November 2015
Volume 56, Issue 12
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Visual Neuroscience  |   November 2015
Visual Pathways in Humans With Ephrin-B1 Deficiency Associated With the Cranio-Fronto-Nasal Syndrome
Author Affiliations & Notes
  • Michael B. Hoffmann
    Department of Ophthalmology Otto-von-Guericke-University, Magdeburg, Germany
    Center for Behavioural Brain Sciences, Magdeburg, Germany
  • Hagen Thieme
    Department of Ophthalmology Otto-von-Guericke-University, Magdeburg, Germany
  • Karin Liedecke
    Department of Ophthalmology Otto-von-Guericke-University, Magdeburg, Germany
  • Synke Meltendorf
    Department of Ophthalmology Otto-von-Guericke-University, Magdeburg, Germany
  • Martin Zenker
    Institute for Human Genetics, Otto-von-Guericke-University, Magdeburg, Germany
  • Ilse Wieland
    Institute for Human Genetics, Otto-von-Guericke-University, Magdeburg, Germany
  • Correspondence: Michael B. Hoffmann, Universitäts-Augenklinik, Visual Processing Laboratory, Leipziger Str. 44, 39120 Magdeburg, Germany; michael.hoffmann@med.ovgu.de
Investigative Ophthalmology & Visual Science November 2015, Vol.56, 7427-7437. doi:10.1167/iovs.15-17705
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      Michael B. Hoffmann, Hagen Thieme, Karin Liedecke, Synke Meltendorf, Martin Zenker, Ilse Wieland; Visual Pathways in Humans With Ephrin-B1 Deficiency Associated With the Cranio-Fronto-Nasal Syndrome. Invest. Ophthalmol. Vis. Sci. 2015;56(12):7427-7437. doi: 10.1167/iovs.15-17705.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Numerous animal studies demonstrated the importance of components of the ephrin/Eph system for correct visual system development. Analogous investigations in humans are entirely missing. Here, we examined the visual system in humans with ephrin-B1 deficiency, which is x-linked and associated with the cranio-fronto-nasal syndrome (CFNS) in heterozygous females.

Methods: For one male hemizygous for ephrin-B1 deficiency and three affected heterozygous females with molecular-genetically confirmed mutations, the integrity of the partial decussation of the optic nerves was assessed with visual evoked potentials (VEPs) and compared with albinotic, achiasmic, and control participants with healthy vision. Further, retinal morphology and function and the gross-retinotopic representation of the primary visual cortex were examined with spectral-domain optical coherence tomography (SD-OCT), ERG, and multifocal (mf) VEPs for the male participant and part of the carriers.

Results: Strabismus and lack of stereovision was evident in the male and two of the females. 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.

