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Show ORIGINAL CONTRIBUTION Ocular Motor Disorders in Mitochondrial Encephalopathy With Lactic Acid and Stroke- Like Episodes With the 3271 ( T- C) Point Mutation in Mitochondrial DNA Yasuhiro Shinmei, MD, PhD, Manabu Kase, MD, PhD, Yasuo Suzuki, MD, PhD, Takuya Nitta, MD, Shinki Chin, MD, PhD, Kazuhiko Yoshida, MD, PhD, Yu- ichi Goto, MD, PhD, Toshiko Nagashima, MD, PhD, and Shigeaki Ohno, MD, PhD Background: Ocular motor function can provide insights into areas of dysfunction within the nervous system. There are no published eye movement recordings in patients with mitochondrial encephalopathy with lactic acid and stroke- like episodes ( MELAS). Our purpose in this study was to analyze the ocular motor features of a family with MELAS with a ( T- C) mutation at nucleotide position 3271 in the mitochondrial tRNA- Leu gene. Methods: The search coil method was used to record visually- guided saccades, antisaccades, and triangular pursuit tasks in the horizontal and vertical planes in three patients in a Japanese family with MELAS. Results: The patients showed saccadic dysmetria and prolonged saccadic reaction times, deficits in the ability to suppress reflex eye movements, and increased reaction time during antisaccades, downbeat nystagmus, square wave jerks, and impairment in pursuit. Conclusions: On the basis of eye movement recordings, patients with MELAS have frontal cortex as well as cerebellar dysfunction. (/ Neuro- Ophthalmol 2007; 27: 22- 28) Department of Ophthalmology and Visual Sciences ( YS, TN, SC, KY, SO), Hokkaido University Graduate School of Medicine, Sapporo, Japan; Departments of Ophthalmology ( MK) and Neurology ( TN), Teine Keijinkai Hospital, Sapporo, Japan; Department of Ophthalmology ( YS), School of Medicine, Sapporo Medical University, Sapporo, Japan; and Department of Mental Retardation and Birth Defect Research ( YG), National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. Address correspondence to Yasuhiro Shinmei, Department of Ophthalmology and Visual Sciences, Hokkaido University Graduate School of Medicine, West 7, North 15, Sapporo 060- 8638, Japan; E- mail: yshinmei@ med. hokudai. ac. jp itochondrial encephalomyopathy is based on biochemical and morphologic abnormalities of the mitochondria in association with mitochondrial DNA ( mtDNA) mutations ( 1,2). It presents clinically as involvement of the central nervous system ( CNS) as well as the skeletal muscle system because these organs require high levels of energy produced in the mitochondria ( 3). The clinically well- defined forms of the disease are classified as mitochondrial encephalopathy with lactic acidosis and stroke- like episodes ( MELAS) ( 4), myoclonus epilepsy with ragged- red fibers ( MERRF) ( 5), and Kearns- Sayre syndrome ( 6). The 3243 mutation is found in 80% of patients with MELAS, and the 3271 mutation is the second most commonly reported type in MELAS, present at a frequency of 7%- 15% ( 7,8). Although more than 10 mtDNA mutations have been reported in MELAS, the clinical features are grossly identical. Ocular changes in MELAS have included reversible scotoma, ophthalmoplegia, and pigmentary retinopathy ( 9,10). We studied eye movements in three members of a Japanese family with MELAS with the 3271 ( T- C) point mutation in mitochondrial DNA. We examined visually guided saccades, antisaccades, and pursuit to investigate cortical, subcortical, and cerebellar function. To our knowledge, there have been no previous reports on eye movement defects in patients with MELAS. METHODS Patients The pedigree and the clinical features of our Japanese family have been reported previously ( 11). Three members in two generations of the family were examined for eye movements: the mother ( Case 1), the older son ( Case 2), and the younger son ( Case 3). Systemic neurologic findings included cerebellar ataxia, dysarthria, tremor/ myoclonus, 22 J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 MELAS J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 slight dementia with memory loss, and photogenic epilepsy, which