A decisive electrophysiological test for human albinism

Electroencephalography and clinical Neurophysiologv, 1983, 55 : 513-531 Elsevier Scientific Publishers Ireland, Ltd.

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A DECISIVE ELECTROPHYSIOLOGICAL TEST FOR HUMAN ALBINISM PATRICIA A P K A R I A N , D I R K REITS, H E N K SPEKREIJSE and D I E U W K E VAN D O R P

The Netherlands Ophthalmic Research Institute, P.O. Box 6411, 1005 EK Amsterdam (The Netherlands) (Accepted for publication: December 8, 1982)

Albinism, a genetically determined biochemical disorder, is characterized by hypopigmentation of the hair, skin and eye and by sensory system anomalies particularly of the visual system. The two major classes of albinism include oculocutaneous albinism characterized by marked reduction in melanin throughout the body and ocular albinism characterized by hypomelanosis of only the iris and the retinal epithelium. Although both oculocutaneous (OCA) and ocular (OA) albinism have been subdivided into as many as 10 different forms based primarily on biochemical assay, phenotypic expression and pedigree analysis (Witkop 1971), all reported forms of albinism share common ophthalmological defects, the clinical features of which are outlined below. (1) Foveal hypoplasia and fundus hypopigmentation (Duke-Elder 1964). (2) Reduced visual acuity and abnormal contrast sensitivity functions (St. John and Timney 1981). (3) Photophobia (Waardenburg 1961). (4) High refractive errors (Fonda et al. 1971). (5) Oculomotor disturbance including nystagmus and strabismus (Wildberger and Meyer 1978). (6) Iris translucency (Jay et al. 1976). (7) Color deviation (Taylor 1976). (8) Abnormal ERGs (Krill and Lee 1963). (9) Aberrant optic pathway projections (Guillery et al. 1975).

i Department of Ophthalmology, Free University of Amsterdam, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands.

The major difficulty in the detection and differential diagnosis of albinism is that for a given albino, not all symptoms, either ophthalmological or cutaneous, may be manifest, whereas several may for a non-albino. To compensate for the wide diversity in albino expression, diagnosis is typically based on the results of combinations of tests. In a detailed genetic, ophthalmological, biochemical, histological and electrophysiological investigation of a large sample of albino probands (N = 78), we have found aberrant optic pathway projections to be the most reliable and measurable albino concomitant. Abnormal projections have also proved to be the only pathognomonic specific to albinism. The abnormal optic pathway projections associated with albinism, now well established anatomically and electrophysiologically in both man and several other species of mammals (Guillery 1969; Guillery and Kaas 1971, 1973; Hubel and Wiesel 1971; Creel et al. 1974, 1981; Lund 1978; Coleman et al. 1979; Cooper and Pettigrew 1979; Gross and Hickey 1980) are a pathological consequence of misrouted temporal retinal fibers. Although we do not yet fully understand the etiology nor the precise topography of the misrouting of retinal regions (for more detail see Cooper and Pettigrew 1979 and Silver and Sapiro 1981) we do know that temporal retinal fibers which should remain ipsilateral erroneously decussate at the optic chiasm, subsequently producing abnormal retinotopic projections at the occipital cortex. Of clinical importance 'for albino diagnosis is that the anomalous retinotopic cortical organization is reflected in the scalp recorded visual evoked potential (VEP). The basic electrophysiological albino feature is that the misrouted optic pathway projec-

0013-4649/83/0000-0000/$03.00 © 1983 Elsevier Scientific Publishers Ireland, Ltd.

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tions produce visual evoked potential asymmetry across the left and right hemispheres. Although previous investigators have reported misrouting evidence in only 70% (Creel et al. 1974) to 78% (Coleman et al. 1979) of the albino patients tested, a more recent investigation (Creel et al. 1981) indicates that the detection rate can be improved by the use of more appropriate stimulus and recording conditions, notably large appearing, disappearing patterns rather than flashes or the more recently used pattern reversal conditions. We have extended this previous investigation in an attempt to (1) determine the optimum test conditions for the detection of misrouting by examining VEP asymmetry across a full range of stimulus and recording conditions and (2) develop a practical clinical diagnostic test of visual pathway anomalies. Under the optimum test conditions we have found that albino misrouting can be detected with 100% accuracy and with zero false positives. Several atypical case histories are also presented to show the reader that VEP asymmetry is a viable and objective measure for albino diagnosis.

P. A P K A R I A N ET AL.

Recording Visual evoked potentials were recorded with t i n n e d / c o p p e r cup electrodes of 8 m m diameter attached to the scalp with collodion and positioned with equal spacing of 3 cm in a horizontal row, 1 cm above the inion, across the left and right occiput. The row consisted of 5 electrodes with the center electrode located at the midline. Reference for all electrodes was linked ears (A 1 and A2); the common ground electrode was located at Cz. Bandwidth of the EEG amplifiers (Medelec/Van Gogh) was set at 0.5-75 Hz. The high cut-off frequency was set by a low-pass fourth order Butterworth filter (Barr and Stroud EF: cut-off frequency 70 Hz) which introduces a phase shift increasing the response latencies by 7 msec. If the reader wishes to estimate peak latency from the responses depicted, this correction should be made. The filtered signals were averaged with an HP2100 computer and displayed in real time. The number of counts or averages was generally 40 to 300, depending on the signal-to-noise ratio.

Subjects Methods

Stimulus The stimulus consisted of white and black checkerboard patterns of approximately 100% contrast generated on a TV screen (Sony CVM1810E, 50 Hz). The mode of stimulus presentation was pattern appearance (300 msec),/disappearance (500 msec) at a constant mean luminance level of 200 asb. These presentation times prevent contamination of the pattern 'onset' response with that of the pattern 'offset' response. Viewing distance was kept constant at 100 cm; check size ranged from about 7' to 220'; a 10' red square centered within the stimulus field served as the fixation spot. Field size configuration included a 20 ° (horizontal)× 15 ° (vertical) field (full-field condition), a 10 ° × 8 ° field (inner-field condition) and an annulus field with inner dimensions of 10 ° x 8 ° and outer of 20 ° x 15 ° (outer-field condition). Non-stimulus areas were replaced by a homogeneous grey field of equal mean luminance.

