtlwlmology 14( M: 135- 140, 1994. © 1994 Raven Press, Ltd., New York Mitochondrial DNA Mutations in Cuban Optic and Peripheral Neuropathy Donald R. Johns, M. D., Michael J. Neufeld, and Thomas R. Hedges III, M. D. Abstract: Objective: To investigate the potential role of mitochondrial DNA ( mtDNA) mutations in the recent outbreak in Cuba of optic neuropathy and peripheral neuropathy ( COPN), Design and Methods: Historical features were reviewed and neuro- ophthalmologic examinations were performed on a sample of COPN patients (/; = 9) and Cuban patients with other forms of optic neuropathy ( n = 2). Molecular genetic methods were then used to test for the presence of 9 mtDNA mutations that were previously associated with Leber's hereditary optic neuropathy ( LHON). Results: Two ( 22%) of 9 COPN patients harbored an LHON- associated mtDNA mutation at nucleotide position 9438 and a novel mutation at nucleotide position 9738 in the cytochrome c oxidase subunit III gene. None of the Cuban patients harbored any of the 8 other LHON- associated mtDNA mutations. Detailed sequence analysis revealed that the Cuban patients could be divided into 7 distinct mtDNA haplotypes and that the 2 COPN patients with mtDNA mutations in the cytochrome c oxidase subunit III gene were not members of the same maternal lineage. Conclusions: The pathogenesis of epidemic COPN is likely complex and multifactorial. Our preliminary results in a small sample of Cuban patients suggest that mtDNA mutations may play a role in some cases. mtDNA mutations may render an individual genetically From the Departments of Neurology and Ophthalmology, Harvard Medical School, Beth Israel Hospital, and the Massachusetts Eye and Ear Infirmary ( D. R. J.); and the Departments of Ophthalmology and Neurology, Tufts University School of Medicine and the New England Eye Center ( T. R. H.), Boston, Massachusetts, U. S. A. This work was supported by grant NS 01359 from the National Institute of Neurological Disorders and Stroke, ( Be-thesda, 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 Plains, 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. susceptible to a variety of factors that impair oxidative phosphorylation, including nutritional deficiency, tobacco, alcohol, and other toxins. Key Words: Optic neuropathy- Mitochondrial DNA- Leber's hereditary optic neuropathy- Toxic- nutritional amblyopia An outbreak of optic and peripheral neuropathy was first noted in Cuba in late 1991 and by June 1993 had affected more than 43,000 people ( 1). The predominant clinical manifestations are decreased visual acuity and central scotomas associated with specific dropout of the papillomacular nerve fibers in a progressive and usually symmetrical manner, as well as lower extremity sensory symptoms ( paresthesias, dysesthesias, and numbness). The initial cases were noted in the Pinar del Rio region of Cuba among adult men who drank alcohol and smoked tobacco. However, the syndrome was subsequently noted throughout Cuba in both men and women and in patients who did not smoke or drink. Leber's hereditary optic neuropathy ( LHON) is a maternally inherited form of acute visual loss that predominantly affects young adult men ( 2). LHON was the first human disease linked to an inherited mutation in mitochondrial DNA ( mtDNA) ( 3). A number of mtDNA mutations have been pathoge-netically linked to LHON ( 4). Genetic factors alone do not account for the clinical course of LHON and the visual loss may be caused by a complex interplay of epigenetic and genetic factors ( 5- 10). The epigenetic factors that may play a role in the clinical expression of the mtDNA mutations include alcohol abuse, tobacco use, and vitamin and nutritional deficiencies. 135 136 D. R. JOHNS ETAL. Due to the clinical and epidemiologic similarity of Cuban optic and peripheral neuropathy ( COPN) and LHON, we investigated the role of mtDNA mutations in the pathogenesis of COPN by using molecular genetic methods. DESCRIPTION OF PATIENTS One of us ( TRH) examined and obtained blood samples from nine Cuban patients with suspected COPN and two Cuban patients who had been referred with suspected COPN but were subsequently determined to have other conditions. The medical records of the patients were reviewed, a brief history was obtained, visual fields were performed at a tangent screen, and funduscopic examinations were performed with white and red free light. Visual evoked potential recordings that had been obtained were reviewed by Samuel Sokol, Ph. D. These 11 patients were among 20 presented to us by Cuban physicians from patients who were being evaluated in the Instituto Oftal-mologico " Ramon Pando Ferrer," the Hospital CQ " Hermanos Ameijeiras," and the Hospital Pinar del Rio in June 1993. Time constraints were such that only 11 patients were available