The optic chiasm is a key structure in the visual system.1 Here, the fate of axons of the retinal ganglion cells is decided during early development such that axons carrying information from the right visual hemifield are guided to the left hemisphere and vice versa. In the human visual system this results in a partial crossing of the optic nerves at the chiasm: fibers from the nasal retina receive input from the ipsilateral visual field and consequently cross the midline separating the hemispheres, and fibers from the temporal retina receive input from the contralateral visual field and consequently remain uncrossed. Remarkably, deviations from this pattern have been observed in specific patient groups (i.e., in albinism with enhanced crossing of the temporal fibres at the chiasm2,3 and in achiasma4,5 with reduced or absent crossing of the nasal fibers). Although it might appear that such misrouting of the optic nerves should completely obstruct vision in the affected individuals, visual function is only partially impaired in these cases.1 Typically, nystagmus associated with reduced visual acuity and strabismus associated with often absent binocular vision are observed, while other visual functions such as pattern detection and visuomotor integration appear largely unaffected. 
Molecular mechanisms shape a number of different processes of optic chiasm formation.6 They promote crossed or uncrossed projections, or guide the general patterning of the optic chiasm. In animal studies, particular attention has been paid to the Eph/ephrin receptor/ligand families of cell surface signalling proteins. There are 14 Eph receptors and eight ephrin ligands in mammals,7 both subdivided into A and B classes. Remarkably, there is promiscuity within each class (i.e., EphA receptors can bind with diverse ephrin-As, and likewise EphB with ephrin-Bs).8 Eph/ephrin interactions are important in development, especially in cell–cell interactions involved in nervous system patterning and axon guidance. Investigations of the development of the visual system demonstrated an importance of A and B class proteins for retinotopic map formation and for chiasm formation.6,810 Some studies suggest specific mechanisms that lead to enhanced crossing of the optic nerves in albinism. They demonstrated the relevance of EphB1-expression, regulated by the transcription factor Zic2,11,12 for the normal formation of the ipsilateral projection at the optic chiasm. Growth cones of retinal axons that are expressing EphB1 are normally repelled by the ligands ephrin-B2 and ephrin-B1 expressing glia at the optic chiasm midline, as demonstrated in EphB1−/− mice and EphB2−/−/EphB1−/Y mice.13,14 Accordingly, during human embryogenesis EphB1 is expressed in the ganglion cells of the temporal retina, which is projecting ipsilaterally.15 Importantly, in animal models of albinism the expression of the transcription factor Zic216 and consequently EphB117 is reduced, which corresponds to the enhanced crossing of the optic nerves at the chiasm in albinism. The relation of these changes to the pigmentation defect in albinism (i.e., the reduction of ocular or oculocutaneous melanin levels) is still under investigation. It is presumably associated with the delaying effect of hypopigmentation on retinal neurogenesis likely mediated via reduced retinal levels of the melanin precursor L-DOPA.1821 
The above highlights the importance of the interaction of ephrin-ligands and Eph receptors for neural guidance during visual system development. However, it must be noted that it is, at present, uncertain, to which extent these mechanisms actually translate to the development of the human optic chiasm, which differs distinctly in its architecture from that of the relevant murine animal models.22 Specific investigations of the visual system in humans are needed, but difficult to conduct as humans with deficiencies in the ephrin/Eph system are extremely rare. In fact, currently only one syndrome associated with relevant deficiencies is known (i.e., the cranio-fronto-nasal syndrome [CFNS MIM 304110]). Remarkably, only a decade ago ephrin-B1 deficiency was demonstrated to be causal for CFNS,23,24 which spurred the notion, that enhanced crossing of the optic nerves might also be evident in CFNS-patients. This patient group might thus provide a key to our understanding of the role of ephrin-B1 in the formation of the human optic chiasm. Cranio-fronto-nasal syndrome is a very rare condition, with an estimated incidence of 1:100,000, and predominantly affects the phenotype of females. Counterintuitively, however, the inheritance is x-chromosomal giving rise to a genetic paradox. The absent or mild phenotype in male carriers is likely due to the promiscuity of the ephrin/Eph ligand/receptor system. In contrast, the more severe manifestation in females may be explained by cellular interferences that are caused by the combination of ephrin/Eph ligand/receptor promiscuity and the consequences of random x-chromosomal inactivation in distinct cellular compartments.25 Cranio-fronto-nasal syndrome is associated with craniofacial dysplasia, body asymmetries, facial asymmetries, hypertelorism, midline defects, skeletal abnormalities, and dermatologic abnormalities. Apart from the description of some peculiarities of visual function, such as strabismus, nystagmus, amblyopia, an in depth analysis of the visual system of the affected individuals is currently missing.25,26 Strabismus, which has been reported to be prevalent in CFNS (incidence of 40%–89%25,26), is also typical for patients with optic nerve misrouting.27,28 Therefore, a detailed ophthalmologic assessment in CFNS, including the assessment of the integrity of the optic chiasm, appears particularly rewarding. 