were reasonably similar in severity in the three family members except for dementia, which was present only in Case 1 ( Table 1). Case 1 presented with the inability to walk; the patient used a wheelchair. Case 2 showed shaky walking using a cane. Case 3 could walk. All patients had sufficient intelligence to understand our tasks. Ophthalmic examinations revealed that all subjects had good visual acuity and no abnormalities of ocular fundi. Their visual fields were normal on Goldmann perimetry. No blepharoptosis or paresis of the extraocular muscles was observed. Brain MRI was normal in each patient. Lactate and pyruvate levels in serum and cerebrospinal fluid ( CSF) were elevated in each case. Biopsy of the biceps brachii muscle showed ragged- red fibers; mtDNA analysis revealed a heteroplasmic ( T- C) point mutation at position 3271 in the mitochondrial tRNA- Leu gene ( UUR) ( 12) in each patient. Six age- matched healthy subjects ( mean age = 30.5 years, SD = 10.7, four men) served as control subjects. The study was approved by our institutional human subjects committee and followed the tenets of the Declaration of Helsinki. All subjects gave their informed consent for genetic testing and ophthalmic examinations. Eye Movement Recording Equipment Eye movements were measured three dimensionally with two orthogonal magnetic fields ( the side length of cubic field coils was 89 cm) and a double- loop search coil ( Skalar Medical, Delft, the Netherlands) on the right eye ( 13), and data were digitized at 1000 Hz. The double- loop search coil was calibrated by monitoring on a protractor device that could be rotated in horizontal, vertical, and torsional planes ( in vitro calibration). The in vitro calibration gave the lengths of the two coil vectors and their TABLE 1. Neurologic features MELAS of our three patients with Case Age at onset ( yrs) Age at examination ( yrs) Photogenic epilepsy Lactic acidosis Ataxia Dementia Myoatrophy Deafness Tremor and myoclonus 1 27 51 + + + + + + + + 2 17 23 + + + + - + + + 3 17 20 + + + - + - + relative angles. The length of each vector represents the relative sensitivity of each coil in the two orthogonal magnetic fields. The sensitivity of the first coil wound in the horizontal plane was used to calibrate horizontal- vertical orientation of the eye ( 14). Each subject sat upright in a chair with his or her head fixed with a bite bar at the center of the cubic field coils. We then carefully checked voltage offsets of the coil signals during each interval between recording sessions and compensated for them if necessary ( in vivo calibration). Testing Paradigms Visually Guided Saccades Nine light- emitting diodes ( LEDs) 0.2° in diameter aligned horizontally and vertically at intervals of 10° on a tangent board were used for visually guided saccades. Subjects were instructed to look at a target moving in a stepwise manner from a fixation point to an eccentric point with amplitudes of 10 and 20° in horizontal and vertical planes. The target was turned on for 2- 5 seconds at random, with the target lit as soon as the other target was extinguished. This task was carried out more than 40 times. Antisaccades In this task, the visual target was presented in the same manner as in the visually guided saccade task. Subjects were instructed to make a horizontal saccade to an imaginary point opposite to the side of the visual target presented in the peripheral field. We provided detailed preparatory explanation for this task. Pursuit A laser projector was used to present a target spot of 0.2° in diameter on the screen for the smooth pursuit task. The target was presented between 4 and 207s with an amplitude of ± 20° in a triangular waveform pattern ( 4- 6 cycles). Pursuit responses were tested in the horizontal and vertical planes to calculate steady- state pursuit gain ( eye velocity divided by target velocity) during tracking of the target. Data Analysis Two components of eye position traces ( vertical and horizontal) were displayed on a computer display for visual inspection, and periods with blinks were discarded. The onset of saccades