Visual evoked responses were recorded from 112 subjects ranging in age from 5 to 65 years. For both the albino and normal controls this age range yields electrophysiological results which are only weakly influenced by maturational processes (De Vries-Khoe and Spekreijse 1982). Our subject sample included 78 albinos, 54 of whom were clinically classified as oculocutaneous tyrosinase positive, 13 as oculocutaneous tyrosinase negative, and 11 as ocular albino. Of the remaining 34 subjects, 9 were obligate heterozygote (7 female carriers, 2 male) and 4 were albino siblings. As no detectable differences in visual evoked potential profiles were found for the obligate heterozygotes and non-albinotic siblings, data from these subjects were combined with those of 20 normal controls. One of the subjects of the remaining 34, referred as an ocular albino, was subsequently diagnosed as suffering from X-chromosomal Nyctalopia. His results are presented in the section on Clinical Application, case III. All albinos and family members tested electrophysiologically underwent a complete ophthalmic examination, phenotypic evaluation and pedigree

ELECTROPHYSIOLOGICAL TEST FOR HUMAN ALBINISM

analysis. Additional tests to aid in the albino classification included hair bulb incubation, tyrosinase assay and melanin and macromelanosome analysis from skin biopsy. All subjects were tested with best corrected visual acuity. In the case of a poor recording session due to, among others, subject fatigue, inattention, high myogenic artefact or computer failure, the subjects were retested. Retesting, however, was necessary with less than 10% (8) of the 113 subjects studied. One of our suspected albino observers WFIN, age 30, who was difficult to diagnose by phenotypic expression and ophthalmic examination, even upon retest, showed idiopathic VEP response profiles. They differed from normal and albino response profiles in that the early component of the contrast response (CI) could only be measured by manipulation of the electrode montage (vertical along midline). Furthermore, all responses were of small amplitude and, whether binocular or monocular were lateralized to the left hemisphere. Tests of WFIN's oculocutaneous albino siblings, however, revealed typical albino responses (see e.g. Fig. 3A). During a fourth retest with WFIN we learned that he had suffered from head trauma at the age of 5. His skull was fractured left to the inion resulting in an impression contusion; treatment involved removal of splintered bone. Due to these unusual circumstances, this subject was excluded from our subject pool.

Procedure Subjects were tested while seated comfortably in an electrically shielded room and observed by the experimenter with the aid of a closed TV monitoring system or with difficult-to-test subjects, by direct observation. Binocular, left, and right eye responses were recorded for each stimulus condition. Monocular recordings were obtained with total occlusion of the fellow-eye. Though we were frequently able to test albino subjects on all 3 field conditions, at minimum each subject was tested on either the inner-field or the full-field condition. Subjects difficult to test, especially younger children, were tested with only the full field. Duration of the test and number of stimulus conditions was subject dependent.

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Data analysis

Qualitative analysis For practical consideration we found that VEP asymmetry could be determined by visual inspection of the left eye response compared to that of the right, as seen from the schematic in Fig. 1 (left). Each trace depicts the monopolar response derived from each of the 5 electrodes positioned in a horizontal row across the scalp. The two upper traces in this schematic and all remaining figures represent responses derived from electrodes positioned over the left occiput (left from the midline, trace 3); fourth and fifth traces are derived from electrodes positioned across the right occiput (right from the midline). Bottom-most traces represent the difference potential obtained by subtracting trace 4 (from the right hemisphere), from trace 2 (from the left hemisphere). The pattern onset (300 msec, see time scale) VEP components are designated, in accordance with previous investigators (Jeffreys and Axford 1972a,b; Spekreijse et al. 1973), as CI (positive), CII (negative) and CIII (positive). To determine the presence of asymmetry, the distributions of electrical potential over the 5 electrodes following whole-field left eye and right eye stimulation are compared. During our investigation we found that the most reliable time window for detecting a shift in the potential distribution is from 80 to 110 msec after pattern onset (for more detail see Results). A comparison of the two monocular potential distributions, for example, at 90 msec (see dashed line), shows a clear example of a shift in the peak of the distribution from the right hemisphere to the left following left and right eye stimulation, respectively. An additional indication of hemispheric laterality which can also be determined by visual inspection is the polarity of the difference potential. For the qualitative analysis, right hemispheric laterality at 90 msec (see arrow) is expressed as a negative appearing difference potential, left as positive. As seen more clearly from a replot of the left and right eye difference potentials (analysis I, Fig. 1, upper right), a change in sign from negative to positive upon left and right eye whole-field stimulation reflects the VEP asymmetry. A more quantitative approach to determine VEP asymmetry can be

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taken b y m e a s u r i n g the location of the peak of the p o t e n t i a l d i s t r i b u t i o n across the scalp (expressed as distance from the midline) at a given time i n s t a n t of the response. A n e x a m p l e of such a m e a s u r e at 90 msec is p r e s e n t e d in Fig. 1 (lower right) u n d e r analysis II. S u p e r i m p o s i n g b o t h left eye a n d right eye i n t e r p o l a t e d location distrib u t i o n s reveals that an analysis of this type can p r o v i d e i n f o r m a t i o n regarding not only the presence b u t also the degree of hemispheric laterality. R a t h e r than c o m p a r i n g left a n d right eye hemispheric laterality b y direct m e a s u r e m e n t of the l o c a t i o n of the p e a k s of the c o r r e s p o n d i n g distributions, for d a t a s u m m a r i e s (e.g., Figs. 4 - 8 ) , we

derive, instead, the difference in location between the two. This expression, the difference between the location of the left eye response minus the l o c a t i o n of the right eye response at a given time, m a y be used as an objective i n d i c a n t of a s y m m e try. The a d v a n t a g e of this p r o c e d u r e is that it is i n d e p e n d e n t of h e m i s p h e r i c a s y m m e t r y (e.g., see Figs. 3 a n d 4) d u e to ocular d o m i n a n c e (which w o u l d affect an a l b i n o response profile) or individual variability in cortical t o p o g r a p h y . F u r t h e r more, the p o s i t i o n of the midline electrode as d e t e r m i n e d b y clinical E E G s t a n d a r d s (Jasper 1958) could be at variance with that of a given subject's occipital midline. The left minus right