Noninvasive electrophysiology is an indispensable tool for the assessment of the integrity of the visual system and function in humans. Full-field ERGs allow for the assessment of global retinal damage and conventional visual evoked potentials (cVEP) for the assessment of the integrity of the visual pathways in their entirety.29,30 The latter can be combined with the multifocal stimulation technique for a spatially resolved assessment of cortical visual field representations yielding multifocal VEPs (mfVEPs31,32). Conventional VEPs have been adapted to detect malformations of the optic chiasm yielding the “misrouting VEP.”2 For this purpose, the interhemispheric difference VEPs are determined and compared for left and right eye stimulation. If they have the same polarity for left and right eye stimulation, this is evidence for a normal lateralization pattern of the representation of the visual hemifields, if they have the opposite polarity, this is evidence for enhanced or reduced crossing of the optic nerves. This paradigm serves as a clinical routine tool for the detection of misrouting of the optic nerves33 and can also be combined with mfVEPs to identify localized optic nerve misroutings.3436 Enhanced crossing of the optic nerves has long been considered as a pathognomonic sign of albinism. As a consequence, the misrouting VEP has since served as an important diagnostic in the detection of albinism as the cause of ocular symptoms specified above. In fact, the abnormality is absent in human carriers of albinism34,35,37 and patients without albinism, but with some ocular symptoms observed in albinism, such as foveal hypoplasia,38 dissociated vertical deviation and missing stereopsis,3941 and congenital nystagmus.42,43 Further, normal projections were observed in patients with unilateral anophthalmia or severe microphthalmia22 and in patients with generalized lateralization abnormalities (i.e., situs inversus in the Kartagener syndrome, and in primary ciliary dyskinesia in general).36 However, it must be noted that comparatively rare incidences of enhanced crossing in the absence of albinism and the presence of other syndromes are also known.4446 
Here, we present a small case series, comprising one male hemizygous and three females heterozygous for ephrin-B1 deficiency of this rare patient group and thus provide the first account assessing visual pathways in patients with ephrin-B1 deficiency. We give a detailed description of the visual system in the participants with a particular focus on the assessment of the integrity of the cortical visual field representations. Remarkably, while deficits of visual function were evident, there was an absence of major representation abnormalities at the level of the visual cortex. 
Methods
Participants
Four participants with defective ephrin-B1 (aged 6–50 years, including one male) and the following reference datasets from previous studies were included in the study: (1) 10 visually healthy control participants (aged 18–60 years) with decimal visual acuity greater than or equal to 1.0 of a previous study,36 (2) 14 albinotic participants (aged 7–47 years; 6 male; 13 participants of a previous study47), and (3) one participant without optic chiasm (aged 22; detailed in Hoffmann et al.5). Binocular visual function in the participants with defective ephrin-B1 was assessed in a dedicated examination by an orthoptist, including alternating cover-test to identify strabismus, and Lang- and TNO-test, and Worth test to identify deficits in binocular visual function (i.e., lack of stereo vision and fusion, respectively). Conventional VEPs were recorded for all participants, mfVEPs only for the CFNS patients and included for the control group from Hoffmann et al.36 The procedures followed the tenets of the declaration of Helsinki48 and the protocol was approved by the ethics committee of the Otto-von-Guericke-University of Magdeburg. All participants gave their informed written consent prior to the study. It should be noted that investigations based on magnetic resonance imaging (e.g., fMRI) though of promise for the investigation of abnormal visual field projections,4951 could not be conducted in the patients with ephrin-B1 deficiency included in the present study due to an MRI-incompatibility, lack of compliance, or low age. 
Ephrin-B1 mutations were confirmed in the patients by molecular genetic analysis. The male participant (P1) and his affected daughter (P4) carried the familial mutation p. T111I previously functionally analyzed in tissue culture experiments.23,52 The other two affected females, mother (P3) and daughter (P2), carried the novel mutation c.374A>G, p.(E125G). Both mutations are located in the extracellular ephrin domain. No abnormalities of fundus, macula, iris, or optic nerve head were observed during an ophthalmologic examination and spectral-domain optical coherence tomography (SD-OCT; Spectralis OCT, Heidelberg Engineering, Germany). To test the integrity of retinal processing, ERGs were recorded according to the International Society for Clinical Electrophysiology of Vision (ISCEV)-Standard53 in the patient cohort (Retiport, Roland Consult, Brandenburg, Germany) and healthy scotopic and photopic ERG responses were obtained. Refraction corrected monocular decimal visual acuities, binocular visual function, and other patient details are given in the Table
Table
 