was identified by an interactive computer program with velocity and acceleration criteria ( velocity > 40/ s, acceleration > l, 200/ s2). Saccadic reaction time was calculated by subtracting the eye movement onset from target onset. Pursuit gain during triangular constant velocity waveform tracking was based on the average eye velocity after removal of saccades. The results were examined for 23 J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 Shinmei et al statistical significance by analysis of variance ( ANOVA) and the Tukey- Kramer test. RESULTS Visually Guided Saccades Saccades in all three patients showed abnormalities even on the simplest paradigm of visually guided saccades ( Fig. 1). The reaction times and saccadic gains are shown in Fig. 2. The reaction times of saccades in all three patients were significantly prolonged compared with those of control subjects in each direction ( P < 0.01) ( Fig. 2A). Furthermore, the reaction times of downward saccades in Case 1 were significantly longer than those in Cases 2 and 3 ( P < 0.01). Visually guided saccades were also characterized by dysmetria ( Fig. 1). The primary saccades were dysmetric, falling short of the visual target in both horizontal and vertical planes and were followed by corrective saccades with reaction times that ranged from 149 to 307 ms. The gains of 10° saccades are shown in Fig. 2B. Hypermetric saccades ( gain > 110%) occurred frequently in Case 1 ( Fig. 1A) appearing in 50% of rightward, 100% of leftward, 17% of upward, and 67% of downward saccades. On the other hand, the majority of visually guided saccades in Case 2 ( Fig. IB) and Case 3 ( Fig. IC) were hypometric ( gain < 90%). Saccadic velocities were normal in all patients. Antisaccades The error rates and reaction times for the normal control subjects were similar to those reported previously ( 15). On the other hand, two major deficits were observed during performance of the antisaccade paradigm in all patients ( Table 2). The patients had a tendency to make a saccade ( prosaccade) to the visual target although they were instructed to execute a saccade in the direction opposite to the visual target presented in the peripheral field. The inappropriate prosaccade was followed by an appropriate antisaccade in each trial ( Fig. 3). These corrective saccades indicated that the patients understood the antisaccade task. These reflexive saccade reaction times were also significantly longer than those in control subjects ( P < 0.01). The error rates indicated that the ability to suppress a prosaccade to the visual target was severely impaired in the three patients with MELAS. Pursuit Downward smooth pursuit showed low gain with interruption of catch- up saccades ( Fig. 4). These average gains in the patients were low for all directions and decreased with increases in target velocity ( Table 3). In contrast, the pursuit gains in control subjects were almost 1 for all directions when target speed was lower than 127s. Case 1 Case 2 Case 3 tow Time in milliseconds FIG. 1. Trajectories of leftward visually guided saccades in our three patients with MELAS. Every three saccades to visual target shifts of 10 and 20° are superimposed with alignment of target onset. Note that saccades in Cases 2 and 3 are hypometric, whereas those in Case 1 are hypermetric. R, right; L, left. 24 © 2007 Lippincott Williams & Wilkins MELAS JNeuro- Ophthalmol, Vol. 27, No. 1, 2007 Reaction Times Controls Case 1 Case 2 Saccadic Gains Case 3 • Rjghtwrd H Leftward • Upward Q Downward • Rjghtwrd El Leftward • Upward Q Downward Controls Case 1 Case 2 Case 3 FIG. 2. Reaction times and saccadic gains in 10° visually guided horizontal and vertical saccades. Saccadic gain is defined as the amplitude of initial eye movement divided by the amplitude of target movement. Each bar shows SD. * P < 0.01 in comparison with control subjects. The gains for downward pursuit were clearly different from those for upward pursuit in each patient. There were no significant differences between rightward and leftward movements at the same velocity ( P > 0.05). Downbeat nystagmus and square