for complete examination and blood drawing. Evaluation of seven patients confirmed the presence of bilateral, subacute or progressive optic neuropathy involving the papillomacular bundle ( COPN 1- 7), two of whom had mild peripheral neuropathy as well ( COPN 2 and 4). Two other patients had predominantly a peripheral neuropathy, one with mild visual complaints ( COPN 8), and one who denied visual problems ( COPN 9). COPN 1- 8 had very poor diets, and many had lost weight ( Tables 1 and 2). Two other patients were examined, one with typical demyelinating optic neuritis and another with inconsistent visual findings suggestive of hysteria and malingering. All patients had been treated with vitamins prior to the ophthalmologic examination. MOLECULAR GENETIC METHODS We initially searched for nine mtDNA mutations that were previously associated with LHON. Total DNA was extracted from dried blood spots obtained in the field in Cuba. This DNA was then used as the template DNA for polymerase chain reaction amplification of the mtDNA region of interest. The oligonucleotide primers, restriction enzyme digestions, and molecular genetic confirmation methods used were as previously described ( 11) for the following nucleotide positions: 3394, 3460, 11778, 13708, 14484, 15257, 15812. The two most recently discovered mutations in the cytochrome c oxidase subunit III ( COX III) gene were detected as follows: 9438 ( G78S) ( Stu I site loss) and 9804 ( A200T) ( Mae III site gain) ( 12). After two independent COPN probands were found to harbor the 9438 mtDNA mutation in the COX III gene, the entire COX III gene was sequenced in all 11 Cuban patients using the amplification and sequencing strategy previously described ( 12) by direct sequence analysis of polymerase chain reaction- amplified mtDNA. TABLE 1. Clinical history in Cuban optic and peripheral neuropathy patients Age 23 43 60 30 53 38 51 44 51 Sex M M M M M F M M F Occupation Laborer Dancer Contractor NA Metal worker Social worker Farmer Farmer Homemaker Initial symptoms/ progress Blurring 11/ 92- 3/ 93 Blurring 12/ 92- 1/ 93 Blurring 2/ 92- 3/ 92 Blurring 11/ 92- 12/ 92 Blurring 3/ 93 Leg numbness 3/ 93 Blurring 6/ 93- 7/ 93 Blurring 10/ 92 Leg pain, numb feet 4/ 92 Other symptoms NA Legs numb, cramps 3/ 92 NA Leg cramps Stomach pains, cramps Arm and leg cramps, ataxia Hearing loss, leg numbness NA Loss of contrast 11/ 92 Diet Poor Poor Poor Poor Poor Good Poor Poor Poor Weight loss Some NA 40 lb NA 28 lb 9 lb NA None 60 lb Tobacco No 1/ 2 ppd 1 ppd NA 1/ 2 ppd 1/ 4 ppd 5 cigars/ day 1 ppd 1 ppd Alcohol ( rum) 1/ 4 L/ day No Social NA Social Social No Yes No Treatment Oral vitamins Oral vitamins IV vitamins Oral vitamins Oral vitamins Oral vitamins Oral vitamins Oral vitamins IV vitamins Improvement Moderate Slight Moderate NA Mild None Moderate Moderate Vision only NA, not available; ppd, packs of 20 cigarettes per day; Pt no., COPN patient number; IV, intravenous hydrocobalamin, lb, pound; L, liters. | Nniro- Ophllmlimil, Vol. 14. No. .1, 1994 mtDNA IN CUBAN OPTIC NEUROPATHY 137 TABLE 2. Clinical findings in Cuban optic and peripheral neuropathy patients Pt. no. 1 2 3 4 5 6 7 8 9 Metric acuity 0.2, 0.1 0.08, 0.08 0.5, 0.6 0.3, 0.3 0.8, 0.8 1.0, 1.0 0.2, 0.2 0.2, 0.4 1.0, 1.0 Color defect NA Red- green Red- green NA Red- green None Red- green Red- green Red- green Ophthalmic Visual field abnormalities Central scotomas Central scotomas Central scotomas Central scotomas Central scotomas Normal Central/ peripheral loss Central scotomas Normal findings Fundi m- p NFL loss m- p NFL loss m- p NFL thin m- p NFL loss m- p NFL thin Normal m- p NFL loss m- p NFL thin m- p NFL thin VEP Abn both eyes Abn both eyes Long latency NA Mild abn NA Abn both eyes NA Mild abn Sensory Normal Loss in legs Normal Normal Normal Loss in legs Loss in legs Normal Loss in legs Neurologi Motor Normal Normal Normal Normal Normal Normal Normal Normal Normal c findings Other Normal Normal Normal Normal Normal Abn gait Hearing loss Normal Normal mtDNA mutations None None None None None 9438/ 9738 None None 9438/ 9738 m- p NFL, maculopapillary nerve fiber layer; NA, not available; abn, abnormal; VEP, visual evoked potential. Color vision tested with Ishihara or American Optical Hardy- Rand- Rittler plates. RESULTS Two of the nine COPN probands harbored the 9438 mtDNA mutation. None of the 11 Cuban patients had any of the eight LHON- associated mutations. Direct sequence analysis of the COX III gene revealed that both COPN probands with the 9438 mutation also harbored a missense mutation ( i. e., a mutation that results in the substitution of one amino acid for another) at nucleotide position 9738 that resulted in the substitution of threonine for evolutionarily conserved alanine at amino acid position 178 ( A178T) in the COX III gene ( Table 