Characteristics of Ephrin-B1 Deficient Participants
Table
 
Characteristics of Ephrin-B1 Deficient Participants
VEP Investigations
Rationale of VEP-Detection of Albinotic Misrouting of the Optic Nerves.
In albinism, each eye projects predominantly to its contralateral hemisphere. Monocular stimulation of the central visual field is therefore expected to elicit greater VEPs on the hemisphere contralateral to the stimulated eye than on the ipsilateral hemisphere. As a consequence, the polarity of the interhemispheric VEP difference is inverted for left compared with right eye stimulation in participants with albinism.2 In controls, interhemispheric activation differences are also observed, even for bilateral cortical activations, which is due to asymmetries of the occipital cortex. In contrast to albinism, however, in controls the polarity differences do not depend on the eye stimulated (see Fig. 2). Supplementing this paradigm with a correlation analysis simplifies the approach and enhances its objectivity. In albinism, the interhemispheric activation differences obtained for right and left eye stimulation are, due to the polarity inversion of the traces, likely to be negatively correlated. In contrast, for control participants (i.e., in the absence of such a polarity inversion) they are likely to be positively correlated.36,44,5456 This correlation approach supports an objective analysis even of small signals. To sample the visual field for representation abnormalities in a spatial resolved manner, the above VEP-paradigm for the detection of misrouted optic nerves can be combined with the multifocal stimulation technique using mfVEPs.34 Multifocal VEPs are recorded to nonbilateral stimuli at distinct visual field locations in either hemifield. Therefore, the cortical responses in controls are asymmetrical leading to sizable interhemispherical mfVEPs, which, as for the misrouting cVEPs, do not depend in their polarity on the eye stimulated, which contrasts to albinism.3436 
Procedure.
The recording and analysis procedures for cVEPs and mfVEPs followed procedures described earlier.36 In a dimly lit room cVEPs and mfVEPs were recorded in successive sessions separated by a break. The entire recording session including preparation and breaks took around 2.5 hours. Left and right eyes were stimulated in separate blocks, while the respective fellow eye was patched. The blocks were presented in a balanced interleaved sequence (‘a-b-b-a’-scheme). 
Conventional VEPs (cVEPs) – Recording.
The electroencephalogram (EEG) was recorded with gold-cup electrodes at Oz, OL, and OR (4 cm left and right from Oz, respectively), referenced to Fz.57 The ground electrode was attached to Fpz. The EEG was amplified with a physiological amplifier (Grass, ×50,000), analogue filtered in the range of 0.3 to 100 Hz and digitized at a rate of 1 kHz with 12-bit resolution. Stimulation (frame rate 75 Hz) and recording employed the “EP2000 Evoked Potentials System”58 running on a G4 Power Macintosh (Apple, Inc., Cupertino, CA, USA). This program presented the stimuli while stepping through the check size sequence, acquired the signals, displayed them on-line, checked for and discarded artefacts (using an amplitude window of generally ±50 μV and repeating sweeps where this was exceeded), displayed on-line averages, and saved the records for off-line processing. To ensure participant alertness, random digits from 0 to 9 appeared in random intervals at the center of the screen and were reported by the participants. 
For visual stimulation black-and-white checkerboard patterns were presented monocularly at a viewing distance of 114 cm in pattern-onset-offset mode (40 ms on, 440 ms off).59 The central visual field (19° × 15°) was stimulated with a checkerboard using three different check sizes that were presented in an interleaved manner (2.0°, 1.0°, and 0.5°). A total of 160 responses per condition were obtained. The participants were instructed to maintain fixation at a central target (3° diameter) and wore optimal refractive correction. The recordings were performed for 98% stimulus contrast twice for each eye in an interleaved sequence and then repeated at 20% stimulus contrast, again twice for each eye. The stimulus had a mean luminance of 45 cd/m2
cVEP – Analysis.
The offline analysis was performed using IGOR 6.22 (WaveMetrics, Inc., Lake Oswego, OR, USA). The difference VEPs (OL − OR) for each eye were digitally low-pass filtered (40 Hz cutoff) and correlated with each other to obtain Pearson's correlation coefficient (r; ranging between −1 and 1). In accordance with previous studies36,56 a time window from 50 to 250 ms was used for this correlation. The correlation allows for the distinction of normal and abnormal projections of the optic nerves: positively correlated traces indicate that both eyes project to the same cortical regions, while negatively correlated traces indicate that both eyes project to opposite hemispheres54,56 (see above). 