wave jerks frequently contaminated fixation and pursuit eye movements in Cases 1 and 2. The frequency and amplitude of the abnormal eye movements were larger in Case 1 than in Case 2. Amplitudes of fast- phase eye movements in downbeat nystagmus during fixation with the front target in the TABLE 2. Case 1 Case 2 Case 3 Controls * P < Error rates and reaction times in antisaccade task 0.01 Error rate 90.3% 81.6% 53.1% 11 ± 8% Reaction time ( ms) Succeeded Failed 923 ± 501* 400 ± 101* 471 ± 71* 294 ± 69* 509 ± 93* 298 ± 77* 284 ± 71 197 ± 24 , compared with control subjects. dark for Cases 1 and 2 averaged 2.2 ± 0.6 and 1.3 ± 0.3°, respectively. Slow- phase velocities averaged 1.4 ± 0.127s in Case 1 and 0.49 ± 0.097s in Case 2. Square wave jerks had average amplitudes of 0.95 ± 0.43° and average frequencies of 1.3 ± 0.27 Hz in Case 1 and 0.71 ± 0.27 and 1.0 ± 0.26 Hz in Case 2. On the other hand, neither downbeat nystagmus nor square wave jerks were observed in Case 3. DISCUSSION The present study demonstrated identical abnormalities of ocular motor function in three members of a family with MELAS associated with a mutation at nucleotide position 3271. Two abnormal parameters in the saccades were observed consistently even in a simple task to generate visually guided saccades. The reaction times of saccades to a visual target presented in the peripheral field were significantly prolonged in both horizontal and vertical directions compared with those of normal control subjects, particularly in Case 1 in whom reaction times of up to more than 300 ms were observed in all directions. The saccade amplitude-maximum saccade velocity relationships for all three patients 25 J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 Shinmei et al Case 1 & 40 20 0 - 2D - 40 20 0 - 20 - 40 p Target atop tp Left 200 w SCO eoo 20 0 - 20 - 40 " ^ p Target » top to Right 1000 Case 2 200 400 600 eoo 1000 L x Tweet steo to Left \ f^ ' v ft . 200 4C0 600 800 1000 ^ r P Target step to Right 4oo eoo 8oo Time in milliseconds Antisaccade Paradigm Right OFF 200 4oo eoo eoo Time in milliseconds OFF I 1000 Target Center H! Eye Left Right Center Left OFF / 1 "\ \ ,*- Failure FIG. 3. Per- trial saccadic trajectories during performance of the antisaccade task in our three patients with MELAS. The left-hand columns show saccades when the visual target was shifted from the center to the left, and the right- hand columns show when the target was shifted to the right. In the antisaccade paradigm ( bottom), the dotted line indicates failure, as exemplified by a reflexive saccade in the direction of the visual target presented in the right visual field, followed by an appropriate saccade in the antisaccade direction. The solid eye line indicates a successful antisaccade. were normal, indicating no impairment of the pulse generator in the brainstem involved in ocular motor function. Another affected parameter was saccadic accuracy. Visually guided saccades were dysmetric for all directions. Most of the dysmetric saccades were hypometric and were followed by single or a few corrective saccades with intersac-cadic intervals ranging from 140 to 307 ms. Hypometric saccades are common in subjects with cerebellar dysfunction ( 16). A striking ocular motor deficit in these patients was observed in the antisaccade task. All three patients had difficulty suppressing reflexive saccades to the visual target. The frequency of directional error was greatest in Case 1, with an error rate of 90.3%. An appropriate antisaccade occurred only three times in a sequential trial, in which reaction times exhibited a large degree of scatter. Moreover, the percentages of errors were 81.6% in Case 2 and 53.1% in Case 3. These results suggested that the frequency of directional error became higher as the disease progressed. Even in Case 3, a 20- year- old patient with minimal neurologic manifestations, errors were observed in about half of the trials. The antisaccade task requires the subject to suppress reflexive or anticipatory saccades toward the target. The potential sources of such suppression are the frontal eye fields ( FEFs) and