ELECTROPHYS1OLOGICAL TEST FOR HUMAN ALBIN 1SM

location estimate is a relative measure and thus compensates for this possible source of error. A detailed description of the location analysis is represented in the next section, Quantitative Analysis; its application is further demonstrated in the Results. To ascertain the presence of asymmetry in the response profiles of our albino subjects, an asymmetry scaling procedure was used which was based upon visual inspection. The degree and reliability of the asymmetry was based on quantitative measures. Three independent asymmetry ratings (by visual inspection) for a standard experimental response triad (binocular, left and right eye responses) were determined as follows. A rater, blind as to whether the data were from albinos or nonalbino controls, was presented with the two monocular responses and was required to state which of the two was derived from left eye stimulation, which from right. A 5-point rating system was used. If the rater was correct a score of 1 (definite asymmetry) or 2 (probable asymmetry) was indicated. If unable to distinguish left eye response from right, a score of 4 (probable no asymmetry) or 5 (definite no asymmetry) was indicated. A score of 3 (unable to determine) was reserved for poor, unreliable response profiles. The latter score of 3 occurred in less than 10% of the subjects tested. Following retests a score of 3 remained for only one subject F H O R , who presented with extremely low acuity of the right eye (OD = 0.07; OS = 0.1). After F H O R was retested with a larger (110') than standard (55') pattern size, his rating changed to 2 (probable asymmetry). Response triads falling into categories 1 and 2 were classified as albino, those in 4 or 5 as non-albino. Results for the 3 separate ratings which were in direct accord indicated a 100% hit rate for albino detection. Moreover, zero false positives were obtained for the non-albino control group.

Quantitative analysis To obtain a quantitative estimate of the degree of asymmetry and the voltages of the different components in the response the following analysis was performed. The baseline of the responses was determined by averaging the first 60 msec across

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all of the 5 recorded VEPs. In our stimulus situation the responses upon pattern presentation do not occur before 60 msec after the onset of the stimulus. Voltages at all sample points were measured against this zero line. One disadvantage of this procedure is that the baseline is sensitive to artefacts. However, as the EEG derivations are not disturbed by eye blinks or eye movements, we have assumed that for the group averages the artefact effects are not dramatic. Across subjects as many positive as negative artefacts will occur. The main effect is an increase in the variances of our data. To determine the location of the peak of the potential distribution at every time instant, a 3rd order polynomial fit was calculated between 4 of the 5 monopolar VEPs. The midline derivation was excluded from analysis (other than visual inspection) because of bone tissue thickness in this skull region. Due to sagittal bone sutures, the skull at the midline is on average about 2 times thicker (9.3 +_ 2.3 mm) than at loci further than 1 cm from the midline (4.7 _+ 1.2 mm) (Van Veenendaal 1982). As a consequence the midline response may evince a reduced amplitude. The mean ratio of the midline to lateral bone tissue thickness is a factor of 2; this ratio, however, can vary from 1 to 4 in different subjects. Although amplitude reduction of the midline response is not a consistent feature across subjects, its occurrence necessitates the above consideration. Another point of consideration for our analysis was the occurrence of asymmetry values greater than _+ 12 cm. As our most lateral electrodes were positioned only _+6 cm from the midline, the locations of the peak potentials determined upon left and right eye stimulation were excluded when the differences exceeded + 12 cm. Their inclusion for further analysis would have resulted in a significant bias in peak estimation; a 3rd order polynomial fit to points lying at such an extreme is inappropriate. The last m a j o r point of consideration for our analysis was that the estimated potential distribution could be derived from a single dipole in the brain. Thus, for the two monocular responses a single peak was estimated from the potential distribution at a given time instant. If a single peak

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estimate could be determined at the same time instant for both the left and the right eye responses, then the distance between these peaks (see Fig. 1, lower right) was used to determine the degree of asymmetry. In our analysis, the location of the peak of the potentials is independent of the baseline value of the responses. A shift in baseline does not affect the estimated location of the peak nor do artefacts from the common reference electrodes. The estimated degree of asymmetry is also independent of response amplitude. For the location analysis, signals were sampled every 5 msec and the stimuli were presented every 800 msec. Thus a maximum of 160 left minus right distances per stimulus condition may be determined. In practise this number is much smaller due to the above mentioned criteria. The direct left and right peak locations may be used to estimate asymmetry as seen in Fig. 2, but we have found that deriving the left minus right location differences provides the most optimum description of the data as seen in Figs. 4-8. For the group averages (e.g. Fig. 5), the mean and standard deviation of the asymmetries of all available points per time instant were calculated, t values of the number of data points at each time instant were calculated to determine the 95% and 99% confidence levels.

Results

Individual albino profiles The basic albino electrophysiological feature compared to normal can be seen in Fig. 2. In Fig. 2A is an example of binocular (first column), and

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monocular (left eye, second column; right eye, third column) pattern appearance/disappearance responses from a normal control. In Fig. 2B, responses from an albino observer are presented. The main point of interest to note in Fig. 2A is that in the normal observer the maximum response or peak of the potential distribution across the 5 electrodes at a given instant of time is relatively symmetric about the midline. Although a decrease in response amplitude occurs at the right-most electrode, the location of the peak of the potential response does not change with monocular stimulation. This can also be seen by inspection of the difference potential. As described above and previously reported (Creel et al. 1981), the polarity of the difference potential can be used as an indicant of hemispheric laterality. Note that there is no change in sign (see arrows). In fact, the only major change in the binocular to monocular response profiles is a slight reduction in amplitude. The stability in the peak of the potential distribution in the monocular response is as expected due to the fact that under monocular, whole-field stimulation, both the left and the right hemisphere receive nearly symmetric input from the temporal and nasal retinal fiber projections. For an objective indication of where across the hemisphere the peak responses are located, the quantitative location analysis (see Methods for more detail) was applied to these data. The results are presented below. The calculated location of the peak of the distribution is expressed in centimeters from the midline at time intervals of 5 msec. The zero line represents the midline. Points falling above this line represent maximum responses localized at the left hemisphere. Points falling be-