Multifocal VEPs (mfVEPs) – Recording.
Multifocal VEPs were recorded from six gold cup electrodes referenced to the inion: Electrodes were placed at OL and OR, as defined above, and 8-cm left and right to the location 1 cm above the inion (lateral occipital sites) and 5 cm left and right to POz (lateral parietal sites57). The EEG was amplified with a physiological amplifier (Grass; ×100,000), band-pass filtered (low and high frequency cut-offs 3 and 100 Hz), and digitized at 1200 Hz. VERIS 5.01.10X (Electro-Diagnostic Imaging [EDI], San Mateo, CA, USA) was used for stimulus delivery and electrophysiological recordings. Supported by a chin rest, participants viewed the stimuli that were presented at a distance of 36 cm on a computer monitor driven with a frame rate of 75 Hz. They were requested to fixate the center of a central black cross of 3.0° diameter. The stimulus display, a circular dartboard pattern (diameter 44°; mean luminance 64 cd/m2; contrast 98%), was subdivided into 60 individual fields, each comprising a checkerboard of 4 × 4 checks. The radial extent of the fields was scaled with eccentricity from 1.5° in the center to 7° in the periphery. The fields were stimulated independently with an m-sequence. M-sequences consist of a pseudorandom succession of 0 and 1 states. For the pattern-reversal stimulation34,60 applied, these two states were represented by two contrast-inverted checkerboard fields. The minimal duration of one state lasted one frame (i.e., 13.3 ms). Stimuli were presented monocularly in two separate blocks for either eye, yielding a total of four blocks of mfVEP recording. A single block of pattern-reversal stimulation lasting 7 minutes consisted of an m-sequence with 215-1 (i.e., 32.767) elements. The blocks were divided into 16 overlapping segments each lasting approximately 27 seconds. Multifocal VEPs were recorded to pattern-reversal stimulation, as pattern-reversal mfVEPs exceed pattern-onset mfVEPs at stimulus eccentricities beyond 10°.61 It must be noted that, while pattern-onset stimulation is mandatory for conventional misrouting VEPs,30,37 for mfVEPs to pattern-onset and pattern-reversal stimulation similar accuracies were determined previously for the differentiation of normal and abnormal response lateralizations.34,35 This is likely related to the finding of similar wave shapes in mfVEPs to pattern-reversal and pattern-onset stimulation,61 which is in contrast to cVEPs. 
mfVEP – Analysis.
First order kernels were extracted using VERIS 5.01 (EDI). Spatial smoothing and artefact rejection features available in VERIS were not used. All subsequent analyses were performed with IGOR 5.0 (WaveMetrics, Inc.). The traces were, in accordance with previous studies,34,35 digitally low-pass filtered with a high frequency cut-off of 30 Hz. To assess the lateralization of the responses, we calculated the difference potentials between each of the three electrodes on one hemisphere and its corresponding electrode on the other hemisphere. These difference potentials entered the further analysis. 
To assess signal presence we evaluated the signal-to-noise ratio (SNR) as described by Hood et al.62 using a “mean noise-window SNR.” First, the records from the two blocks for each stimulus were averaged. Then the SNR for each i-th sector (of the n = 60 total sectors) of participant j was defined as    
The denominator in Equation 1 is the average of the individual RMS values of n = 60 sectors in the noise window (325–430 ms after stimulus onset). An estimate of false positive rates was obtained from the distribution of SNR values following Hood and Greenstein32 and showed for the control 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%. We therefore applied an SNR threshold of 1.75 to exclude ‘silent' visual field locations (i.e., without recordable signals) from our analyses. In our quantitative analyses we compared two stimulus conditions (i.e., left and right eye stimulation). Each stimulus location was required to evoke suprathreshold responses in at least one of the two conditions to enter the analysis (logical OR-operator).36 
For further analysis we selected for each visual field location the difference potential for the pair of electrodes on opposing hemispheres, which yielded the greatest SNR during stimulation of either eye.62 This ensured that the same electrode pair was selected for left and right eye stimulation. Next, similar to the analysis of the cVEPs, the difference VEPs obtained for each eye were correlated with each other to obtain Pearson's correlation coefficient (r). For this correlation the ‘signal time window' (45–150 ms; see above) was used in accordance with previous studies.3436 It should be noted that the correlation approach is a more objective approach than a single peak 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. 
Figure 1
 
