Visual Pathways in Humans with Ephrin-B1 Deficiency Associated

Visual Neuroscience Visual Pathways in Humans With Ephrin-B1 Deﬁciency Associated With the Cranio-Fronto-Nasal Syndrome

Michael B. Hoffmann,1,2 Hagen Thieme,1 Karin Liedecke,1 Synke Meltendorf,1 Martin Zenker,3 and Ilse Wieland3

1Department of Ophthalmology, Otto-von-Guericke-University, Magdeburg, Germany 2Center for Behavioural Brain Sciences, Magdeburg, Germany 3Institute for Human Genetics, Otto-von-Guericke-University, Magdeburg, Germany

Correspondence: Michael B. Hoff- PURPOSE. Numerous animal studies demonstrated the importance of components of the mann, Universitäts-Augenklinik, Vi- ephrin/Eph system for correct visual system development. Analogous investigations in sual Processing Laboratory, Leipziger humans are entirely missing. Here, we examined the visual system in humans with ephrin-B1 Str. 44, 39120 Magdeburg, Germany; deficiency, which is x-linked and associated with the cranio-fronto-nasal syndrome (CFNS) in [email protected]. heterozygous females. Submitted: July 15, 2015 Accepted: October 6, 2015 METHODS. For one male hemizygous for ephrin-B1 deficiency and three affected heterozygous females with molecular-genetically confirmed mutations, the integrity of the partial Citation: Hoffmann MB, Thieme H, decussation of the optic nerves was assessed with visual evoked potentials (VEPs) and Liedecke K, Meltendorf S, Zenker M, compared with albinotic, achiasmic, and control participants with healthy vision. Further, Wieland I. Visual pathways in humans with ephrin-B1 deficiency associated retinal morphology and function and the gross-retinotopic representation of the primary with the cranio-fronto-nasal syn- visual cortex were examined with spectral-domain optical coherence tomography (SD-OCT), drome. Invest Ophthalmol Vis Sci. ERG, and multifocal (mf) VEPs for the male participant and part of the carriers. 2015;56:7427–7437. DOI:10.1167/ RESULTS. Strabismus and lack of stereovision was evident in the male and two of the females. iovs.15-17705 Other characteristics of the visual system organization and function were normal: (1) retina: SD-OCT and funduscopy indicated normal foveal and optic nerve head morphology. Electroretinograms indicated normal retinal function, (2) optic chiasm: conventional (c)VEP showed no evidence for misrouting and mfVEPs were only suggestive of, if any, very minor local misrouting, and (3) visual cortex: mfVEP characteristics indicated normal retinotopic gross-representations of the contralateral visual hemifield in each hemisphere.

CONCLUSIONS. While ephrin-B1 deﬁciency leads to abnormal visual pathways in mice, it leaves the human visual system, apart from deﬁcits in binocular vision, largely normal. We presume that other components of the ephrin-system can substitute the lack of ephrin-B1 in humans. Keywords: optic chiasm, misrouting, ephrin, eph, guidance molecules, visual cortex

he optic chiasm is a key structure in the visual system.1 Molecular mechanisms shape a number of different pro- T Here, the fate of axons of the retinal ganglion cells is cesses of optic chiasm formation.6 They promote crossed or decided during early development such that axons carrying uncrossed projections, or guide the general patterning of the information from the right visual hemifield are guided to the left optic chiasm. In animal studies, particular attention has been hemisphere and vice versa. In the human visual system this paid to the Eph/ephrin receptor/ligand families of cell surface results in a partial crossing of the optic nerves at the chiasm: signalling proteins. There are 14 Eph receptors and eight fibers from the nasal retina receive input from the ipsilateral ephrin ligands in mammals,7 both subdivided into A and B visual field and consequently cross the midline separating the classes. Remarkably, there is promiscuity within each class (i.e., hemispheres, and fibers from the temporal retina receive input EphA receptors can bind with diverse ephrin-As, and likewise from the contralateral visual field and consequently remain EphB with ephrin-Bs).8 Eph/ephrin interactions are important uncrossed. Remarkably, deviations from this pattern have been in development, especially in cell–cell interactions involved in observed in specific patient groups (i.e., in albinism with nervous system patterning and axon guidance. Investigations of enhanced crossing of the temporal fibres at the chiasm2,3 and in the development of the visual system demonstrated an achiasma4,5 with reduced or absent crossing of the nasal fibers). importance of A and B class proteins for retinotopic map Although it might appear that such misrouting of the optic formation and for chiasm formation.6,8–10 Some studies suggest nerves should completely obstruct vision in the affected specific mechanisms that lead to enhanced crossing of the optic individuals, visual function is only partially impaired in these nerves in albinism. They demonstrated the relevance of EphB1- cases.1 Typically, nystagmus associated with reduced visual expression, regulated by the transcription factor Zic2,11,12 for acuity and strabismus associated with often absent binocular the normal formation of the ipsilateral projection at the optic vision are observed, while other visual functions such as chiasm. Growth cones of retinal axons that are expressing pattern detection and visuomotor integration appear largely EphB1 are normally repelled by the ligands ephrin-B2 and unaffected. ephrin-B1 expressing glia at the optic chiasm midline, as