Fig. 2. Binocular (OU), left eye (OS) and right eye (OD) responses to an appearing/disappearing checkerboard pattern in a normal observer (A) and an albino (B). Check size equals 55', field size 20 ° × 15 °. A: for normal observer M K E U , the peak of the potential distribution across the scalp does not change from binocular to monocular stimulation. Stability in the location of the peak of the potential distribution can be seen from the difference potentials (note that a change in sign from left to right eye stimulation is not present) and from the direct location analysis. The location analyses (below) represent the location in centimeters across the scalp of the peak of the potential distribution at every possible time instant of the response. The location analysis shows a slight left hemispheric dominance in the binocular response profiles which does not change, however, with left and right eye stimulation. B: for tyrosinase positive oculocutaneous albino TOR, the location of the peak of the potential distribution within an early response component changes dramatically with left to right eye stimulation. Note from the bipolar derivation the change in sign at 100 msec (see arrows) from left to right eye stimulation. The location analysis (below) indicates a right hemispheric dominance with binocular stimulation. With left eye stimulation the right hemispheric laterality is apparent but shifts to the left hemisphere upon right eye stimulation.

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low represent responses localized across the right hemisphere. Again note that the difference in location of the left eye and right eye responses at any given time instant is negligible. If~ for example, we take the location of the peak of the potential distribution of the left eye response at 100 msec which is approximately + 2.7 cm and subtract this from the location of the peak c,f the potential distribution of the right eye response at 100 msec, which is also about + 2.7 cm, we obtain an asymmetry value close to zero. These results, typical for our normal observers, are in strong contrast to the response profiles and results of the location analyses obtained in the albino observer as seen in Fig. 2B. The binocular responses from each electrode (first column) show the maximum response to occur primarily at the right hemisphere. This is also the case for the left eye responses (second column). Binocular asymmetry, however, is not unique to the albino response but is frequently observed in normal observers as well as in observers with optic tract pathology such as chiasmal compression, lesions, etc. (Holder 1978; Blumhardt et al. 1982; see also below). With such strong asymmetry occurring even binocularly, it is the right eye response in comparison to the left eye response which reveals the albino feature. One can clearly see from this example that the right eye response', (third column) shows peak potentials localized across the left hemisphere. The albino asymmetry in the potential distribution can also be clearly seen in the difference potential (bottom-most traces). As opposed to the normal observer, there is a large difference response which reverses in sign from left to right eye (see arrows). From the direct quantitative location analysis of the data (below) the degree of' left and right hemispheric asymmetry can be determined. For this albino observer, the location of the peak of the potential distribution of the left eye response at 100 msec is approximately +5.6 cm whereas the location of the right eye response is approximately - 3 . 6 cm. The difference between the two yields an asymmetry value of + 9.2 cm as compared to the value of nearly zero obtained in the non-albino control of Fig. 2A. To further demonstrate the importance of com-

P. APKAR1AN ET AL.

paring the left eye response to that of the right we present Fig. 3. In Fig. 3A and B are the binocular and monocular response profiles of two albinos whose early response components under binocular stimulation are symmetric about the midline. The monocular responses clearly reflect the albino misrouting; the potential distributions shift over the hemisphere with left and fight eye stimulation. (That albino asymmetry is restricted primarily to the early component as seen here and in Fig. 2B is described in more detail below. For the present discussion we will concentrate our attention on the first 125 msec of the response.) For albino K K O S (Fig. 3B), it is of interest to note that there is a reduced amplitude of the midline response. This may be due to the thickness of the underlying bony structure in this scalp region as previously discussed in Methods. A reduction in midline response amplitude from an electrode positioned near the inion for the early component is an occasional feature for all subjects, normal or albino. An additional interesting feature of the responses in Fig. 3A and B is the presence of an off-component. The 'offset' response of albinos in this and previous investigations (Creel et al. 1981) is frequently absent (see Figs. 2B, 3C, 3D in contrast to 2A). Its presence, however, does not reflect reliable asymmetry. Across our albino sample, we have found that the offset response, if present, may reflect near zero asymmetry, positive asymmetry (.typically less robust than that of the onset response, e.g. Fig. 5) or as seen in Fig. 3A and B, reverse asymmetry, i.e., negative asymmetry. This later response, therefore, cannot be used as a reliable indicant of misrouting. Two examples of hemispheric asymmetry following binocular stimulation are presented in Fig. 3C and D. Left hemispheric dominance is depicted in 3C, right hemispheric dominance in 3D. In Fig. 3C one can see that as expected for a left eye response reflecting misrouting, the peak potential distribution of the early component shifts to the right hemisphere with left eye stimulation. However, the right eye responses, though consistent with misrouting predictions, are undistinguishable from those of the binocular. In the case of right hemispheric dominance (Fig. 3D), the left eye responses are now undistinguishable from the bin-

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Fig. 3. Symmetric (A and B) and asymmetric (C and D) response profiles following binocular (OU) stimulation in 4 albinos to an appearing/disappearing checkerboard pattern. Each pattern element subtended 55' of visual angle, field size 10° × 8°. Below each response the standard error of the mean at every time instant is indicated. Note that the variance is always constant as a function of time. A: binocular (OU), left (OS) and right (OD) eye responses from tyrosinase positive oculocutaneous albino, FINI. Following binocular stimulation the potential distribution appears symmetric about the midline. B: responses from tyrosinase positive oculocutaneous albino, KKOS. Although the peak potential distribution around the first 100 msec after pattern onset following binocular stimulation is symmetric across the left and right hemisphere, amplitude attenuation is present for the response derived from the midline electrode. C: responses from tyrosinase negative oculocutaneous albino, SLA. Following binocular stimulation a left hemispheric dominance is revealed. D: responses from tyrosinase negative oculocutaneous albino, WVEN. For this albino a right hemispheric dominance following binocular stimulation is present similar to that observed for albino TOR (Fig. 2A).