the supplementary eye fields ( SEFs) ( 16). A recent animal study demonstrated that the majority of 26 © 2007 Lippincott Williams & Wilkins MELAS J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 Time FIG. 4. Representative observations of vertical smooth pursuit eye movements at target velocities of 16, 8, and 4°/ s in Case 2. Downward pursuit shows low gain with interruption by catch- up saccades. The upper trace, indicated by a solid line, shows vertical eye movements. The lower trace, indicated by a dotted line, shows target movement with amplitude of ± 20°. The entire time scale is 10 seconds. eye movement- related neurons in SEFs fired significantly more preceding antisaccades than prosaccades ( 17). Similar defects in generating antisaccades have been reported in patients with schizophrenia ( 18), Parkinson disease ( 19), Huntington disease ( 20), progressive supranuclear palsy ( 21), and Alzheimer disease ( 22). All three patients also showed low gain pursuit. Cases 1 and 2 exhibited downbeat nystagmus. A vertical vestibular tone asymmetry, a neural integrator failure, and an imbalance of vertical smooth- pursuit signals have been proposed as mechanisms of downbeat nystagmus ( 23- 25). Zee et al ( 25) proposed a model based on asymmetric vertical pursuit signals, in which the overbalance of upward visual velocity commands results in spontaneous upward drift. In a recent healthy human study, Marti et al ( 26) reported that vertical pursuit imbalance led to downbeat nystagmus in darkness. In our study, the minimal slow-phase velocities ( 1.4 ± 0.12 and 0.49 ± 0.097s) were not sufficient to cause the lower downward pursuit gains in Cases 1 and 2. A large asymmetry in vertical gains has been proposed as a possible mechanism for downbeat nystagmus ( 25). However, this hypothesis cannot apply to our Case 3. The assumption of an asymmetry of vertical pursuit signals is based on the observation of the tracking behavior of patients with cerebellar disease, who show relatively smooth upward but saccadic downward tracking during pursuit stimulation. Our results suggest that downward pursuit with low gain and downbeat nystagmus may be caused by effects on cerebellar control, given that cerebellar ataxia was observed in all three of our patients. Acknowledgments We thank Dr. D. A. Suzuki and Dr. K. Fukushima for valuable comments on the manuscript. TABLE 3 Case 1 Case 2 Case 3 Control Theg * P < Eye velocity gains subjects ain is 0.01, the Target velocity 47s 87s 127s 207s 47s 87s 127s 207s 47s 87s 127s 207s 47s 87s 127s 207s during smooth pursuit at each Rig htward pursuit gain 0.91 0.84 0.77 0.70 0.86 0.76 0.82 0.88 0.77 0.78 0.73 0.71 0.99 0.99 0.99 0.95 ± 0.11 ± 0.16* ± 0.21* ± 0.09* ± 0.25 ± 0.11* ± 0.21* ± 0.14 ± 0.09* ± 0.12* ± 0.17* ± 0.19* ± 0.03 ± 0.05 ± 0.02 ± 0.09 velocity of eye movement divided by the velocity compared with control subjects. target speed Leftward pursuit gain 0.94 0.84 0.84 0.77 0.88 0.80 0.79 0.77 0.90 0.84 0.72 0.79 0.99 0.99 0.99 0.97 ± 0.16 ± 0.12* ± 0.15* ± 0.10* ± 0.19 ± 0.17* ± 0.14* ± 0.19* ± 0.18 ± 0.15* ± 0.14* ± 0.26* ± 0.05 ± 0.06 ± 0.03 ± 0.03 of the target. Values Upward pursuit gain 0.78 0.76 0.70 0.66 0.78 0.69 0.64 0.66 0.90 0.94 0.92 0.94 0.97 0.95 0.98 0.90 + + + + + + + + + + + + + + + + are means ± SD. 0.19* 0.21* 0.21* 0.08* 0.11* 0.17* 0.15* 0.15* 0.18 0.18 0.31 0.18 0.09 0.15 0.17 0.17 Downward pursuit gain 0.84 0.52 0.42 0.35 0.33 0.32 0.31 0.17 0.63 0.69 0.42 0.38 0.99 0.97 0.99 0.89 ± 0.19* ± 0.09* ± 0.15* ± 0.12* ± 0.24* ± 0.18* ± 0.12* ± 0.07* ± 0.08* ± 0.17* ± 0.31* ± 0.31* ± 0.09 ± 0.15 ± 0.13 ± 0.18 27 J Neuro- Ophthalmol, Vol. 27, No. 1, 2007 Shinmei et al REFERENCES 1. Holt IJ, Harding AE, Morgan- Hughes JA. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 1988; 331: 717- 9. 2. Goto Y, Nonaka I, Horai S. A mutation in the tRNA( Leu) ( UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 1990; 348: 651- 3. 3. Blass JP, Sheu RK, Cedarbaum JM. Energy metabolism in disorders of the nervous system. Rev Neurol ( Paris) 1988; 144: 543- 63. 4. 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