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demonstrated in EphB1/ mice and EphB2//EphB1/Y hemifields, if they have the opposite polarity, this is evidence mice.13,14 Accordingly, during human embryogenesis EphB1 for enhanced or reduced crossing of the optic nerves. This is expressed in the ganglion cells of the temporal retina, which paradigm serves as a clinical routine tool for the detection of is projecting ipsilaterally.15 Importantly, in animal models of misrouting of the optic nerves33 and can also be combined albinism the expression of the transcription factor Zic216 and with mfVEPs to identify localized optic nerve misroutings.34–36 consequently EphB117 is reduced, which corresponds to the Enhanced crossing of the optic nerves has long been enhanced crossing of the optic nerves at the chiasm in considered as a pathognomonic sign of albinism. As a albinism. The relation of these changes to the pigmentation consequence, the misrouting VEP has since served as an defect in albinism (i.e., the reduction of ocular or oculocuta- important diagnostic in the detection of albinism as the cause neous melanin levels) is still under investigation. It is of ocular symptoms specified above. In fact, the abnormality is presumably associated with the delaying effect of hypopig- absent in human carriers of albinism34,35,37 and patients mentation on retinal neurogenesis likely mediated via reduced without albinism, but with some ocular symptoms observed retinal levels of the melanin precursor L-DOPA.18–21 in albinism, such as foveal hypoplasia,38 dissociated vertical The above highlights the importance of the interaction of deviation and missing stereopsis,39–41 and congenital nystag- ephrin-ligands and Eph receptors for neural guidance during mus.42,43 Further, normal projections were observed in visual system development. However, it must be noted that it patients with unilateral anophthalmia or severe microphthal- is, at present, uncertain, to which extent these mechanisms mia22 and in patients with generalized lateralization abnormal- actually translate to the development of the human optic ities (i.e., situs inversus in the Kartagener syndrome, and in chiasm, which differs distinctly in its architecture from that of primary ciliary dyskinesia in general).36 However, it must be the relevant murine animal models.22 Specific investigations of noted that comparatively rare incidences of enhanced crossing the visual system in humans are needed, but difficult to in the absence of albinism and the presence of other conduct as humans with deficiencies in the ephrin/Eph system syndromes are also known.44–46 are extremely rare. In fact, currently only one syndrome Here, we present a small case series, comprising one male associated with relevant deficiencies is known (i.e., the cranio- hemizygous and three females heterozygous for ephrin-B1 fronto-nasal syndrome [CFNS MIM 304110]). Remarkably, only deficiency of this rare patient group and thus provide the first a decade ago ephrin-B1 deficiency was demonstrated to be account assessing visual pathways in patients with ephrin-B1 23,24 causal for CFNS, which spurred the notion, that enhanced deficiency. We give a detailed description of the visual system crossing of the optic nerves might also be evident in CFNS- in the participants with a particular focus on the assessment of patients. This patient group might thus provide a key to our the integrity of the cortical visual field representations. understanding of the role of ephrin-B1 in the formation of the Remarkably, while deficits of visual function were evident, human optic chiasm. Cranio-fronto-nasal syndrome is a very there was an absence of major representation abnormalities at rare condition, with an estimated incidence of 1:100,000, and the level of the visual cortex. predominantly affects the phenotype of females. Counterintu- itively, however, the inheritance is x-chromosomal giving rise to a genetic paradox. The absent or mild phenotype in male METHODS carriers is likely due to the promiscuity of the ephrin/Eph ligand/receptor system. In contrast, the more severe manifes- Participants tation in females may be explained by cellular interferences that are caused by the combination of ephrin/Eph ligand/ Four participants with defective ephrin-B1 (aged 6–50 years, receptor promiscuity and the consequences of random x- including one male) and the following reference datasets from chromosomal inactivation in distinct cellular compartments.25 previous studies were included in the study: (1) 10 visually Cranio-fronto-nasal syndrome is associated with craniofacial healthy control participants (aged 18–60 years) with decimal dysplasia, body asymmetries, facial asymmetries, hypertelor- visual acuity greater than or equal to 1.0 of a previous study,36 ism, midline defects, skeletal abnormalities, and dermatologic (2) 14 albinotic participants (aged 7–47 years; 6 male; 13 abnormalities. Apart from the description of some peculiarities participants of a previous study47), and (3) one participant of visual function, such as strabismus, nystagmus, amblyopia, without optic chiasm (aged 22; detailed in Hoffmann et al.5). an in depth analysis of the visual system of the affected Binocular visual function in the participants with defective individuals is currently missing.25,26 Strabismus, which has ephrin-B1 was assessed in a dedicated examination by an been reported to be prevalent in CFNS (incidence of 40%– orthoptist, including alternating cover-test to identify strabis- 89%25,26), is also typical for patients with optic nerve mus, and Lang- and TNO-test, and Worth test to identify deficits misrouting.27,28 Therefore, a detailed ophthalmologic assess- in binocular visual function (i.e., lack of stereo vision and ment in CFNS, including the assessment of the integrity of the fusion, respectively). Conventional VEPs were recorded for all optic chiasm, appears particularly rewarding. participants, mfVEPs only for the CFNS patients and included Noninvasive electrophysiology is an indispensable tool for for the control group from Hoffmann et al.36 The procedures the assessment of the integrity of the visual system and followed the tenets of the declaration of Helsinki48 and the function in humans. Full-field ERGs allow for the assessment of protocol was approved by the ethics committee of the Otto- global retinal damage and conventional visual evoked poten- von-Guericke-University of Magdeburg. All participants gave tials (cVEP) for the assessment of the integrity of the visual their informed written consent prior to the study. It should be pathways in their entirety.29,30 The latter can be combined noted that investigations based on magnetic resonance imaging with the multifocal stimulation technique for a spatially (e.g., fMRI) though of promise for the investigation of resolved assessment of cortical visual field representations abnormal visual field projections,49–51 could not be conducted yielding multifocal VEPs (mfVEPs31,32). Conventional VEPs in the patients with ephrin-B1 deficiency included in the have been adapted to detect malformations of the optic chiasm present study due to an MRI-incompatibility, lack of compli- yielding the ‘‘misrouting VEP.’’2 For this purpose, the ance, or low age. interhemispheric difference VEPs are determined and com- Ephrin-B1 mutations were confirmed in the patients by pared for left and right eye stimulation. If they have the same molecular genetic analysis. The male participant (P1) and his polarity for left and right eye stimulation, this is evidence for a affected daughter (P4) carried the familial mutation p. T111I normal lateralization pattern of the representation of the visual previously functionally analyzed in tissue culture experi-