ocular. These results clearly d e m o n s t r a t e that the d e t e r m i n a t i o n of m i s r o u t i n g is reliable only by left a n d right eye c o m p a r i s o n s . T h e results of the q u a n t i t a t i v e left eye a n d right eye difference analyses for the d a t a in Fig. 3 A - D are d e p i c t e d in Fig. 4. R a t h e r t h a n presenting the direct b i n o c u l a r a n d m o n o c u l a r l o c a t i o n values as in Fig. 2, the d a t a of Fig. 4 simply represent the location of the p o t e n t i a l d i s t r i b u t i o n at a given instant of time for the right eye s u b t r a c t e d from that of the left. N o t e that regardless of the presence or direction of the b i n o c u l a r l y o b s e r v e d a s y m m e t r y , all 4 a l b i n o sub-

j e c t s show clear left eye minus right eye hemispheric a s y m m e t r y . The a s y m m e t r y is positive (for o u r objective analysis p o t e n t i a l d i s t r i b u t i o n s p e a k ing at the left h e m i s p h e r e are expressed as negative with respect to midline; those to the right as positive) a n d is restricted p r i m a r y to an early ' o n s e t ' response. F o r the left m i n u s right location analysis in this a n d all r e m a i n i n g figures, positive a s y m m e t r y values are r e p r e s e n t e d above the zero line; negative a s y m m e t r y values are r e p r e s e n t e d below.

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Fig. 4. Individual left minus right location analyses for the left eye and right eye responses from the 4 albinos presented in Fig. 3 A - D . Values falling on the zero line indicate that the location of the peak of the potential distribution for the left eye at a given time instant is equal to that of the right eye at a corresponding time instant. Positive asymmetry values indicate contralateral asymmetry, i.e., the peak of the potential distribution following left eye stimulation is located more to the right than the peak of the potential distribution following right eye stimulation. Negative values typically indicate ipsilateral asymmetry. Note that for all 4 albinos the early appearance response evinces positive asymmetry. Values occurring at longer latencies scatter randomly around the midline.

Population profiles When we apply our location analysis to the response profiles of both an albino sample and a group of non-albino controls plotting the difference in location of the left eye potential distribution minus the right as a function of time for computer measurable responses from all subjects within each group we obtained Fig. 5. In performing this group analysis we made no a priori assumptions regarding asymmetry for either albinos or normals and simply asked two questions. Does the left minus the right peak of the potential distribution differ from zero for either sample and if so, where? The data presented in Fig. 5A and B were obtained under the standard test protocol, i.e., full-field, 55' checkerboard, appearance/dis-

appearance condition. The results from our normal controls in this condition are shown to the left (Fig. 5A). The ordinate is expressed as the left minus the right distance in centimeters. The length of each datum line represents + and - 1 standard error of the mean. The dotted lines indicate a confidence level of 95% and of the 160 possible values only 10 can be seen to deviate from zero. Although one sees several values at this confidence level, the degree of asymmetry is not large. Furthermore, by probability alone, one would expect about 8 values to appear randomly. At a 99% confidence level, at no time instant in the response does asymmetry from the normal control group differ from zero. For our albino sample we see a striking dif-

ELECTROPHYSIOLOGICALTEST FOR HUMAN ALBINISM ference (Fig. 5B). At a 95% confidence level, two clusters of clear asymmetry are apparent. At the 99% confidence level (solid lines) only the left-most cluster remains. The cluster which manifests a high degree of asymmetry (from approximately + 2 to + 6 cm), appears within a narrow time window between about 80 and 110 msec. Most of the values occurring at much longer latencies are not significant at the 99% level. From this analysis we concluded that (a) normals do not have significant left/right hemispheric asymmetry whereas albinos definitely do with a peak value of about 6 cm, and (b) it appears as though the largest asymmetry observed occurs at about 100 msec after stimulus onset (see arrow) which corresponds with the first positive component in the contrast onset evoked potential (CI). As seen from this and previous investigations (Coleman et al. 1979; Creel et al. 1981), the early component of the pattern onset response shows the clearest positive hemispheric asymmetry. It is of interest to note that this component (CI) is presumed to originate from primary visual cortex (Jeffreys and Axford 1972a; Spekreijse et al. 1973) from dipoles oriented perpendicularly to the cortex around the calcarine fissure. Since the albino response profile frequently shows an absence of both the CII and pattern disappearance response, we have confirmed CI positive hemispheric asymmetry (contralateral asymmetry) by half-field stimulation in non-albino controls (Apkarian et al. 1983). With the half-field paradigm, positive asymmetry occurs within 80-110 msec after pattern onset, longer latency responses show reverse (ipsilateral) asymmetry.

Effects of field size The lower figure to the left (Fig. 5C) illustrates the results of testing albino observers with an inner-field ( 1 0 ° x 8 °) condition as compared to the full-field (20 ° x 15 °) condition of Fig. 5A and B. In Fig. 5D, data from albinos tested with either an inner or full-field condition are combined. For these subjects tested with both conditions, selection of which of the two conditions would be included for this figure was based on the t values of each. That is, the field size response selected for a given subject was that which yielded the most

523 significant difference from normal controls. Note that all 3 albino population analyses (inner-field condition, full, or the best of inner or full field) are comparable. In tests with more peripheral stimulation (outer 10°-20 ° field) we did find, however, a decrease in the degree of measurable asymmetry. To examine the effects of field size and to determine the optimum field conditions for the albino population as a whole, we compared the degree of asymmetry for 3 field configurations in a matched sample of 20 albinos who were tested on each condition. Left minus right asymmetry values averaged over four 5 msec time intervals around the peak of our asymmetry window (90-105 msec) for an inner-field, full-field and outer-field condition are presented in Fig. 6a. This narrower analysis window was selected to reduce intersubject variability. Although the inner-field (10 ° x 8 °) condition yields the highest asymmetry values compared to the full-field (20 ° x 15 °) or outer-field (10-20 °) conditions, we have found that the fullfield condition is more reliable as seen from the results of t test comparisons presented in Fig. 6b. That is, one can expect more than a 25% improvement in measurable responses by testing with the larger field condition. The increased 'hit rate' cannot be attributed to the number of averages but is most probably due to the advantages of a larger field for albinos with a high degree of nystagmus and for subjects, particularly younger, with attention difficulties. As a possible explanation of the reduced degree of asymmetry with more peripheral stimulation, we suggest that this may be due to the topography of retino-cortical projections of the CI component, i.e., the dipoles yielding the smaller response lie deeper within the calcarine fissure (Jeffreys and Axford 1972a, b).