3). Although no other missense mutations were found in the COX III gene among the other Cuban patients, six silent mutations ( i. e., mutations that do not alter the encoded amino acid) were detected that allowed the patients to be divided into seven distinct mtDNA haplotypes ( Fig. 1). Four of these TABLE 3. Evolutionary conservation of Cox III mutations Species Man ( optic neuropathy) Man ( Cambridge)" Cow Mouse Rat Chicken Frog ( Xenopus laevis) Carp Fruit fly ( Drosophila yakuba) Sea urchin ( Paracentrotus lividus) Ref." 13 14 15 16 17 18 19 20 21 AA residue no. 78 9438 position S G G G G G G G G G AA residue no. 178 9738 position T A A A A A A A A A S, serine; T, threonine; G, glycine; A, alanine. a Cambridge refers to the canonical human mtDNA sequence ( 13). 6 mtDNA sequences. silent mutations were uniquely associated with the Cuban samples, and two had been noted earlier ( 12). Direct sequence analysis of the mtDNA non-coding D- loop of patients COPN 6 and COPN 9 indicated that they shared eight mutations but differed at two nucleotide positions ( Neil Howell, Ph. D., unpublished observation). DISCUSSION The pathogenesis of the recent outbreak of optic neuropathy and peripheral neuropathy in Cuba is likely to be complex and multifactorial. Two of nine independent COPN probands harbored an mtDNA mutation, at nucleotide position 9438 in the COX III gene, which had been previously noted only in five Caucasian LHON probands ( 12). This mutation has not been previously seen in more than 800 Caucasian controls ( 22), causes the mutation of a highly conserved amino acid residue, and is therefore likely to be pathogenetically significant. The second missense mutation in the COX III gene, at nucleotide position 9738, that was also harbored by probands COPN 6 and COPN 9 was subsequently found in 2 of 5 LHON probands with the 9438 mutation ( Donald R. Johns and Michael J. Neufeld, unpublished observation). Our data are limited to a small number of COPN probands and other Cuban patients and the prevalence of the LHON- associated mtDNA mutations in the Cuban population is unknown. However, out preliminary data suggest that mtDNA mutations may play a pathogenetic role in some cases of COPN. A brief review of the clinical and epidemiologic features of LHON may help clarify the potential role of mtDNA mutations in COPN. Pathogenetic mtDNA mutations explain the strict maternal inheritance of LHON but do not account for a number of other clinical features, such as the marked / Neuro- Ophthalmol, Vol. 14, No. 3, 1994 138 D. R. JOHNS ET AL. 9540 + WT COPN- 2- 5,7,8 CTL- 2 9221 + COPN- 2,3,4 WT COPN- 1,6,9 CTL- 1 9438 9738 , , COPN- 5,7,8 CTL- 2 COPN- 6,9 WTWT COPN- 1 CTL- 1 9647 9758 + + WTWT 9950 WT COPN- 7 COPN- 5,8 CTL- 2 COPN- 1 CTL- 1 9545 + WT CTL- 2 COPN- 5,8 FIG. 1. Haplotype analysis of Cuban mitochondrial DNA. Sequence analysis of the COX III gene revealed 8 point mutations that allowed the 11 patients ( 9 COPN patients and 2 non- COPN Cuban patients) to be divided into 7 independent mtDNA haplotypes. The highlighted box contains the 2 COPN patients who harbor the missense mutations. The numerals refer to the nucleotide position of each mtDNA mutation according to the canonical Cambridge human mtDNA sequence ( 13). COPN, Cuban optic neuropathy and peripheral neuropathy patients; CTL, non- COPN Cuban control patients; +, mutation present; WT, wild type, mutation absent. male predominance in a predominantly Caucasian population and the acute nature of the visual loss after a long latency period ( mean age at onset: 28) ( 8). The core clinical feature of LHON is painless, subacute loss of central vision. Groups of patients with each pathogenetic LHON- associated mtDNA mutation have distinguishing clinical features that allow a genotype- specific LHON phenotype to be formulated for each mutation ( 5- 9). Among the mutations studied to date, the phenotype associated with the 15,257 mutation, in the apocy-tochrome b gene, most closely resembles that seen in COPN ( 7). In both groups of patients the optic neuropathy is frequently associated with a peripheral neuropathy ( 1,7). One of the Cuban patients who harbored the COX III mutations ( COPN 9) had predominantly a peripheral neuropathic presentation, but none of the LHON- associated mtDNA mutations has been systematically screened for in patients with peripheral neuropathy. An LHON- associated mtDNA mutation renders an individual genetically susceptible to visual loss but is not the sole determinant. The acute precipitation of optic neuropathy and visual loss in LHON patients is not well understood, but a number of epigenetic factors may contribute ( 5- 10). These factors include alcohol and tobacco use ( 23), nutritional and vitamin deficiencies, diabetes mel-litus, environmental exposures ( 24), and head trauma. The