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TABLE. Characteristics of Ephrin-B1 Deﬁcient Participants

Visual Acuity Stereo-Tests

Subject Sex Age RE LE Lang TNO Strabismus Dom. Eye Amblyopia Nystagmus ERG

P1 M 49 0.8 0.8 Esotropia OS n P2 F 27 0.32 0.8 vert. s. OS OD [ P3 F 50 1.0 0.08 S. div. þ vert. s. OD OS /epn n P4 F 6 (0.25) (0.4) [[Orthotropia [ [ n, normal; ‘‘’’, negative or absent; [, not determined; m, male (hemizygous ephrin-B1 deﬁciency); f, female (heterozygous ephrin-B1 deﬁciency); s. div, strabismus divergens; vert. s., vertical strabismus; dom. eye, dominant eye; epn, end point nystagmus.

ments.23,52 The other two affected females, mother (P3) and while the respective fellow eye was patched. The blocks were daughter (P2), carried the novel mutation c.374A>G, presented in a balanced interleaved sequence (‘a-b-b-a’- p.(E125G). Both mutations are located in the extracellular scheme). ephrin domain. No abnormalities of fundus, macula, iris, or Conventional VEPs (cVEPs) – Recording. The electro- optic nerve head were observed during an ophthalmologic encephalogram (EEG) was recorded with gold-cup electrodes examination and spectral-domain optical coherence tomogra- at Oz, OL, and OR (4 cm left and right from Oz, respectively), phy (SD-OCT; Spectralis OCT, Heidelberg Engineering, Ger- referenced to Fz.57 The ground electrode was attached to Fpz. many). To test the integrity of retinal processing, ERGs were The EEG was amplified with a physiological amplifier (Grass, recorded according to the International Society for Clinical 350,000), analogue filtered in the range of 0.3 to 100 Hz and Electrophysiology of Vision (ISCEV)-Standard53 in the patient digitized at a rate of 1 kHz with 12-bit resolution. Stimulation cohort (Retiport, Roland Consult, Brandenburg, Germany) and (frame rate 75 Hz) and recording employed the ‘‘EP2000 healthy scotopic and photopic ERG responses were obtained. Evoked Potentials System’’58 running on a G4 Power Macintosh Refraction corrected monocular decimal visual acuities, (Apple, Inc., Cupertino, CA, USA). This program presented the binocular visual function, and other patient details are given stimuli while stepping through the check size sequence, in the Table. acquired the signals, displayed them on-line, checked for and discarded artefacts (using an amplitude window of generally VEP Investigations 650 lV and repeating sweeps where this was exceeded), displayed on-line averages, and saved the records for off-line Rationale of VEP-Detection of Albinotic Misrouting of processing. To ensure participant alertness, random digits from the Optic Nerves. In albinism, each eye projects predomi- 0 to 9 appeared in random intervals at the center of the screen nantly to its contralateral hemisphere. Monocular stimulation and were reported by the participants. of the central visual field is therefore expected to elicit greater For visual stimulation black-and-white checkerboard pat- VEPs on the hemisphere contralateral to the stimulated eye terns were presented monocularly at a viewing distance of 114 than on the ipsilateral hemisphere. As a consequence, the cm in pattern-onset-offset mode (40 ms on, 440 ms off).59 The polarity of the interhemispheric VEP difference is inverted for central visual field (1983158) was stimulated with a left compared with right eye stimulation in participants with checkerboard using three different check sizes that were albinism.2 In controls, interhemispheric activation differences presented in an interleaved manner (2.08, 1.08, and 0.58). A are also observed, even for bilateral cortical activations, which total of 160 responses per condition were obtained. The is due to asymmetries of the occipital cortex. In contrast to participants were instructed to maintain fixation at a central albinism, however, in controls the polarity differences do not target (38 diameter) and wore optimal refractive correction. depend on the eye stimulated (see Fig. 2). Supplementing this The recordings were performed for 98% stimulus contrast paradigm with a correlation analysis simplifies the approach twice for each eye in an