Fundus photographs and OCT scans for the left (LE) and right eye (RE) of P1 (hemizygous male) and P2 (heterozygous female patient).
Figure 1
 
Fundus photographs and OCT scans for the left (LE) and right eye (RE) of P1 (hemizygous male) and P2 (heterozygous female patient).
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.
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.
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. 
Figure 3
 
Individual interhemispheric activation-difference cVEPs for the ephrin-B1–deficient patients for three check sizes (2.0°, 1.0°, and 0.5°, as indicated by the icons) as obtained for the right (black traces) and left eye (gray traces). Parallel traces were evident for all patients.
Figure 3
 
Individual interhemispheric activation-difference cVEPs for the ephrin-B1–deficient patients for three check sizes (2.0°, 1.0°, and 0.5°, as indicated by the icons) as obtained for the right (black traces) and left eye (gray traces). Parallel traces were evident for all patients.
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 detailed in Methods. As a reference, data for controls, albinism, and achiasma (examples shown in Fig. 2) are included. While there was a strong overlap of r values between controls and 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 of sizable misrouting of the optic nerves. Further, for none of the ephrin-B1–deficient patients consistently reduced r values were obtained for all conditions. 
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 (2.0°, 1.0°, and 0.5°). 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.
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 (2.0°, 1.0°, and 0.5°). 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.
Multifocal Misrouting VEP
In order to characterize the topography of the cortical visual field representations mfVEPs were recorded in P1, P2, and P3 and compared with control data. In Figure 5 the individual trace arrays (interhemispherical mfVEP differences) and the assessment of intraindividual interocular trace similarities with interocular correlation coefficients, r, are depicted. Suprathreshold (SNR ≥ 1.75) responses, allowing for the assessment of the response lateralization's, were obtained for 72%, 80%, and 60% of the visual field locations tested, in P1, P2, and P3, respectively. Parallel (i.e., positively correlated [filled symbols in Fig. 5]), traces are indicative of normal routing, while antiparallel (i.e., negatively correlated [open symbols in Fig. 5]), traces are indicative of abnormal routing (see also Fig. 1 in Hoffmann et al.31). As for controls,3436 for most suprathreshold visual field locations positively correlated responses were obtained. For only very few locations the r values were less than −0.5, which is indicative of antiparallel traces for the corresponding traces (i.e., in P1, P2, and P3 for two, one, and two locations, respectively). It should be noted, however, that the recordings in P1 were heavily contaminated with alpha waves, which is likely to be associated with the occasional seemingly antiparallel traces. Consequently, also mfVEPs corroborate the notion of normal optic nerve projections in ephrin-B1–hemizygous patients. For P2 only one suprathreshold location was less than −0.5. This paramacular location was indeed associated with antiparallel traces for the two eyes. This suggests, if any, a minor, much localized potential misrouting in this ephrin-B1–heterozygous patient. For P3 noise levels in the data were high and the responses were particularly small for the left eye due to its strong amblyopia. Only two suprathreshold locations were less than −0.5, again suggesting, if any, a minor localized potential misrouting in ephrin-B1–heterozygous patients. These findings are supported by the quantitative analysis of the frequency distributions of the r values depicted in Figure 6. Moreover, it should also be noted in Figure 6, that although for all ephrin-B1–heterozygous patients (P2 and P3) the distribution is shifted to the right (i.e., to smaller correlation values) the frequency of clearly negative coefficients (<−0.50) does not exceed 6%. This is taken as an indication of noise in the data, but also of the absence of sizable misrouting, which would result in higher frequencies of r values less than −0.50 (for reference see, e.g., Fig. 4 in Hoffmann et al.34 or Fig. 5 in Hoffmann et al.32). 
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 ‘+'. 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.
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 ‘+'. 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.
Figure 6
 
Individual r value distributions for controls (n = 10) and P1, P2, and P3. The frequency of r values less than 1.00, 0.75, 0.50, 0.00, −0.50, and −0.75 is given. Although the distribution is shifted to the right for all ephrin-B1–deficient patients, the frequency of clearly negative coefficients (<−0.50) does not exceed 6%.
Figure 6
 