Effects of pattern size The results presented thus far indicate that albino misrouting is primarily reflected in the early pattern onset component (CI). The CI albino asymmetry specificity across the albino population suggests that the presence or degree of evoked potential asymmetry will remain relatively unaffected by changes in the element size of the stimulus. However, if the presence or degree of asymmetry is based on element sizes which are too small

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relative to the visual resolution capabilities of the subject, the resultant small amplitude evoked responses will not be easily measurable, regardless of the presence or absence of asymmetry. Therefore, to determine the optimum pattern size for detecting misrouting, we examined the degree of asymmetry with checks ranging from about 7' to 220'. Left minus right asymmetry values indicated that the optimum check size for detecting misrouting is from about 25' to 110'; the most reliable asymmetry occurred for 55' checks. A summary of these results is presented in Fig. 7 in which group averages of left minus right asymmetry are plotted as a function of check size. Closed circles represent the mean, error bars + and - 1 standard error of the mean, obtained from left minus right values across 80 110 msec. Open circles represent median values.

As predicted from a CI involvement, albino asymmetry as a function of pattern size is broadly tuned, with asymmetry values remaining relatively stable over more than a two octave range. In the few albinos who had better than average albino acuity, measurable responses were also obtained for checks subtending less than 15'. Note, in addition to the high variability the degree of asymmetry with small checks is reduced by as much as 50%. For the largest check size tested (220') one also sees greater variability most probably due to an increasing luminance component. In testing with luminance flash stimulus we found, indeed, that the misrouting detection rate dropped by more than 50%. As further support for CI involvement, we present the asymmetry results for 3 albinos as a

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Fig. 5. Group average left eye minus right eye location analyses for non-albinos (A) and for albinos (B, C, D). The mean of the left minus right locations calculated across subjects at every possible time instant of the response is at the midpoint of each vertical data line. Length of the line represents + 1 S.E. around the mean. Dotted lines indicate a significance level of P < 0.05; solid lines, P < 0.01. Vertical arrow is placed at 100 msec. A: left minus right asymmetry values for a group of normals. Pattern size equals 55', field size 2 0 ° x 15 °. B: left minus right asymmetry values for a group of albinos tested under full-field conditions ( 2 0 ° x 15°). C: left minus right asymmetry values for a group of albinos tested under inner-field conditions (10°× 8°). D: left minus right asymmetry for a group of albinos tested with either the full-field condition or the inner-field condition whichever yielded the highest t value per subject. Note that all 3 albino group averages (B, C and D) are comparable.

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Clinical application As discussed in the Introduction, difficulties in diagnosis arise from the absence of one or more albino symptoms in a proband who is an albino or from the presence of typical albino features in a patient who is not an albino. For example, an oculocutaneous albino diagnosed as tyrosinase negative and with negative results on a hair bulb incubation test may, in fact, have skin which tans, no apparent nystagmus and relatively high acuity (van Dorp et al. 1982). An oculocutaneous albino diagnosed as tyrosinase positive may have pigmentation of the retina, difficult to detect foveal hypoplasia and no photophobia (Krill 1977). Ocular albinos particularly those of dark Caucasian or black origin may show normal melanosome structure (even those diagnosed as X-linked, van Dorp, unpublished observation), non-detectable hypopigmentation of skin, hair or eyes and non-detectable iris translucency (Wirtschafter et al. 1973). A nonalbino blond individual with a yellow-white fundus coloration a n d / o r with nystagmus may easily be diagnosed as an albino while a dark haired albino with macular reflex, non-albinotic fundus and nystagmus may be given the diagnosis of congenital nystagmus. During the course of our investigation, however, we have found that under appropriate test conditions as described in detail above, the electrophysiological response profiles of left eye and right eye can be used as the decisive clinical test for albino detection and differential diagnosis. (Applicability of this test for patients under 5 years of age is now under investigation.) To support this conclusion case histories, clinical and electrophysiological results of 3 patients who were difficult to diagnose with standard clinical procedures are presented. Case L J . v . H . , born 05-03-61, male. This boy has two brothers suffering from ocular albinism, whereas he was reported not to be affected. He accompanied his brothers when they visited our

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responses to an appearing/disappearing checkerboard pattern (55') in normal patient, FINM, case II. Albino asymmetry is not present. The amplitude of the potential distribution across the scalp does not change from binocular, left eye or right eye stimulation. Note from the bipolar derivation that there is no change in sign (see arrows) from left to right eye stimulation.

eye clinic. On examination, however, we also diagnosed him as an O C U L A R ALBINO, on the following criteria: Ophthalmological examination: visual acuity for OD and OS: 0.5 (20/40) corrected with hard contact lenses (S-2.5 resp. S-3.75). He suffered from photophobia and the brown irides showed marked translucency. The fundi showed a mottled pigmentation with clearly visible choroidal vessels; macular and foveal reflexes were absent. Nystagmus and strabismus were not apparent. Hair: the color of the hair was black. The hairbulb was black before incubation with tyrosine (TP+). Tyrosinase activity was present. Skin: the skin was normally pigmented, except for the belly, which showed patches of hypopigmentation and hyperpigmentation, which according to anamnesis is more pronounced after sun exposure. The patient has an ability to get a suntan. Electron microscopic examination revealed the presence of macromelanosomes. Electrophysiology: positive albino asymmetry (see Fig. 9). Case II. M. McH., born 07-03-68, male. When we saw this patient, at first sight we thought him to be an albino. On further examination, however, he appeared to be NORMAL:

Ophthalmological examination: visual acuity for OD and OS: 1.25 (20/16): emmetropic. He suffered from slight photophobia and the blue irides showed no translucency. The fundi showed a total absence of pigmentation; however, macular and foveal reflexes were normal. Nystagmus and strabismus were absent. Hair: the color of the hair had been white at birth and was very light blond on examination. The hairbulb test gave dubious reaction (TP _+ ) on tyrosinase. Tyrosinase activity was negligible. Skin: the skin was white and showed some freckles and moles. On sun exposure the skin would burn and only tan minimally. Electrophysiology: non-albino response profile (see Fig. 10). Case III. H.J.J., born 04-10-59, male. This patient was referred from Finland, where he was diagnosed as suffering from ocular albinism, having the Forsius-Eriksson syndrome. On examination, however, we diagnosed him as a person suffering from X-CHROMOSOMAL NYCTALOPIA A N D MYOPIA, and not as an ocular albino, according to the following criteria: Ophthalmological examination: visual acuity for OD, 0.25 (20/80) with S-6.5 = C-4.25 30 °, OS, 0.33 (20/60) with S-6.5 = C-4.75 115 °. He suffered

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from mild photophobia and the gray irides showed no translucency. The fundi showed myopic papillae and choroidal sclerosis. There was substantial pigment in the fundi and also in the periphery; with red-free light the choroidal vessels were clearly seen. There was a poor macular reflex and the right macula showed depigmentation. Foveal reflexes were normal. An esophory was present with a preference for the left eye. Strabismus was absent. Visual fields showed no defects, nor did color vision examination. Dark adaptation was disturbed up to 1.2 log units. A nystagmus latens was present. Hair: the color of the hair was darkblond. The hairbulb test was black before incubation with tyrosinase (TP+). Tyrosinase activity was present. Skin." the skin had a normal appearance and could tan on sun exposure. Electrophysiology: non-albino response profiles (see Fig. 11).

Discussion Evidence for misrouting of the optic fibers as reflected in the visual evoked potential upon mon-

ocular stimulation was found in all 78 albinos examined in this investigation. By testing a large sample of albinos with various genotypes and phenotypic expression, our results indicate that the early component (CI) in the pattern onset EP shows positive asymmetry and is a decisive pathognomonic of albinism. Lower detection rates of misrouting by previous investigators (Creel et al. 1974; Coleman et al. 1979) are most probably due to non-optimal stimulus and recording conditions. As a diagnostic measure for clinical purposes, optimum detection parameters established in this study include large (about 55'), appearing (300 msec onset), disappearing (500 msec offset) checkerboard patterns of high contrast. Due to the frequent presence of hemispheric asymmetry upon binocular stimulation, we found it necessary to obtain both left eye and right eye responses for the determination of asymmetry. By placing a row of, at minimum, 4 electrodes across the left and right occiput (equally spaced at 3 cm distances from the midline), it is possible to detect the presence of misrouting by visual inspection alone. A bipolar derivation between an electrode positioned to the left of the midline and that to the right provides an additional indication of hemispheric laterality; in albinos the polarity of this difference potential reverses from left to right eye stimulation. The assessment should be made by restricting the qualitative analysis to the first 120 msec (if the luminance level is that of a standard TV) of the pattern onset response as it is the early CI component which reflects the contralateral albino asymmetry. Cortical area 17, from which the CI component is derived, receives direct input from the lateral geniculate nucleus (LGN). If an abnormal ipsilateral field representation of the L G N is present, the aberrant afferent organization has to be maintained at the cortical level at least in humans as evidenced in the present study. In this study we found that albino VEP asymmetry is a reflection of neural projections primarily from the temporal retina. However, we were unable to delineate the demarcation line between misrouted ipsilateral and normal contralateral projections, reported previously to occur at a temporal eccentricity of 15-20 ° (Kaas and Guillery 1973; Shatz 1977; Coleman et

ELECTROPHYS1OLOGICAL TEST FOR HUMAN ALBINISM

al. 1979). This failure to define a decussation boundary cannot be attributed to the difficulties associated with stimulating a local retina region in subjects with nystagmus as it was also not possible to detect a sharp boundary in albinos with no detectable nystagmus. Rather these results are consistent with the previous anatomical results of Cooper and Pettigrew (1979) who reported an increasing gradient of the percentage of misrouted fibers as one moved more temporally. From their investigation of the total retinofugal pathway in Siamese cats they found that crossed and uncrossed retinothalamic fibers in the temporal retina were mixed and that the division between ipsilateral and temporal projections was smeared. This gradual rather than sharp decussation line between ipsilateral and contralateral afferents may be due to a regional melanin deficiency in the optic stalk cells which, during a particular stage of embryological development, affects the entire contingent of ipsilaterally destined axons. The involvement of selective but transient pigmentation of the optic stalk tissue for guiding the direction and orientation of axonal outgrowth has recently been reported by Silver and Sapiro (1981). Their results suggest that a malfunction of melanin production at an early period of outx~ard migration of optic nerve fibers may be the mechanism responsible for the contralateral shunting of those ganglion cells which would otherwise remain ipsilateral. Rather than abnormal melanin production as the precursor to aberrant optic pathway projections, Strongin and Guillery (1981) suggest that it is the abnormal degenerative changes of melanin which relate to the pattern of axonal growth. Although the precise nature of the relationship between pigment cells of the eye stalk and the course of retinofugal axons is yet to be determined, apparently the albino mutation which precludes normal melanin production or degeneration as a consequence of metabolic disturbances also precludes normal contralateral and ipsilateral projection.