interleaved sequence and then and enhances its objectivity. In albinism, the interhemispheric repeated at 20% stimulus contrast, again twice for each eye. activation differences obtained for right and left eye stimula- The stimulus had a mean luminance of 45 cd/m2. tion are, due to the polarity inversion of the traces, likely to be cVEP – Analysis. The offline analysis was performed using negatively correlated. In contrast, for control participants (i.e., IGOR 6.22 (WaveMetrics, Inc., Lake Oswego, OR, USA). The in the absence of such a polarity inversion) they are likely to be difference VEPs (OL OR) for each eye were digitally low-pass positively correlated.36,44,54–56 This correlation approach filtered (40 Hz cutoff) and correlated with each other to obtain supports an objective analysis even of small signals. To sample Pearson’s correlation coefficient (r; ranging between 1 and 1). the visual field for representation abnormalities in a spatial In accordance with previous studies36,56 a time window from resolved manner, the above VEP-paradigm for the detection of 50 to 250 ms was used for this correlation. The correlation misrouted optic nerves can be combined with the multifocal allows for the distinction of normal and abnormal projections stimulation technique using mfVEPs.34 Multifocal VEPs are of the optic nerves: positively correlated traces indicate that recorded to nonbilateral stimuli at distinct visual field locations both eyes project to the same cortical regions, while negatively in either hemifield. Therefore, the cortical responses in correlated traces indicate that both eyes project to opposite controls are asymmetrical leading to sizable interhemispherical hemispheres54,56 (see above). mfVEPs, which, as for the misrouting cVEPs, do not depend in Multifocal VEPs (mfVEPs) – Recording. Multifocal VEPs their polarity on the eye stimulated, which contrasts to were recorded from six gold cup electrodes referenced to the albinism.34–36 inion: Electrodes were placed at OL and OR, as defined above, Procedure. The recording and analysis procedures for and8-cmleftandrighttothelocation1cmabovetheinion cVEPs and mfVEPs followed procedures described earlier.36 In (lateral occipital sites) and 5 cm left and right to POz (lateral a dimly lit room cVEPs and mfVEPs were recorded in parietal sites57). The EEG was amplified with a physiological successive sessions separated by a break. The entire recording amplifier (Grass; 3100,000), band-pass filtered (low and high session including preparation and breaks took around 2.5 frequency cut-offs 3 and 100 Hz), and digitized at 1200 Hz. hours. Left and right eyes were stimulated in separate blocks, VERIS 5.01.10X (Electro-Diagnostic Imaging [EDI], San Mateo,

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FIGURE 1. Fundus photographs and OCT scans for the left (LE) and right eye (RE) of P1 (hemizygous male) and P2 (heterozygous female patient).

CA, USA) was used for stimulus delivery and electrophysio- cross of 3.08 diameter. The stimulus display, a circular logical recordings. Supported by a chin rest, participants dartboard pattern (diameter 448; mean luminance 64 cd/m2; viewed the stimuli that were presented at a distance of 36 cm contrast 98%), was subdivided into 60 individual fields, each on a computer monitor driven with a frame rate of 75 Hz. comprising a checkerboard of 4 3 4 checks. The radial extent They were requested to fixate the center of a central black of the fields was scaled with eccentricity from 1.58 in the

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FIGURE 2. Misrouting cVEP. Righthand panels show schematics and lefthand panels data from individuals for the detection of normal partial crossing at the optic chiasm (C1 and C2), enhanced crossing as typical for albinism (A1), and reduced crossing as typical for achiasma (Ach1). Interhemispherical difference cVEPs were calculated in each individual for three different check sizes as detailed in Methods. Parallel interhemispherical difference cVEPs indicate normal optic nerve projections, antiparallel indicate a deviation from the normal partial decussation.