Individual r value distributions for controls (n = 10) and P1, P2, and P3. The frequency of r values less than 1.00, 0.75, 0.50, 0.00, −0.50, and −0.75 is given. Although the distribution is shifted to the right for all ephrin-B1–deficient patients, the frequency of clearly negative coefficients (<−0.50) does not exceed 6%.
It should be noted that for the vertical derivation (i.e. Oz versus Iz) the well-known polarity inversion for lower versus upper visual hemifield mfVEPs was also evident in the above patients (Supplementary Fig. S1). This is evidence for the representation of these hemifields on opposing banks of the calcarine. Taken together with the polarity inversion for left versus right hemifield mfVEPs, this indicates a normal gross-retinotopic representation (i.e., the representation of left and right hemifields on opposing hemispheres) and of upper and lower hemifields on opposing banks of the calcarine sulcus. 
Discussion
We studied participants with ephrin-B1 deficiency from two independent families with two different ephrin-B1 mutations and report deficits of visual function, but a general integrity of the cortical visual field representations. This is taken as evidence that neither functional deficiency of ephrin-B1 itself nor cellular interference is sufficient to induce major misrouting of the optic nerves of the human chiasm and indicates the importance of other embryonic guidance mechanisms in humans. 
Optic Nerve Projections and Visual Field Representations in Ephrin-B1 Deficiency
In ephrin-B1–deficient animal models, that is, ephrin-B1−/Y (hemizygous ephrin-B1–deficient mice) and ephrin-B1−/− (ephrin-B1–deficient mice) the optic nerve crossing is stronger than normal, in fact the proportion of ipsilaterally projecting retinal ganglion cells is decreased by 31%.14 Here, we report for the misrouting-cVEP the absence of misrouting in the four participants tested and for the misrouting-mfVEP weak evidence of possible local misrouting for less than 6% visual field locations. It is concluded that sizable misrouting is absent in ephrin-B1–deficiency. 
Furthermore, the patterns of mfVEP response signatures are clearly in accordance with a normal gross-retinotopy of the cortical visual field representations. They support a representation of the left and right visual field on opposing hemispheres and a representation of the upper and lower visual fields on opposite banks of the calcarine sulcus. It is concluded that cortical visual field representations in ephrin-B1–deficient patients do not diverge from the general principle of retinotopy. 
Visual Function in Ephrin-B1–Deficient Patients
No fundus abnormalities were evident in the individuals 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 adult participants of the study and not fully tested in the 6-year old. These deficits are in accordance with previous reports on ephrin-B10–hemizygous and –heterozygous participants,22,23 and are also likely related to the observation of amblyopia in the heterozygous adults of the study. Importantly, the VEP findings of the present study clearly argue against optic chiasm malformations or a disruption of the retinotopic representations in the visual cortex as a cause of these binocular vision deficits. Taken together, there are some specific deficits in visual function in ephrin-B1–deficient patients, however, they are not related to gross-misrouting at the optic chiasm. Their cause is unknown and might be due to more subtle alterations in the visual system not accessible to the methods of the present study. 
Mechanisms for Optic Chiasm Formation in Humans and Animal Models
Animal studies in transgenic mice highlight the importance of both ephrin-B1 and ephrin-B2 for axonal pathfinding at the optic chiasm. Remarkably, the complete absence of ephrin-B1 in humans usually leads to a largely normal phenotype. The case of the present study had deficits in binocular vision, which are not the rule for ephrin-B1 hemizygosity, but still normal visual pathways as tested within the scope of the present study. It is concluded that ephrin-B1 itself does not appear to be indispensable for normal nerve routing at the human optic chiasm. In contrast to hemizygosity, haploinsufficiency of ephrin-B1 in heterozygous humans usually leads to the abnormal phenotype typical for CFNS, highlighting the severe consequences of competing ephrin-B1 and ephrin-B1–deficient populations in a cellular mosaic. This however, does not appear to affect optic nerve routing. In conclusion, as neither complete loss nor haploinsufficiency of ephrin-B1combined with cellular interference appear to affect the partial decussation, ephrin-B1 does not appear to be involved in this process in humans. This highlights the fact that there are significant differences in the mechanisms guiding optic chiasm formation in the murine model and humans, as has been suggested previously. Indeed, midline interactions at the optic chiasm are more important in the former. Moreover, the anatomy of the chiasm itself differs. In humans uncrossed axons are confined laterally and do not mix in each hemichiasm, while in rodents and ferrets, retinotopic order is lost in the proximal optic nerve and the two projections from each 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 resolved whether the occurrence of misrouting in these CSNB patients might be related to mild forms of albinism, in particular ocular albinism OA1, as it is also associated with an x-linked locus. On the other hand, enhanced nerve fiber crossing at the optic chiasm in the absence of albinism has been demonstrated for patients with foveal hypoplasia, optic nerve decussation defects, and in some cases anterior segment dysgenesis.46 This syndrome has recently been termed FHONDA, which is associated with recessive mutations in the putative glutamine transporter SLC38A8 located within a 3.1-Mb interval containing 33 genes on chromosome 16q23.3-24.1.63,64 The question of the association of these two conditions with albinism deserves particular attention and is currently under investigation.65 In contrast and importantly, Ephrin-B1 deficiency in humans and CFNS, do not add another exception to the power of the misrouting VEP in detecting albinism. There appears to be no relevant misrouting in the cases reported in the present study, which underlines the high specificity of the misrouting VEP paradigms for the identification of albinism in clinically ambiguous and unresolved cases. 
Limitations and Outlook
A major limitation of the present study arises from the rarity of patients with ephrin-B1 mutations causing functional ephrin-B1 deficiency. Although female patients with their pronounced CFNS-phenotype are rare, they are at least readily recognized. Hemizygous males are also rare, but due to their usually mild phenotype they are commonly only detected via their female descendants. Consequently, only a small group of participants was included. This has a number of consequences for the present study. Firstly, the findings described for ephrin-B1 hemizygosity are based on a single case. However, this is the first report on visual pathways in this condition and it provides clear evidence of the absence of enhanced optic nerve crossing, as it is typically found in albinism, even though the participant had deficits in binocular visual function. Secondly, the findings for ephrin-B1 heterozygosity are based on only three cases. It must be noted, however, that they provide concurrent evidence of the absence of gross misrouting and of only very minor local misrouting, if any, in this condition. Thirdly, the study was limited to ophthalmologic and noninvasive electrophysiological investigations, but did not make use of MRI and fMRI as the participants did not fulfil the respective inclusion criteria. Therefore, only physiological evidence from retinal and cortical signals, as provided by ERGs, cVEP, and mfVEPs was available. Clearly, these methods allowed for the assessment of the integrity of retina and optic nerve routing and to some extent of retinotopic features of the cortical representations. However, detailed cortical retinotopic maps and signals from subcortical projection targets (e.g., the lateral geniculate nucleus and the superior colliculus), and brain anatomy are the domain of MRI and fMRI. Therefore, future studies should, besides increasing the sample size for the presented findings, aim at MRI and fMRI investigations to complement the present description of the visual system in human ephrin-B1 deficiencies at the cortical and subcortical level. This is of great promise to resolve the discrepancy between deficits specifically of binocular visual function and the reported largely normal visual pathways in human ephrin-deficiency. 
Acknowledgments
The authors thank the study participants for their support. 
Supported by grants from the German Research Foundation (DFG; HO2002/10-2; Germany) 
Disclosure: M.B. Hoffmann, None; H. Thieme, None; K. Liedecke, None; S. Meltendorf, None; M. Zenker, None; I. Wieland, None 
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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).
Figure 1
 