Summary A b n o r m a l decussation of temporal retinogeniculostriate projections associated with albi-

529

nism is reflected in the potential distribution of the visual evoked potential (VEP). Following monocular stimulation misrouted optic pathway projections produce VEP asymmetry across the occipital left and fight hemispheres. With recordings from 5 electrodes positioned at equal spaces of 3 cm in a horizontal row across the scalp, VEP asymmetry in 78 albino probands and 34 non-albino controls was assessed by two methods. The first method which was qualitative consisted of visual inspection of the potential distribution across the scalp within an early time period of the response. As an adjunct to visual inspection, the reverse in sign of a difference potential from a left minus right hemispheric response following both left eye and right eye stimulation was also used. The second method which was quantitative included analysis that was based on estimation of the peak of the potential distribution across the electrode array at every time instant of the response. For this latter method the midline derivation was excluded. Several experimental parameters (e.g. mode of stimulus presentation, pattern size, field size) were investigated to determine the optimum stimulus and recording parameters for detecting albino misrouting. Under optimum test conditions, evidence of misrouting by visual inspection was found in all of the 78 albino probands tested. The quantitative analysis which provides an indication of both the presence and the degree of VEP asymmetry was used to establish population norms for various test conditions. Results of the quantitative analysis provided evidence that the degree of asymmetry in a sample of non-albino controls (including heterozygote family members) was not significant. The degree of asymmetry for albinos, however, was highly significant and was primarily restricted to a narrow time window of the response between about 80 and 110 msec following the appearance of a checkerboard pattern with elements subtending 55' of arc. The early time course of the asymmetry and the effects of pattern size and defocus suggest that albino misrouting is reflected in the CI component of the pattern appearance response. As the CI component is thought to be generated by dipoles lying within cortical area 17, we can assume that the reflected misrouting is of a similar origin. The 100% detection rate of VEP asymmetry in

530

albinos and 0 false positives in normal controls, heterozygote family members and non-albino patients with comparable albino symptoms (e.g. nystagmus, reduced acuity, retinal hypopigmentation), indicates that VEP asymmetry is a decisive clinical measure for the diagnosis of albinism and for differential diagnosis. The protocol for electrophysiological diagnosis and the results of its application in several atypical patients is also presented.

R~sum~

Un test klectrophysiologique infailhble de dktection de l'albinisme chez l'homme La drcussation anormale des projections r6ticulogrniculostri~es associre/t l'albinisme se rrpercute sur la distribution des potentiels 6voqurs visuels (PEV). Lors de la stimulation monoculaire, les voies de projections optiques ~ trajet anormal sont /l l'origine d'une asymrtrie des PEV sur toute l'rtendue de la rrgion occipitale des hrmisphrres droit et gauche. En enregistrant avec 5 61ectrodes placres/a 6gale distance (3 cm) sur une couronne horizontale entourant le scalp, l'asym6trie des PEV a 6t6 6valu6e chez 78 sujets reconnus comme albinos et chez 34 trmoins non-albinos, par 2 techniques. La premirre, qualitative, consistait en un examen visuel de la distribution des potentiels sur le scalp pour la partie prrcoce de la rrponse. On a de plus utilis6 l'inversion de signe de la diffrrenee de potentiel entre les rrponses des 2 hrmisphrres, consrcutives /~ la stimulation de l'oeil droit et de l'oeil gauche. La seconde technique, quantitative, comportait une analyse fondre sur l'estimation du pic de la distribution des potentiels le long de la rangre d'61ectrodes/l chaque instant de la r~ponse. Pour cette dernirre technique, on a exclu la drrivation de la ligne mrdiane. On a 6tudi6 plusieurs paramrtres exprrimentaux (par exemple le mode de prrsentation du stimulus, la taille du pattern prrsent6, la taille du champ) afin de drterminer le stimulus optimal et les meilleurs param6tres d'enregistrement pour drtecter le trajet anormal des albinos. Dans les meilleures conditions, cette

P. A P K A R I A N ET AL.

anomalie a 6t6 mise en 6vidence par l'examen visuel chez t o u s l e s sujets albinos. On a utilis6 l'analyse quantitative, qui donne une indication/l la fois sur la prrsence et le degr6 de l'asymrtrie des PEV, pour 6tablir des normes de populations pour les diffrrentes conditions exprrimentales. Les rrsultats de cette analyse ont montr6 que le degr6 d'asymrtrie dans un 6chantillon de trmoins non-albinos (comprenant des membres hrtrrozygotes de la famille) n'rtait pas significatif. Le degr6 d'asymrtrie chez les albinos, toutefois, 6tait hautement significatif, et essentiellement limit6 / t u n e 6troite fen&re temporelle de la rrponse entre environ 80 et 110 msec aprrs l'apparition d'un damier dont les 616ments 6taient distants de 55'. La datation prrcoce de l'asymrtrie et les effets de la taille du pattern et de la drfocalisation suggrrent que l'anomalie du trajet optique est reflrtre par la composante CI de ta rrponse /a l'apparition du pattern. Puisque l'on pense que cette composante est engendrre par des diprles siturs dans l'aire corticale 17, on peut estimer que i'anomalie drcelre est d'origine similaire. La drtection /l 100% de l'asymrtrie des PEV chez les albinos et l'absence totale de faux positifs chez les trmoins normaux, les membres hrt6rozygotes de la famille et les patients non-albinos prrsentant des symptrmes comparables a ceux de l'aibinisme (c'est-h-dire nystagmus, diminution de l'acuitr, hypopigmentation rrtinienne), indiquent que l'asymrtrie des PEV est une mesure clinique drterminan~e pour le diagnostic de l'albinisme et pour le diagnostic diffrrentiel. On prrsente 6galement le protocole du diagnostic 61ectrophysiologique et les resultats de son application chez diffrrents patients atypiques. The authors thank J.W. Delleman, M.D., for his continuing clinical support and T. Kasamatsu, Ph.D., for his helpful discussions and criticism. The authors wish to acknowledge the expert photographic assistance of N.C.M. Bakker and skillful secretarial assistance of E. Borghols and B. Bastiaenen. Special thanks are due to the albino participants whose cheerful and willing collaboration made this investigation possible.

References Apkarian, P., Reits, D. and Spekreijse, H. Component specificity in albino VEP asymmetry. In: R.H. Nodar and C. Barber (Eds.), Evoked Potentials, 1983: in press.

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