center to 78 in the periphery. The fields were stimulated reversal and pattern-onset stimulation,61 which is in contrast independently with an m-sequence. M-sequences consist of a to cVEPs. pseudorandom succession of 0 and 1 states. For the pattern- mfVEP – Analysis. First order kernels were extracted using reversal stimulation34,60 applied, these two states were VERIS 5.01 (EDI). Spatial smoothing and artefact rejection represented by two contrast-inverted checkerboard fields. features available in VERIS were not used. All subsequent The minimal duration of one state lasted one frame (i.e., 13.3 analyses were performed with IGOR 5.0 (WaveMetrics, Inc.). The traces were, in accordance with previous studies,34,35 ms). Stimuli were presented monocularly in two separate digitally low-pass filtered with a high frequency cut-off of 30 blocks for either eye, yielding a total of four blocks of mfVEP Hz. To assess the lateralization of the responses, we calculated recording. A single block of pattern-reversal stimulation the difference potentials between each of the three electrodes 15 lasting 7 minutes consisted of an m-sequence with 2 -1 on one hemisphere and its corresponding electrode on the (i.e., 32.767) elements. The blocks were divided into 16 other hemisphere. These difference potentials entered the overlapping segments each lasting approximately 27 seconds. further analysis. Multifocal VEPs were recorded to pattern-reversal stimula- To assess signal presence we evaluated the signal-to-noise tion, as pattern-reversal mfVEPs exceed pattern-onset mfVEPs ratio (SNR) as described by Hood et al.62 using a ‘‘mean noise- at stimulus eccentricities beyond 108.61 It must be noted that, window SNR.’’ First, the records from the two blocks for each while pattern-onset stimulation is mandatory for conventional stimulus were averaged. Then the SNR for each i-th sector (of 30,37 the n ¼ 60 total sectors) of participant j was defined as misrouting VEPs, for mfVEPs to pattern-onset and ÂÃ pattern-reversal stimulation similar accuracies were deter- SNRij ¼ RMSijð45150 msÞ= RiRMSijð325430 msÞ=n ð1Þ mined previously for the differentiation of normal and abnormal response lateralizations.34,35 This is likely related The denominator in Equation 1 is the average of the to the finding of similar wave shapes in mfVEPs to pattern- individual RMS values of n ¼ 60 sectors in the noise window

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analysis and is particularly useful for dealing with small signal amplitudes.

RESULTS Ophthalmologic Status in Ephrin-B1 Deficiency As illustrated in Figure 1 for P1 and P2, fundus, optic nerve head and foveal development were normal in all participants with ephrin-B1 deficiency. Scotopic and photopic visual function was assessed in P1 and P3 with full-field ERGs and found to be within normal range. In contrast, for visual function the picture was more diverse as shown in the Table. Severe monocular reductions of visual acuity were evident for the right and left eye of P2 and P3, respectively. These were attributed to amblyopia due to strabismus (P2 and P3). Strabismus was evident in all patients but P4.

Conventional Misrouting VEP From the obtained cVEPs difference VEPs were calculated to determine the interhemispherical activation difference as depicted in Figure 2 for reference subjects and Figure 3 for the patients with ephrin-B1 deficiency. Notably in the latter parallel trace pairs were obtained for each condition. This demonstrates similar response lateralization’s for both eyes, and thus indicates the absence of large scale misrouting of the optic nerves, which is typical for albinism and achiasma (Fig. 2). While the large interhemispheric response differences for P4 might indicate pronounced interhemispherical asymmetries in cortical folding, the responses from both eyes are similarly affected underlining the absence of relevant optic nerve misrouting. A quantitative analysis of the similarity of the difference VEPs obtained for right and left eye stimulation is provided in Figure 4. Trace similarity was assessed via the correlation coefficient, r (see Methods), for each trace pair in Figure 3, as FIGURE 3. Individual interhemispheric activation-difference cVEPs for detailed in Methods. As a reference, data for controls, albinism, the ephrin-B1–deficient patients for three check sizes (2.08, 1.08, and and achiasma (examples shown in Fig. 2) are included. While 0.58, as indicated by the icons) as obtained for the right (black traces) there was a strong overlap of r values between controls and and left eye (gray traces). Parallel traces were evident for all patients. patients with ephrin-B1 deficiency, there was none between albinism/achiasma and CFNS (even though one of the albinotic individuals had particularly small evidence of misrouting, i.e., positive correlation coefficients). This underlines the absence (325–430 ms after stimulus onset). An estimate of false positive of sizable misrouting of the optic nerves. Further, for none of rates was obtained from the distribution of SNR values the ephrin-B1–deficient patients consistently reduced values 32 r following Hood and Greenstein and showed for the control were obtained for all conditions. participants that the probability of SNR greater than or equal to 1.75 to be part of the noise distribution was smaller than 5.4%. Multifocal Misrouting VEP We therefore applied an SNR threshold of 1.75 to exclude ‘silent’ visual field locations (i.e., without recordable signals) In order to characterize the topography of the cortical visual from our analyses. In our quantitative analyses we compared field representations mfVEPs were recorded in P1, P2, and P3 two stimulus conditions (i.e., left and right eye stimulation). and compared with control data. In Figure 5 the individual Each stimulus location was required to evoke suprathreshold trace arrays (interhemispherical mfVEP differences) and the responses in at least one of the two conditions to enter the assessment of intraindividual interocular trace similarities with analysis (logical OR-operator).36 interocular correlation coefficients, r, are depicted. Supra- For further analysis we selected for each visual field location threshold (SNR ‡ 1.75) responses, allowing for the assessment of the response lateralization’s, were obtained for 72%, 80%, the difference potential for the pair of electrodes on opposing and 60% of the visual field locations tested, in P1, P2, and P3, hemispheres, which yielded the greatest SNR during stimula- 62 respectively. Parallel (i.e., positively correlated [filled symbols tion of either eye. This ensured that the same electrode pair in Fig. 5]), traces are indicative of normal routing, while was selected for left and right eye stimulation. Next, similar to antiparallel (i.e., negatively correlated [open symbols in Fig. the analysis of the cVEPs, the difference VEPs obtained for each 5]), traces are indicative of abnormal routing (see also Fig. 1 in eye were correlated with each other to obtain Pearson’s Hoffmann et al.31). As for controls,34–36 for most suprathresh- correlation coefficient (r). For this correlation the ‘signal time old visual field locations positively correlated responses were window’ (45–150 ms; see above) was used in accordance with obtained. For only very few locations the r values were less previous studies.34–36 It should be noted that the correlation than 0.5, which is indicative of antiparallel traces for the approach is a more objective approach than a single peak corresponding traces (i.e., in P1, P2, and P3 for two, one, and