Fundus photographs and OCT scans for the left (LE) and right eye (RE) of P1 (hemizygous male) and P2 (heterozygous female patient).
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.
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.
Figure 3
 
Individual interhemispheric activation-difference cVEPs for the ephrin-B1–deficient patients for three check sizes (2.0°, 1.0°, and 0.5°, as indicated by the icons) as obtained for the right (black traces) and left eye (gray traces). Parallel traces were evident for all patients.
Figure 3
 
Individual interhemispheric activation-difference cVEPs for the ephrin-B1–deficient patients for three check sizes (2.0°, 1.0°, and 0.5°, as indicated by the icons) as obtained for the right (black traces) and left eye (gray traces). Parallel traces were evident for all patients.
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 (2.0°, 1.0°, and 0.5°). 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.
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 (2.0°, 1.0°, and 0.5°). 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.
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 ‘+'. 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.
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 ‘+'. 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.
Figure 6
 
Individual r value distributions for controls (n = 10) and P1, P2, and P3. The frequency of r values less than 1.00, 0.75, 0.50, 0.00, −0.50, and −0.75 is given. Although the distribution is shifted to the right for all ephrin-B1–deficient patients, the frequency of clearly negative coefficients (<−0.50) does not exceed 6%.
Figure 6
 
Individual r value distributions for controls (n = 10) and P1, P2, and P3. The frequency of r values less than 1.00, 0.75, 0.50, 0.00, −0.50, and −0.75 is given. Although the distribution is shifted to the right for all ephrin-B1–deficient patients, the frequency of clearly negative coefficients (<−0.50) does not exceed 6%.
Table
 
Characteristics of Ephrin-B1 Deficient Participants
Table
 
Characteristics of Ephrin-B1 Deficient Participants
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