importance of nongenetic factors is illustrated by a pair of identical twin brothers who harbor the identical homoplasmic 11,778 mtDNA mutation. One brother became blind 6V2 years ago, while his twin has remained visually normal ( 24). In a study of LHON- associated mtDNA mutations in patients diagnosed as having tobacco-alcohol amblyopia, we found that 2 ( 17%) of 12 patients harbored such a mutation ( 23). The pathogenesis of tobacco- alcohol amblyopia is unknown, but may involve nutritional and vitamin deficiencies, direct toxic effects of the substances, or both. Alcohol and tobacco were suspected to contribute to COPN, and most of the patients in the early phase of the epidemic consumed both. One toxin that has been postulated to contribute to the deleterious effects of tobacco smoke is cyanide, which is a potent inhibitor of the cytochrome c oxidase component of the electron transport chain ( 25). Cyanide was suspected as a contributing factor in COPN because it is present in both tobacco smoke and the cassava plant, which is consumed frequently in Cuba. The presence of mtDNA mutations in the COX III gene, one of the three mito-chondrial- encoded subunits of cytochrome c oxidase, could render an individual more vulnerable to toxins that interfere with oxidative phosphorylation. Nutritional and vitamin deficiencies may be operative in COPN patients, due to shortages of food I Neuro- Ophlhahnol, Vol. 14, No. .3, 1994 mtDNA IN CUBAN OPTIC NEUROPATHY 139 exacerbated by geopolitical forces, including loss of trade with the former Soviet Union and the United States trade embargo ( 26). Deficiencies of calories, protein, and water- soluble and fat- soluble vitamins have been documented among COPN patients ( 1,26). The presence of a variety of mtDNA mutations could render a subset of the population genetically susceptible to COPN and thus explain the nonuniformity of the epidemic. MtDNA analysis of the Cuban patients revealed that seven independent Cuban mtDNA haplo-types could be distinguished based on the complete sequence of the COX III gene from each patient ( Fig. 1). This indicates that there are a variety of mtDNA backgrounds in this population and that the COPN patients do not appear to have a recent, common maternal ancestor. The two Cuban patients ( COPN 6 and COPN 9) who harbor missense mutations in the COX III gene cluster within the same mtDNA haplotype, but further mtDNA sequence analysis revealed that they are not members of the same maternal lineage. The COX III mutations at nucleotide positions 9438 and 9738 are present in two maternally unrelated COPN probands, and thus these mutations have arisen independently on two distinct Cuban mtDNA backgrounds. The etiology of COPN is unknown, but our preliminary evidence suggests that inherited mtDNA mutations may play a pathogenetic role in some cases. Analysis of mtDNA mutations in patients suspected of having COPN might be considered a way of distinguishing this group of patients from the majority of patients in the epidemic, just as one might exclude those patients with demyelinating optic neuritis. However, we feel that the occurrence of these mtDNA mutations suggests a common underlying pathophysiologic abnormality in oxidative phosphorylation. mtDNA mutations may render some patients within the Cuban population genetically susceptible to several factors that inhibit oxidative phosphorylation and cause specific dysfunction within retina ganglion cells and peripheral nerves. The relative role of these inhibitors may vary from patient to patient and may include nutritional deficiency, inability to detoxify cyanide from tobacco or certain foods, or an unidentified neurotoxin. Only a small number of COPN patients and Cuban controls were available for clinical and molecular genetic analysis. Therefore any preliminary conclusions based on our data, particularly regarding an epidemic of this magnitude, must be drawn with caution and explicit recognition of the limitations imposed by such a small sample size. Further studies of epidemiologic, clinical, and molecular genetic factors in a larger number of COPN patients are needed to elucidate the relative importance of these factors in this disease. Additional molecular genetic data are also needed about mtDNA and its variant in the Cuban population to verify that there is an increased prevalence of the COX III mutations in the COPN population. Acknowledgments: We would like to acknowledge Ms. Mary Murray and the U. S. + Cuba Medical Project ( 198 Broadway, New York, N. Y.) for arranging the visit to Cuba; Dr. Samuel Sokol, New England Eye Center for reviewing the visual evoked potential data; Professor Marcelino Rios and Dra. Carolina Salazar Campos, Institute Oftalmologico " Ramon Pando Ferrer"; Dra. 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