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FIGURE 4. Individual interocular correlation coefficients (r) of interhemispheric activation differences given in separate columns for albinotic/ achiasmic participants, controls, and ephrin-B1–deficient patients (hemizygous male, P1 and heterozygous females, P2–4) for the three check sizes used (2.08, 1.08, and 0.58). There is no overlap of the r values of controls and ephrin-B1–deficient patients with those of the albinotic/achiasmic participants. Further, ephrin-B1–deficient patients and controls had consistently positive r values across conditions.

two locations, respectively). It should be noted, however, that and report deficits of visual function, but a general integrity of the recordings in P1 were heavily contaminated with alpha the cortical visual field representations. This is taken as waves, which is likely to be associated with the occasional evidence that neither functional deficiency of ephrin-B1 itself seemingly antiparallel traces. Consequently, also mfVEPs nor cellular interference is sufficient to induce major misrout- corroborate the notion of normal optic nerve projections in ing of the optic nerves of the human chiasm and indicates the ephrin-B1–hemizygous patients. For P2 only one suprathresh- importance of other embryonic guidance mechanisms in old location was less than 0.5. This paramacular location was humans. indeed associated with antiparallel traces for the two eyes. This suggests, if any, a minor, much localized potential misrouting in Optic Nerve Projections and Visual Field this ephrin-B1–heterozygous patient. For P3 noise levels in the Representations in Ephrin-B1 Deficiency data were high and the responses were particularly small for the left eye due to its strong amblyopia. Only two suprathresh- In ephrin-B1–deficient animal models, that is, ephrin-B1/Y old locations were less than 0.5, again suggesting, if any, a (hemizygous ephrin-B1–deficient mice) and ephrin-B1/ minor localized potential misrouting in ephrin-B1–heterozy- (ephrin-B1–deficient mice) the optic nerve crossing is stronger gous patients. These findings are supported by the quantitative than normal, in fact the proportion of ipsilaterally projecting analysis of the frequency distributions of the r values depicted retinal ganglion cells is decreased by 31%.14 Here, we report in Figure 6. Moreover, it should also be noted in Figure 6, that for the misrouting-cVEP the absence of misrouting in the four although for all ephrin-B1–heterozygous patients (P2 and P3) participants tested and for the misrouting-mfVEP weak the distribution is shifted to the right (i.e., to smaller evidence of possible local misrouting for less than 6% visual correlation values) the frequency of clearly negative coeffi- field locations. It is concluded that sizable misrouting is absent cients (<0.50) does not exceed 6%. This is taken as an in ephrin-B1–deficiency. indication of noise in the data, but also of the absence of Furthermore, the patterns of mfVEP response signatures are sizable misrouting, which would result in higher frequencies of clearly in accordance with a normal gross-retinotopy of the r values less than 0.50 (for reference see, e.g., Fig. 4 in cortical visual field representations. They support a represen- Hoffmann et al.34 or Fig. 5 in Hoffmann et al.32). tation of the left and right visual field on opposing hemispheres It should be noted that for the vertical derivation (i.e. Oz and a representation of the upper and lower visual fields on versus Iz) the well-known polarity inversion for lower versus opposite banks of the calcarine sulcus. It is concluded that upper visual hemifield mfVEPs was also evident in the above cortical visual field representations in ephrin-B1–deficient patients (Supplementary Fig. S1). This is evidence for the patients do not diverge from the general principle of representation of these hemifields on opposing banks of the retinotopy. calcarine. Taken together with the polarity inversion for left versus right hemifield mfVEPs, this indicates a normal gross- Visual Function in Ephrin-B1–Deficient Patients retinotopic representation (i.e., the representation of left and right hemifields on opposing hemispheres) and of upper and No fundus abnormalities were evident in the individuals lower hemifields on opposing banks of the calcarine sulcus. assessed, nor were scotopic and photopic ERGs reduced. Importantly, deficits in binocular vision, such as strabismus and the absence of stereo vision, were clearly evident in the three DISCUSSION adult participants of the study and not fully tested in the 6-year old. These deficits are in accordance with previous reports on We studied participants with ephrin-B1 deficiency from two ephrin-B10–hemizygous and –heterozygous participants,22,23 independent families with two different ephrin-B1 mutations and are also likely related to the observation of amblyopia in

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FIGURE 5. Comparison of right and left eye mfVEPs (interhemispherial activation differences [horizontal derivations] as described in Methods) to pattern-reversal stimulation for ephrin-B1–deficient patients P1, P2, and P3. Traces and symbols are arranged corresponding to the spatial layout of the visual field locations that evoked them, but traces and symbols from different eccentricities are arranged in an equidistant manner, while the actual stimulus layout is approximately m-scaled. On the left interhemispheric mfVEP difference traces after right (black traces) and left eye stimulation (gray traces) are depicted. As for controls, the responses varied across the visual field, specifically responses from opposing hemifields tend to be inverted in their polarity (for P1 and P2 arrows highlight the polarity inversion of traces on opposite sides of the vertical meridian). Importantly, for a particular visual field location similar trace signatures were obtained for both eyes and only few visual field locations are associated with inverted polarities for left and right eye stimulation, as depicted on the righthand panels with correlations of the interhemispheric mfVEP differences to stimulation of the left and right eye. The strength of the correlation is coded by the diameter of the circles, which scale linearly with the absolute correlation coefficient (r) obtained (filled symbols, positive correlation, i.e., normal projection; open symbols, negative correlation, i.e., abnormal projection). Subthreshold responses (SNR < 1.75) are indicated by ‘þ’. As for controls, for most suprathreshold visual field locations positively correlated responses were obtained. For only few locations the r values were less than 0.5 (for two, one, and two locations in P1, P2, and P3, respectively), which indicated antiparallel traces.

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eye mix through each hemichiasm prior to separating.20 These differences from murine models prompt the question of which molecular mechanisms other than ephrin-B1 are of importance for the normal formation of the partial decussation at the human optic chiasm. We presume that other components of the ephrin-system can substitute the lack of ephrin-B1 in humans. As ephrin-B2 has been identified, again in murine models, as a ligand of strong importance for the formation of the partial decussation,13 it is a promising candidate to be tested for its relevance for optic chiasm formation in humans in future studies. However, to our knowledge patients with ephrin-B2 deficiency have not been identified yet.

Specificity of Misrouting-VEPs Enhanced crossing of the optic nerves at the chiasm has long been considered to be a highly specific pathognomonic sign for albinism.33 Two exceptions, however, have to be noted. On the one hand, sizable projection abnormalities were demonstrated for some cases of congenital stationary nightblindness (CSNB), especially x-linked CSNB.44,45 It is at present not fully FIGURE 6. Individual r value distributions for controls (n ¼ 10) and P1, resolved whether the occurrence of misrouting in these CSNB P2, and P3. The frequency of r values less than 1.00, 0.75, 0.50, 0.00, patients might be related to mild forms of albinism, in 0.50, and 0.75 is given. Although the distribution is shifted to the particular ocular albinism OA1, as it is also associated with right for all ephrin-B1–deficient patients, the frequency of clearly an x-linked locus. On the other hand, enhanced nerve fiber negative coefficients (<0.50) does not exceed 6%. crossing at the optic chiasm in the absence of albinism has been demonstrated for patients with foveal hypoplasia, optic the heterozygous adults of the study. Importantly, the VEP nerve decussation defects, and in some cases anterior segment findings of the present study clearly argue against optic chiasm dysgenesis.46 This syndrome has recently been termed malformations or a disruption of the retinotopic representa- FHONDA, which is associated with recessive mutations in tions in the visual cortex as a cause of these binocular vision the putative glutamine transporter SLC38A8 located within a deficits. Taken together, there are some specific deficits in 3.1-Mb interval containing 33 genes on chromosome 16q23.3- visual function in ephrin-B1–deficient patients, however, they 24.1.63,64 The question of the association of these two are not related to gross-misrouting at the optic chiasm. Their conditions with albinism deserves particular attention and is cause is unknown and might be due to more subtle alterations currently under investigation.65 In contrast and importantly, in the visual system not accessible to the methods of the Ephrin-B1 deficiency in humans and CFNS, do not add another present study. exception to the power of the misrouting VEP in detecting albinism. There appears to be no relevant misrouting in the Mechanisms for Optic Chiasm Formation in cases reported in the present study, which underlines the high Humans and Animal Models specificity of the misrouting VEP paradigms for the identifica- tion of albinism in clinically ambiguous and unresolved cases. Animal studies in transgenic mice highlight the importance of both ephrin-B1 and ephrin-B2 for axonal pathfinding at the Limitations and Outlook optic chiasm. Remarkably, the complete absence of ephrin-B1 in humans usually leads to a largely normal phenotype. The A major limitation of the present study arises from the rarity of case of the present study had deficits in binocular vision, patients with ephrin-B1 mutations causing functional ephrin- which are not the rule for ephrin-B1 hemizygosity, but still B1 deficiency. Although female patients with their pronounced normal visual pathways as tested within the scope of the CFNS-phenotype are rare, they are at least readily recognized. present study. It is concluded that ephrin-B1 itself does not Hemizygous males are also rare, but due to their usually mild appear to be indispensable for normal nerve routing at the phenotype they are commonly only detected via their female human optic chiasm. In contrast to hemizygosity, haploinsuffi- descendants. Consequently, only a small group of participants ciency of ephrin-B1 in heterozygous humans usually leads to was included. This has a number of consequences for the the abnormal phenotype typical for CFNS, highlighting the present study. Firstly, the findings described for ephrin-B1 severe consequences of competing ephrin-B1 and ephrin-B1– hemizygosity are based on a single case. However, this is the deficient populations in a cellular mosaic. This however, does first report on visual pathways in this condition and it provides not appear to affect optic nerve routing. In conclusion, as clear evidence of the absence of enhanced optic nerve neither complete loss nor haploinsufficiency of ephrin- crossing, as it is typically found in albinism, even though the B1combined with cellular interference appear to affect the participant had deficits in binocular visual function. Secondly, partial decussation, ephrin-B1 does not appear to be involved the findings for ephrin-B1 heterozygosity are based on only in this process in humans. This highlights the fact that there three cases. It must be noted, however, that they provide are significant differences in the mechanisms guiding optic concurrent evidence of the absence of gross misrouting and of chiasm formation in the murine model and humans, as has only very minor local misrouting, if any, in this condition. been suggested previously. Indeed, midline interactions at the Thirdly, the study was limited to ophthalmologic and optic chiasm are more important in the former. Moreover, the noninvasive electrophysiological investigations, but did not anatomy of the chiasm itself differs. In humans uncrossed make use of MRI and fMRI as the participants did not fulfil the axons are confined laterally and do not mix in each hemi- respective inclusion criteria. Therefore, only physiological chiasm, while in rodents and ferrets, retinotopic order is lost in evidence from retinal and cortical signals, as provided by the proximal optic nerve and the two projections from each ERGs, cVEP, and mfVEPs was available. Clearly, these methods

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allowed for the assessment of the integrity of retina and optic 13. Williams SE, Mann F, Erskine L, et al. Ephrin-B2 and EphB1 nerve routing and to some extent of retinotopic features of the mediate retinal axon divergence at the optic chiasm. Neuron. cortical representations. However, detailed cortical retinotopic 2003;39:919–935. maps and signals from subcortical projection targets (e.g., the 14. Chenaux G, Henkemeyer M. Forward signaling by EphB1/ lateral geniculate nucleus and the superior colliculus), and EphB2 interacting with ephrin-B ligands at the optic chiasm is brain anatomy are the domain of MRI and fMRI. Therefore, required to form the ipsilateral projection. Eur J Neurosci. future studies should, besides increasing the sample size for 2011;34:1620–1633. the presented findings, aim at MRI and fMRI investigations to 15. Lambot MA, Depasse F, Noel JC, Vanderhaeghen P. Mapping complement the present description of the visual system in labels in the human developing visual system and the human ephrin-B1 deficiencies at the cortical and subcortical evolution of binocular vision. J Neurosci. 2005;25:7232–7237. level. This is of great promise to resolve the discrepancy 16. Herrera E, Brown L, Aruga J, et al. Zic2 patterns binocular between deficits specifically of binocular visual function and vision by specifying the uncrossed retinal projection. Cell. the reported largely normal visual pathways in human ephrin- 2003;114:545–557. deficiency. 17. Rebsam A, Bhansali P, Mason CA. Eye-specific projections of retinogeniculate axons are altered in albino mice. J Neurosci. Acknowledgments 2012;32:4821–4826. The authors thank the study participants for their support. 18. Ilia M, Jeffery G. 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